LILRB4在免疫中的功能与作用

目的 单细胞转录组层面解析T细胞大颗粒淋巴细胞白血病（T-LGLL）免疫谱系变化，探索其致病机制。 方法 收集2019年6月至2020年12月中国医学科学院血液病医院收治的5例T-LGLL患者治疗前后及3名健康志愿者外周血，应用10×Genomics技术进行单细胞转录组建库测序，比较患者与健康志愿者的免疫细胞差异基因并进行通路富集。 结果 通过对67 237个免疫细胞分析，发现T-LGLL患者疾病状态下：①效应CD8+ T细胞数量增多、细胞毒性及增殖能力增强，免疫抑制治疗有效后效应CD8+ T细胞增殖能力及效应功能均下降（P<0.05）；②调节性T（Treg）细胞比例减少且凋亡增加，免疫抑制治疗有效后Treg细胞比例升高，凋亡通路下调（P<0.05）；③抗原提呈细胞（APC）功能增强，单核细胞、树突状细胞均可富集到抗原合成与呈递通路，B细胞抗原结合能力增强，富集到与T细胞活化相关通路（P<0.05）；④自然杀伤（NK）细胞的毒性杀伤功能减弱，但对T细胞的调节能力增强（P<0.05）。 结论 T-LGLL患者存在特征性免疫谱系，表现为免疫稳态的失衡，突出的特征为效应CD8+ T细胞异常活化伴数量增加，Treg细胞数量减少且功能失调；APC及NK细胞正向调控T淋巴细胞激活、分化和增殖。

酪氨酸磷酸化在T细胞的信号转导过程中发挥着重要的作用,然而其丰度较低,鉴定困难。生物组织样本中分离获得的原代T细胞数量较少,培养和扩增的难度较大,因而常使用永生化细胞系来研究T细胞的酪氨酸磷酸化介导的信号转导过程,这通常会导致获得与原代T细胞相差较大的结论。因此,本研究发展了一种解析原代T细胞酪氨酸磷酸化修饰信号的高灵敏度的蛋白质组学方法。首先,针对原代T细胞数量有限的问题,本研究优化了一套从小鼠脾脏中分离、活化和扩增T细胞的完整流程,第4天时T细胞数量扩增到7倍以上;其次,针对酪氨酸磷酸化修饰丰度较低、不易被质谱检测的难题,本研究利用SH2超亲体(SH2-superbinder)亲和富集和固定化钛离子亲和色谱(Ti4+-IMAC)技术对抗CD3和抗CD28单克隆抗体共刺激条件下的原代T细胞多肽样品进行了酪氨酸磷酸化多肽富集,并结合纳升液相色谱-串联质谱(nanoLC-MS/MS)进行解析。最终成功从1 mg蛋白质中鉴定到282个酪氨酸磷酸化位点,其中包含T细胞受体膜蛋白CD3胞内区的免疫受体酪氨酸激活序列(ITAM)上的多个酪氨酸磷酸化位点,以及信号转导相关蛋白ZAP70、LAT、VAV1等的重要位点信息。综上,本研究发展了一套深度解析原代T细胞中的酪氨酸磷酸化修饰的高灵敏度蛋白质组学分析流程,有望应用于绘制更接近生理状态下的信号转导网络。

Immune checkpoint blockade therapy has been successful in treating some types of cancer but has not shown clinical benefits for treating leukaemia1. This result suggests that leukaemia uses unique mechanisms to evade this therapy. Certain immune inhibitory receptors that are expressed by normal immune cells are also present on leukaemia cells. Whether these receptors can initiate immune-related primary signalling in tumour cells remains unknown. Here we use mouse models and human cells to show that LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor and a marker of monocytic leukaemia, supports tumour cell infiltration into tissues and suppresses T cell activity via a signalling pathway that involves APOE, LILRB4, SHP-2, uPAR and ARG1 in acute myeloid leukaemia (AML) cells. Deletion of LILRB4 or the use of antibodies to block LILRB4 signalling impeded AML development. Thus, LILRB4 orchestrates tumour invasion pathways in monocytic leukaemia cells by creating an immunosuppressive microenvironment. LILRB4 represents a compelling target for the treatment of monocytic AML. The receptor LILRB4 on monocytic leukaemia cells suppresses T cell activity and support the infiltration of tumour cells into tissues.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells that accumulate in the tumor microenvironment (TME). MDSCs have been shown to dampen antitumor immune responses and promote tumor growth; however, the mechanisms of MDSC induction and their role in promoting immune suppression in cancer remain poorly understood. Here, we characterized the phenotype and function of monocytic MDSCs (M-MDSC) generated by coculture of human peripheral blood mononuclear cells with SK-MEL-5 cancer cells in vitro. We selected the SK-MEL-5 human melanoma cell line to generate M-MDSCs because these cells form subcutaneous tumors rich in myeloid cells in humanized mice. M-MDSCs generated via SK-MEL-5 coculture expressed low levels of human leukocyte antigen (HLA)-DR, high levels of CD33 and CD11b, and suppressed both CD8+ T-cell proliferation and IFNγ secretion. M-MDSCs also expressed higher levels of immunoglobulin-like transcript 3 (ILT3, also known as LILRB4) and immunoglobulin-like transcript 4 (ILT4, also known as LILRB2) on the cell surface compared with monocytes. Therefore, we investigated how ILT3 targeting could modulate M-MDSC cell function. Treatment with an anti-ILT3 antibody impaired the acquisition of the M-MDSC suppressor phenotype and reduced the capacity of M-MDSCs to cause T-cell suppression. Finally, in combination with anti-programmed cell death protein 1 (PD1), ILT3 blockade enhanced T-cell activation as assessed by IFNγ secretion. Implications: These results suggest that ILT3 expressed on M-MDSCs has a role in inducing immunosuppression in cancer and that antagonism of ILT3 may be useful to reverse the immunosuppressive function of M-MDSCs and enhance the efficacy of immune checkpoint inhibitors.

Tumor-associated myeloid cells such as myeloid-derived suppressor cells (MDSC) accumulate in the tumor microenvironment (TME) and suppress anti-tumor immune responses in a broad range of cancers. LILRB4 (Leukocyte Immunoglobulin-Like Receptor Subfamily B Member 4), an emerging myeloid target, is specifically expressed in tumor-associated myeloid cells and suppresses the immune response, thus promoting cancer progression and metastasis. ABL407 is a First-in-Class bispecific antibody designed to simultaneously target LILRB4 and 4-1BB, enabling LILRB4 expression-dependent 4-1BB activation. By antagonizing LILRB4 signaling, ABL407 inhibited LILRB4 mediated immunosuppression, restoring T cell activity. In addition, ABL407 inhibited fibronectin, a potential LILRB4 ligand, -mediated suppression of myeloid cells. To evaluate the efficacy of ABL407, we established EL4 murine tumor model overexpressing LILRB4 (EL4/LILRB4) in h4-1BB transgenic mice and hLILRB1/4-h4-1BB triple transgenic mice. We observed LILRB4 was overexpressed in M-MDSC in the blood and liver-metastasized tumors. A significant decrease of M-MDSC population and tumor growth inhibition was observed in mice treated with anti-LILRB4 antibody and ABL407. ABL407 treatment resulted in a proportion increment of T cells. However, the percentage of Treg cells and M-MDSC in the liver and blood was reduced by ABL407 treatment in hLILRB1/4-h4-1BB triple transgenic mice. Mice with complete remission (CR) were further protected from the rechallenge of previously exposed tumor after 3 months of cessation of ABL407 treatment, implicating that immunological memory might be generated. In conclusion, our findings suggest that ABL407, LILRB4x4-1BB bispecific antibody, may be a promising strategy for the treatment of cancer. It can be applied as a "LILRB4 antagonist" as well as a "LILRB4-dependent 4-1BB agonist" across a broad spectrum of cancers. Citation Format: Minkyu Seon, Yangsoon Lee, Hyunseong Youn, Yelim Park, Sora Kim, Kyungjin Park, Jonghwa Won. ABL407, a LILRB4x4-1BB bispecific antibody with a wild type Fc, exhibits potent antitumor activity by modulating immune system in multiple ways involving T cells, myeloids, and regulatory T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5304.

Abstract LILRB4 is expressed in AML M4/M5 cells and negatively regulates immune cell activation via T-cell suppression. Its expression and role in chronic myelomonocytic leukemia (CMML) and myelodysplastic syndrome (MDS) are unknown. We investigated LILRB4 expression in 19 CMML and 27 MDS patients and correlated it with response to subsequent hypomethylating agent (HMA) therapy. LILRB4 RNA expression was increased in CMML patients when compared to MDS patients and healthy controls (q < 0.1) and slightly increased in patients who responded to HMAs (q > 0.1). Pathway analysis revealed upregulation of PD-1 signaling, CTLA-4 signaling, and inflammatory response, and gene correlates were positively associated with CTLA-4 expression. Given current modest results with immunotherapy in myeloid malignancies, further investigation of LILRB4 as an immune checkpoint inhibitor target is needed. With the positive correlation between LILRB4 and CTLA-4 expression, combining anti-LILRB4 and anti-CTLA-4 agents may be a novel therapeutic approach in myeloid malignancies that warrants larger studies.

Background: ILT3 (LILRB4) is expressed in various suppressive myeloid cells including tumor associated macrophages (TAM), myeloid derived suppressor cells (MDSCs) and tolerogenic dendritic cells (DCtol). The binding of ILT3 to its ligands induces an immunosuppressive phenotype in myeloid cells, mediates the inhibition of T cells activity and generates an immunosuppressive tumor microenvironment (TME) which supports tumor growth. The inhibition of ILT3 can restore the anti-tumor activity of myeloid cells and T cells, and thus remodulate the TME from immunosuppressive to pro-inflammatory. Here we describe preclinical characterization of BND-35, a humanized IgG4, ILT3 antagonist antibody developed for the treatment of solid tumors. Methods: BND-35 binding to ILT3 was evaluated by flow cytometry, ELISA and surface plasmon resonance. We investigated the ability of BND-35 to block ILT3 interactions with APOE and fibronectin, to enhance the pro inflammatory activity of various myeloid cells and reverse ILT3-mediated immune suppression of T cells by different suppressive myeloid cells using ELISA and in vitro and ex vivo cell-based assays. The anti-tumor activity of BND-35 was also evaluated in vivo in hILT3 transgenic mouse tumor models as well as in tumoroid systems generated from cancer patients. Results: BND-35 binds ILT3, but not other ILT-family receptors, with low nanomolar affinity and blocks its interaction with APOE and fibronectin in a concentration-dependent manner. ILT3 blockade with BND-35 restored the pro-inflammatory activity of FcR-stimulated DCs and THP1 cells inhibited by fibronectin. BND-35 was also shown to restore an M1 phenotype in cancer patients’ derived monocytes differentiated in the presence of autologous tumor cells. In addition, BND-35 restored T cell activity (both, CD4 and CD8 T cells) inhibited by either DCtol, MDSCs or M2 cells as a single agent and in combination with anti PD-1. BND-35 enhanced immune activity in a unique system of patient-derived tumoroids as evidenced by the secretion of pro-inflammatory cytokines. In vivo, blocking ILT3 activity with BND-35 resulted in decreased tumor growth and induced a pro-inflammatory phenotype in tumor resident T cells and myeloid cells populations as a single agent and in combination with anti PD-1. Conclusions: BND-35 is an anti-ILT3 antagonist antibody that was shown to induce potent pro inflammatory activity of myeloid cells and enhance T cells activity inhibited by ILT3 expressing myeloid suppressive cells in multiple in vitro, ex vivo and in vivo models. By doing so, BND-35 can lead to TME remodelling from immunosuppressive to proinflammatory. Safety, tolerability, and anti-tumor activity of BND-35 will be explored in a first-in-human clinical trial in cancer patients with solid tumors. Citation Format: Tsuri Peretz, Yoav Pizem, Liat Iancovici, Ella Peled, Motti Hakim, Sharon Hashmueli, Ilana Mandel, Yair Sapir, Tehila Ben Moshe. BND-35, a novel anti-ILT3 antibody for remodulation of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3920.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of clonal malignant B cells and heterogeneous clinical outcomes. This study explored the role of the immune checkpoint receptor LILRB4 (ILT3), previously implicated in acute myeloid leukemia, in the pathogenesis and progression of CLL. Analysis of 126 CLL patient samples revealed aberrantly elevated LILRB4 expression on leukemic B cells, with higher levels observed in advanced disease stages. Elevated LILRB4 expression was correlated with shorter time-to-treatment intervals and reduced overall survival, highlighting its potential as a prognostic marker. Mechanistically, LILRB4 was enriched in a proliferative leukemic subpopulation driven by the TLR9 and BTK signaling pathways. Functional assays demonstrated that LILRB4 promotes immune evasion by suppressing T and NK cell-mediated cytotoxicity, while its blockade restored immune effector activity and impaired leukemic cell migration. Furthermore, elevated levels of soluble LILRB4 (sLILRB4) in patient serum are associated with poor clinical outcomes, including an increased incidence of secondary malignancies. Receiver operating characteristic (ROC) analysis demonstrated the high sensitivity and specificity of sLILRB4 as a biomarker for disease progression. Collectively, these findings identify membrane-bound LILRB4 as a mediator of immune suppression and disease progression in CLL, while highlighting sLILRB4 as a potential biomarker associated with poor clinical outcomes. Further investigations into LILRB4-directed therapies could pave the way for improved patient stratification and novel treatment strategies in CLL patients.

Natural killer (NK) cells play pivotal roles in innate immunity as well as in anti-tumor responses via natural killing, while their activity is tightly regulated by cell-surface inhibitory receptors. Immunoglobulin (Ig)-like transcript 3/leukocyte Ig-like receptor B4 (ILT3/LILRB4, also known as gp49B in mice) is an inhibitory receptor expressed on activated NK cells as well as myeloid-lineage cells. The common physiologic ligand of human LILRB4 and gp49B is identified very recently as fibronectin (FN), particularly the N-terminal 30 kDa domain (FN30). We hypothesized that LILRB4 could bind FN on target cells in trans together with integrin, a classical FN receptor, in cis and deliver an inhibitory signal in NK cells, leading to attenuated natural killing. Flow cytometric and confocal microscopic analyses of NK cell-surface gp49B and integrin suggested that these novel and classical FN receptors, respectively, co-engage FN immobilized on a culture plate. Biochemical analyses indicated that tyrosine phosphorylation of spleen tyrosine kinase was augmented in gp49B-deficient NK cells upon binding to the immobilized FN. While surface FN-poor YAC-1 cells were evenly sensitive as to natural killing of both gp49B-positive and -negative NK cells, the killing of FN-rich Lewis lung carcinoma cells, but not the FN30-knockout cells, was augmented among gp49B-deficient NK cells. These results suggest that the natural cytotoxicity of NK cells is negatively regulated through LILRB4/gp49B sensing FN on target cells, which sheds light on the unexpected role of LILRB4 and FN as a potential attenuator of NK cell cytotoxicity in the tumor microenvironment.

Immunoglobulin-like transcript (ILT) 3 is an immunosuppressive molecule that negatively regulates myeloid cell activation. ILT3 overexpression in tumor cells induces immune escape of solid tumors and facilitates invasion of monocytic acute myelocytic leukemia cells. However, the expression and function of ILT3 in non-small cell lung cancer (NSCLC) cells remain elusive. Herein, we found that ILT3 was enriched in human NSCLC cells, and predicted advanced disease and poor overall survival. ILT3 overexpression enhanced the migration and invasion of NSCLC cells and tubule formation of human umbilical vein endothelial cells by upregulating and interacting with its ligand apolipoprotein E (ApoE) in vitro. Mechanistically, ILT3 recruited SHP2 and SHIP1, and subsequently activated ERK1/2 signaling mediating epithelial-mesenchymal transition (EMT) and increasing vascular endothelial growth factor (VEGF)-A expression in NSCLC cells, which are responsible for tumor cell motility and angiogenesis, respectively. Using murine metastasis models, we further confirmed ILT3 promoted NSCLC metastasis and explored the exact correlation of ILT3 with ApoE, EMT, and VEGF-A in vivo. These results unraveled novel mechanisms for ILT3-induced tumor progression and proposed ILT3 as a potential therapeutic target and prognostic biomarker for NSCLC patients.

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e14647 Background: The low response of immune checkpoint inhibitors against PD-1/PD-L1 in cancer patients calls for further development of new immune targets. Immunoglobulin-like transcript (ILT)3 and its ligand ApoE have been demonstrated to be highly expressed in a variety of malignant tumor cells and involved in tumor biological behaviors, including invasion and angiogenesis. ILT3 targeting drugs are undergoing increasing preclinical and clinical trials. However, the role of tumor-derived ILT3 in immune regulation is unclear. The aim of this study was to investigate the immunomodulatory effect of tumor-derived ILT3 and its immunotherapy therapeutic efficacy that combination with PD-L1 blockade in NSCLC. Methods: The correlations between ILT3/ApoE expression and prognosis of NSCLC patients were analyzed basing on public databases. Multiplex immunofluorescence was performed to analyze the correlations between tumor-derived ILT3/ApoE expression and intratumoral immune cells infiltration in human adenocarcinoma tissues. The impact of ILT3 and ApoE on tumor-associated macrophage (TAM) recruitment and polarization were evaluated by transwell migration assay, flow cytometry, and RT-PCR, while their direct impact on T cell survival and cytotoxicity was detected by flow cytometry. Using lung cancer xenograft murine models, we further confirmed the pro-tumoural activity of tumor-derived gp49B and analyzed its correlations with immune cells in vivo. Tumor immunotherapy models targeting at gp49B and/or PD-L1 were established in C57BL/6 mice inoculated with LLC cells. Gp49b inhibition was implemented by infection of specific knockdown lentivirus and PD-L1 was blocked using mouse neutralizing antibodies. Flow cytometry was performed to detect the frequency and phenotype of macrophages and T cells in the tumors, spleen, and blood of the mice. Results: Patients with high expression of ILT3 had significantly worse prognosis in LUAD, but not in LUSC. In vitro assays demonstrated that tumor-derived ILT3/ApoE promoted recruitment and M2-like polarization of TAMs and directly inhibited the proliferation and cytotoxicity of T cells, which had been further confirmed in vivo models. Moreover, ILT3 (gp49B) inhibition enhanced anti-tumor immunity and suppressed tumor progression by counteracting TAM- and dysfunctional T cell- induced immunosuppressive TME; the combined inhibition of gp49B and PD-L1 showed the most dramatic tumor retraction in LLC xenograft models. Conclusions: Tumor-derived ILT3 overexpression suppressed anti-tumor immunity by recruiting M2-like TAMs and directly impairing T cell activities, while ILT3 inhibition counteracted above immunosuppression and potentiated the efficacy of PD-L1 blockade in LUAD. Our study verified that ILT3 may be a promising novel immunotherapeutic target for LUAD patients.

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Immunoglobulin-like transcript 3 (ILT3) is an inhibitory receptor that is expressed by mature monocytes, dendritic cells (DC), plasma blasts, and the malignant cells arising thereof including acute myeloid leukemia (AML), B cell lymphoma and multiple myeloma (MM). We developed an anti-ILT3 blocking monoclonal antibody (mAb) 17G8 to test the hypothesis that blockade of ILT3 on hematopoietic malignant cells might drive anti-cancer responses through several mechanisms such as cell migration, ADCC, and T cell driven immune responses. Blockade of ILT3 using 17G8 without Fc effector function impaired AML cell line THP1 dissemination into distant sites in vivo and using 17G8 with enhanced Fc function through afucosylation further reduced the tumor burden. This effect of afucosylated 17G8 appears to be mediated by a mechanism other than ADCC, since afucosylated 17G8 showed modest tumor inhibition in ICR mice carrying ILT3 positive AML and MM xenografts. In immune competent mice carrying murine syngeneic AML cell C1498 expressing human ILT3, 17G8 also showed modest anti-tumor effects as a single agent, however, when combined with anti-PD1, afucosylated 17G8 increased CD8+ effector memory T cells, reduced proportion of cells with an exhausted phenotype and significantly improved survival when compared to different controls. Since T cells don’t express ILT3, the effects of 17G8 on T cells are hypothesized to be mediated by blocking ILT3 expressed on myeloid cells. The expression of ILT3 on immature human monocyte-derived DC (moDC) is higher than on mature moDC. Blocking ILT3 with afucosylated 17G8 on immature moDC potentiated maturation of moDC when compared to the controls in vitro. Surprisingly, afucosylated 17G8 stimulated IFNγ production from moDC during maturation. The enhanced DC function induced by afucosylated 17G8 was subsequently found to be translated into enhanced T cell function in an allogeneic mixed lymphocyte reaction (allo-MLR) assay, in which afucosylated 17G8 significantly increased IFNγ production when combined with anti-PD1. The activation of effector T cells was also monitored through a cytotoxicity assay by mixing THP-1 cells with human PBMC in culture. Afucosylated 17G8 induced strong cytotoxicity on THP-1 cells with minimal effects on normal ILT3 positive cells such as monocytes or moDC, suggesting that blockade of ILT3 with afucosylated 17G8 induced more potent T cell mediated allogeneic cytotoxicity than innate cell mediated ADCC killing. In summary, ILT3 is a highly selective M4/M5 AML target. Developing a blocking antibody with enhanced Fc function could add an additional therapeutic strategy of inducing host anti-tumor immunity to current AML treatments which induce direct tumor killing, such as chemotherapy, targeted therapy, and antibody drug conjugate. Citation Format: Shiming Ye, Dong Zhang, Min-Zu Wu, Harini Raghu, Diane Cohen, Mally Romero, Siu-Sze Tan, Donghee Choi, Jonathan Hickson, John Engelhardt, Alex Shoemaker. Blocking inhibitory receptor ILT3 by an antibody with enhanced Fc function promoted adaptive immunity against hematopoietic malignancy independent of ADCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1363.

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In this study, the fibronectin–ILT3 interaction is shown to represent a “stromal checkpoint” through which the extracellular matrix actively suppresses tumor-associated myeloid cells. Therapeutics targeting this interaction can potentially reprogram suppressive myeloid cells, increasing antitumor immune responses. Suppressive myeloid cells inhibit antitumor immunity by preventing T-cell responses. Immunoglobulin-like transcript 3 (ILT3; also known as LILRB4) is highly expressed on tumor-associated myeloid cells and promotes their suppressive phenotype. However, the ligand that engages ILT3 within the tumor microenvironment and renders tumor-associated myeloid cells suppressive is unknown. Using a screening approach, we identified fibronectin as a functional ligand for ILT3. The interaction of fibronectin with ILT3 polarized myeloid cells toward a suppressive state, and these effects were reversed with an ILT3-specific antibody that blocked the interaction of ILT3 with fibronectin. Furthermore, ex vivo treatment of human tumor explants with anti-ILT3 reprogrammed tumor-associated myeloid cells toward a stimulatory phenotype. Thus, the ILT3–fibronectin interaction represents a “stromal checkpoint” through which the extracellular matrix actively suppresses myeloid cells. By blocking this interaction, tumor-associated myeloid cells may acquire a stimulatory phenotype, potentially resulting in increased antitumor T-cell responses.

Extracellular matrix (ECM) is the basis for virtually whole cellular processes and is also related to tumor metastasis. Fibronectin (FN), a major ECM macromolecule expressed by different cell types and also present in plasma, consists of multiple functional modules that bind to ECM-associated, plasma, and cell-surface proteins such as integrin and FN itself, thus ensuring its cell-adhesive and modulatory role. Here we show that FN constitutes an immune checkpoint. Thus, FN was identified as a physiological ligand for a tumor/leukemia/lymphoma- as well as autoimmune-associated checkpoint, ILT3/LILRB4 (B4, CD85k). Human B4 and the murine ortholog, gp49B, bound FN with sub-micromolar affinities as assessed by bio-layer interferometry. The major B4-binding site in FN was located at the N-terminal 30-kDa module (FN30), which is apart from the major integrin-binding site present at middle of the molecule. Blockade of B4-FN binding such as with B4 antibodies or a recombinant FN30-Fc fusion protein paradoxically ameliorated autoimmune disease in lupus-prone BXSB/Yaa mice. These unexpected nature of the B4-FN checkpoint in autoimmunity is discussed, referring to its potential role in tumor immunity.

Immunoglobulin like transcript 3 (ILT3) was previously identified as an inhibitory receptor to induce T cell anergy in tranplantation, autoimmunity and allergy. Here we aimed to investigate the expression of ILT3 in colorectal cancer, analyze the association between ILT3 expression and clinicopathological variables and prognosis, and evaluate the correlation between the expression of ILT3 and CD45RO+ T cells density. Expression of ILT3 was identified on the cell membrane and/or in the cytoplasm. High expression ILT3 was identified in 55 of 85 (64.7%) tumor specimens, which was significantly higher than that in the adjacent normal tissues(5/30) (P < 0.001). High ILT3 expression was significantly associated with positive lymph node metastasis (N1-2; P = 0.03), advanced disease (stage III-IV; P = 0.03), and reduced OS in patients. The ILT3 expression level was an independent prognostic factor (P = 0.004) and inversely correlated with the number of CD45RO+ T cells (P = 0.019). In the present study, high ILT3 expression was observed in colorectal cancer and inversely associated with CD45RO+ T cells density and prognosis, suggesting that ILT3 played an important role in tumor progression by possible influence on CD45RO+ T cells in the tumor microenvironment.

Key Points The onset and evolution of EAE in C57BL/6 mice is significantly delayed by ILT3.Fc. ILT3.Fc inhibits T cell production of IL-17 and IFN-γ and migration through BMEC. Deletion of CD8 T cells abrogates the inhibitory effect of ILT3.Fc. Multiple sclerosis (MS) is a chronic autoimmune disease of the CNS that is characterized by demyelination, axonal loss, gliosis, and inflammation. The murine model of MS is the experimental autoimmune encephalopathy (EAE) induced by immunization of mice with myelin oligodendrocyte glycoprotein (MOG)35–55. Ig-like transcript 3 (ILT3) is an inhibitory cell surface receptor expressed by tolerogenic human dendritic cells. In this study, we show that the recombinant human ILT3.Fc protein binds to murine immune cells and inhibits the release of proinflammatory cytokines that cause the neuroinflammatory process that result in paralysis. Administration of ILT3.Fc prevents the rapid evolution of the disease in C57BL/6 mice and is associated with a profound reduction of proliferation of MOG35–55–specific Th1 and Th17 cells. Inhibition of IFN-γ and IL-17A in mice treated with ILT3.Fc is associated with delayed time of onset of the disease and its evolution to a peak clinical score. Neuropathological analysis shows a reduction in inflammatory infiltrates and demyelinated areas in the brains and spinal cords of treated mice. These results indicate that inhibition of Th1 and Th17 development provides effective suppression of EAE and suggests the feasibility of a clinical approach based on the use of ILT3.Fc for treatment of MS. Furthermore, our results open the way to further studies on the effect of the human ILT3.Fc protein in murine experimental models of autoimmunity and cancer.

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BACKGROUND Kawasaki disease (KD) is an acute, systemic vasculitis syndrome that occurs in children. The clinical symptoms and epidemiological features of KD strongly suggest that KD is triggered by unidentified infectious agents in genetically predisposed patients. In addition, a number of studies have described the role of B cells in the development of KD. OBJECTIVE To obtain a mechanistic insight into the humoral immune response of B-lineage cells in KD patients, we examined peripheral blood antibody secreting cells (ASCs) and inhibitory immunoreceptors, ILT/LILR, on each B cell subpopulation. METHODS Eighteen Japanese KD patients and thirteen healthy control subjects were recruited for this study. Their peripheral blood mononuclear cells (PBMCs) were examined by flow cytometry for the number of CD19 B cells, the size of each B cell subset, and the expression of the inhibitory isoforms of ILT/LILR on the B cell subset. RESULTS The frequency of CD19CD27 ASCs, was significantly increased in the acute-phase of KD and reduced after high-dose intravenous immunoglobulin (IVIG) treatment. Interestingly, while ILT2/LILRB1 expression was ubiquitously observed on every B cell/ASCs subset and the level was not significantly different after IVIG, ILT3/LILRB4 (B4) was uniquely expressed on only ASCs, and its expression was significantly decreased after IVIG. CONCLUSION In the acute-phase of KD, the frequency of ASCs is high with augmented B4 expression, whereas it is lower with decreased B4 expression after IVIG. Further studies of B4 expression on ASCs in autoimmune and infectious diseases will be needed to confirm the significance of our findings.

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Immune inhibitory receptors play an important role in organ transplantation, autoimmune diseases and cancers. Immunoglobulin-like transcript (ILT)2 and ILT3 belong to the inhibitory receptors of the ILT family, which have been reported to regulate a broad range of cellular functions involved in the immune response. They contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which are related to immune regulation. Although ILT receptors have been studied in dendritic cells (DCs), T cells, NK cells and other cell types, the expression and clinical significance of ILT2 and ILT3 in gastric cancer have yet to be elucidated. Here, the expression of ILT2 and ILT3 in gastric cancer cell lines and pathologic tissues, as well as their effects on the cytotoxicity of NK92MI against the gastric cancer cell lines MKNI with ILT2lowILT3low and HGC-27 with ILT2highILT3high were detected. The results suggest that ILT2 and ILT3 are expressed with diverse degrees in gastric cancer cells and tissues, and the expression of ILT2 is related with differentiation and size of tumors. Furthermore, the cytotoxic activity of NK92MI against the MKNI cell line was stronger than that against HGC-27. This study indicates that ILT2 and ILT3 play a key role in gastric cancer immune escape, and ILT2 may be a new target in the clinical diagnosis and treatment of gastric cancer.

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The myeloid inhibitory receptor LILRB4 (also called ILT3, LIR-5, CD85k), a member of the leukocyte immunoglobulin-like receptors (LILRs/LIRs), is an important mediator of immune tolerance. Up-regulated on tolerogenic dendritic cells, it has been shown to modulate immune responses via induction of T cell anergy and differentiation of CD8+ T suppressor cells and may play a role in establishing immune tolerance in cancer. Consequently, characterizing the molecular mechanisms involved in LILRB4 function and in particular its structure and ligands is a key aim but has remained elusive to date. Here we describe the production, crystallization, and structure of the LILRB4 ectodomain to 1.7 Å using an expression strategy involving engineering of an additional disulfide bond in the D2 domain to enhance protein stability. LILRB4 comprises two immunoglobulin domains similar in structure to other LILRs; however, the D2 domain, which is most closely related to the D4 domains of other family members, contains 310 helices not previously observed. At the D1-D2 interface, reduced interdomain contacts resulted in an obtuse interdomain angle of ∼107°. Comparison with MHC class I binding Group 1 LILRs suggests LILRB4 is both conformationally and electrostatically unsuited to MHC ligation, consistent with LILRB4 status as a Group 2 LILR likely to bind novel non-MHC class I ligands. Finally, examination of the LILRB4 surface highlighted distinctive surface patches on the D1 domain and D1D2 hinge region, which may be involved in ligand binding. These findings will facilitate our attempts to precisely define the role of LILRB4 in the regulation of immune tolerance.

No abstract available

Leukocyte immunoglobulin like receptor B4 (LILRB4) was considered to promote tumor progression and immunosuppression in various malignancies. As a murine homolog of LILRB4, gp49B has been employed in numerous mouse models to investigate the immunosuppressive properties of LILRB4. However, gp49B differs significantly from LILRB4 in its amino acid sequence and intracellular domains. In this study, we developed a conditional mouse model that overexpresses LILRB4 specifically in myeloid cells to investigate its effects on solid tumors and hematological malignancies. Our results showed that the physiological structure and overall immune system of LILRB4L/L; Cre mice were normal. LL2 tumors in LILRB4L/L; Cre mice exhibited increased size and weight, with elevated levels of immunosuppressive markers programmed cell death protein 1 (PD-1) and T cell immunoglobulin and mucin-domain containing-3 (TIM-3) on infiltrating CD3+ T cells, alongside a shift in tumor-associated macrophages (TAMs) from M1-type to M2-type. In the C1498 model, LILRB4 overexpression promoted tumor progression and metastasis, evidenced by increased bioluminescence and enhanced infiltration of monocytic myeloid-derived suppressor cells (M-MDSCs). Real-time PCR analysis showed upregulation of immunosuppressive mRNAs, including colony-stimulating factor 1 (CSF1), arginase1 (Arg1), macrophage galactose N-acetyl-galactosamine specific lectin 2 (Mgl2) and interleukin-1β (IL-1β) while downregulating pro-inflammatory markers like nitric oxide synthase 2 (Nos2). These findings indicate that LILRB4 fosters an immunosuppressive microenvironment that supports tumor progression. LILRB4L/L; Cre mice may serve as a promising tool for studying targeted LILRB4 tumor immunotherapy.

Multiple myeloma (MM) remains an incurable hematologic malignancy. Despite tremendous advances in the treatment of this disease, about 10% of patients still have very poor outcomes with a median overall survival of less than 24 months. Our study aimed to underscore the critical mechanisms pertaining to rapid disease progression and provide novel therapeutic choices for these ultrahigh-risk patients. We utilized single-cell transcriptomic sequencing to dissect the characteristic bone marrow niche of patients who survived less than 2 years (EM24). Notably, enrichment of a LILRB4high pre-mature plasma-cell cluster was observed in EM24 patients compared to patients with durable remission. This cluster exhibited aggressive proliferation and a drug-resistance phenotype. High levels of LILRB4 promoted MM clonogenicity and progression. Clinically, high expression of LILRB4 was correlated with poor prognosis in both newly diagnosed MM patients and relapsed/ refractory MM patients. ATAC-sequencing analysis identified that pronounced chromosomal accessibility caused the elevation of LILRB4 on MM cells. CRISPR-Cas9 deletion of LILRB4 alleviated the growth of MM cells, inhibited the immunosuppressive function of myeloid-derived suppressive cells (MDSC), and further rescued T-cell dysfunction in the MM microenvironment. Greater infiltration of MDSC was observed in EM24 patients. We therefore generated an innovative T-cell receptor-based chimeric antigen receptor T cell, LILRB4-STAR-T. Cytotoxicity experiments demonstrated that LILRB4-STAR-T cells efficaciously eliminated tumor cells and impeded MDSC function. In conclusion, our study elucidates that LILRB4 is an ideal biomarker and promising immunotherapy target for high-risk MM. LILRB4-STAR-T-cell immunotherapy is promising against both tumor cells and the immunosuppressive tumor microenvironment in MM.

Leukocyte immunoglobulin‐like receptor subfamily B member 4 (LILRB4/ILT3) is an up‐and‐coming molecule that promotes immune evasion. We have previously reported that LILRB4 facilitates myeloid‐derived suppressor cells (MDSCs)‐mediated tumor metastasis in mice. This study aimed to investigate the impact of the LILRB4 expression levels on tumor‐infiltrating cells on the prognosis of non‐small cell lung cancer (NSCLC) patients.

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Acute monocytic leukaemia, a subtype of acute myeloid leukaemia (AML), is a highly aggressive malignancy characterised by a poor prognosis, primarily due to the ability of leukaemic cells to evade immune surveillance. In this study, we demonstrate that homoharringtonine (HHT), an FDA‐approved therapeutic agent for chronic myeloid leukaemia (CML), inhibits this immune evasion by targeting the FTO/m6A/LILRB4 signalling pathway in monocytic AML. Utilising RNA sequencing (RNA‐seq) and various functional assays, we reveal that HHT treatment significantly reduces LILRB4 expression at both the RNA and protein levels, suggesting that the effects of HHT on LILRB4 are distinct from its well‐established role as a protein synthesis inhibitor. Mechanistically, HHT treatment markedly increases global levels of RNA m6A in THP‐1 cells by promoting the degradation of FTO, which subsequently diminishes the expression of its downstream targets, MLL1 and LILRB4. Furthermore, in vitro and in vivo analyses employing monocytic AML cell lines, mouse‐derived AML xenograft models, and patient samples collectively support the conclusion that HHT suppresses immune evasion in monocytic AML by reducing LILRB4 expression. Importantly, the downregulation of LILRB4 resulting from HHT treatment enhances the susceptibility of THP‐1 cells to CD8+ T cell cytotoxicity, accompanied by increased markers of immune activation. Overall, our findings position HHT as a promising clinical agent for enhancing CD8+ T cell‐based cancer immunotherapy by mitigating immune evasion in monocytic AML.

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Summary LILRB4 is an immunosuppressive receptor, and its targeting drugs are undergoing multiple preclinical and clinical trials. Currently, the absence of a functional LILRB4 ligand in solid tumors not only limits the strategy of early antibody screening but also leads to the lack of companion diagnostic (CDx) criteria, which is critical to the objective response rate in early-stage clinical trials. Here, we show that galectin-8 (Gal-8) is a high-affinity functional ligand of LILRB4, and its ligation induces M-MDSC by activating STAT3 and inhibiting NF-κB. Significantly, Gal-8, but not APOE, can induce MDSC, and both ligands bind LILRB4 noncompetitively. Gal-8 expression promotes in vivo tumor growth in mice, and the knockout of LILRB4 attenuates tumor growth in this context. Antibodies capable of functionally blocking Gal-8 are able to suppress tumor growth in vivo. These results identify Gal-8 as an MDSC-driving ligand of LILRB4, and they redefine a class of antibodies for solid tumors.

Leukocyte immunoglobulin-like receptor B4 (LILRB4) plays a significant role in regulating immune responses. LILRB4 in microglia might influence the infiltration of peripheral T cells. However, whether and how LILRB4 expression aggravates brain damage after acute ischemic stroke remains unclear. This study investigates the role of LILRB4 in modulating the immune response and its potential protective effects against ischemic brain injury in mice. Microglia-specific LILRB4 conditional knockout (LILRB4-KO) and overexpression transgenic (LILRB4-TG) mice were constructed by a Cre-loxP system. Then, they were used to investigate the role of LILRB4 after ischemic stroke using a transient middle cerebral artery occlusion (tMCAO) mouse model. Spatial transcriptomics analysis revealed increased LILRB4 expression in the ischemic hemisphere. Single-cell RNA sequencing (scRNA-seq) identified microglia-cluster3, an ischemia-associated microglia subcluster with elevated LILRB4 expression in the ischemic brain. Flow cytometry and immunofluorescence staining showed increased CD8+ T cell infiltration into the brain in LILRB4-KO-tMCAO mice. Behavioral tests, cortical perfusion maps, and infarct size measurements indicated that LILRB4-KO-tMCAO mice had more severe functional deficits and larger infarct sizes compared to Control-tMCAO and LILRB4-TG-tMCAO mice. T cell migration assays demonstrated that LILRB4-KD microglia promoted CD8+ T cell recruitment and activation in vitro, which was mitigated by CCL2 inhibition and recombinant arginase-1 addition. The scRNA-seq and spatial transcriptomics identified CCL2 was predominantly secreted from activated microglia/macrophage and increased CCL2 expression in LILRB4-KD microglia, suggesting a chemokine-mediated mechanism of LILRB4. LILRB4 in microglia plays a crucial role in modulating the post-stroke immune response by regulating CD8+ T cell infiltration and activation. Knockout of LILRB4 exacerbates ischemic brain injury by promoting CD8+ T cell recruitment. Overexpression of LILRB4, conversely, offers neuroprotection. These findings highlight the therapeutic potential of targeting LILRB4 and its downstream pathways to mitigate immune-mediated damage in ischemic stroke.

Leukocyte Ig-like receptor B family 4 (LILRB4) as an immune checkpoint on myeloid cells is a potential target for tumor therapy. Extensive osteolytic bone lesion is the most characteristic feature of multiple myeloma. It is unclear whether ectopic LILRB4 on multiple myeloma regulates bone lesion. The conditioned medium (CM) from LILRB4-WT and -KO cells was used to analyze the effects of LILRB4 on osteoclasts and osteoblasts. Xenograft, syngeneic and patient derived xenograft models were constructed, and micro-CT, H&E staining were used to observe the bone lesion. RNA-seq, cytokine array, qPCR, the activity of luciferase, Co-IP and western blotting were used to clarify the mechanism by which LILRB4 mediated bone damage in multiple myeloma. We comprehensively analyzed the expression of LILRB4 in various tumor tissue arrays, and found that LILRB4 was highly expressed in multiple myeloma samples. The patient’s imaging data showed that the higher the expression level of LILRB4, the more serious the bone lesion in patients with multiple myeloma. The conditioned medium from LILRB4-WT not -KO cells could significantly promote the differentiation and maturation of osteoclasts. Xenograft, syngeneic and patient derived xenograft models furtherly confirmed that LILRB4 could mediate bone lesion of multiple myeloma. Next, cytokine array was performed to identify the differentially expressed cytokines, and RELT was identified and regulated by LILRB4. The overexpression or exogenous RELT could regenerate the bone damage in LILRB4-KO cells in vitro and in vivo. The deletion of LILRB4, anti-LILRB4 alone or in combination with bortezomib could significantly delay the progression of bone lesion of multiple myeloma. Our findings indicated that LILRB4 promoted the bone lesion by promoting the differentiation and mature of osteoclasts through secreting RELT, and blocking LILRB4 singling pathway could inhibit the bone lesion.

BACKGROUND Glioblastoma (GBM), the most aggressive primary brain tumor, exhibits a profoundly immunosuppressive tumor microenvironment (TME) dominated by M2-like tumor-associated macrophages (TAMs). The leukocyte immunoglobulin-like receptor B4 (LILRB4) has emerged as a critical immunoregulatory molecule implicated in cancer progression, yet its role in GBM remains poorly understood. This study investigated the expression, prognostic significance, and functional role of LILRB4 in TAM-mediated immunosuppression and GBM progression. METHODS Expression patterns and prognostic significance of LILRB4 were analyzed using TCGA and validation datasets. Single-cell RNA sequencing (scRNA-seq), spatial transcriptomics (ST), and proteogenomics analyses identified cellular sources and spatial distribution of LILRB4. THP-1 cells were differentiated into TAM-like macrophages using PMA and tumor-conditioned medium (TCM) from GBM cells. LILRB4 was silenced using shRNA, and macrophage polarization markers were assessed by RT-qPCR, western blot, and ELISA. RESULTS LILRB4 was identified as an independent prognostic factor in glioma, with high expression correlating with poor survival. Multiple bioinformatics approaches revealed strong LILRB4-M2 macrophage associations. ST revealed predominant LILRB4 expression in macrophage clusters with strongest tumor cell co-occurrence, identifying LAIR1 as a potential receptor. Proteogenomics analysis showed strong LILRB4 protein-mRNA correlations and associations with M2 markers. LILRB4 regulated macrophage polarization through the STAT3/IL10 axis. Knockdown reversed M2-like phenotype toward M1-like state, decreasing CD163, IL10, and TGFB1 while increasing IL1B and TNFA. TCM from LILRB4 knockdown macrophages significantly inhibited GBM cell proliferation. CONCLUSION LILRB4 might functions as a critical regulator of the immunosuppressive TME in GBM by promoting M2 macrophage polarization through the STAT3/IL10 axis. Targeting LILRB4 represents a promising approach for enhancing immunotherapeutic efficacy in GBM.

Leukocyte immunoglobulin-like receptor subfamily B member 4 (LILRB4), an emerging immune checkpoint molecule, exhibits therapeutic potential in acute myeloid leukemia (AML). While single nucleotide polymorphisms (SNPs) of immune checkpoint genes have been extensively investigated in AML, the association between LILRB4 genetic polymorphisms and clinical outcomes remains unexplored. We investigated SNPs within the LILRB4 immunoglobulin domain and immunoreceptor tyrosine-based inhibitory motif (ITIM) regions in 151 AML patients and 203 controls. The rs1048801 G allele was significantly associated with increased LILRB4 mRNA expression, higher disease susceptibility, and reduced overall survival. Functional studies revealed that the G allele enhanced AML cell proliferation and colony formation. Furthermore, protein-protein interaction network analysis identified CD4 as a pivotal downstream mediator of LILRB4. Flow cytometry revealed elevated LILRB4 expression in CD45+ leukocytes and CD45+CD33+CD14+ monocytic AML cells from G allele carriers, concomitant with reduced CD3+CD4+ T cell populations and impaired proliferation. Collectively, these findings establish rs1048801 as a critical modulator of AML progression through LILRB4-mediated CD4+ T cells suppression, providing new insights for personalized therapeutic strategies.

No abstract available

The anti-tumor immune response is greatly hindered by the protumor polarization of tumor-associated macrophages (TAMs). Cancer-related inflammation plays a central role in TAMs protumor polarization. Our study explored the unique positive feedback loop between inflammasome and complement in TAMs. The present study identified NOD-like receptors family pyrin domain containing 12 (NLRP12) formed positive feedback with C1qA and drove TAMs protumor polarization via the LILRB4/NF-κB pathway. In addition, NLRP12 was predominantly expressed in TAMs and was associated with poorer prognosis in lung adenocarcinoma (LUAD) patients. Knocking down LILRB4 inhibited TAMs protumor polarization. NLRP12-overexpressing TAMs promoted tumor cells’ malignant progression and inhibited T cells’ proliferation and cytotoxic function. Lastly, NLRP12 knockout (NLRP12−/−) reversed macrophage polarization, enhanced T-cell anti-tumor immunity, and suppressed tumor growth. Our findings highlighted the essential role of NLRP12/C1qA positive feedback loop and the LILRB4/NF-κB pathway in promoting TAMs protumor polarization. Inhibition of NLRP12 suppressed tumor development and promoted immune response. NLRP12 may be a promising target for LUAD immunotherapy.

Introduction Multiple myeloma (MM) is the second most common hematologic malignancy. Although multiple targets on MM cells are discovered and applied in clinical treatment, relapse is almost inevitable in MM patients. Our previous study found a high-risk cell cluster from MM patients with overall survival less than 2 years by single-cell RNA sequencing (scRNA seq). In this specific cluster, leukocyte immunoglobulin-like receptor B4 (LILRB4) was highly expressed, indicating the critical role of LILRB4 in myelomagenesis and drug resistance. However, the mechanisms of LILRB4 in MM development has not been fully understood. Here, we investigated the role of LILRB4 in tumorigenesis and MM cell proliferation. Methods and Results Our clinical data showed that MM patients with LILRB4 overexpression had poor prognosis and decreased overall survival. Compared with newly-diagnosed MM patients, expression of LILRB4 was higher in the MM patients relapsed after treatment, indicating the significance of LILRB4 in MM progression and drug resistance. In vitro experiment showed that MM cells with LILRB4 overexpression enhanced the cell colony-forming ability and promoted cell proliferation. Conversely, knocking-out LILRB4 induced cell apoptosis. We used MM cells with LILRB4 overexpression to establish myeloma xenograft model and results showed that compared with LILRB4neg MM cells, mice injected with LILRB4high MM cells displayed higher tumor formation rate and lower survival rate. Then, we analyzed RNA-seq data to investigate the mechanisms of LILRB4 in MM proliferation. Transcriptome analysis indicated that overexpression of LILRB4 in MM cell lines activated NF-kB, glycolysis, hypoxia and MTORC1 signaling pathways. Among these, NF-kB pathway has been proved in previous studies. More important, RNA-seq data showed that PIM kinase, correlating with cancer cell proliferation and apoptosis, was significantly upregulated in MM cells with LILRB4 overexpression, which has been confirmed in our in vitro experiments. We also found that LILRB4 upregulated the expression of PIM through activating Src-homology 2-containing protein tyrosine phosphatase (SHP) and signal transducer and activator of transcription (STAT) protein, indicating the underlying mechanism of MM cell proliferation. Interestingly, transcription factors promoting B cell program were upregulated, and the immunophenotypic marker related to B cell differentiation was downregulated after LILRB4 overexpression. These findings indicated that LILRB4 may induce myeloma cell dedifferentiation and promote tumorigenesis. Conclusion In conclusion, LILRB4 is highly associated with poor prognosis of MM patients and has the ability of tumorigenesis. LILRB4 promotes MM cell proliferation by activating SHP and STAT to upregulate the expression of PIM kinase, which plays critical roles in MM pathogenesis.

Recent advancements in the treatment of multiple myeloma (MM), including immunomodulatory agents, monoclonal antibodies, and T cell-redirecting therapies, have significantly improved patient outcomes. However, MM remains incurable. The interaction between MM cells and non-malignant cells within the immunosuppressive tumor immune microenvironment (TiME) plays a critical role in disease progression, but it has not been fully elucidated. Our previous study reported that myeloid-derived suppressor cells (MDSCs), dysfunctional dendritic cells (DCs), and tumor-promoting osteoclasts (OCs) are enriched in the bone marrow of myeloma patients and play critical roles in T cell dysfunction. However, the underlying mechanisms remain incompletely understood (Front Immunol. 2022). A myeloid inhibitory receptor, LILRB4, was identified as highly expressed in a super high-risk subgroup of MM cells by single-cell RNA sequencing of patient samples. LILRB4 expression facilitated the generation of monocytic MDSCs and was associated with poor outcomes in MM patients (Haematologica 2024 & 2025). These findings suggest a potential mechanism by which LILRB4 drives an immunosuppressive microenvironment through the induction of immunosuppressive myeloid cells. Here, we further observed a significant reduction in erythroid-lineage commitment in MM patients with high LILRB4 expression, and we explored its molecular and cellular impact on the interaction between myeloma cells and the tumor microenvironment (TME). We report that LILRB4 is highly expressed in MM and activates the SHP2/ERK/AP-1 signaling cascade, thereby enhancing the expression of AP-1-associated chemokines and promoting the transdifferentiation of erythroid cells into immunosuppressive erythroid-derived myeloid cells (EDMCs). Through in vivo and in vitro analyses, we demonstrate that CD45⁺ erythroid progenitor cells (EPCs) in the MM microenvironment lose their erythroid differentiation potential and instead transdifferentiate into EDMCs. This reprogramming is driven by elevated levels of CCL3 secreted by LILRB4⁺ MM cells. Compared with the control group, deletion of CCL3 significantly inhibited myeloma cell growth, increased peripheral blood hemoglobin levels, reduced EDMC proportions in the bone marrow, enhanced IFN-γ secretion by CD8⁺ T cells, decreased the frequency of regulatory T cells (Tregs), and reduced the proportion of macrophages. Additionally, the ratio of monocytic MDSCs (M-MDSCs), which preferentially differentiate into tumor-associated macrophages (TAMs), to granulocytic MDSCs (G-MDSCs) was also decreased. EDMCs emerged as another major source of tumor-associated myeloid cells, further contributing to the suppressive TiME. These results provide a novel model of hematopoietic diversion in which myeloma “hijacks” CD45⁺ erythroid progenitor cells, redirecting them toward a myeloid fate to evade immune surveillance. EDMCs, as potent immunosuppressive players, strongly inhibit CD8⁺ T cell function and accelerate disease progression. Additionally, our findings establish a mechanistic link between LILRB4 and MM-associated anemia, a well-known poor prognostic factor.Our study therefore suggests that LILRB4 overexpression induces key molecular changes that enable macrophage inflammatory protein-1 alpha (MIP-1α/CCL3)-mediated formation of an immunosuppressive myeloid tumor microenvironment, thereby promoting MM cell growth and survival.

Inflammation and pyroptosis are pivotal in myocardial ischemia-reperfusion injury. Although leukocyte immunoglobulin-like receptor subfamily B4 (LILRB4) modulates inflammation in conditions such as myocardial hypertrophy, its involvement in myocardial ischemia-reperfusion injury (MIRI) remains ambiguous. Recombinant adenoviral vectors were designed to induce the overexpression or knockdown of LILRB4 in H9C2 cardiomyocytes or rat myocardial tissue. H9C2 cells were subjected to hypoxia for 2 h and reoxygenation for 4 h (H/R), while myocardial tissue experienced 30 min of ischemia followed by 2 h of reperfusion (I/R). Biochemical assay, histopathology, ELISA, and other tests were performed on myocardial cells and tissues. Meanwhile, LILRB4 knockout mice were used for further validation. LILRB4 promotes the release of inflammatory factors induced by I/R and H/R, thereby increasing the size of myocardial infarction and leading to functional impairment. Downregulation of LILRB4 suppressed SHP-2 phosphorylation and activation, consequently reducing the expression of the thioredoxin-interacting protein (TXNIP), NOD-like receptor protein 3 (NLRP3), Caspase-1, and Gasdermin D (GSDMD). The SHP2 inhibitor PHPS1 mitigated the inflammatory and pyroptotic effects of LILRB4 in cardiomyocytes induced by H/R. Compared to wild-type mice, LILRB4-/- mice exhibited markedly diminished myocardial tissue swelling, decreased release of inflammatory cytokines, and a notable reduction in the expression of p-SHP2, TXNIP, NLRP3, Caspase-1, and GSDMD proteins after I/R induction.LILRB4 promotes inflammation and pyroptosis, which in turn worsens MIRI, likely through activating the TXNIP/NLRP3 signaling pathway, providing mechanistic insights into the MIRI.

Lactylation plays a pivotal role in the metabolic reprogramming, proliferation, migration and immune evasion of tumour cells. However, its specific impact on prostate cancer (PCa) remains poorly understood. This study aimed to investigate the role of lactylation related genes (LRGs) in PCa. LRGs were identified and analysed using data from The Cancer Genome Atlas (TCGA), DKFZ2018, GSE46602 and GSE70768 cohorts. Unsupervised clustering was employed to categorise patients with PCa into two distinct clusters. Prognostic models for PCa were developed using multiple machine learning techniques. LRGs signature was established and validated through training and validation sets. The role of leukocyte immunoglobulin‐like receptor B4 (LILRB4) in PCa was examined both in vitro and in vivo. Analysis of LRG expression and prognosis in patients with PCa revealed two distinct clusters with differing survival rates and immune responses. Machine learning models demonstrated the ability to predict survival risks, potentially aiding in the development of personalised treatment strategies. Additionally, LILRB4, a key LRG, promotes PCa progression by modulating the NF‐κB and PI3K/AKT pathways, highlighting its potential as a therapeutic target. LRGs exert a pivotal influence on PCa, impacting patient prognosis, immune response and drug sensitivity. The LRGs signature emerges as an essential prognostic tool and a promising therapeutic target for PCa.

Objective: Microglia, the central nervous system's immune cells, are crucial in neuroinflammation after ischemic brain injury. Previous single-cell and spatial transcriptomics showed that LILRB4 was upregulated in microglia after MCAO in C57BL/B6J mice, but its role is unclear. This study investigates LILRB4's function and mechanism in ischemic injury. Methods: In C57BL/B6J mice, LILRB4 expression was analyzed at various time points post-MCAO using Q-PCR, Western blot, and flow cytometry. A microglia-specific LILRB4 knockout mouse model (LILRB4-cKO) and control mice (LILRB4 fl/fl ) were established, and MCAO was induced. Infarct volume and behavioral changes were assessed by MAP2 staining, mNSS scoring, grip strength, gait, and rotarod tests. qPCR and Western blot were used to detect brain inflammatory factors and DAMPs, while microglial morphology, cell counts, and RIPK3 levels were analyzed by immunofluorescence and flow cytometry. In vitro, LILRB4 expression changes post-OGD were validated, and LILRB4 knockdown BV2 cells (sh-LILRB4 BV2) were constructed. Necroptosis was induced using RIPK3 inhibitor and OGD, and necroptosis markers were detected. The JASPAR database was used to identify ATF2 as a potential RIPK3 transcription factor, and its role in gene regulation was validated by transfecting ATF2 plasmid into sh-LILRB4 BV2 cells. Results: LILRB4 expression in microglia was upregulated after MCAO, peaking at day 3. LILRB4-cKO mice showed larger infarct volumes, greater neurological deficits, and higher pro-inflammatory cytokines (TNF, IL-6, IL-1β) than LILRB4 fl/fl mice. Microglia from LILRB4-cKO mice had larger cell bodies, shorter processes, reduced numbers, and more RIPK3 + necroptotic cells. In primary microglia and sh-LILRB4 BV2 cells, OGD increased LILRB4, RIPK3, MLKL, and cytokines, which were suppressed by GSK-872. The JASPAR database identified ATF-2 as a potential RIPK3 transcription factor, and its role in promoting RIPK3 transcription was confirmed. The JASPAR database identified ATF-2 as a potential RIPK3 transcription factor, and its role in promoting RIPK3 transcription was confirmed. Knockdown of ATF-2 reduced the elevated levels of inflammatory cytokines, DAMPs, and necroptosis markers induced by LILRB4 deletion. Conclusion: LILRB4 inhibits microglial necroptosis through the ATF2/RIPK3 pathway, reducing neuroinflammation and offering neuroprotection. Therefore, LILRB4 may serve as a potential therapeutic target for ischemic brain injury

Epidemiological studies have shown that circadian rhythm disruption (CRD) is associated with the risk of breast cancer. However, the role of CRD in mammary gland morphology and aggressive basal mammary tumorigenesis and the molecular mechanism underlying CRD-induced carcinogenesis remain unknown. To investigate the effect of CRD on aggressive tumorigenesis, a genetically engineered mouse model of aggressive breast cancer was used. The impact of CRD on the tumor microenvironment was investigated using the tumors from LD12:12 and CRD mice via scRNA-seq, flow cytometry, multiplexing immunostaining, and realtime PCR. The effect of LILRB4-immunotherapy on CRD-induced tumorigenesis was also investigated. Here we investigated and identified the impact of CRD on basal tumorigenesis and mammary gland morphology. We found that chronic CRD disrupted mammary gland morphology, increased lung metastasis, and induced an immunosuppressive tumor microenvironment by enhancing LILRB4 expression. Furthermore, targeted immunotherapy against LILRB4 reduced CRD-induced immunosuppressive microenvironment and lung metastasis. Finally, we showed that LILRB4 regulates CRD-induced mammary tumorigenesis via a non-canonical WNT signaling pathway. These findings identify and implicate LILRB4 as a link between CRD and aggressive mammary tumorigenesis and establish the potential role of the targeted LILRB4a immunotherapy as an inhibitor of CRD-induced lung metastasis.

Although multiple myeloma (MM) responds well to immunotherapeutic treatment, certain portions of MM are still unresponsive or relapse after immunotherapy. Other immune molecules are needed for the immunotherapy of MM. Here, we revealed that leukocyte immunoglobulin-like receptor B4 (LILRB4) was highly expressed in multiple myeloma cell lines and patient samples and that the expression of LILRB4 was adversely correlated with the overall survival of MM patients. Knockdown of LILRB4 efficiently delayed the growth of MM cells both in vitro and in vivo. Mechanistically, IKZF1 transactivated LILRB4 expression to trigger the downstream of STAT3-PFKFB1 pathways to support MM cell proliferation. Blockade of LILRB4 signaling by blocking antibodies can effectively inhibit MM progression. Our data show that targeting LILRB4 is potentially an additional therapeutic strategy for the immunotherapeutic treatment of MM.

Immune diseases are caused by the imbalance of immune regulation. This imbalance is regulated by many factors, both negative and positive. Leukocyte immunoglobulin-like receptor B4 (LILRB4) is a member of leukocyte immunoglobulin-like receptors (LILRs). LILRs are expressed constitutively on the surface of multiple immune cells which associate with membrane adaptors to signal through multi- ple cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) or immunoreceptor tyro-sine-based activation motifs (ITAMs). Through ITIM, LILRB4 could recruit the src homology domain type-2-containing tyrosine phosphatase 1 or 2 (SHP-1 or SHP-2) into the cell membrane. In addition, many factors can induce the expression of LILRB4, such as vitamin D, interferon and so on. Studies have demonstrated that LILRB4 had a negative regulatory role in various of immune diseases. The present review intends to expound the structure and function of LILRB4, as well as its regulators and receptors in the immune cells, so as to provide a theoretical basis for immune disease therapy.

Hemorrhagic shock (HS) leads to intestinal damage and subsequent multiple organ dysfunction syndrome. Intestinal barrier dysfunction is the main cause of multiple organ failure associated with HS. Leukocyte immunoglobulin-like receptor B4 (Lilrb4) belongs to the Ig superfamily and is a vital natural immunomodulatory receptor. The purpose of this study was to identify the role and molecular mechanism of Lilrb4 in HS-induced ileal injury. In this work, HS was established by femoral artery cannula and 90 min of HS (blood pressure, 35-40 mmHg), followed by resuscitation. RNA sequencing analysis showed that Lilrb4 was highly expressed in the ileum of HS rats. As observed, HS rats exhibited severe ileal injury, characterized by enlarged subepithelial space, edema, exfoliation and extensive loss of villi. Whereas, lentivirus system-mediated Lilrb4 overexpression considerably mitigated these alterations. HS led to increased release of markers associated with intestinal injury, which was effectively reversed by Lilrb4 overexpression. In addition, after resuscitation, Lilrb4 overexpression inhibited HS-triggered inflammatory response, as evidenced by decreased levels of proinflammatory cytokines. Lilrb4 also inhibited the activation of NF-κB signal induced by HS. Notably, Lilrb4 modulated the balance of regulatory T (Treg)-T helper 17 (Th17) cells in the mesenteric lymph node (MLN), which may also contribute to its protective role in HS progression. In aggregate, these findings confirmed that Lilrb4 overexpression protected against ileal injury caused by HS, indicating that Lilrb4 may be a potential candidate for the treatment of HS.

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Background: In contrast to leukemia, where the cancer stem cell model has clearly explained tumor hierarchies, multiple myeloma (MM) lacks a unified understanding of its cellular hierarchy, impeding both biological understanding and therapeutic advances. Early attempts to classify MM hierarchies relied on morphological criteria or surface markers. However, these studies failed to establish a developmental continuum linking MM subtypes to normal plasma cell maturation. The putative origin of MM hierarchies‒whether from myeloma stem cells (MSCs), myeloma-initiating cells (MICs), or clonotypic memory B cells‒remains contentious. The lack of consensus in defining myeloma developmental hierarchies—particularly their divergence from normal plasma cell maturation—has hindered the development of MIC-targeting therapies for MM. To address this gap and develop therapies against MICs, we reconstructed the MM cellular hierarchy based on normal plasma cell development, functionally identified putative MICs, and discovered a promising MIC-targeting signaling axis. Methods: We utilized both open-access and self-generated single-cell transcriptomic data of normal and MM plasma cells to construct a developmental blueprint. Computational analysis of stemness and developmental progression identified surface markers of myeloma populations resting at earlier developmental stage, which were then sorted by flow cytometry for functional validation. An epigenetic drug library was screened to identify compounds capable of depleting primitive myeloma populations. CUT&Tag was employed to investigate molecular mechanisms. Results: First, we established an eight-stage developmental framework for normal plasma cells and computationally mapped single-cell transcriptomes from MM specimens, thereby constructing a comprehensive MM hierarchy blueprint. Based on this framework, deconvolution analysis of bulk RNA-seq data of MM patients categorized them into six distinct subgroups according to their distribution pattern of the eight developmental stages. The six subgroups correlated with cytogenetic profiles and presented significant prognostic values. Further, we identified two stages scored highest in stemness including: 1) cycling plasma cells in the peripheral blood and bone marrow (PC-cyclingPB/BM), 2) low-CXCR4 plasma cells in the PB/BM (PC-lowC4PB/BM), as did their corresponding PC-cyclingPB/BM -like and PC-lowC4PB/BM -like myeloma cell counterparts. Surface marker profiling identified LILRB4 as a consistent marker of the two high-stemness stages in both normal and malignant plasma cells. Patients with higher LILRB4 expression showed poorer prognosis. Relapsed patients demonstrated increased LILRB4+ cells compared to newly diagnosed cases. We next validated the functional output of LILRB4+ cells. Single LILRB4+ MM cells generated both LILRB4+ and LILRB4-colonies, whereas LILRB4- cells produced only LILRB4-ones. Furthermore, LILRB4+ MM cells exhibited greater colony-forming unit (CFU) capacity and established stronger tumor burden in vivo. Following bortezomib (BTZ) treatment, LILRB4+ MM cells demonstrated higher viability than their LILRB4- counterparts. These suggest LILRB4 as a putative marker of MICs resistant to BTZ. Finally, we utilized an epigenetic drug library to identify compounds capable of inducing differentiation in LILRB4+ MM cells. EP300 inhibitors emerged as the top candidates that downregulated LILRB4 expression in these cells, simultaneously abolishing BTZ resistance. Animal studies evaluating EP300 inhibitors as a key differentiation-inducing therapy in combination with BTZ are currently in development. Mechanistically, CUT&Tag-seq revealed that EP300 does not directly bind to the LILRB4 promoter, but binds to transcription factor TBX2, highly expressed in LILRB4+ cells. Consistently, EP300 inhibition resulted in downregulation of TBX2. TBX2-knockdown reduced LILRB4 expression, subsequently decreased survival following BTZ treatment, reduced CFU potential, and diminished tumorigenic capacity in vivo. These changes phenocopied the effects observed with EP300 inhibitors. Conclusions: Our study provides the first comprehensive blueprint of MM hierarchy, identifying functionally validated LILRB4+ MICs residing at the apex of the developmental hierarchy as potential drivers of disease initiation and relapse, and nominates EP300 inhibition as a promising differentiation-based therapeutic strategy through TBX2 downregulation.

No abstract available

Immune receptors expressed on TAMs are intriguing targets for tumor immunotherapy. In this study, we found inhibitory receptor LILRB4 on a variety of intratumoral immune cell types in murine tumor models and human cancers, most prominently on TAMs. LILRB4, known as gp49B in mice, is a LILRB family receptor. Human and murine LILRB4 have two extracellular domains but differ in the number of intracellular ITIMs (three versus two). We observed a high correlation in LILRB4 expression with other immune inhibitory receptors. After tumor challenge, LILRB4-/- mice and mice treated with anti-LILRB4 antibody showed reduced tumor burden and increased survival. LILRB4-/- genotype or LILRB4 blockade increased tumor immune infiltrates and the effector (Teff) to regulatory (Treg) T cell ratio and modulated phenotypes of TAMs toward less suppressive, CD4+ T cells to Th1 effector, and CD8+ T cells to less exhausted. These findings reveal that LILRB4 strongly suppresses tumor immunity in TME and that alleviating that suppression provides antitumor efficacy.

Therapeutic strategies are urgently needed for patients with acute myeloid leukemia (AML). Leukocyte immunoglobulin-like receptor B4 (LILRB4), which suppresses T-cell activation and supports tissue infiltration of AML cells, represents an attractive drug target for anti-AML therapeutics. Here, we report the identification and development of an LILRB4-specific humanized mAb that blocks LILRB4 activation. This mAb, h128-3, showed potent activity in blocking the development of monocytic AML in various models including patient-derived xenograft mice and syngeneic immunocompetent AML mice. MAb h128-3 enhanced the anti-AML efficacy of chemotherapy treatment by stimulating mobilization of leukemia cells. Mechanistic studies revealed four concordant modes of action for the anti-AML activity of h128-3: (i) reversal of T-cell suppression, (ii) inhibition of monocytic AML cell tissue infiltration, (iii) antibody-dependent cellular cytotoxicity, and (iv) antibody-dependent cellular phagocytosis. Therefore, targeting LILRB4 with antibody represents an effective therapeutic strategy for treating monocytic AML.

Cervical cancer (CC) is the most common gynecological malignancy and is strongly linked to human papillomavirus (HPV) infection. Currently, immune checkpoint blockade therapy has shown limited clinical benefits for CC, highlighting the need to find more effective therapeutic targets. LILRB4, a member of the leukocyte immunoglobulin-like receptor superfamily, is considered a key mediator of cancer immunosuppression. However, its role in the CC immune microenvironment remains unclear. Here, LILRB4 expression was upregulated in CC tissues, and high expression levels were positively associated with advanced disease and immunosuppressive genes in tumors. In an immunocompetent mouse model, LILRB4 expression in CC tumors increased with tumor growth, whereas blocking LILRB4 reduced tumor growth. Flow cytometry analysis revealed that blockade of LILRB4 reduced CD8

The identification and characterization of regulatory and suppressor T cells that control immune responsiveness to self and non-self antigens has become the focus of innumerable studies. There are two broad categories of naturally occurring and induced CD4(+)CD25(+) regulatory T cells. Naturally occurring T(R) are antigen non-specific and interact directly with other T cells inhibiting their activation. Induced T(R) are either CD4(+)CD25(+) or CD8(+), produce immunosuppressive cytokines such as IL-10, act directly on other T cells or APC and are antigen specific in some but not in all systems. Finally, a distinct subset of T suppressor cells, characterized by their CD8(+)CD28(-) phenotype have been shown to be antigen-specific, recognizing HLA class I/peptide complexes. T(S) act directly on APC inducing the up-regulation of inhibitory receptors ILT3 and ILT4, which render the APC tolerogenic. Tolerized APC, which expresses high ILT3 and ILT4, trigger the generation of antigen-specific CD4(+) T(R) propagating antigen-specific suppression. Up-regulation of ILT3 and ILT4 appears to be a general characteristic of tolerogenic DC since it is also induced by use of vit D3, IL-10 and/or IFN-alpha. The clinical relevance of these inhibitory receptors is in the maintenance of transplantation tolerance as well as in progression of AIDS has been demonstrated.

The direct interaction between antigen specific CD8(+) CD28(-) T suppressor cells (T(S)) with dendritic cells (DC) results in the tolerization of DC by inducing the upregulation of immunologlobulin like transcript 3 (ILT3) and ILT4. We show here that such tolerogenic DC anergize alloreactive CD4(+) CD45RO(+) CD25(+) T cells converting them into regulatory T cells (T(R)), which in turn, continue the cascade of suppression by tolerizing other DC. Interleukin 10 (IL-10) and interferon-alpha (IFN-alpha) also induce ILT3 and ILT4 upregulation in DC, rendering them tolerogenic. This implies a common mechanism of DC-mediated suppression. This finding and the observation that in organ allograft recipients quiescence is associated with the presence in the circulation of donor-specific T(S) and T(R) emphasize the importance of the cross talk between tolerogenic DC and T cells in suppression of the immune response.

Myeloid-derived suppressor cells (MDSCs) play an important role in immune suppression and accumulate under pathologic conditions such as cancer and chronic inflammation. They comprise a heterogeneous population of immature myeloid cells that exert their immunosuppressive function via a variety of mechanisms. Immunoglobulin-like transcript 3 (ILT3) is a receptor containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) that can be expressed on antigen-presenting cells and is an important regulator of dendritic cell tolerance. ILT3 exists in a membrane-bound and a soluble form and can interact with a yet unidentified ligand on T cells and thereby induce T-cell anergy, regulatory T cells, or T suppressor cells. In this study, we analyzed freshly isolated peripheral blood mononuclear cells (PBMCs) of 105 patients with non-small cell lung cancer and 20 healthy controls and demonstrated for the first time that ILT3 is expressed on MDSCs. We show that increased levels of circulating MDSCs correlate with reduced survival. On the basis of ILT3 cell surface expression, an ILT3

The rapid Ab responses observed after primary and secondary immunizations are mainly derived from marginal zone (MZ) and memory B cells, respectively, but it is largely unknown how these responses are negatively regulated. Several inhibitory receptors have been identified and their roles have been studied, but mainly on follicular B cells and much less so on MZ B, and never on memory B cells. gp49B is an Ig superfamily member that contains two ITIMs in its cytoplasmic tail, and it has been shown to negatively regulate mast cell, macrophage, and NK cell responses. In this study, we demonstrate that gp49B is preferentially expressed on memory and MZ B cells. We show that gp49B(-/-) mice produce more IgM after a primary immunization and more IgM and IgG1 after a secondary immunization than gp49B(+/+) mice in T cell-dependent immune responses. Memory and MZ B cells from gp49B(-/-) mice also produce more Abs upon in vitro stimulation with CD40 than those from gp49B(+/+) mice. The in vitro IgM production by MZ B cells from gp49B(+/+), but not gp49B(-/-), mice is suppressed by interaction with a putative gp49B ligand, the integrin αvβ3 heterodimer. In addition, gp49B(-/-) mice exhibited exaggerated IgE production in the memory recall response. These results suggest that plasma cell development from memory and MZ B cells, as well as subsequent Ab production, are suppressed via gp49B. In memory B cells, this suppression also prevents excessive IgE production, thus curtailing allergic diseases.

Endothelial cells have been shown to activate T cell responses to alloantigens, triggering transplant rejection. However, they may also play a role in tolerance induction. Using RT-PCR we show here that alloantigen specific CD8(+)CD28(-) T suppressor cells generated in vitro are FOXP3 positive and interact with human endothelial cells. This interaction results in the induction of inhibitory receptors and down-regulation of costimulatory and adhesion molecules, thus rendering endothelial cells tolerogenic. In turn, tolerized endothelial cells elicit the differentiation of CD8(+)CD28(-) FOXP3(+) T suppressor cells. Taken together our data demonstrate a functional and phenotypic overlap between tolerogenic dendritic cells and endothelial cells. Furthermore, alloantigen specific CD8(+)CD28(-) FOXP3(+) T cells, which trigger the upregulation of inhibitory receptors in endothelial cells, are present in the circulation of heart allograft recipients in quiescence as demonstrated by flow cytometry, RT-PCR and luciferase transcription assays. Their detection facilitates the identification of patients who may benefit from partial or complete cessation of immunosuppressive therapy, a goal of obvious importance given the morbidity and mortality associated with chronic immunosuppression. Modulation of endothelial cells in favor of promoting tolerance may be important for long-term survival of organ allografts.

Allergen-specific venom immunotherapy (VIT) represents the only rational-based option to treat allergic sensitizations against bee and wasp venom. So far, there is not much knowledge about early induction of protective and tolerogenic pathways during VIT. To identify the earliest markers for protective mechanisms against allergic reactions in the peripheral blood during the build-up phase of VIT. PBMC and monocytes were isolated, and serum samples were taken before and during a five day build-up phase from 65 hymenoptera venom allergic patients. Expression level of tolerogenic markers was analyzed on mRNA and protein level. Serum levels of different soluble tolerogenic factors were measured. We observed significantly enhanced tryptophan degradation, elevated ILT4 expression of monocytes as well as IL-10 production of CD3(+) T cells only a few hours after the first injection on day 1, followed by increased IL-10 serum levels, monocyte apoptosis and elevated intracellular cAMP levels of monocytes on day 3 combined with a higher ILT3 protein expression and IL-10 secretion of monocytes on day 5. From these data, we conclude that tryptophan depletion, ILT3/4-mediated inhibition, higher IL-10 production as well as intracellular cAMP might contribute to early induction of protective mechanisms against allergic reactions during the build-up phase of VIT.

Tryptophan catabolism through IDO activity can cause nonresponsiveness and tolerance acting on T cells. Given the crucial importance of dendritic cells (DCs) in the initiation of a T cell response, surprisingly little is known about the impact of IDO activity and tryptophan deprivation on DCs themselves. In the present study, we show that human DCs differentiated under low-tryptophan conditions acquire strong tolerogenic capacity. This effect is associated with a markedly decreased Ag uptake as well as the down-regulation of costimulatory molecules (CD40, CD80). In contrast, the inhibitory receptors ILT3 and ILT4 are significantly increased. Functionally, tryptophan-deprived DCs show a reduced capacity to stimulate T cells, which can be restored by blockade of ILT3. Moreover, ILT3(high)ILT4(high) DCs lead to the induction of CD4(+)CD25(+) Foxp3(+) T regulatory cells with suppressive activity from CD4(+)CD25(-) T cells. The generation of ILT3(high)ILT4(high) DCs with tolerogenic properties by tryptophan deprivation is linked to a stress response pathway mediated by the GCN2 kinase. These results demonstrate that tryptophan degradation establishes a regulatory microenvironment for DCs, enabling these cells to induce T regulatory cells. The impact of IDO thus extends beyond local immune suppression to a systemic control of the immune response.

The Ig-like transcript (ILT)3 is crucial to the tolerogenic activity acquired by dendritic cells exposed to allospecific T suppressor (Ts) cells. We have explored the immunomodulatory property of the extracellular region of ILT3 using a cytoplasmic deletion mutant of ILT3 (ILT3delta), expressed as membrane-bound ILT3 on KG1 cells, and a rILT3-Fc fusion protein. We found that both membrane-bound and soluble ILT3 inhibited T cell proliferation in primary and secondary MLC inducing anergy in CD4+ Th cells and suppressing the differentiation of IFN-gamma-producing CD8+ CTL. Furthermore, membrane-bound and soluble ILT3 induced the differentiation of CD8+ FOXP3+ Ts cells in primary 7-day MLC. The suppressive activity of these CD8+ Ts cells is alloantigen specific and mediated by their capacity to induce the up-regulation of ILT3 and down-regulation of costimulatory molecules such as CD86 in APC from the stimulator used for priming, but not on control HLA-mismatched APC. Our finding that ILT3-Fc has potent immunosuppressive activity in vitro and that it acts on T cells only upon activation suggests the possibility that this agent may be of use for specific suppression of the immune response in autoimmunity or transplantation.

Tolerogenic antigen presenting cells (APC) are characterized by high expression of the inhibitory receptors ILT3 and ILT4. We have engineered ILT3 and ILT4 cytoplasmic deletion mutants (ILT3delta and ILT4delta), which were transfected in the dendritic-like cell line KG1, to investigate ILT3 and ILT4's capacity to signal extracellularly. KG1.ILT3delta, similar to untruncated ILT3, inhibits T cell responses such as proliferation and cell-mediated cytotoxicity. In contrast, KG1.ILT4delta lost the suppressive activity of untruncated ILT4. This indicates that the inhibitory function of ILT4 relies entirely on the cytoplasmic region containing ITIM motifs. We further demonstrated that recombinant soluble ILT3 inhibits T helper and cytotoxic function while inducing the differentiation of CD8(+) Ts cells. Hence, Ts modulate APC function inducing inhibitory receptors, which in turn elicit the generation of Ts.

Microglia help limit the progression of Alzheimer's disease (AD) by constraining amyloid-β (Aβ) pathology, effected through a balance of activating and inhibitory intracellular signals delivered by distinct cell surface receptors. Human leukocyte Ig-like receptor B4 (LILRB4) is an inhibitory receptor of the immunoglobulin (Ig) superfamily that is expressed on myeloid cells and recognizes apolipoprotein E (ApoE) among other ligands. Here, we find that LILRB4 is highly expressed in the microglia of patients with AD. Using mice that accumulate Aβ and carry a transgene encompassing a portion of the

Acute liver injury (ALI) is an important global health concern, primarily caused by widespread hepatocyte cell death, coupled with a complex immune response and a lack of effective remedies. This study explores the underlying mechanisms, immune infiltration patterns, and potential targets for intervention and treatment ALI. The datasets of acetaminophen (APAP), carbon tetrachloride (CCl4), and lipopolysaccharide (LPS)-induced ALI were obtained from the GEO database. Differentially expressed genes (DEGs) were individually identified using the limma packages. Functional enrichment analysis was performed using KEGG, GO, and GSEA methods. The overlapping genes were extracted from the three datasets, and hub genes were identified using MCODE and CytoHubba algorithms. Additionally, PPI networks were constructed based on the String database. Immune cell infiltration analysis was conducted using ImmuCellAI, and the correlation between hub genes and immune cells was determined using the Spearman method. The relationship between hub genes, immune cells, and biochemical indicators of liver function (ALT, AST) was validated using APAP and triptolide (TP) -induced ALI mouse models. Functional enrichment analysis indicated that all three ALI models were enriched in pathways linked to fatty acid metabolism, drug metabolism, inflammatory response, and immune regulation. Immune analysis revealed a significant rise in macrophage infiltration. A total of 79 overlapping genes were obtained, and 10 hub genes were identified that were consistent with the results of the biological information analysis after screening and validation. Among them, Clec4n, Ms4a6d, and Lilrb4 exhibited strong associations with macrophage infiltration and ALI.

Toxoplasma gondii infection can cause adverse pregnancy outcomes, such as recurrent abortion, fetal growth restriction and infants with malformations, among others. Decidual myeloid-derived suppressor cells (dMDSCs) are a novel immunosuppressive cell type at the fetal-maternal interface which play an important role in sustaining normal pregnancy that is related to their high expression of the inhibitory molecule leukocyte immunoglobulin-like receptor B4 (LILRB4). It has been reported that the expression of LILRB4 is downregulated on decidual macrophages after T. gondii infection, but it remains unknown whether T. gondii infection can induce dMDSC dysfunction resulting from the change in LILRB4 expression. LILRB4-deficient (LILRB4 Toxoplasma gondii infection was observed to reduce STAT3 phosphorylation, resulting in decreased LILRB4 expression on dMDSCs. The levels of the main functional molecules (arginase-1 [Arg-1], interleukin-10 [IL-10]) and main signaling molecules (phosphorylated Src-homology 2 domain-containing protein tyrosine phosphatase [p-SHP2], phosphorylated signal transducer and activator of transcription 6 [p-STAT6]) in dMDSCs were all significantly reduced in human and mouse dMDSCs due to the decrease of LILRB4 expression induced by T. gondii infection. SHP-2 was found to directly bind to STAT6 and STAT6 to bind to the promoter of the Arg-1 and IL-10 genes during T. gondii infection. The downregulation of LILRB4 expression on dMDSCs induced by T. gondii infection could regulate the expression of Arg-1 and IL-10 via the SHP-2/STAT6 pathway, resulting in the dysfunction of dMDSCs, which might contribute to adverse outcomes during pregnancy by T. gondii infection.

The tolerogenic phenotype of human dendritic cells is characterized by high cell surface expression of the inhibitory receptor ILT3. ILT3 signals both intracellularly inhibiting tyrosine phosphorylation, NF-kappaB and MAPK p38 activity, transcription of certain co-stimulatory molecules, secretion of cytokines and chemokines, and extracellularly into the T cells with which the dendritic cells interact. Both ILT3(high) tolerogenic dendritic cells and soluble ILT3 induce CD4 Th anergy and differentiation of antigen specific CD8 T suppressor cells. Recombinant ILT3-Fc protein has important immunotherapeutic potential acting directly on activated T cells and promoting the induction of immunological tolerance.

Attempts to ameliorate the poor prognosis of pancreatic cancer have been largely unsuccessful. Interventions to enhance patients' immune responses to malignancies have been also largely unsuccessful. We now describe an immune-escape mechanism mediated by the inhibitory receptor immunoglobulin-like transcript 3 (ILT3) which may be responsible for such failures. Using a humanized severe combined immunodeficiency (SCID) mouse model, we demonstrate that soluble and membrane ILT3 induce CD8+ T suppressor cells and prevent rejection of allogeneic tumor transplants. Furthermore, we found that patients with carcinoma of the pancreas produce the soluble ILT3 protein, which induces the differentiation of CD8+ T suppressor cells and impairs T cell responses in mixed lymphocyte culture. These responses are restored by anti-ILT3 mAb or by depletion of sILT3 from the serum. Immunohistochemical staining of biopsies from the tumors and metastatic lymph nodes suggest that CD68+ tumor associated macrophages represent the major source of soluble ILT3. Alternative splicing, resulting in the loss of the ILT3 transmembrane domain may contribute to the release of ILT3 in the circulation. These data suggest that ILT3 depletion or blockade is crucial to the success of immunobiotherapy, particularly in pancreatic cancer.

CD8+ T suppressor cells differentiate both in vivo and in vitro upon chronic exposure of responding T cells to allogeneic APC. These Ts are allospecific and exhibit their function interacting directly with priming APC which they render tolerogenic. Tolerogenicity of professional and non-professional human APC, such as dendritic cells and endothelial cells, respectively is due to the upregulation of the inhibitory receptors ILT3 and ILT4. ILT3 signals both intracellularly, inhibiting NF-kappaB activation, and transcription of costimulatory molecules, and extracellularly, inducing anergy and regulatory function in T cells with cognate specificity. Both membrane and soluble ILT3 are proteins with potent immunosuppressive activity which are of importance for treatment of rejection, autoimmunity and cancer.

IVIG is frequently used in the 'pre-conditioning' regimens for higher risk transplants; its effects are attributed in part to induction of Tregs. We have identified regulatory T cell (Treg) epitopes, now known as Tregitopes, in IgG, the main component of intravenous immunoglobulin therapy (IVIg). Tregitopes provide one explanation for the expansion and activation of Treg cells following IVIg treatment. Tregitopes are peptides that exhibit high affinity binding to multiple human HLA Class II DR; they are conserved across IgG isotypes and mammalian species. In vitro and in vivo, for human PBMC and in animal models, Tregitopes activate Tregs. Studies to delineate the mechanism of action have shown that Tregitopes' effects are very similar to IVIg in vitro. Here we demonstrate that Tregitopes induce Tregs to produce IL-10, leading to modulation of dendritic cell phenotype (down-regulation of Class II, CD80 and CD86 and up-regulation of ILT3), and describe the effects of Tregitopes in the ABM-TCR-transgenic skin transplantation model. The discovery of Tregitopes in IgG and other autologous proteins may contribute to improved understanding of the mechanism of action of IVIg and lead to the application of these powerful immunomodulators to improve transplantation success and suppress autoimmune disease, in the future.

T cell responses against leukemia-associated antigens have been reported in chronic lymphocytic leukemia (CLL). However, the relentless accumulation of CLL B cells in some patients indicates that anti-tumor immune responses are inefficient. Inhibitory receptors from the Ig-like transcript (ILT) family, such as ILT3 and ILT4, are crucial to the tolerogenic activity of antigen presenting cells. In this study, we examined the expression of ILT3 on CD5+ B cells obtained from 47 patients with CLL. Using flow cytometry and RT-PCR, we found that B CLL cells from 23 of 47 patients expressed ILT3 protein and mature ILT3 mRNA. ILT3 protein and mRNA were not found in normal B cells obtained from donors without CLL. Expression of ILT4 in normal and B CLL cells showed a pattern similar to ILT3. The frequency of ILT3 positive CLL B cells was higher in patients with lymphoid tissue involvement, suggesting that ILT3 may have prognostic value in CLL. Our findings indicate that expression of ILT3 and ILT4 on CLL B cells represents a phenotypic abnormality that may play a role in tolerization of tumor-specific T cells.

Immunoglobulin-like transcript (ILT) 3 is a novel cell surface molecule of the immunoglobulin superfamily, which is selectively expressed by myeloid antigen presenting cells (APCs) such as monocytes, macrophages, and dendritic cells. The cytoplasmic region of ILT3 contains putative immunoreceptor tyrosine-based inhibitory motifs that suggest an inhibitory function of ILT3. Indeed, co-ligation of ILT3 to stimulatory receptors expressed by APCs results in a dramatic blunting of the increased [Ca2+]i and tyrosine phosphorylation triggered by these receptors. Signal extinction involves SH2-containing protein tyrosine phosphatase 1, which is recruited by ILT3 upon cross-linking. ILT3 can also function in antigen capture and presentation. It is efficiently internalized upon cross-linking, and delivers its ligand to an intracellular compartment where it is processed and presented to T cells. Thus, ILT3 is a novel inhibitory receptor that can negatively regulate activation of APCs and can be used by APCs for antigen uptake.

Hyperactivity of the type I interferon (IFN) pathway is involved in the pathogenesis of systemic lupus erythematosus (SLE). Immunoglobulin like transcript (ILT3) is an immunohibitory transmembrane molecule which is induced by type I IFNs. ILT3 is expressed by plasmacytoid dendritic cells (PDCs), monocytoid dendritic cells (MDCs), and monocytes/macrophages. Given the pathogenic role of IFN in SLE, we hypothesised that the IFN-induced immunosuppressive ILT3 receptor may be dysfunctional in human SLE. 132 European-derived and 79 Hispanic-American SLE patients were genotyped for two coding-change single nucleotide polymorphisms (SNPs) predicted to interfere with protein folding in ILT3 (rs11540761 and rs1048801). 116 control DNA samples and sera from healthy controls were also studied. We detected associations between ILT3 genotype and serum cytokine profiles. ILT3 expression levels on PDCs and MDCs from 18 patients and 10 controls were studied by flow cytometry. The rs11540761 SNP in the extracellular region was associated with decreased cell surface expression of ILT3 on circulating MDCs and to a lesser extent PDCs in SLE patients. The cytoplasmically located rs1048801 SNP was not associated with a change in dendritic cells expression of ILT3. Both SNPs were significantly and independently associated with increased levels of serum type I IFN activity in SLE patients. The rs1048801 SNP was also associated with increased serum levels of TNF-α. Loss-of-function polymorphisms in ILT3 are associated with increased inflammatory cytokine levels in SLE, supporting a biological role for ILT3 in SLE.

No abstract

Antigen-specific CD8 suppressor T cells (CD8(+) Ts) are adaptive regulatory T cells that are induced in vivo and in vitro by chronic antigenic stimulation of human T cells. CD8(+) Ts induce the upregulation of the inhibitory receptors ILT3 and ILT4 on monocytes and dendritic cells rendering these antigen presenting cells (APCs) tolerogenic. Tolerogenic APCs induce CD4(+) T helper anergy and elicit the differentiation of CD4(+) and CD8(+) T regulatory/suppressor cells. Overexpression of membrane ILT3 in APC results in inhibition of NF-κB activation, transcription of inflammatory cytokines and costimulatory molecules. Soluble ILT3-Fc which contains only the extracellular, Ig-like domain linked to mutated IgG1 Fc, is strongly immunosuppressive. ILT3-Fc, induces the differentiation of human CD8(+) Ts which inhibit CD4(+) Th and CD8(+) CTL effector function both in vitro and in vivo. The acquisition of Ts' function by primed CD8(+) T cells treated with ILT3-Fc was demonstrated to be the effect of the significant upregulation of BCL6, a transcriptional repressor of IL-2, IFN-gamma, IL-5 and granzyme B. The upregulated expression of BCL6, SOCS1 and DUSP10 is integral to the signature of ILT3-Fc-induced CD8(+) Ts. These genes are known inhibitors of cytokine production and TCR signaling and are targeted by miRNAs which are suppressed by ILT3-Fc. ILT3-Fc induces tolerance to allogeneic human islets and reverses rejection after its onset in a humanized NOD/SCID mouse model. Based on these findings we postulate that ILT3-Fc may become an important new agent for treatment of autoimmunity and transplant rejection.

1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) is a secosteroid hormone that renders dendritic cells (DCs) tolerogenic, favoring the induction of regulatory T cells. Induction of DCs with tolerogenic properties by 1,25(OH)(2)D(3) is associated with increased selective expression of immunoglobulin-like transcript 3 (ILT3), suggesting its involvement in the immunoregulatory properties of this hormone. Here we show an in vivo correlate of the increased ILT3 expression on DCs in healing psoriatic lesions following topical treatment with the 1,25(OH)(2)D(3) analog calcipotriol. Analysis of DC subsets reveals a differential regulation of ILT3 expression by 1,25(OH)(2)D(3), with a marked up-regulation in myeloid DCs but no effect on its expression by plasmacytoid DCs. A regulatory role for ILT3 expressed on DCs is indicated by the increased interferon-gamma (IFN-gamma) secretion promoted by anti-ILT3 addition to cultures of DCs and T cells, but this effect is blunted in 1,25(OH)(2)D(3)-treated DCs, suggesting ILT3-independent mechanisms able to regulate T-cell activation. Although ILT3 expression by DCs is required for induction of regulatory T cells, DC pretreatment with 1,25(OH)(2)D(3) leads to induction of CD4(+)Foxp3(+) cells with suppressive activity irrespective of the presence of neutralizing anti-ILT3 monoclonal antibody (mAb), indicating that ILT3 expression is dispensable for the capacity of 1,25(OH)(2)D(3)-treated DCs to induce regulatory T cells.

Immunoglobulin-like transcript (ILT)-3 is a transmembrane receptor, which belongs to the immunoglobulin superfamily. In previous studies, we showed that allospecific CD8+CD28- T suppressor cells (Ts) induce the expression of ILT3 in human endothelial cells (EC) rendering them tolerogenic. Using a polymerase chain reaction (PCR)-based approach, we now demonstrate by cell fractionation and sequencing studies that ILT3 precursor RNA is expressed and retained in nuclei of resting EC. Ts interaction with EC or exposure of EC to interleukin-10 (IL-10) and interferon alpha (IFN-alpha) triggers processing of ILT3 pre-mRNA. Western blot analysis showed that the expression of the mature ILT3 transcript is accompanied by production of ILT3 protein.

The low response rate of current immune checkpoint inhibitors in cancer has necessitated the development of new immune targets. Survival and public databases analyses were performed to determine the clinical significance of immunoglobulin-like transcript 3 (ILT3). The impact of ILT3 and apolipoprotein E (APOE) on tumor-associated macrophage (TAM) recruitment and polarization were evaluated by transwell assay, flow cytometry (FCM), and real-time PCR, while their impact on T cell survival and cytotoxicity was detected by CFSE, apoptotic assay, FCM and ELISA. These pro-tumoural activity of (an ortholog of ILT3 in mouse) were verified in vivo models. Survival and public databases analyses revealed that high ILT3 expression was significantly associated with worse prognosis in lung adenocarcinoma (LUAD), but not in squamous cell carcinoma. The same association was observed with its ligand, APOE. In vitro assays demonstrated that tumor-derived ILT3/APOE promoted recruitment and M2-like polarization of TAMs in LUAD and directly inhibited T cell proliferation and cytotoxicity. In vivo knockdown of gp49b enhanced anti-tumor immunity and suppressed tumor progression by counteracting TAM- and dysfunctional T cell-induced tumor microenvironment immunosuppression. Furthermore, combined inhibition of gp49b and programmed cell death ligand 1 (PD-L1) showed the most drastic tumor regression in C57BL/6 mice models. Tumor-derived ILT3 overexpression suppresses anti-tumor immunity by recruiting M2-like TAMs and impairing T cell activities, while ILT3 inhibition counteracts this immunosuppression and enhances the efficacy of PD-L1 blockade in LUAD. Thus, ILT3 could be a promising novel immunotherapeutic target for combined immunotherapy.

Immune activation needs to be tightly regulated to control immune-mediated tissue damage. Inhibitory pathways serve to terminate an immune response and resolve inflammation. Persistent exposure to antigens can drive development of adaptive regulatory cells. Similarly exposure of activated T cells to the recombinant ILT3-Fc molecule during priming triggers the differentiation of CD8 T suppressor cells and the induction of CD4 T helper anergy. Ts express high levels of immunoregulatory signature genes together with low levels of microRNA which control their function. Analysis of microRNA contained by exosomes from cultures in which T cells were alloactivated in the presence or absence of ILT3.Fc, demonstrated that this agent inhibits the release of inflammatory microRNA. The source of such inflammatory microRNA was found to reside in alloactivated CD4 T cells, since exosomes from MLC primed CD4 T cells were shown to diminish the suppressive activity of ILT3-Fc-induced CD8(+) Ts at high effector to suppressor T cell ratios. This indicates that inflammatory exosomes can swing the balance between effector and regulatory T cells in favor of immunity. These data suggest that isolation and characterization of micro-RNA containing exosomes in patients' circulation may be of use for treatment, prevention and monitoring of immune activation.

Acute myeloid leukemia (AML) is a hematologic malignancy with poor overall survival (OS). The immunosuppressive microenvironment significantly impacts AML development and chemoresistance. Despite new immunotherapeutic strategies entering standard clinical care for various tumors, progress in AML remains poor. Multi-omics analyses, such as single-cell transcriptomics, have revealed many potential new targets to improve AML prognosis from an immunological perspective. DNA from 307 AML patients and 316 healthy individuals were extracted. We detected nine single nucleotide polymorphisms (SNPs) in five immunosuppression-related genes (CIITA, CD200, CD163, MRC1 and LILRB4) in these samples. SNP genotyping was performed on the MassARRAY platform. We then analyzed the relationship between these SNPs and AML susceptibility, treatment response, and prognosis. Our findings indicated that rs4883263 in the CD163 gene is a protective factor for AML susceptibility and chromosomal karyotype abnormalities. Additionally, rs4883263 in CD163 was related to low PLT count at diagnosis, while rs2272022 in CD200 was protective against low PLT count. rs4780335 in CIITA was associated with high WBC count at diagnosis and worse OS. Furthermore, rs1048801 in LILRB4 was linked to worse AML treatment response, lower OS, and may be an independent prognostic risk factor for AML. Lastly, expressions of CD163, CIITA, LILRB4, and CD200 were higher in AML patients than that in normal controls. Our findings on SNP associations in AML immunosuppression-related genes provide important reference points for predicting treatment outcomes in AML patients.

Mesenchymal stromal cell (MSC) transplantation has achieved significant clinical benefits for many diseases, such as systemic lupus erythematosus (SLE) and inflammatory diseases. However, the detailed therapeutic mechanism of MSCs is not fully understood. Here, in the SLE treatment, we show that MSC transplantation triggers recipient bone marrow neutrophil aggregation to generate an endogenous extracellular vesicle (EV) storm in the circulation via the TNFα/ICAM-1/Rab11b axis. Interestingly, blockade of the EV storm abolishes the MSC-mediated therapeutic effect for SLE. The level of EV storm is positively associated with the therapeutic effect of MSCs in SLE patients. Mechanistically, aggregated neutrophils-derived EV storms equalize Th17 and T-regulatory (Treg) cells to promote immune tolerance and disease remission via the DHA/LILRB4/STAT5/STAT3 pathway in the MSC treatment for SLE. Taken together, our findings reveal a new immune-modulating function of MSCs through the induction of endogenous neutrophil aggregation in the bone marrow, which results in the secretion of EV storms for immune tolerance in SLE mice and patients. In addition, this study revealed a previously unknown role of the recipient EV storm in determining the therapeutic effect of MSC in SLE, and the recipient EV storm can be used to predict the therapeutic efficacy of MSCs in SLE therapy.

AbstractImmune homeostasis is critically regulated by the balance between activating and inhibitory receptors expressed on various immune cells such as T and B lymphocytes, and myeloid cells. The inhibitory receptors play a fundamental role in the immune checkpoint pathway, thus maintaining peripheral tolerance. We recently found that expression of leukocyte immunoglobulin-like receptor (LILR)B4, an inhibitory member of the human LILR family, is augmented in auto-antibody-producing plasmablasts/plasma cells of systemic lupus erythematosus (SLE) patients. However, the mechanism behind the 'paradoxical' up-regulation of this inhibitory receptor upon pathogenic antibody-secreting cells is yet to be known. To this end, in this study, we examined if glycoprotein 49B (gp49B), the murine counterpart of human LILRB4, is also elevated in auto-antibody-producing cells in several SLE mouse models, and tried to clarify the underlying mechanism. We found that gp49B is expressed on plasma cells of lupus-prone models but not of healthy C57BL/6 mice, and the level was positively correlated to the anti-double-stranded DNA IgG titer in serum. Gp49B genetic deletion, however, did not abolish the serum auto-antibodies or fully ameliorate the lethal glomerulonephritis, indicating that gp49B is not the sole regulator of lupus but a pathogenic element in the disease. We conclude that the elevated expression of this inhibitory receptor on pathogenic plasma cells was also relevant upon the murine SLE model. The mechanism of gp49B underlying the disease progression in lupus-prone mice has been discussed.

The periodontal pathogen Porphyromonas gingivalis (Pg) is a potent inducer of the production of pro-inflammatory cytokines by neutrophils, monocytes, and macrophages, and can desensitize immune cells in vitro and in vivo. We analyzed the ability of Pg lipopolysaccharide (LPS) to induce endotoxin tolerance. Treatment of dendritic cells (DC), the human macrophage cell line THP-1, and monocytes (antigen-presenting cells, APC) with Pg.LPS inhibited APC maturation assessed by CD80 and CD86 expression, and inhibited chemokine (CCL3 and CCL5) production. Pre-treatment with glucocorticoids (GC) and interleukin-10 (IL-10) abolished the effect of Pg.LPS on CD80, CD83, and CD86, and on CCL3 and CCL5 production. We also showed that Pg.LPS enhanced the tolerogenic properties of APCs and up-regulated ILT-3 and B7-H1 expression.

Dendritic cells (DCs) can acquire unique features or phenotypes in different tissue microenvironments and decide whether immunity or tolerance develops. DCs observed within the decidua have been implicated in pregnancy maintenance. However, the precise distribution of decidual DC subsets and their phenotypic characteristics are largely unknown. Using flow cytometry, we identified three DC subsets in normal human first-trimester decidua: BDCA-1+ CD19- CD14(-) myeloid DC type 1 (MDC1), BDCA-3+ CD14- myeloid DC type 2 (MDC2) and BDCA-2+ CD123+ plasmacytoid DC (PDC). The percentage of MDC1 to mononuclear cells in the decidua was similar to that in the peripheral blood controls. The percentage of MDC2 in the decidua was significantly higher than that in the peripheral blood controls, whereas the percentage of PDC was significantly lower. Both MDC1 and MDC2 subsets expressed human leucocyte antigen D-related, CD86 and CD80 at low levels, suggesting a characteristic of immature myeloid DCs. Immunoglobulin-like transcript 3, suggested to be involved in immune tolerance induction, was also expressed on decidual MDC1 and MDC2 subsets. In addition, as gestational age increased from 6 to 9 weeks, the numbers of MDC1 decreased but MDC2 increased significantly. This is the first study to demonstrate the presence of three previously unidentified BDCA-1+, BDCA-3+ and BDCA-2+ DC subsets in human decidua, these decidual DCs might play important role in the maintenance of pregnancy.

Resveratrol is a polyphenol that acts on multiple molecular targets important for cell differentiation and activation. Dendritic cells (DCs) are a functionally diverse cell type and represent the most potent antigen-presenting cells of the immune system. In this study, we investigated resveratrol-induced effects on DCs during their differentiation and maturation. Our results show that resveratrol induces DC-associated tolerance, particularly when applied during DC differentiation. Costimulatory molecules CD40, CD80 and CD86 were down-regulated, as was the expression of major histocompatibility complex (MHC) class II molecules. Surface expression of inhibitory immunoglobulin-like transcript 3 (ILT3) and ILT4 molecules was induced, while human leucocyte antigen (HLA)-G expression was not affected. Resveratrol-treated DCs lost the ability to produce interleukin (IL)-12p70 after activation, but had an increased ability to produce IL-10. Such DCs were poor stimulators of allogeneic T cells and had lowered ability to induce CD4(+) T-cell migration. Furthermore, treated cells were able to generate allogeneic IL-10-secreting T cells, but were not competent in inducing FoxP3 expression These tolerogenic effects are probably associated with the effect of resveratrol on multiple molecular targets through which it interferes with DC differentiation and nuclear factor (NF)-kappaB translocation. Our data provide new insights into the molecular and functional mechanisms of the tolerogenic effects that resveratrol exerts on DCs.

The nonclassical HLA class I antigen HLA-G is an inhibitory molecule involved in immune tolerance and immune escape. HLA-G exerts its inhibitory functions via interaction with inhibitory receptors ILT2, ILT4, and KIR2DL4, differentially expressed by NK, T, and antigen-presenting cells. Cells expressing HLA-G and cells expressing its receptors are often found in the vicinity of each other, but the mechanisms responsible for this colocalization are still unknown. We report that ILT2, ILT3, ILT4, and KIR2DL4 expression is up-regulated by HLA-G in antigen-presenting cells, NK cells, and T cells. Because this up-regulation seems not to require antigenic costimulation, it might precede an immune response. Functionally, up-regulation of inhibitory receptors in immune cells before stimulation might increase their activation thresholds and participate in immune escape mechanisms.

The leukocyte immunoglobulin-like receptors (LIRs, also known as ILTs, CD85, and LILRs) comprise a family of related immunoregulatory receptors encoded within the leukocyte receptor cluster (LRC) on human chromosome 19. LIRs are transmembrane proteins containing either two or four extracellular immunoglobulin domains, and most family members are expressed predominantly on myeloid cell lineages. Although the inhibitory receptors LIR-1 and LIR-2 are known to bind to a broad range of class I MHC molecules and are thought to play important roles in immune regulation, the majority of LIRs are currently of unknown structure and their ligands remain unidentified. In this study, we describe recombinant production and characterisation of the extracellular portion of LIR-5 (ILT3), a poorly understood inhibitory receptor that transduces tolerising signals to dendritic cells. The two extracellular immunoglobulin domains of LIR-5 were expressed in Escherichia coli to a high level and were found to accumulate in inclusion bodies. Inclusion bodies were purified, solubilised, and receptor then renatured by dilution refolding, with acceptable yields. Size exclusion chromatography and SDS-PAGE analyses confirmed the extracellular portion behaved as a monomer in solution, and purified protein was antibody-reactive. LIR-5 is representative of a subset of LIR receptors that on the basis of structural and sequence comparisons with LIR-1 seem unlikely to bind class I MHC molecules. Successful prokaryotic generation of correctly folded LIR-5 in high levels has implications for production of other LRC receptors and should greatly facilitate attempts to define the structure and ligands of this important regulator of dendritic cell function.

Attempts to enhance patients' immune responses to malignancies have been largely unsuccessful. We now describe an immune-escape mechanism mediated by the inhibitory receptor Ig-like transcript 3 (ILT3) that may be responsible for such failures. Using a humanized SCID mouse model, we demonstrate that soluble and membrane ILT3 induce CD8(+) T suppressor cells and prevent rejection of allogeneic tumor transplants. Furthermore, we found that patients with melanoma, and carcinomas of the colon, rectum, and pancreas produce the soluble ILT3 protein, which induces the differentiation of CD8(+) T suppressor cells and impairs T cell responses in MLC. These responses are restored by anti-ILT3 mAb or by depletion of soluble ILT3 from the serum. Immunohistochemical staining of biopsies from the tumors and metastatic lymph nodes suggests that CD68(+) tumor-associated macrophages represent the major source of soluble ILT3. Alternative splicing, resulting in the loss of the ILT3 transmembrane domain, may contribute to the release of ILT3 in the circulation. These data suggest that ILT3 depletion or blockade is crucial to the success of immunotherapy in cancer. In contrast, the inhibitory activity of soluble ILT3 on T cell alloreactivity in vitro and in vivo suggests the potential usefulness of rILT3 for immunosuppressive treatment of allograft recipients or patients with autoimmune diseases.

The role of T-cells within the immune system is to confirm and assess anomalous situations and then either respond to or tolerate the source of the effect. To illustrate how these mechanisms can be harnessed to solve real-world problems, we present the blueprint of a T-cell inspired algorithm for computer security worm detection. We show how the three central T-cell processes, namely T-cell maturation, differentiation and proliferation, naturally map into this domain and further illustrate how such an algorithm fits into a complete immune inspired computer security system and framework.

In immune system simulation there are two competing simulation approaches: System Dynamics Simulation (SDS) and Agent-Based Simulation (ABS). In the literature there is little guidance on how to choose the best approach for a specific immune problem. Our overall research aim is to develop a framework that helps researchers with this choice. In this paper we investigate if it is possible to easily convert simulation models between approaches. With no explicit guidelines available from the literature we develop and test our own set of guidelines for converting SDS models into ABS models in a non-spacial scenario. We also define guidelines to convert ABS into SDS considering a non-spatial and a spatial scenario. After running some experiments with the developed models we found that in all cases there are significant differences between the results produced by the different simulation methods.

We continue the study of asynchrony immunity in cellular automata (CA), which can be considered as a weaker version of correlation immunity in the context of vectorial Boolean functions. The property could have applications as a countermeasure for side-channel attacks in CA-based cryptographic primitives, such as S-boxes and pseudorandom number generators. We first give some theoretical results on the necessary conditions that a CA rule must satisfy in order to meet asynchrony immunity, the most important one being center permutivity. Next, we perform an exhaustive search of all asynchrony immune CA rules of neighborhood size up to $5$, leveraging on the discovered theoretical properties to greatly reduce the size of the search space.

Over the last decade, a new idea challenging the classical self-non-self viewpoint has become popular amongst immunologists. It is called the Danger Theory. In this conceptual paper, we look at this theory from the perspective of Artificial Immune System practitioners. An overview of the Danger Theory is presented with particular emphasis on analogies in the Artificial Immune Systems world. A number of potential application areas are then used to provide a framing for a critical assessment of the concept, and its relevance for Artificial Immune Systems.

A combined Short-Term Learning (STL) and Long-Term Learning (LTL) approach to solving mobile robot navigation problems is presented and tested in both real and simulated environments. The LTL consists of rapid simulations that use a Genetic Algorithm to derive diverse sets of behaviours. These sets are then transferred to an idiotypic Artificial Immune System (AIS), which forms the STL phase, and the system is said to be seeded. The combined LTL-STL approach is compared with using STL only, and with using a handdesigned controller. In addition, the STL phase is tested when the idiotypic mechanism is turned off. The results provide substantial evidence that the best option is the seeded idiotypic system, i.e. the architecture that merges LTL with an idiotypic AIS for the STL. They also show that structurally different environments can be used for the two phases without compromising transferability

Artificial Immune Systems have been used successfully to build recommender systems for film databases. In this research, an attempt is made to extend this idea to web site recommendation. A collection of more than 1000 individuals web profiles (alternatively called preferences / favourites / bookmarks file) will be used. URLs will be classified using the DMOZ (Directory Mozilla) database of the Open Directory Project as our ontology. This will then be used as the data for the Artificial Immune Systems rather than the actual addresses. The first attempt will involve using a simple classification code number coupled with the number of pages within that classification code. However, this implementation does not make use of the hierarchical tree-like structure of DMOZ. Consideration will then be given to the construction of a similarity measure for web profiles that makes use of this hierarchical information to build a better-informed Artificial Immune System.

It has previously been shown that a recommender based on immune system idiotypic principles can out perform one based on correlation alone. This paper reports the results of work in progress, where we undertake some investigations into the nature of this beneficial effect. The initial findings are that the immune system recommender tends to produce different neighbourhoods, and that the superior performance of this recommender is due partly to the different neighbourhoods, and partly to the way that the idiotypic effect is used to weight each neighbours recommendations.

Network Intrusion Detection Systems (NDIS) monitor a network with the aim of discerning malicious from benign activity on that network. While a wide range of approaches have met varying levels of success, most IDS's rely on having access to a database of known attack signatures which are written by security experts. Nowadays, in order to solve problems with false positive alters, correlation algorithms are used to add additional structure to sequences of IDS alerts. However, such techniques are of no help in discovering novel attacks or variations of known attacks, something the human immune system (HIS) is capable of doing in its own specialised domain. This paper presents a novel immune algorithm for application to an intrusion detection problem. The goal is to discover packets containing novel variations of attacks covered by an existing signature base.

The INTERSPEECH 2020 Deep Noise Suppression Challenge is intended to promote collaborative research in real-time single-channel Speech Enhancement aimed to maximize the subjective (perceptual) quality of the enhanced speech. A typical approach to evaluate the noise suppression methods is to use objective metrics on the test set obtained by splitting the original dataset. Many publications report reasonable performance on the synthetic test set drawn from the same distribution as that of the training set. However, often the model performance degrades significantly on real recordings. Also, most of the conventional objective metrics do not correlate well with subjective tests and lab subjective tests are not scalable for a large test set. In this challenge, we open-source a large clean speech and noise corpus for training the noise suppression models and a representative test set to real-world scenarios consisting of both synthetic and real recordings. We also open source an online subjective test framework based on ITU-T P.808 for researchers to quickly test their developments. The winners of this challenge will be selected based on subjective evaluation on a representative test set using P.808 framework.

The absence of fixed momentum excitations in a theory with Lifshitz scale invariance gives rise to exponential suppression of spectral weight in the low-frequency limit. In the holographic dual, this suppression arises as a consequence of a tunneling barrier that decouples the horizon from the boundary. We compute the spin-1/2 holographic Green's function and show that the form of the barrier is identical to that of the scalar case. We furthermore demonstrate that the suppression factor is universal in the $\hatω\to0$ limit where $\hatω=ω/|\vec k|^z$. In particular, it depends only on $\hatω$ and the critical exponent $z$, and is independent of scaling dimension and spin.

The combination of immune checkpoint blockade and chemotherapies is the standard of care for triple-negative breast cancer (TNBC). However, initially, responsive tumors can still develop recurrences, suggesting acquired resistance mechanisms that remain poorly understood. Herein, we discover that TNBC cells surviving anti-programmed cell death protein-1 (anti-PD-1) and chemotherapy treatment accumulate neutral lipids. Disrupting lipid droplet formation in cancer cells reverses resistance and mitigates the immunosuppressive microenvironment. Single-cell RNA sequencing reveals a subset of neutrophils exhibiting a lipid-laden phenotype similar to adjacent tumor cells. Mechanistically, tumor-derived extracellular vesicles carrying lipids, including arachidonic acid (AA), mediate neutrophil reprogramming. Blocking dietary intake of omega-6 fatty acids or inhibiting fatty acid elongation for AA synthesis restores anti-tumor immunity and re-sensitizes the resistant tumors to anti-PD-1 and chemotherapy treatment. In human patients, AA metabolism-related pathways correlates with neutrophil enrichment. Overall, we demonstrate how lipid accumulation in TNBC cells leads to immune suppression and therapy resistance.

No abstract available

Dihydroorotate dehydrogenase (DHODH)-mediated ferroptosis defense is a targetable vulnerability in cancer. Currently, only a few DHODH inhibitors have been utilized in clinical practice. To further enhance DHODH targeting, we introduced the mitochondrial targeting group triphenylphosphine (TPP) to brequinar (BRQ), a robust DHODH inhibitor, resulting in the creation of active molecule B2. This compound exhibits heightened anticancer activity, effectively inhibiting proliferation in various cancer cells, and restraining tumor growth in melanoma xenografts in mice. B2 achieves these effects by targeting DHODH, triggering the formation of reactive oxygen species (ROS), promoting mitochondrial lipid peroxidation, and inducing ferroptosis in B16F10 and A375 cells. Surprisingly, B2 significantly downregulates PD-L1 and alleviates immune suppression. Importantly, B2 exhibits no apparent adverse effects in mice. Collectively, these findings highlight that enhancing the mitochondrial targeting capability of the DHODH inhibitor is a promising therapeutic approach for melanoma treatment.

The intricate interplay between immune and stromal cells within the tumour microenvironment (TME) significantly influences tumour progression. Myeloid cells, including tumour-associated macrophages (TAMs), neutrophils (TANs), and myeloid-derived suppressor cells (MDSCs), contribute to immune suppression in the TME 1,2. This poses a significant challenge for novel immunotherapeutics that rely on host immunity to exert their effect. This systematic review explores the preclinical evidence surrounding the inhibition of phosphoinositide 3-kinase gamma (PI3Kγ) as a strategy to reverse myeloid-driven immune suppression in solid tumours. EMBASE, MEDLINE, and PubMed databases were searched on 6th October 2022 using keyword and subject heading terms to capture relevant studies. The studies, focusing on PI3Kγ inhibition in animal models, were subjected to predefined inclusion and exclusion criteria. Extracted data included tumour growth kinetics, survival endpoints, and immunological responses which were meta-analysed. PRISMA and MOOSE guidelines were followed. A total of 36 studies covering 73 animal models were included in the review and meta-analysis. Tumour models covered breast, colorectal, lung, skin, pancreas, brain, liver, prostate, head and neck, soft tissue, gastric, and oral cancer. The predominant PI3Kγ inhibitors were IPI-549 and TG100-115, demonstrating favourable specificity for the gamma isoform. Combination therapies, often involving chemotherapy, radiotherapy, immune checkpoint inhibitors, biological agents, or vaccines, were explored in 81% of studies. Analysis of tumour growth kinetics revealed a statistically significant though heterogeneous response to PI3Kγ monotherapy, whereas the tumour growth in combination treated groups were more consistently reduced. Survival analysis showed a pronounced increase in median overall survival with combination therapy. This systematic review provides a comprehensive analysis of preclinical studies investigating PI3Kγ inhibition in myeloid-driven tumour immune suppression. The identified studies underscore the potential of PI3Kγ inhibition in reshaping the TME by modulating myeloid cell functions. The combination of PI3Kγ inhibition with other therapeutic modalities demonstrated enhanced antitumor effects, suggesting a synergistic approach to overcome immune suppression. These findings support the potential of PI3Kγ-targeted therapies, particularly in combination regimens, as a promising avenue for future clinical exploration in diverse solid tumour types. Graphical abstract

The relevance of biopterin metabolism in resistance to immune checkpoint blockade (ICB) therapy remains unknown. We demonstrate that the deficiency of quinoid dihydropteridine reductase (QDPR), a critical enzyme regulating biopterin metabolism, causes metabolite dihydrobiopterin (BH2) accumulation and decreases the ratio of tetrahydrobiopterin (BH4) to BH2 in pancreatic ductal adenocarcinomas (PDACs). The reduced BH4/BH2 ratio leads to an increase in reactive oxygen species (ROS) generation and a decrease in the distribution of H3K27me3 at CXCL1 promoter. Consequently, myeloid-derived suppressor cells are recruited to tumor microenvironment via CXCR2 causing resistance to ICB therapy. We discovered that BH4 supplementation is capable to restore the BH4/BH2 ratio, enhance anti-tumor immunity, and overcome ICB resistance in QDPR-deficient PDACs. Tumors with lower QDPR expression show decreased responsiveness to ICB therapy. These findings offer a novel strategy for selecting patient and combining therapies to improve the effectiveness of ICB therapy in PDAC.

Increased glucose metabolism and uptake are characteristic of many tumors and used clinically to diagnose and monitor cancer progression. In addition to cancer cells, the tumor microenvironment (TME) encompasses a wide range of stromal, innate, and adaptive immune cells. Cooperation and competition between these cell populations supports tumor proliferation, progression, metastasis, and immune evasion. Cellular heterogeneity leads to metabolic heterogeneity because metabolic programs within the tumor are dependent not only on the TME cellular composition but also on cell states, location, and nutrient availability. In addition to driving metabolic plasticity of cancer cells, altered nutrients and signals in the TME can lead to metabolic immune suppression of effector cells and promote regulatory immune cells. Here we discuss how metabolic programming of cells within the TME promotes tumor proliferation, progression, and metastasis. We also discuss how targeting metabolic heterogeneity may offer therapeutic opportunities to overcome immune suppression and augment immunotherapies.

Sepsis arises from diverse and incompletely understood dysregulated host response processes following infection that leads to life-threatening organ dysfunction. Here we showed that neutrophils and emergency granulopoiesis drove a maladaptive response during sepsis. We generated a whole-blood single-cell multiomic atlas (272,993 cells, n  = 39 individuals) of the sepsis immune response that identified populations of immunosuppressive mature and immature neutrophils. In co-culture, CD66b^+ sepsis neutrophils inhibited proliferation and activation of CD4^+ T cells. Single-cell multiomic mapping of circulating hematopoietic stem and progenitor cells (HSPCs) (29,366 cells, n  = 27) indicated altered granulopoiesis in patients with sepsis. These features were enriched in a patient subset with poor outcome and a specific sepsis response signature that displayed higher frequencies of IL1R2 ^+ immature neutrophils, epigenetic and transcriptomic signatures of emergency granulopoiesis in HSPCs and STAT3-mediated gene regulation across different infectious etiologies and syndromes. Our findings offer potential therapeutic targets and opportunities for stratified medicine in severe infection. Knight and colleagues report altered granulopoiesis and increased frequency of immature neutrophil subsets with immunosuppressive properties in a subset of patients with sepsis with poor outcome.

SARS-CoV-2 adaptation to humans is evidenced by the emergence of variants of concern (VOCs) with distinct genotypes and phenotypes that facilitate immune escape and enhance transmission frequency. Most recently Omicron subvariants have emerged with heavily mutated spike proteins which facilitate re-infection of immune populations through extensive antibody escape driving replacement of previously-dominant VOCs Alpha and Delta. Interestingly, Omicron is the first VOC to produce distinct subvariants. Here, we demonstrate that later Omicron subvariants, particularly BA.4 and BA.5, have evolved an enhanced capacity to suppress human innate immunity when compared to earliest subvariants BA.1 and BA.2. We find that, like previously dominant VOCs, later Omicron subvariants tend to increase expression of viral innate immune antagonists Orf6 and nucleocapsid. We show Orf6 to be a key contributor to enhanced innate immune suppression during epithelial replication by BA.5 and Alpha, reducing innate immune signaling through IRF3 and STAT1. Convergent VOC evolution of enhanced innate immune antagonist expression suggests common pathways of adaptation to humans and links VOC, and in particular Omicron subvariant, dominance to improved innate immune evasion.

No abstract available

BACKGROUND & AIMS Immune checkpoint blockade (ICB) therapy only benefits a small subset of colorectal cancer (CRC) patients and identification of CRC-intrinsic events modulating ICB efficacy is an unmet need. Here, we revealed AlkB Homolog 5 (ALKBH5), an RNA N6-methyladenosine (m6A) eraser, drives immunosuppression and is a molecular target to boost ICB therapy in CRC. METHODS Clinical significance of ALKBH5 was evaluated in human samples (n=205). Function of ALKBH5 was investigated in allografts, CD34+ humanized mice, and Alkbh5 knockin mice. Immunity change was determined by flow cytometry, immunofluorescence, and functional investigation. MeRIP-seq and RNA-seq were employed to identify ALKBH5 targets. Vesicle-like nanoparticles (VNPs)-encapsulated ALKBH5-siRNA was constructed for targeting ALKBH5 in vivo. RESULTS High ALKBH5 expression predicts poor prognosis in CRC. ALKBH5 induced myeloid-derived suppressor cells (MDSCs) accumulation but reduced natural killer cells and cytotoxic CD8+ T cells to induce colorectal tumorigenesis in allografts, CD34+ humanized mice and intestine-specific Alkbh5 knockin mice. Mechanistically, AXIN2, a Wnt suppressor, was identified as a target of ALKBH5. ALKBH5 binds and demethylates AXIN2 mRNA, which caused its dissociation from m6A reader IGF2BP1 and degradation, resulting in hyperactivated Wnt/β-catenin. Subsequently, Wnt/β-catenin targets including Dickkopf-related protein 1 (DKK1) were induced by ALKBH5. ALKBH5-induced DKK1 recruited MDSCs to drive immunosuppression in CRC, and this effect was abolished by anti-DKK1 in vitro and in vivo. Finally, VNPs-encapsulated ALKBH5-siRNA, or anti-DKK1 potentiated anti-PD1 treatment in suppressing CRC growth by enhancing anti-tumor immunity. CONCLUSIONS Our study identifies an ALKBH5-m6A-AXIN2-Wnt-DKK1 axis in CRC which drives immune suppression to facilitate tumorigenesis. Targeting of ALKBH5 is a promising strategy for sensitizing CRC to immunotherapy.

Myeloid-derived suppressor cells (MDSCs), which are immature heterogeneous bone marrow cells, have been described as potent immune regulators in human and murine cancer models. The distribution of MDSCs varies across organs and is divided into three subpopulations: granulocytic MDSCs or polymorphonuclear MDSCs (G-MDSCs or PMN-MDSCs), monocytic MDSCs (M-MDSCs), as well as a recently identified early precursor MDSC (eMDSCs) in humans. Activated MDSCs induce the inactivation of NK cells, CD4+, and CD8+ T cells through a variety of mechanisms, thus promoting the formation of tumor immunosuppressive microenvironment. ER stress plays an important protecting role in the survival of MDSC, which aggravates the immunosuppression in tumors. In addition, ferroptosis can promote an anti-tumor immune response by reversing the immunosuppressive microenvironment. This review summarizes immune suppression by MDSCs with a focus on the role of endoplasmic reticulum stress-mediated immune suppression in cancer and infectious disease, in particular leprosy and tuberculosis.

Immune checkpoint inhibitor (ICI) therapy is effective against many cancers for a subset of patients; a large percentage of patients remain unresponsive to this therapy. One contributing factor to ICI resistance is accumulation of monocytic myeloid-derived suppressor cells (M-MDSCs), a subset of innate immune cells with potent immunosuppressive activity against T lymphocytes. Here, using lung, melanoma, and breast cancer mouse models, we show that CD73-expressing M-MDSCs in the tumor microenvironment (TME) exhibit superior T cell suppressor function. Tumor-derived PGE2, a prostaglandin, directly induces CD73 expression in M-MDSCs via both Stat3 and CREB. The resulting CD73 overexpression induces elevated levels of adenosine, a nucleoside with T cell–suppressive activity, culminating in suppression of antitumor CD8+ T cell activity. Depletion of adenosine in the TME by the repurposed drug PEGylated adenosine deaminase (PEG-ADA) increases CD8+ T cell activity and enhances response to ICI therapy. Use of PEG-ADA can therefore be a therapeutic option to overcome resistance to ICIs in cancer patients.

Background Management of immune-related adverse events (irAEs) is important as they cause treatment interruption or discontinuation, more often seen with combination immune checkpoint inhibitor (ICI) therapy. Here, we retrospectively evaluated the safety and effectiveness of anti-interleukin-6 receptor (anti-IL-6R) as therapy for irAEs. Methods We performed a retrospective multicenter study evaluating patients diagnosed with de novo irAEs or flare of pre-existing autoimmune disease following ICI and were treated with anti-IL-6R. Our objectives were to assess the improvement of irAEs as well as the overall tumor response rate (ORR) before and after anti-IL-6R treatment. Results We identified a total of 92 patients who received therapeutic anti-IL-6R antibodies (tocilizumab or sarilumab). Median age was 61 years, 63% were men, 69% received anti-programmed cell death protein-1 (PD-1) antibodies alone, and 26% patients were treated with the combination of anti-cytotoxic T lymphocyte antigen-4 and anti-PD-1 antibodies. Cancer types were primarily melanoma (46%), genitourinary cancer (35%), and lung cancer (8%). Indications for using anti-IL-6R antibodies included inflammatory arthritis (73%), hepatitis/cholangitis (7%), myositis/myocarditis/myasthenia gravis (5%), polymyalgia rheumatica (4%), and one patient each with autoimmune scleroderma, nephritis, colitis, pneumonitis and central nervous system vasculitis. Notably, 88% of patients had received corticosteroids, and 36% received other disease-modifying antirheumatic drugs (DMARDs) as first-line therapies, but without adequate improvement. After initiation of anti-IL-6R (as first-line or post-corticosteroids and DMARDs), 73% of patients showed resolution or change to ≤grade 1 of irAEs after a median of 2.0 months from initiation of anti-IL-6R therapy. Six patients (7%) stopped anti-IL-6R due to adverse events. Of 70 evaluable patients by RECIST (Response Evaluation Criteria in Solid Tumors) V.1.1 criteria; the ORR was 66% prior versus 66% after anti-IL-6R (95% CI, 54% to 77%), with 8% higher complete response rate. Of 34 evaluable patients with melanoma, the ORR was 56% prior and increased to 68% after anti-IL-6R (p=0.04). Conclusion Targeting IL-6R could be an effective approach to treat several irAE types without hindering antitumor immunity. This study supports ongoing clinical trials evaluating the safety and efficacy of tocilizumab (anti-IL-6R antibody) in combination with ICIs (NCT04940299, NCT03999749).

Abstract Background Cancer‐associated fibroblasts (CAFs) are potential targets for cancer therapy. Due to the heterogeneity of CAFs, the influence of CAF subpopulations on the progression of lung cancer is still unclear, which impedes the translational advances in targeting CAFs. Methods We performed single‐cell RNA sequencing (scRNA‐seq) on tumour, paired tumour‐adjacent, and normal samples from 16 non‐small cell lung cancer (NSCLC) patients. CAF subpopulations were analyzed after integration with published NSCLC scRNA‐seq data. SpaTial enhanced resolution omics‐sequencing (Stereo‐seq) was applied in tumour and tumour‐adjacent samples from seven NSCLC patients to map the architecture of major cell populations in tumour microenvironment (TME). Immunohistochemistry (IHC) and multiplexed IHC (mIHC) were used to validate marker gene expression and the association of CAFs with immune infiltration in TME. Results A subcluster of myofibroblastic CAFs, POSTN + CAFs, were significantly enriched in advanced tumours and presented gene expression signatures related to extracellular matrix remodeling, tumour invasion pathways and immune suppression. Stereo‐seq and mIHC demonstrated that POSTN + CAFs were in close localization with SPP1 + macrophages and were associated with the exhausted phenotype and lower infiltration of T cells. POSTN expression or the abundance of POSTN + CAFs were associated with poor prognosis of NSCLC. Conclusions Our study identified a myofibroblastic CAF subpopulation, POSTN + CAFs, which might associate with SPP1 + macrophages to promote the formation of desmoplastic architecture and participate in immune suppression. Furthermore, we showed that POSTN + CAFs associated with cancer progression and poor clinical outcomes and may provide new insights on the treatment of NSCLC.

Summary FoxP3-expressing regulatory T cells (Tregs), whether naturally generated in the immune system or unnaturally induced from conventional T cells (Tconvs) in the laboratory, have much therapeutic value in treating immunological diseases and establishing transplantation tolerance. Natural Tregs (nTregs) can be selectively expanded in vivo by administration of low-dose IL-2 or IL-2 muteins for immune suppression. For adoptive Treg cell therapy, nTregs can be expanded in vitro by strong antigenic stimulation in the presence of IL-2. Synthetic receptors such as CAR can be expressed in nTregs to equip them with a particular target specificity for suppression. In addition, antigen-specific Tconvs can be converted in vitro to functionally stable Treg-like cells by a combination of antigenic stimulation, FoxP3 induction, and establishment of the Treg-type epigenome. This review discusses current and prospective strategies for Treg-based immune suppression and the issues to be resolved for achieving stable antigen-specific immune suppression and tolerance induction in the clinic by targeting Tregs.

Background Myeloid-derived suppressor cells (MDSCs) are crucial mediators of tumor-associated immune suppression. Targeting the accumulation and activation of MDSCs has been recognized as a promising approach to enhance the effectiveness of immunotherapies for different types of cancer. Methods The MC38 and B16 tumor-bearing mouse models were established to investigate the role of Fgl2 during tumor progression. Fgl2 and FcγRIIB-deficient mice, adoptive cell transfer, RNA-sequencing and flow cytometry analysis were used to assess the role of Fgl2 on immunosuppressive activity and differentiation of MDSCs. Results Here, we show that fibrinogen-like protein 2 (Fgl2) regulates the differentiation and immunosuppressive functions of MDSCs. The absence of Fgl2 leads to an increase in antitumor CD8+ T-cell responses and a decrease in granulocytic MDSC accumulation. The regulation mechanism involves Fgl2 modulating cholesterol metabolism, which promotes the accumulation of MDSCs and immunosuppression through the production of reactive oxygen species and activation of XBP1 signaling. Inhibition of Fgl2 or cholesterol metabolism in MDSCs reduces their immunosuppressive activity and enhances differentiation. Targeting Fgl2 could potentially enhance the therapeutic efficacy of anti-PD-1 antibody in immunotherapy. Conclusion These results suggest that Fgl2 plays a role in promoting immune suppression by modulating cholesterol metabolism and targeting Fgl2 combined with PD-1 checkpoint blockade provides a promising therapeutic strategy for antitumor therapy.

α-Fetoprotein (AFP) is a secreted tumor protein and biomarker with impact on immunity. Fatty acid–bound AFP promotes immune suppression by skewing human dendritic cell metabolism toward glycolysis and reduced immune stimulation.

No abstract available

There is increasing evidence indicating the significant role of DDX5 (also called p68), acting as a master regulator and a potential biomarker and target, in tumorigenesis, proliferation, metastasis and treatment resistance for cancer therapy. However, DDX5 has also been reported to act as an oncosuppressor. These seemingly contradictory observations can be reconciled by DDX5’s role in DNA repair. This is because cancer cell apoptosis and malignant transformation can represent the two possible outcomes of a single process regulated by DDX5, reflecting different intensity of DNA damage. Thus, targeting DDX5 could potentially shift cancer cells from a growth-arrested state (necessary for DNA repair) to apoptosis and cell killing. In addition to the increasingly recognized role of DDX5 in global genome stability surveillance and DNA damage repair, DDX5 has been implicated in multiple oncogenic signaling pathways. DDX5 appears to utilize distinct signaling cascades via interactions with unique proteins in different types of tissues/cells to elicit opposing roles (e.g., smooth muscle cells versus cancer cells). Such unique features make DDX5 an intriguing therapeutic target for the treatment of human cancers, with limited low toxicity to normal tissues. In this review, we discuss the multifaceted functions of DDX5 in DNA repair in cancer, immune suppression, oncogenic metabolic rewiring, virus infection promotion, and negative impact on the human microbiome (microbiota). We also provide new data showing that FL118, a molecular glue DDX5 degrader, selectively works against current treatment-resistant prostate cancer organoids/cells. Altogether, current studies demonstrate that DDX5 may represent a unique oncotarget for effectively conquering cancer with minimal toxicity to normal tissues.

The formation of pre-metastatic niche is a key step in the metastatic burden. The pluripotent factor Lin28B is frequently expressed in breast tumors and is particularly upregulated in the triple negative breast cancer subtype. Here, we demonstrate that Lin28B promotes lung metastasis of breast cancer by building an immune-suppressive pre-metastatic niche. Lin28B enables neutrophil recruitment and N2 conversion. The N2 neutrophils are then essential for immune suppression in pre-metastatic lung by PD-L2 up-regulation and a dysregulated cytokine milieu. We also identify that breast cancer-released exosomes with low let-7s are a prerequisite for Lin28B-induced immune suppression. Moreover, Lin28B-induced breast cancer stem cells are the main sources of low-let-7s exosomes. Clinical data further verify that high Lin28B and low let-7s in tumors are both indicators for poor prognosis and lung metastasis in breast cancer patients. Together, these data reveal a mechanism by which Lin28B directs the formation of an immune-suppressive pre-metastatic niche. The establishment of a pre-metastatic niche is a key step preceding metastasis formation. Here the authors show that tumor-intrinsic Lin28B, a RNA-binding protein, has an essential role in the formation of an immune-suppressive pre-metastatic niche, promoting lung metastasis of breast cancer.

An extensive fibroinflammatory stroma rich in macrophages is a hallmark of pancreatic cancer. In this disease, it is well appreciated that macrophages are immunosuppressive and contribute to the poor response to immunotherapy; however, the mechanisms of immune suppression are complex and not fully understood. Immunosuppressive macrophages are classically defined by expression of the enzyme Arginase 1 (Arg1), which we demonstrated is potently expressed in pancreatic tumor associated macrophages from both human patients and mouse models. While routinely used as a polarization marker, Arg1 also catabolizes arginine, an amino acid required for T cell activation and proliferation. To investigate this metabolic function, we used a genetic and a pharmacologic approach to target Arg1 in pancreatic cancer. Genetic inactivation of Arg1 in macrophages, using a dual recombinase genetically engineered mouse model of pancreatic cancer, delayed formation of invasive disease, while increasing CD8+ T cell infiltration. Treatment of established tumors with the arginase inhibitor CB-1158 exhibited further increased CD8+ T cell infiltration, beyond that seen with the macrophage-specific knockout, and sensitized the tumors to anti-PD1 immune checkpoint blockade. Thus, our data demonstrate that Arg1 is more than simply a marker of macrophage function. Rather, Arg1 is also a driver of immune suppression and represents a promising immunotherapeutic target for pancreatic cancer.

MAPK signaling inhibitor (MAPKi) therapies show limited efficacy for advanced thyroid cancers despite constitutive activation of the signaling correlates with disease recurrence and persistence. Understanding how BRAF pathway stimulates tumorigenesis could lead to new therapeutic targets. Here, through genetic and pathological approaches, we demonstrate that BRAFV600E promotes thyroid cancer development by increasing myeloid-derived suppressor cells (MDSCs) penetrance. This BRAFV600E-induced immune suppression involves re-activation of the developmental factor TBX3, which in turn up-regulates CXCR2 ligands in a TLR2-NFκB dependent manner, leading to MDSCs recruitment into the tumor microenvironment. CXCR2 inhibition or MDSCs repression improves MAPKi therapy effect. Clinically, high TBX3 expression correlates with BRAFV600E mutation and increased CXCR2 ligands, along with abundant MDSCs infiltration. Thus, our study uncovers a BRAFV600E-TBX3-CXCLs-MDSCs axis that guides patient stratification and could be targeted to improve the efficacy of MAPKi therapy in advanced thyroid cancer patients. BRAF-V600E mutation is common in patients with papillary thyroid carcinoma (PTC) and has been associated with an aggressive phenotype. Here the authors show that the mutation supports cancer progression by reactivating the developmental factor TBX3 and promoting the recruitment of myeloid derived suppressive cells.

Indoleamine 2,3 dioxygenase 1 (IDO1) is an attractive target for cancer immunotherapy. However, IDO1 inhibitors have shown disappointing therapeutic efficacy in clinical trials, mainly because of the activation of the aryl hydrocarbon receptor (AhR). Here, we show a post-transcriptional regulatory mechanism of IDO1 regulated by a proteasome-associated deubiquitinating enzyme, USP14, in colorectal cancer (CRC). Overexpression of USP14 promotes tryptophan metabolism and T-cell dysfunction by stabilizing the IDO1 protein. Knockdown of USP14 or pharmacological targeting of USP14 decreases IDO1 expression, reverses suppression of cytotoxic T cells, and increases responsiveness to anti-PD-1 in a MC38 syngeneic mouse model. Importantly, suppression of USP14 has no effects on AhR activation induced by the IDO1 inhibitor. These findings highlight a relevant role of USP14 in post-translational regulation of IDO1 and in the suppression of antitumor immunity, suggesting that inhibition of USP14 may represent a promising strategy for CRC immunotherapy. IDO1-mediated tryptophan metabolism plays an important role in creating an immunosuppressive tumour microenvironment. Here, the authors show that deubiquitinase USP14 regulates immune suppression by inducing IDO1 stabilization and suggest USP14 as a potential therapeutic target to improve immunotherapy in colorectal cancer.

Exosomes are a class of small membrane-bound extracellular vesicles released by almost all cell types and present in all body fluids. Based on the studies of exosome content and their interactions with recipient cells, exosomes are now thought to mediate “targeted” information transfer. Tumor-derived exosomes (TEX) carry a cargo of molecules different from that of normal cell-derived exosomes. TEX functions to mediate distinct biological effects such as receptor discharge and intercellular cross-talk. The immune system defenses, which may initially restrict tumor progression, are progressively blunted by the broad array of TEX molecules that activate suppressive pathways in different immune cells. Herein, we provide a review of the latest research progress on TEX in the context of tumor-mediated immune suppression and discuss the potential as well as challenges of TEX as a target of immunotherapy.

Tumor-associated macrophages (TAM) play a detrimental role in triple-negative breast cancer (TNBC). In-depth analysis of TAM characteristics and interactions with stromal cells, such as cancer-associated fibroblast (CAF), could provide important biological and therapeutic insights. Here we identify at the single-cell level a monocyte-derived-STAB1+TREM2high lipid-associated macrophage (LAM) subpopulation with immune suppressive capacities that is expanded in patients resistant to immune checkpoint blockade (ICB). Genetic depletion of this LAM subset in mice suppressed TNBC tumor growth. Flow cytometry and bulk-RNA sequencing data demonstrated that co-culture with TNBC-derived CAFs led to reprogramming of blood monocytes towards immune suppressive STAB1+TREM2high LAMs, which inhibit T cell activation and proliferation. Cell-to-cell interaction modeling and assays in vitro demonstrated the role of the inflammatory CXCL12-CXCR4 axis in CAF-myeloid cell crosstalk and recruitment of monocytes in tumor sites. Altogether, these data suggest an inflammation model whereby monocytes recruited to the tumor via the CAF-driven CXCL12-CXCR4 axis acquire pro-tumorigenic LAM capacities to support an immunosuppressive microenvironment.

The mRNA SARS-CoV-2 vaccines were brought to market in response to the public health crises of Covid-19. The utilization of mRNA vaccines in the context of infectious disease has no precedent. The many alterations in the vaccine mRNA hide the mRNA from cellular defenses and promote a longer biological half-life and high production of spike protein. However, the immune response to the vaccine is very different from that to a SARS-CoV-2 infection. In this paper, we present evidence that vaccination induces a profound impairment in type I interferon signaling, which has diverse adverse consequences to human health. Immune cells that have taken up the vaccine nanoparticles release into circulation large numbers of exosomes containing spike protein along with critical microRNAs that induce a signaling response in recipient cells at distant sites. We also identify potential profound disturbances in regulatory control of protein synthesis and cancer surveillance. These disturbances potentially have a causal link to neurodegenerative disease, myocarditis, immune thrombocytopenia, Bell's palsy, liver disease, impaired adaptive immunity, impaired DNA damage response and tumorigenesis. We show evidence from the VAERS database supporting our hypothesis. We believe a comprehensive risk/benefit assessment of the mRNA vaccines questions them as positive contributors to public health.

The limited efficacy of immune checkpoint inhibitor treatment in triple-negative breast cancer (TNBC) patients is attributed to sparse or unresponsive tumor-infiltrating lymphocytes, but the mechanisms that lead to a therapy resistant tumor immune microenvironment are incompletely known. Here we show a strong correlation between MYC expression and loss of immune signatures in human TNBC. In mouse models of TNBC proficient or deficient of breast cancer type 1 susceptibility gene (BRCA1), MYC overexpression dramatically decreases lymphocyte infiltration in tumors, along with immune signature remodelling. MYC-mediated suppression of inflammatory signalling induced by BRCA1/2 inactivation is confirmed in human TNBC cell lines. Moreover, MYC overexpression prevents the recruitment and activation of lymphocytes in both human and mouse TNBC co-culture models. Chromatin-immunoprecipitation-sequencing reveals that MYC, together with its co-repressor MIZ1, directly binds promoters of multiple interferon-signalling genes, resulting in their downregulation. MYC overexpression thus counters tumor growth inhibition by a Stimulator of Interferon Genes (STING) agonist via suppressing induction of interferon signalling. Together, our data reveal that MYC suppresses innate immunity and facilitates tumor immune escape, explaining the poor immunogenicity of MYC-overexpressing TNBCs. Tripe-negative breast cancers poorly respond to immune checkpoint inhibition therapy, due to their immune-hostile tumour microenvironment. Authors here show that the oncogene MYC plays a pivotal role in suppressing anti-tumour immunity via directly regulating the transcription of interferon signalling genes.

Macrophages are crucial innate immune cells that maintain tissue homeostasis and defend against pathogens; however, their infiltration into tumors has been associated with adverse outcomes. Tumor-associated macrophages (TAMs) represent a significant component of the inflammatory infiltrate in breast tumors, and extensive infiltration of TAMs has been linked to poor prognosis in breast cancer. Here, we detail how TAMs impede a productive tumor immunity cycle by limiting antigen presentation and reducing activation of cytotoxic T lymphocytes (CTLs) while simultaneously supporting tumor cell survival, angiogenesis, and metastasis. There is an urgent need to overcome TAM-mediated immune suppression for durable anti-tumor immunity in breast cancer. To date, failure to fully characterize TAM biology and classify multiple subsets has hindered advancement in therapeutic targeting. In this regard, the complexity of TAMs has recently taken center stage owing to their subset diversity and tightly regulated molecular and metabolic phenotypes. In this review, we reveal major gaps in our knowledge of the functional and phenotypic characterization of TAM subsets associated with breast cancer, before and after treatment. Future work to characterize TAM subsets, location, and crosstalk with neighboring cells will be critical to counteract TAM pro-tumor functions and to identify novel TAM-modulating strategies and combinations that are likely to enhance current therapies and overcome chemo- and immuno-therapy resistance.

Metastasis is the leading cause of cancer-related deaths, and greater knowledge of the metastatic microenvironment is necessary to effectively target this process. Microenvironmental changes occur at distant sites prior to clinically detectable metastatic disease; however, the key niche regulatory signals during metastatic progression remain poorly characterized. Here, we identify a core immune suppression gene signature in pre-metastatic niche formation that is expressed predominantly by myeloid cells. We target this immune suppression program by utilizing genetically engineered myeloid cells (GEMys) to deliver IL-12 to modulate the metastatic microenvironment. Our data demonstrate that IL12-GEMy treatment reverses immune suppression in the pre-metastatic niche by activating antigen presentation and T cell activation, resulting in reduced metastatic and primary tumor burden and improved survival of tumor-bearing mice. We demonstrate that IL12-GEMys can functionally modulate the core program of immune suppression in the pre-metastatic niche to successfully rebalance the dysregulated metastatic microenvironment in cancer.

Metastasis is the major cause of cancer-related deaths due to the lack of effective therapies. Emerging evidence suggests that certain epigenetic and transcriptional regulators drive cancer metastasis and could be targeted for metastasis treatment. To identify epigenetic regulators of breast cancer metastasis, we profiled the transcriptomes of matched pairs of primary breast tumors and metastases from human patients. We found that distant metastases are more immune inert with increased M2 macrophages compared to their matched primary tumors. The acetyl-lysine reader, cat eye syndrome chromosome region candidate 2 (CECR2), was the top up-regulated epigenetic regulator in metastases associated with an increased abundance of M2 macrophages and worse metastasis-free survival. CECR2 was required for breast cancer metastasis in multiple mouse models, with more profound effect in the immunocompetent setting. Mechanistically, the nuclear factor κB (NF-κB) family member v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA) recruits CECR2 to increase chromatin accessibility and activate the expression of their target genes. These target genes include multiple metastasis-promoting genes, such as TNC, MMP2, and VEGFA, and cytokine genes CSF1 and CXCL1, which are critical for immunosuppression at metastatic sites. Consistent with these results, pharmacological inhibition of CECR2 bromodomain impeded NF-κB–mediated immune suppression by macrophages and inhibited breast cancer metastasis. These results reveal that targeting CECR2 may be a strategy to treat metastatic breast cancer. Description Depletion or inhibition of CECR2 impedes breast cancer metastasis by suppressing NF-κB signaling, migration, invasion, and immune suppression. Modulating metastasis Breast cancer metastasis is thought to be driven through both epigenetic and transcriptional regulators, either of which could be targeted as a therapeutic strategy. Here, Zhang et al. identified the acetyl-lysine reader, CECR2, as an epigenetic regulator required for breast cancer metastasis in mouse models. CECR2 promoted M2 macrophage polarization and increased expression of metastasis-associated signaling molecules. Deletion or pharmacological inhibition of CECR2 prevented breast cancer metastasis in mouse models, suggesting that CECR2 should be explored as a therapeutic target for breast cancer.

This study shows that elevated apolipoprotein E in PDAC mediates immune suppression and high serum apolipoprotein E levels correlate with poor patient survival. Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with few effective therapeutic options. PDAC is characterized by an extensive fibroinflammatory stroma that includes abundant infiltrating immune cells. Tumor-associated macrophages (TAM) are prevalent within the stroma and are key drivers of immunosuppression. TAMs in human and murine PDAC are characterized by elevated expression of apolipoprotein E (ApoE), an apolipoprotein that mediates cholesterol metabolism and has known roles in cardiovascular and Alzheimer's disease but no known role in PDAC. We report here that ApoE is also elevated in peripheral blood monocytes in PDAC patients, and plasma ApoE protein levels stratify patient survival. Orthotopic implantation of mouse PDAC cells into syngeneic wild-type or in ApoE−/− mice showed reduced tumor growth in ApoE−/− mice. Histologic and mass cytometric (CyTOF) analysis of these tumors showed an increase in CD8+ T cells in tumors in ApoE−/− mice. Mechanistically, ApoE induced pancreatic tumor cell expression of Cxcl1 and Cxcl5, known immunosuppressive factors, through LDL receptor and NF-κB signaling. Taken together, this study reveals a novel immunosuppressive role of ApoE in the PDAC microenvironment. Significance: This study shows that elevated apolipoprotein E in PDAC mediates immune suppression and high serum apolipoprotein E levels correlate with poor patient survival. See related commentary by Sherman, p. 4186

Central nervous system trauma is a common cause of morbidity and mortality. Additionally, these injuries frequently occur in younger individuals, leading to lifetime expenses for patients and caregivers and the loss of opportunity for society. Despite this prevalence and multiple attempts to design a neuroprotectant, clinical trials for a pharmacological agent for the treatment of traumatic brain injury (TBI) or spinal cord injury (SCI) have provided disappointing results. Improvements in outcome from these disease processes in the past decades have been largely due to improvements in supportive care. Among the many challenges facing patients and caregivers following neurotrauma, posttraumatic nosocomial infection is a significant and potentially reversible risk factor. Multiple animal and clinical studies have provided evidence of posttraumatic systemic immune suppression, and injuries involving the CNS may be even more prone, leading to a higher risk for in-hospital infections following neurotrauma. Patients who have experienced neurotrauma with nosocomial infection have poorer recovery and higher risks of long-term morbidity and in-hospital mortality than patients without infection. As such, the etiology and reversal of postneurotrauma immune suppression is an important topic. There are multiple possible etiologies for these posttraumatic changes including the release of damage-associated molecular patterns, the activation of immunosuppressive myeloid-derived suppressor cells, and sympathetic nervous system activation. Postinjury systemic immunosuppression, particularly following neurotrauma, provides a challenge for clinicians but also an opportunity for improvement in outcome. In this review, the authors sought to outline the evidence of postinjury systemic immune suppression in both animal models and clinical research of TBI, TBI polytrauma, and SCI.

Metastasis is the primary cause of cancer mortality, and colorectal cancer (CRC) frequently metastasizes to the liver. Our previous studies demonstrated the critical role of KIAA1199 in tumor invasion and metastasis in CRC. In the present study, we described an immune regulatory effect of KIAA1199 that creates a permissive environment for metastasis.

Microglia are critical responders to amyloid beta (Aβ) plaques in Alzheimer's disease (AD). Therefore, the therapeutic targeting of microglia in AD is of high clinical interest. While previous investigation has focused on the innate immune receptors governing microglial functions in response to Aβ plaques, how microglial innate immune responses are regulated is not well understood. Interestingly, many of these microglial innate immune receptors contain unique cytoplasmic motifs, termed immunoreceptor tyrosine‐based activating and inhibitory motifs (ITAM/ITIM), that are commonly known to regulate immune activation and inhibition in the periphery. In this review, we summarize the diverse functions employed by microglia in response to Aβ plaques and also discuss the innate immune receptors and intracellular signaling players that guide these functions. Specifically, we focus on the role of ITAM and ITIM signaling cascades in regulating microglia innate immune responses. A better understanding of how microglial innate immune responses are regulated in AD may provide novel therapeutic avenues to tune the microglial innate immune response in AD pathology.

In thrombosis and hemostasis, the formation of a platelet-fibrin thrombus or clot is a highly controlled process that varies, depending on the pathological context. Major signaling pathways in platelets are well established. However, studies with genetically modified mice have identified the contribution of hundreds of additional platelet-expressed proteins in arterial thrombus formation and bleeding. Using phenotype information of 540 mouse genes, involved in arterial thrombosis and hemostasis, we review current insights into established and novel platelet signaling mechanisms. We discuss pathways involved in platelet adhesion, shape change, integrin activation, intracellular vesicle trafficking and protein processing, granule secretion, aggregate formation and procoagulant activity. Specific attention is paid to the signaling routes used by ITAM-linked, ITIM-linked and G protein-coupled receptors, as well as downstream events feeding into GTPase regulation and protein kinase activation. We further summarize known alterations in platelet responses under conditions of venous, inflammatory and infection-dependent thrombosis, taking into account interactions of platelets with the endothelium, leukocytes and red blood cells. Understanding the genes and proteins involved in platelet signaling in the context of hemostasis, thrombosis and inflammation may lead to improved therapies to prevent and treat thrombotic disorders.

Aberrant expression of CXCR4, a chemokine G protein‐coupled receptor (GPCR), promotes a migratory and invasive phenotype during breast cancer metastasis, however the mechanism is unknown. Here we investigate the role of CXCR4 tyrosine phosphorylation within a novel ITIM motif. Following activation, CXCR4 is normally phosphorylated on Ser/Thr residues, which promotes receptor desensitization and internalization. We found previously that Ser/Thr phosphorylation of CXCR4 is delayed upon SDF gradient sensing, and correlates with sustained signaling to SHP2, a protein that drives hyperproliferation and invasion of breast cancer. Since SHP2 is known to be recruited to tyrosine phosphorylated ITIM motifs (immmunoreceptor tyrosine‐based inhibitory consensus motifs), we investigated if CXCR4 contains an ITIM motif, and if tyrosine phosphorylation within this motif regulates SHP2 binding, signaling, and migration. Specifically, we assessed 1) if CXCR4 is tyrosine phosphorylated as measured by recombinant phosphatase assay, phospho‐Ab development, and tyrosine mutagenesis (YF); 2) if tyrosine mutagenesis alters CXCR4 membrane localization, internalization, and SDF gradient sensing, by ELISA; 3) if tyrosine mutagenesis disrupts CXCR4 interaction with SHP2, by co‐IP; 4) if tyrosine mutagenesis alters CXCR4 sustained signaling to SHP2, by Western; and 5) if tyrosine mutation (YF) within the novel ITIM motif in CXCR4 alters migration of metastatic breast cancer cells, by transwell motility assays. Our results demonstrate that CXCR4 phosphorylation is sensitive to recombinant tyrosine phosphatase treatment and tyrosine mutagenesis, suggesting that CXCR4 is indeed tyrosine phosphorylated within the ITIM motif. We determined that while tyrosine phosphorylation within the ITIM motif is not required for CXCR4 membrane localization, internalization, or SDF gradient sensing, it does regulate CXCR4 binding and signaling to SHP2. Investigating the role in CXCR4 mediated migration is on going. To date, our data support a working model that tyrosine phosphorylation of CXCR4 within an ITIM motif is critical for binding to SHP2 and transducing sustained signaling to SHP2. These data have implications on aggressive breast cancers with dysregulated CXCR4 and SHP2.

B-cell receptor (BCR) signaling plays an important role in the pathogenesis of mantle cell lymphoma (MCL), but the detailed mechanisms are not fully understood. In this study, through a genome-wide loss-of-function screen, we identify carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) as an essential factor in a subset of MCL tumors. Our signal transduction studies reveal that CEACAM1 plays a critical role in BCR activation through involvement in two dynamic processes. First, following BCR engagement, CEACAM1 co-localizes to the membrane microdomains (lipid rafts) by anchoring to the F-actin cytoskeleton through the adaptor protein filamin A. Second, CEACAM1 recruits and increases the abundance of SYK in the BCR complex leading to BCR activation. These activities of CEACAM1 require its cytoplasmic tail and the N-terminal ectodomain. Considering that previous studies have extensively characterized CEACAM1 as an ITIM-bearing inhibitory receptor, our findings regarding its activating role are both surprising and context-dependent, which may have implications for BCR-targeting therapies. Pathological B-cell receptor (BCR) signaling is a key driver of mantle cell lymphoma tumorigenesis. Here, the authors discover that CEACAM1, an immunoglobulin-like transmembrane protein, is essential for a subset of mantle cell lymphoma through activation of the BCR.

Despite the effectiveness of anti-programmed death 1 (PD-1)/PD-1 ligand 1 monoclonal antibodies against various cancers, resistance remains a significant issue among patients. The immunosuppressive T cell immunoreceptor with Ig and ITIM domains (TIGIT)/CD155 axis has emerged as a key mechanism contributing to this resistance. However, the intricacies of CD155 expression are not fully elucidated. In this study, we aimed to identify the key molecules involved in the regulation of CD155 expression and explore their role in modulating CD155 within the tumor microenvironment (TME). By employing clustered regularly interspaced palindromic repeats (CRISPR) screening, we identified dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) as one of the key regulators of CD155 expression. Subsequent inhibition of Dyrk1a through CRISPR/CRISPR-associated protein 9 (Cas9) technology or treatment with DYRK1A inhibitors effectively mitigated PD-1 blockade resistance. Moreover, in certain head and neck squamous cell carcinoma (HNSCC) cell lines, cetuximab-mediated epidermal growth factor receptor blockade reduced CD155 expression by targeting downstream PI3K/Akt signaling. In patients with HNSCC (n = 96), CD155 expression correlated with Akt phosphorylation, particularly impacting PD-1 blockade resistance in those with high CD8+ T cell infiltration. These findings underscore the role of the PI3K/Akt signaling pathway in regulating CD155 expression, which may influence resistance to PD-1 blockade therapies in a variety of cancers, particularly those characterized by an inflamed TME. This study suggests that targeting the PI3K/Akt pathway could overcome resistance, particularly in cancers with an inflamed TME and high CD155 expression.

Introduction: Multiple suppressive mechanisms within the tumor microenvironment (TME) contribute to blunt anti-tumor T cell responses. Among them, tumor-associated T cells have been phenotypically described to be functionally exhausted (or dysfunctional), reflecting to a hyporesponsive state of chronically stimulated T cells that express multiple inhibitory receptors (or immune checkpoint molecules), such as programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), lymphocyte-activation gene 3 (LAG-3), 2B4 (also known as CD244), and T cell immunoreceptor with Ig and ITIM domains (TIGIT), and exhibit a compromised cellular cytotoxicity. While immune checkpoint blockade therapies aimed at reversing the dysfunctional state T cell effector function have demonstrated clinical effectiveness, not all cancer patients achieve long-term disease control. This is due, at least in part, to the refractory nature of what are categorized as terminally exhausted CD8+ T cells to PD-1/PD-L blockade, for example. Considering that CD8+ T cell exhaustion and/or dysfunction is one of the major therapeutic challenges, we aim to uncover pathways that contribute to program T cells toward exhaustion/dysfunction during cancer progression. Methods and experimental procedures: CD47 (a.k.a. integrin-associated protein, IAP) is known for its role as a ‘‘don’t-eat-me’’ signal in malignant, transformed cells. Our recent study found that both human and murine tumor-associated CD8+ T cells derived from lesions of human and murine melanoma, and ovarian cancers exhibit a significant elevation of surface CD47. Surprisingly, the population of tumor-associated CD8+ T cells that express high level of CD47 also express high levels of TOX, the critical factor that drives differentiation of exhausted T (Tex) cells, as well as other known immune checkpoint molecules. Through naïve T cell adoptive transfer experiments paired with single cell-RNA sequencing analysis, we demonstrated that CD47 plays a key role in driving T cell exhaustion. We demonstrated that extracellular matrix protein thrombospondin-1 (TSP-1) is the ligand for CD47-mediated T cell exhaustion during tumor progression by selectively disrupting the interaction between TSP-1 and CD47. Mechanistically, we demonstrate through immunoblot experiments that engagement of TSP-1:CD47 results in activation of calcineurin-NFAT axis, a key modulatory pathway upstream of TOX that drives T cell exhaustion and dysfunction. Summary of new, unpublished data: Our study uncovered a novel role of TSP-1:CD47 interaction, besides chronic antigen stimulation, in driving CD8 T cells exhaustion via the calcineurin-NFAT pathway. Our findings have implications for the mechanism of action in CD47 targeting therapies. Concluding statement: Extracellular matrix protein thrombospondin-1 and CD47 expressed on T cells contributes to promote TOX-driven T cell exhaustion program in cancer. Citation Format: Chien-Huan Weng, Anais Assouvie, Lauren Dong, Jean-Christophe Beltra, Sadna Budhu, Levi Mangarin, Yacine Marouf, Lucia Morgado-Palacin, Cailian Liu, Sébastien Monette, Jonathan Khan, Isabell Schulze, Dmitriy Zamarin, Linda Hamadene, Fadi Samaan, Daniel Hirschhorn, Stephane Pourpe, David Schroder, Roberta Zappasodi, Pamela Holland, Niroshana Anandasabapathy, John Wherry, Jedd D Wolchok, Taha Merghoub. Thrombospondin-1:CD47 signaling contributes to the development of T cell exhaustion in cancer [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B056.

Targeting cytotoxic T lymphocytes (CTLs), as chimeric antigen T cells (CAR-T), T cell receptor-engineered (TCR)-T cells or adoptive cell transfer of tumor infiltrating T cells (TILs) to solid tumors is a major therapeutic challenge. We describe a new strategy to confer these lymphocytes with de novo adhesiveness to surface proteins enriched in the tumor microenvironment. This approach is based on decorating CTLs with monoclonal antibodies (mAbs) specific to any surface protein of interest within the stroma and the extracelullar matrix of solid tumors. For efficient mAb decoration, we have introduced a mAb binding Fc receptor (FcR) scaffold, FcγRIIB1 (CD32B1), which we found to be enriched on B lymphocyte microvilli (MV). This isoform contains an inhibitory ITIM motif within a cytoplasmic tail anchored to the cortical cytoskeleton. We thus generated a non-signaling CD32B1 mutant lacking the ITIM motif (termed ITIM-less CD32B1, or ILCD32B1) and successfully expressed it in human T cells which normally do not express this FcR. The ILCD32B1 expressing lymphocytes bound multiple IgG1 mAbs whose Fc domain was engineered with a 5-residue substitution to reach a nM range of Fc-FcγCR dissociation constants. The mAb decorated ILCD32B1 expressing T cells could readily adhere to a surface-bound cognate antigen. To broaden the utility of this scaffold, we have also generated a new fusion protein in which the entire Fc binding domain was truncated (tILCD32B1) and replaced with a monomeric streptavidin variant, mSA2, via a CD8 hinge. The molecule, termed mSA2-CD8h-tILCD32B1, was also successfully expressed in T cells, readily and stably bound biotinylated IgG mAbs in vitro and once decorated with the biotin labeled mAbs, conferred the T cells with high adhesiveness to multiple surface-coated antigens. mSA2-CD8h-tILCD32B1 expressing human T cells decorated ex vivo with a biotin-labeled mAb retained the antibody for hours after accumulation inside breast tumors implanted in immunodeficient recipient mice. Our results collectively suggest that a non-signaling CD32B1 can be used as a versatile scaffold for mAb decoration of T cells. Our mAb decoration approach can confer new cell adhesive reactivities to improve tumor CTL (CAR-T and TIL) accumulation and retention inside solid tumors.

Ischemia reperfusion (IR) injury increases T cell immunoreceptor with Ig and ITIM domains (TIGIT) on kidney T cells that participate in the pathogenesis of acute kidney injury (AKI) in mice. We investigated the effects of IR injury on TIGIT interacting ligands (CD155, CD112, CD113) and co-signaling molecules (CD226, CD96, PD1). Male C57BL6 mice underwent bilateral kidney IR surgery. TIGIT ligand expression was measured with western blotting at 24h and 18 days post-IR. TIGIT ligands and co-signaling partners were assessed at baseline and 24h post-IR using flow cytometry. Western blotting showed enhanced expression of CD155 (5.6±0.5 fold) and CD112 (2.4±0.5 fold) 24h after AKI. Flow analysis showed increased CD155 (1.5±0.3 vs 26.7±4.7%; P < 0.0001) and CD112 (1.5±0.3 vs 26.7±4.7%; P < 0.0001) expression in CD11b+ cells at 24h compared to control. TIGIT co-stimulatory counterpart CD226 decreased in CD4 (67.1±19.0 vs 46.0±10.3%; P < 0.05), double negative (DN) (92.1±5.1 vs 61.4±10.8%; P < 0.001) and NKT (91.2±3.5 vs 71.2±5.9%; P < 0.001) cells at 24h. TIGIT+ cells expressed PD1 both at baseline and after IR injury in CD4, CD8 and DN T cells. PD1 significantly (P < 0.01) increased at 24h in TIGIT+ CD4, CD8 and DN T cells compared to TIGIT- cells. IR injury increased CD155 and CD112 and decreased co-signaling CD226 expression. TIGIT and PD1 are co-expressed by a subset of kidney T cells. TIGIT ligands and co-signaling partners are promising targets for IR injury to kidney and other organs. This work was supported by NIH/NIDDK grants R01DK132278 and R01DK123342 to SN and HR, respectively. Immune Response Regulation: Cellular Mechanisms (IRC)

Although anti-programmed death 1 (PD-1)/PD-1 ligand 1 monoclonal antibodies are effective against various cancers, resistance remains a significant issue in many patients. The immunosuppressive T cell immunoreceptor with Ig and ITIM domains (TIGIT)/CD155 axis has emerged as one of the crucial mechanisms contributing to this resistance. However, the complexities of CD155 expression remain incompletely understood. In this study, we conducted clustered regularly interspaced palindromic repeats (CRISPR) screening to identify key molecules involved in the regulation of CD155 expression and to investigate their roles in modulating CD155 within the tumor microenvironment (TME). CRISPR screening revealed that dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) as one of the key regulators of CD155 expression via the PI3K/Akt signaling pathway. In mouse models, inhibition of Dyrk1a using CRISPR/CRISPR-associated protein 9 (Cas9) technology or treatment with harmine, a DYRK1A inhibitor reduced CD155 expression, resulting in overcoming the resistance to PD-1 blockade. Moreover, in human head and neck squamous cell carcinoma (HNSCC) cell lines, inhibition of the PI3K/Akt signaling pathway reduced CD155 expression, similar to mouse models. In addition, cetuximab-mediated epidermal growth factor receptor blockade reduced CD155 expression by targeting downstream PI3K/Akt signaling in certain HNSCC cell lines. Clinically, patients with HNSCC harboring high CD155 expression exhibited elevated levels of Akt phosphorylation, consistently indicating that the PI3K/Akt signaling pathway is involved in CD155 expression. Furthermore, high CD155 expression contributed to PD-1 blockade resistance especially in patients who had high CD8+ T cell infiltration. These results emphasized the role of the PI3K/Akt signaling pathway in regulating CD155 expression, which may influence resistance to PD-1 blockade therapies in various cancers, particularly those characterized by an inflamed TME. This study suggests that the reduction of CD155 expression by inhibiting the PI3K/Akt signaling pathway could overcome resistance to PD-1 blockade therapies. Katsushige Kawase, Shusuke Kawashima, Tatsuya Nishi, Takashi Inozume, Takao Morinaga, Masahito Kawazu, Toyoyuki Hanazawa, Yosuke Togashi. PI3K/Akt signaling pathway regulates CD155 expression involved in resistance to cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6172.

RNA viral infections seriously endanger human health. Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP2) suppresses innate immunity against influenza A virus, and pharmacological inhibition of SHP2 provokes hepatic innate immunity. SHP2 binds and catalyzes tyrosyl dephosphorylation of protein zero-related (PZR), but the regulatory effect of PZR on innate immune response to viral infection is unclear. In this study, the transcription and protein level of PZR in host cells were found to be decreased with RNA viral infection, and high level of PZR was uncovered to inhibit IFN signaling mediated by RIG-I and MDA5. Through localizing in mitochondria, PZR targeted and interacted with MAVS (also known as IPS-1/VISA/Cardif), suppressing the aggregation and activation of MAVS. Specifically, Y263 residue in ITIM is critical for PZR to exert immunosuppression under RNA viral infection. Moreover, the recruited SHP2 by PZR that modified with tyrosine phosphorylation under RNA viral infection might inhibit phosphorylation activation of MAVS. In conclusion, PZR and SHP2 suppress innate immune response to RNA viral infection through inhibiting MAVS activation. This study reveals the regulatory mechanism of PZR-SHP2-MAVS signal axis on IFN signaling mediated by RIG-I and MDA5, which may provide new sight for developing antiviral drugs.

CAR T cell therapy achieves impressive remission rates in hematologic malignancies, yet clinical durability remains a persistent challenge. Autologous CAR T cell products require time-intensive individualized manufacture, while allogeneic CAR T cells demonstrate shortened persistence compared to autologous counterparts. Recent mechanistic insights reveal that endogenous TCR signaling through weak self-pMHC interactions provides essential homeostatic cues that support long-term CAR T cell survival and memory formation. However, current manufacturing paradigms treat native TCR signaling as a binary choice: either fully preserved or entirely deleted. We hypothesize that tonic TCR signaling functions as a tunable parameter rather than an on/off switch, and that CD5, an ITIM-bearing inhibitory receptor that marks tonic signal strength, represents a rational therapeutic target for optimizing CAR T cell fitness. Critically, the field lacks clarity on whether CD5 is mechanistically required for CAR T cell durability or merely marks cells with inherently superior developmental history. This distinction is fundamental: if CD5 is mechanistically required, next-generation CAR T therapies can be rationally engineered to fine-tune CD5 expression or target CD5-dependent signaling pathways. If CD5 is predictive only, alternative upstream mechanisms must be identified and targeted. Our work directly addresses this gap by empirically testing whether CD5 manipulation alters CAR T cell fitness and by identifying the specific signaling and transcriptional nodes that CD5 controls. By establishing the mechanistic role of CD5 in tonic signaling, we will deliver quantitative rules for calibrating native TCR input in both autologous and allogeneic CAR T products, enabling the design of safer, more durable, and more accessible CAR T therapies across patient populations. Samuel D. Burciaga, Michael Yarnell, Lily Mussallem, Amanda Novak, Ross Kedl, Terry Fry. Understanding the Role of Tonic TCR Signaling to Optimize CAR-T Efficacy [abstract]. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C005.

Background: T-cell immunoglobulin and ITIM domain (TIGIT) was recently defined as an inhibitory receptor that is expressed on NK cells and T cells. Results: TIGIT/poliovirus receptor (PVR) ligation signaling mediates suppression of IFN-γ production through NF-κB pathway via β-arrestin 2-mediated negative signaling. Conclusion: TIGIT/PVR signaling suppresses IFN-γ production of NK cells. Significance: TIGIT/PVR signaling acts as a potent negative mediator to down-regulate NK cell response for immune homeostasis. Natural killer (NK) cell activation is well orchestrated by a wide array of NK cell receptor repertoire. T-cell immunoglobulin and ITIM domain (TIGIT) receptor was recently defined as an inhibitory receptor that is expressed on NK cells and T cells. TIGIT receptor/poliovirus receptor (PVR) ligand engagement signaling inhibits cytotoxicity mediated by NK and CD8+ T cells. However, it is unclear how TIGIT/PVR signaling regulates cytokine secretion in NK cells. Here we show that TIGIT/PVR engagement suppresses interferon-γ (IFN-γ) production of NK cells. TIGIT transgenic NK cells generate less IFN-γ undergoing TIGIT/PVR ligation. Moreover, TIGIT knock-out NK cells produce much more IFN-γ. TIGIT/PVR ligation signaling mediates suppression of IFN-γ production via the NF-κB pathway. We identified a novel adaptor β-arrestin 2 that associates with phosphorylated TIGIT for further recruitment of SHIP1 (SH2-containing inositol phosphatase 1) through the ITT-like motif. Importantly, SHIP1, but not other phosphatases, impairs the TNF receptor-associated factor 6 (TRAF6) autoubiquitination to abolish NF-κB activation, leading to suppression of IFN-γ production in NK cells.

Summary Many therapeutic antibodies deplete target cells and elicit immunotherapy by engaging activating Fc gamma receptors (FcγRs) on host effector cells. These antibodies are negatively regulated by the inhibitory FcγRIIB (CD32B). Dogma suggests inhibition is mediated through the FcγRIIB immunoreceptor tyrosine-based inhibition motif (ITIM), negatively regulating immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling from activating FcγR. To assess this, we generated experimental models expressing human (h)FcγRIIB on targets or effectors, lacking or retaining ITIM signaling capacity. We demonstrate that signaling through the hFcγRIIB ITIM is dispensable for impairing monoclonal antibody (mAb)-mediated depletion of normal and malignant murine target cells through three therapeutically relevant surface receptors (CD20, CD25, and OX40) affecting immunotherapy. We demonstrate that hFcγRIIB competition with activating FcγRs for antibody Fc, rather than ITIM signaling, is sufficient to impair activating FcγR engagement, inhibiting effector function and immunotherapy.

One of the key challenges for successful cancer therapy is the capacity of tumors to evade immune surveillance. Tumor immune evasion can be accomplished through the induction of T cell exhaustion via the activation of various immune checkpoint molecules. The most prominent examples of immune checkpoints are PD-1 and CTLA-4. Meanwhile, several other immune checkpoint molecules have since been identified. One of these is the T cell immunoglobulin and ITIM domain (TIGIT), which was first described in 2009. Interestingly, many studies have established a synergistic reciprocity between TIGIT and PD-1. TIGIT has also been described to interfere with the energy metabolism of T cells and thereby affect adaptive anti-tumor immunity. In this context, recent studies have reported a link between TIGIT and the hypoxia-inducible factor 1-α (HIF1-α), a master transcription factor sensing hypoxia in several tissues including tumors that among others regulates the expression of metabolically relevant genes. Furthermore, distinct cancer types were shown to inhibit glucose uptake and effector function by inducing TIGIT expression in CD8+ T cells, resulting in an impaired anti-tumor immunity. In addition, TIGIT was associated with adenosine receptor signaling in T cells and the kynurenine pathway in tumor cells, both altering the tumor microenvironment and T cell-mediated immunity against tumors. Here, we review the most recent literature on the reciprocal interaction of TIGIT and T cell metabolism and specifically how TIGIT affects anti-tumor immunity. We believe understanding this interaction may pave the way for improved immunotherapy to treat cancer.

Background Neoplastic myeloma cells show a wide variety of mechanisms that induce the microenvironment, allowing immune evasion and promoting their proliferation, survival, migration and drug resistance. In this context, immunocheckpoints are particularly important, representing the modulators of signal pathways responsible for immune tolerance, a mechanism that prevents the destruction of cancer cells by the immune system. T cell Ig and ITIM domains (TIGIT), a member of the poliovirus (PVR) /nectin receptor family, is a new immune checkpoint that negatively regulates T cell functions. In Multiple Myeloma (MM), TIGIT could play a relevant role leading to immunological escape. TIGIT performs its task mainly through binding ligands: CD155 and CD112, through which it inhibits the cytotoxicity of CD8+ T cells, as well as that of NK. In This Study, we evaluate the presence of TIGIT in bone marrow using instant digital pathology. AIMS Aims of this study are to develop a valid and reproducible workflow for instant digital pathology, to demonstrate the presence of TIGIT-positive T lymphocytes in bone marrow of patients with MM and to evaluate its correlation with clinical features PATIENTS and Methods 18 patients with newly diagnosed MM not previously treated, were evaluated in this study. The sample was enrolled from November 2022 to May 2023. Bone marrow blood was loaded on a porous matrix able to retain cells (Cytomatrix, UCS Diagnostics srl, Rome) for real time assessment of sample adequacy, morphological details preservation, and composition of hematopoietic subpopulations, using an ex-vivo fluorescent confocal microscope (FCM). Expert pathologists compared the digital FCM images with permanent Formalin Fixed Paraffin Embedded (FFPE) sections of the matrix and trephine bone marrow biopsy. Immunohistochemistry for TIGIT (TG-1 Clone, OncoDianova) was performed on matrix FFPE sections. RESULTS Our results showed that 15/17 (88%) patients resulted positive for TIGIT and 2/17 (12%) were negative. TIGIT expression was found mainly on CD8+ T lymphocytes, TIGIT positivity was correlated with parameters that define the aggressiveness of myeloma disease such as PC > 60% or FLC > 100, ISS score, R-ISS score, cytogenetic risk and LDH > 220mU/ml. Among the patients defined as “ultra-High Risk”, 9/10 (90%) resulted positive for TIGIT. Comparing TIGIT with the MM International Staging System (ISS), we observed that all patients with scores II and III were positive for TIGIT (100%) while 78% of them with ISS I results positive. Conclusions Our results suggest that FCM can be applied immediate evaluation of bone marrow aspirates, preserving the cellular sample for final histological analysis. “Furthermore, our data shows the positivity of TIGIT in 88% of patients, demonstrating the presence of this immune checkpoint in the bone marrow microenvironment. These data will need to be confirmed in a larger group of patients, but they open up the possibility of new target treatments in multiple myeloma

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Siglecs, membrane-bound lectins of the sialic acid-binding immunoglobulin superfamily, inhibit immune responses by recruiting tyrosine phosphatases (e.g., SHP-1 and SHP-2) through their cytoplasmic immunoreceptor tyrosine-based inhibition motif (ITIM) domain. The role of Siglecs in infection has been extensively studied, but downstream signaling through the ITIM domain remains unclear. Here, we used a GST pull-down assay to identify additional proteins associated with the ITIM domain during bacterial infection. Gdi2 bound to ITIM under normal homeostasis, but Rab1a was recruited to ITIM during bacterial infection. Western blot analysis confirmed the presence of SHP-1 and SHP-2 in eluted ITIM-associated proteins under normal homeostasis. We confirmed the association of ITIM with Gdi2 or Rab1a by transfection of corresponding expression vectors in 293T cells followed by immunoprecipitation-western blot assay. Thus, ITIM's role in the inhibition of the immune response during bacterial infection may be regulated by interaction with Gdi2 and Rab1a in addition to SHP-1 and SHP-2.

Protein Zero Related (PZR) is a type I transmembrane glycoprotein encoded by the MPZL1 gene and a member of the immunoglobulin superfamily (IgSF). Despite sharing 46 % sequence homology in its extracellular domain with myelin P0 protein (MPZ), PZR exhibits distinct functional specialization. It undergoes alternative splicing to generate three isoforms (PZR, PZRa, PZRb) with tissue-specific expression patterns, predominantly enriched in cardiovascular, renal, and pancreatic tissues, and localized to cell-cell contact sites and migration-associated domains, consistent with its roles in adhesion and motility. Functionally, PZR serves as a multifunctional signaling hub, acting as both a receptor for concanavalin A (ConA) and a regulator of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) and Src family kinases. ConA binding triggers c-Src activation, leading to PZR autophosphorylation and subsequent recruitment of SHP-2. Its intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) further mediate interactions with Src kinases and SHP-2, driving oncogenesis, immunomodulation, and antiviral responses. Post-translational modifications, including phosphorylation and glycosylation, enhance its protein-binding capacity, enabling broad influence over physiological and pathological processes, particularly in tumor microenvironment signaling and cellular fate regulation. Initially implicated in Noonan syndrome and schizophrenia, recent studies highlight PZR as a critical regulator of cancer cell adhesion and migration, with dysregulation accelerating disease progression. This review systematically analyzes the structural and functional properties of PZR, explores its disease-associated molecular mechanisms, and integrates emerging evidence to propose PZR as a promising therapeutic target. By delineating its signaling networks and pathophysiological roles, we provide a framework for advancing diagnostic and therapeutic strategies targeting PZR-related disorders.

Colorectal cancer (CRC) is rising alarmingly in younger populations, potentially arising from additional risk factors such as obesity, pro-inflammatory microbiomes and accumulation of toxic metabolites. However, how metabolites such as ammonia impact key signaling pathways to promote CRC remains unclear. Our study investigates a critical link between gut microbiome alterations, ammonia, and their toxic effects on the TGF-β signaling pathway, to drive CRC progression. Through human TCGA-CRC data analyses, with animal model studies, molecular docking and signaling studies, we examine how gut microbiome alterations, particularly ammonia production, modulate key oncogenic pathways involving Smad3 adaptor, SPTBN1, CEACAM1, and TGF-β. Our work builds on two of our recent publications (Cell Rep.2024.43(9):114676 and Sci Transl Med.2021.13(624): eabk2267). Results We observe altered microbial populations in an obesity induced mouse model of cancer, where ammonia promotes caspase-3-mediated cleavage of the SMAD3 adaptor βII-spectrin (SPTBN1). Cleaved SPTBN1 fragments form adducts with ammonia that induce pro-inflammatory cytokine expression and disrupt TGF-β signaling. Extending on AlphaFold docking simulations, we identified that ammonia interacts with six polar residues at SPTBN1 (S553, Y556, S663, Y666, N986, and T1178) to form hydrogen bonds that disrupt downstream SMAD3 signaling, altering TGF-β signaling to a protumor genic phenotype. Blocking SPTBN1, through an SPTBN1-specific siRNA blocks ammonia toxicity and restores normal SMAD3/TGF-β signaling by reducing the abundance of SPTBN1-cleaved fragments in SW480 and HCT116 (CRC) cell lines. In addition, our research establishes crosstalk between TGF-β signaling and a microbial sensor, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is significantly overexpressed in CRC patients. We identified CEACAM1-SPTBN1 interactions at specific residues (E517 and Y520) within the immunoreceptor tyrosine-based inhibitory motif (ITIM) of CEACAM1 cytoplasmic domain, identifying a potential axis that is harnessed by the altered microbiome. Our study identifies mechanistic insights into how microbial metabolites such as ammonia target TGF-β, a major signaling pathway, to promote CRC. These findings underscore the therapeutic potential of targeting SPTBN1 to restore tumor-suppressive TGF-β signaling and improve outcomes for CRC patients. Krishanu Bhowmick, Puja Ghosh, Xiyan Xiang, Xiaochun Yang, Taj Mohammad, Bibhuti Mishra, Md. Imtaiyaz Hassan, Adrian R Krainer, Srinivasan Dasarathy, Keith A Crandall, Lopa Mishra. Microbial metabolite ammonia disrupts CEACAM1 TGF-β signaling to drive colon cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6533.

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Leukocyte immune-type receptors (LITRs) represent a polymorphic and polygenic family of immunoregulatory proteins originally discovered in channel catfish (Ictalurus punctatus; IpLITRs). Belonging to the immunoglobulin superfamily (IgSF), IpLITRs are generally classified as stimulatory or inhibitory types based on their utilization of various intracellular tyrosine-based signaling motifs. While research has shown that IpLITRs can activate as well as abrogate different immune cell effector responses including phagocytosis, recent identification of LITRs within the zebrafish genome (Danio rerio; DrLITRs) revealed the existence of fish LITR-types uniquely containing counteracting stimulatory and inhibitory cytoplasmic tail (CYT) region motifs (i.e., an immunoreceptor tyrosine-based activation motif; ITAM, and immunoreceptor tyrosine-based inhibitory motif; ITIM) within the same receptor. This arrangement is unusual as these motifs typically exist on separate stimulatory (i.e., ITAM-containing) or inhibitory (i.e., ITIM-containing) immunoregulatory receptors that then co-engage to fine-tune cellular signaling and effector responses. Using a flow cytometric-based phagocytosis assay, we show here that engagement of DrLITR 1.2-expressing cells with antibody coated 4.5 μm beads causes a robust ITAM-dependent phagocytic response and reveal that its tandem ITIM motif surprisingly enhances the DrLITR 1.2-induced phagocytic activity while simultaneously decreasing the receptors ability to bind the beads. Confocal microscopy studies also revealed that the ITIM-associated inhibitory signaling molecule SHP-2 is localized to the phagocytic synapse during the phagocytic response. Overall, these results provide the first functional characterization of teleost immune receptors containing a tandem ITAM and ITIM and allow for the proposal of an intracytoplasmic tail signaling model for ITIM-mediated enhancement of ITAM-dependent cellular activation.

Context: TIGIT has grown to be an increasingly important target for immunotherapy of cancer. However, it is unclear if there is an association between abundance of TIGIT expressing immune cells in tumor microenvironment and effectiveness of TIGIT-based intervention. This study utilizes an exploratory novel duplex immunohistochemical (IHC) assay to assess if the distribution patterns and/or the abundance of TIGIT-positive immune cells are related to the expression levels of TIGIT’s receptor PVR in tumor. Methods: Individual formalin-fixed, paraffin-embedded surgical resections and tissue microarray cores of 75 cervical, 84 esophageal, 79 non-small cell lung , and 81 small cell lung carcinoma specimens were stained using a duplex chromogenic IHC assay that detects PVR and TIGIT sequentially on a single slide. The slides were examined for (1) overall intensity and percentage of tumor staining positive for PVR, (2) percentage of tumor area occupied by TIGIT-expressing immune cells, and (3) distribution pattern of TIGIT-positive cells. For each specimen, overall PVR intensity was multiplied by percentage of tumor positive for PVR to generate the PVR Expression Score. Results: PVR and TIGIT staining at any intensity was widely observed in all four tumor types ranging from 91% to 100% of specimens positive for PVR and 77% to 100% of specimens positive for TIGIT. Little to no differences in the average percentages of PVR tumor positivity or TIGIT tumor area positivity were found among the four tumor types. However, individual tumor specimens differed widely in these two parameters. Among all 319 tumor specimens evaluated, the percentages of tumor expressing PVR at any intensity ranged from 0 to 100% with approximately 1/3 of the specimens having less than 50% and 3% showing no PVR expression. Percentages of tumor area occupied by TIGIT-positive cells ranged from 0% to 30% with approximately 40% of the specimens having less than 1%. Distribution patterns of TIGIT-expressing cells in each specimen was classified as peritumoral, intratumoral, or mixed. Specimens classified as peritumoral exhibited higher PVR Expression Scores than those classified as intratumoral. Specimens classified as having a mixed TIGIT-positive cell distribution pattern had an average PVR Expression Score that was intermediate between specimens having intratumoral and peritumoral TIGIT distribution patterns. Interestingly, no correlation was found between tumor PVR Expression Scores and abundance of TIGIT expressing cells in tumor area. Conclusion: The correlation between high tumor PVR expression levels and peritumoral distribution patterns of TIGIT-positive cells suggests PVR/TIGIT signaling pathway activation status could be important in infiltration of T cells into tumor parenchyma. Citation Format: Sara A. Moore, Julie Cheung, Aram B. Cholanians, June Clements, Jessica L. Baumann, Tsu-Shuen Tsao. Association between poliovirus receptor (PVR) expression in tumor and exclusion of immune cells expressing T cell immunoreceptor with Ig and ITIM domain (TIGIT) from tumor area [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6631.

Background and Significance: Initiation of B-cell receptor (BCR) signaling, and subsequent antigen-encounter in germinal centers represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL) and lymphoma also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells, the significance of its expression on B-cells was unclear. High expression levels on the surface of B-ALL cells are associated with poor clinical outcomes in pediatric (COG P9906) and adult (ECOG E2993) B-ALL cohorts. In DLBCL, aggressive disease is associated with CD25-inactivation by shedding and high serum levels of soluble CD25. Results: Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCδ, immunoreceptor tyrosine-based inhibitory motif (ITIM)-bearing receptors and inhibitory phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCδ and phosphatases, conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early stages of B-cell development (e.g. B-ALL) CD25-loss induced hyperactivation of oncogenic signaling followed by cell death. In contrast, genetic deletion of CD25 in models for mature B-cell lymphoma accelerated proliferation of malignant cells. Biochemical and interactome studies revealed a critical role of CD25 in feedback regulation of oncogenic signaling in B-cell malignancies: Signaling from BCR-ABL1 and RAS-oncogenes in B-ALL as well as oncogenic BCR-signaling in DLBCL induced PKCδ-mediated phosphorylation of CD25 on its cytoplasmic tail (S 268). Importantly, CD25-phosphorylation stabilized its expression at the cell membrane and opposed both inactivation by CD25-shedding and internalization. Moreover CD25-S 268 tail-phosphorylation enabled CD25 to recruit inhibitory receptors, including CD22, that bear ITIM-motifs for the activation of inhibitory phosphatases (e.g. SHP1, Figure 1). Thereby, the positively charged cytoplasmic tail of CD25 engages negatively charged residues in the cytoplasmic tails of the BCR signaling chain CD79B and the inhibitory CD22-ITIM. Molecular dynamics simulations revealed that the ternary complex between CD25, CD79B and CD22 enables recruitment and activation of the phosphatase SHP1 within reach of its substrate, the BCR signaling chain CD79B. Thereby, the SH2-domain of SHP1 engages the CD22-ITIM motif, while its phosphatase domain makes contact with the CD79B-ITAM to dephosphorylate and terminate BCR-signaling. This complex, while engaging and activating SHP1, is held together by CD25 ( Figure 1). To test functional consequences of destabilization of this complex, we generated a knockin mouse model carrying a single point mutation (S268A) in the cytoplasmic tail of CD25. The CD25 S268A knockin mutation abolished recruitment and activation of SHP1. In addition, the S268A-mutant failed to assemble CD22 and CD79B in complex with SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function and failure to terminate oncogenic signaling resulted in constitutive activation of ERK and NF-κB, autonomous Ca 2+-oscillations. Hyperactivation of oncogenic signaling resulted in exhaustion and cell death in B-ALL cells, as opposed to excessive proliferation and acceleration of disease in mature B-cell lymphoma. Conclusions: While CD25 has an established function in IL2 signal transduction in T- and NK-cells, these findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and provides an explanation for the common feature of CD25 shedding and high serum levels of functionally inactive soluble CD25 in aggressive B-cell lymphomas.

Background Microglia plays crucial roles in Alzheimer’s disease (AD) development. Triggering receptor expressed on myeloid cells 2 (TREM2) in association with DAP12 mediates signaling affecting microglia function. Here we study the negative regulation of TREM2 functions by leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2), an inhibitory receptor bearing ITIM motifs. Methods To specifically interrogate LILRB2-ligand (oAβ and PS) interactions and microglia functions, we generated potent antagonistic LILRB2 antibodies with sub-nanomolar level activities. The biological effects of LILRB2 antagonist antibody (Ab29) were studied in human induced pluripotent stem cell (iPSC)–derived microglia (hMGLs) for migration, oAβ phagocytosis, and upregulation of inflammatory cytokines. Effects of the LILRB2 antagonist antibody on microglial responses to amyloid plaques were further studied in vivo using stereotaxic grafted microglia in 5XFAD mice. Results We confirmed the expression of both LILRB2 and TREM2 in human brain microglia using immunofluorescence. Upon co-ligation of the LILRB2 and TREM2 by shared ligands oAβ or PS, TREM2 signaling was significantly inhibited. We identified a monoclonal antibody (Ab29) that blocks LILRB2/ligand interactions and prevents TREM2 signaling inhibition mediated by LILRB2. Further, Ab29 enhanced microglia phagocytosis, TREM2 signaling, migration, and cytokine responses to the oAβ-lipoprotein complex in hMGL and microglia cell line HMC3. In vivo studies showed significantly enhanced clustering of microglia around plaques with a prominent increase in microglial amyloid plaque phagocytosis when 5XFAD mice were treated with Ab29. Conclusions This study revealed for the first time the molecular mechanisms of LILRB2-mediated inhibition of TREM2 signaling in microglia and demonstrated a novel approach of enhancing TREM2-mediated microglia functions by blocking LILRB2-ligand interactions. Translationally, a LILRB2 antagonist antibody completely rescued the inhibition of TREM2 signaling by LILRB2, suggesting a novel therapeutic strategy for improving microglial functions.

Understanding how membrane composition influences the dynamics and function of transmembrane proteins is crucial for the comprehensive elucidation of cellular signaling mechanisms and the development of targeted therapeutics. In this study, we employed all-atom molecular dynamics simulations to investigate the impact of different membrane compositions on the conformational dynamics of the NKG2A/CD94/HLA-E immune receptor complex, a key negative regulator of natural killer cell cytotoxic activity. Our results reveal significant variations in the behavior of the immune complex structure across five different membrane compositions, which include POPC, POPA, DPPC, and DLPC phospholipids, and a mixed POPC/cholesterol system. These variations are particularly evident in the intracellular domain of NKG2A, manifested as changes in mobility, tyrosine exposure, and interdomain communication. Additionally, we found that a large concentration of negative charge at the surface of the POPA-based membrane greatly increased the number of contacts with lipid molecules and significantly decreased the exposure of intracellular NKG2A ITIM regions to water molecules, thus likely halting the signal transduction process. Furthermore, the DPPC model with a membrane possessing a high transition temperature in a gel-like state became curved, affecting the exposure of one ITIM region. The decreased membrane thickness in the DPLC model caused a significant transmembrane domain tilt, altering the linker protrusion angle and potentially disrupting the hydrogen bonding network in the extracellular domain. Overall, our findings highlight the importance of considering membrane composition in the analysis of transmembrane protein dynamics and in the exploration of novel strategies for the external modulation of their signaling pathways.

No abstract available

Background and Significance: CD25 (IL2RA) has been extensively studied as IL2-receptor chains expressed on T- and NK-cells. However, CD25 is sharply upregulated upon BCR-signaling. Mature B-cell lymphoma subtypes (CLL, MCL, DLBCL) that depend on oncogenic BCR-signaling inactivate CD25 by proteolytic cleavage and aggressiveness of disease is associated with low expression CD25 surface levels and high serum levels of cleaved CD25. Results: Here, we discovered a mechanistic framework of dynamic BCR-feedback control and its dependency on coordinated activity of PKCδ and CD25. To study the role of CD25 in a model of aggressive B-cell lymphoma, we propagated splenic B-cells from CD25-fl/fl mice and transduced them with the CARD11-L232LI oncogene. Induction of Cre-activity in this model resulted in rapid loss of CD25 surface expression and dramatic acceleration of proliferation of CD25-/- B-cell lymphoma cells. Conditional ablation of CD25 during normal B-cell development in vivo (Cd25-fl/fl x Mb1-Cre) resulted in the development of B-cell autoimmunity with pervasive autoantibody production, development of spontaneous germinal centers. RNA-seq and mass spectrometry-based global phosphoproteomic studies revealed that acute ablation of CD25 in CARD11-L232LI B-cell lymphoma resulted in prominent activation of NF-kB gene expression programs, hyperphosphorylation of multiple substrates in the BCR-signaling pathway, including SYK, Src-family kinases, BLNK, NF-kB-components and PKCδ. Conversely, CD25-deletion caused loss of phosphorylation of inhibitory phosphatases SHP1 and SHIP1. Imbalances of control of BCR-signalling following CD25-deletion were confirmed by Western blot, namely hyperphosphorylation of SYK BLNK, ERK, NFKBIA and PKCδ as well as loss of SHP1- and SHIP1-phosphorylation. In addition to Cre-mediated deletion of CD25 in murine B-cell lymphoma cells, we confirmed these results by CRISPR-mediated deletion of CD25 in patient-derived mantle cell lymphoma xenografts (PDX). Mechanistic studies revealed that oncogenic BCR signaling induced PKCδ-dependent CD25-phosphorylation on its cytoplasmic tail (S268, T271). Genetic deletion of PKCδ and mutations of CD25 S268/T271 residues demonstrated that PKCδ-mediated CD25-phosphorylation was critical for CD25-dependent feedback control of oncogenic BCR signaling. Our genetic and interactome studies revealed that BCR-signaling induced PKCδ-mediated phosphorylation and recruitment of CD25 to the BCR. Instead of transducing IL2-signals, CD25 directed ITIM-mediated SHP1-activation towards BCR-signaling molecules, within range of SHP1-mediated dephosphorylation. In proximity of the BCR, CD25 attracted ITIM-receptor nanoclusters, resulting in local concentration and activation of SHP1 for negative feedback control of BCR-signaling. Defective BCR-feedback control in human CD25-/- germinal center B-cells could be restored by a novel bispecific antibody to direct ITIM-dependent SHP1 activation towards BCR-proximal signaling molecules. BCR-dependent B-cell lymphomas frequently harbor deleterious mutations of PKCδ and CD25 is typically inactivated by proteolytic cleavage. To investigate the interplay between PKCδ and CD25, we introduced genetic CD25 knockin alleles using HDR templates encoding GFP and wildtype CD25 or CD25-S268A/T271V (AV) mutations in primary human germinal center (GC) B-cells cultured on YK6 follicular dendritic cells. Interestingly, CD25-AV mutant knockin GC B-cells failed to terminate BCR-signaling and showed autonomous Ca2+ oscillations. In addition, CD25-AV knockin GC B-cells expressed increased levels of the activation markers CD69, CD80 and CD86 and exhibited constitutive activation of the NF-kB pathway. In coculture experiments determining how CD25 regulates competitive fitness, CD25-AV knockin GC B-cells rapidly outcompeted their CD25-wildtype counterparts. Conclusions: While CD25 has an established function in IL2 signal transduction in T- and NK-cells, our findings highlight the previously unrecognized role of PKCδ and CD25 in assembling inhibitory phosphatases to control BCR signaling. Also in the context of oncogenic BCR-signaling in B-cell lymphoma, CD25 and PKCδ cooperate as negative regulators. These findings are consistent with frequent inactivation of CD25 by proteolytic cleavage as well as PKCδ-mutations in B-cell lymphomas.

Key Points Siglec-F does not dampen A. fumigatus–elicited lung eosinophilia. Siglec-F cross-linking enhances mediator release from IL-33–stimulated eosinophils. This effect depends on the cytoplasmic tail, but not the ITIM/ITIM-like motifs. Visual Abstract Eosinophils are potent innate effector cells associated mainly with type 2 immune responses elicited by helminths and allergens. Their activity needs to be tightly controlled to prevent severe inflammation and tissue damage. Eosinophil degranulation and secretion of inflammatory effector molecules, including cytokines, chemokines, and lipid mediators, can be regulated by activating and inhibitory receptors on the cell surface. In this study, we investigated the modulation of proliferation, apoptosis, gene expression, and cytokine/chemokine secretion from IL-33–activated Mus musculus eosinophils on cross-linking of the transmembrane receptor Sialic acid–binding Ig-like lectin F (Siglec-F). Siglec-F contains an ITIM plus an ITIM-like motif in its intracellular tail and is mainly regarded as an inhibitory and apoptosis-inducing receptor. In vitro costimulation of bone marrow–derived eosinophils with anti–Siglec-F and IL-33 compared with treatment with either alone led to enhanced STAT6 phosphorylation, stronger induction of hypoxia/glycolysis-related proinflammatory genes, and elevated secretion of type 2 cytokines (IL-4, IL-13) and chemokines (CCL3, CCL4) with only minor effects on proliferation and apoptosis. Using a competitive mixed bone marrow chimera approach with wild-type and Siglec-F–deficient eosinophils, we observed no evidence for Siglec-F–regulated inhibition of Aspergillus fumigatus–elicited lung eosinophilia. Truncation of the Siglec-F cytoplasmic tail, but not mutation of the ITIM and ITIM-like motifs, ablated the effect of enhanced cytokine/chemokine secretion. This provides evidence for an ITIM phosphorylation-independent signaling pathway from the cytoplasmic tail of the Siglec-F receptor that enhances effector molecule release from activated eosinophils.

Background and Objective Persistent infection of hepatitis B virus (HBV) and liver damage in immune active chronic hepatitis B (CHB) could be partly due to the overreaction of natural killer (NK) cells, including pro-inflammatory cytokine secretion and cytotoxicity. An immunosuppressive receptor, T-cell immunoglobulin and immunoreceptor tyrosine–based inhibitory motif (ITIM) domain (TIGIT) is specifically expressed in NK cells. This study aims to investigate the role of the TIGIT signaling pathway in regulating NK cell functions in patients with CHB. Method We comparatively assessed the expression of TIGIT in NK cells of patients with immune active CHB (CHB-IA), carriers of immune control chronic HBV (CHB-IC), and healthy controls (HCs), and then explored mechanisms of the TIGIT signaling pathway in regulating NK cell-mediated liver injury by different molecular assessments. Result The expression of TIGIT in NK cells was enhanced in CHB-IC but was reduced in CHB-IA compared with the HC group. In patients with CHB-IA, the expression of TIGIT was inversely correlated with intensity of the liver damage. Moreover, TIGIT-NK cells show higher IFN-γ secretion capability, degranulation activity, and cytotoxicity but lower apoptosis than TIGIT+ NK cells. Blockade of the TIGIT pathway with anti-TIGIT antibody increased NK cell function, while activation of the TIGIT pathway with TIGIT Fc and CD155 Fc chimera protein down-regulated NK cell function. Conclusion Our data showed that the TIGIT signaling pathway participates in NK cell impairment, which could be used as a new therapeutic target to protect patients with chronic HBV infection from severe liver injury.

Leukocyte immunoglobulin (Ig)-like receptors (LILRs) on human chromosome 19q13.4 encode 11 immunoglobulin superfamily receptors, exhibiting genetic diversity within and between human populations. Among the LILR genes, the genomic region surrounding LILRB3 and LILRA6 has yet to be fully characterized due to their significant sequence homology, which makes it difficult to differentiate between them. To examine the LILRB3 and LILRA6 genomic region, a tool named JoGo-LILR CN Caller, which can call copy number from short-read whole genome sequencing (srWGS) data, was applied to an extensive international srWGS dataset comprising 2,504 samples. During this process, a previously unreported loss of both LILRB3 and LILRA6 was detected in three samples. Using long-read sequencing of these samples, we have discovered a novel large deletion (33,692 bp) in the LILRB3 and LILRA6 genomic regions in the Japanese population. This deletion spanned three genes, LILRB3, LILRA6, and LILRB5, resulting in LILRB3 exons 12-13 being located immediately downstream of LILRB5 exons 1-12 with the loss of LILRA6, suggesting the potential expression of a hybrid gene between LILRB5 and LILRB3 (LILRB5-3). Transcription and subsequent translation of the LILRB5-3 hybrid gene were also verified. The hybrid junction was located within the intracellular domain, resulting in an LILRB5 extracellular domain fused to a partial LILRB3 intracellular domain with three immunoreceptor tyrosine-based inhibitory motifs (ITIMs), suggesting that LILRB5-3 acquired a novel signaling function. Further application of the JoGo-LILR tool to srWGS samples suggested the presence of the LILRB5-3 hybrid gene in the CEU population. Our findings provide insight into the genetic and functional diversity of the LILR family.

Immune modulators play a crucial role in carcinogenesis and cancer progression by impairing cancer cell-targeted immune responses. T-cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) regulates T-cell function and cancer cell recognition and was therefore identified as a promising target for cancer immunotherapy. TIGIT is expressed in T cells and natural killer (NK) cells and has three ligands: CD155, CD112 and CD113. CD155 binds TIGIT with the highest affinity and promotes direct and indirect downregulation of T-cell response. TIGIT signalling further inhibits NK function and secretion of proinflammatory cytokines. An association between TIGIT expression and poor survival was identified in multiple cancer entities. Blocking TIGIT with monoclonal antibodies, and a combination of TIGIT and programmed cell death protein 1 blockade in particular, prevented tumour progression, distant metastasis and tumour recurrence in in vivo models. Inhibition of TIGIT is currently evaluated in first clinical trials.

No abstract available

Thymic homing of hematopoietic progenitor cells (HPCs) is tightly regulated for proper T cell development. Previously we have identified a subset of specialized thymic portal endothelial cells (TPECs), which is important for thymic HPC homing. However, the underlying molecular mechanism still remains unknown. Here, we found that signal regulatory protein alpha (SIRPα) is preferentially expressed on TPECs. Disruption of CD47-SIRPα signaling in mice resulted in reduced number of thymic early T cell progenitors (ETPs), impaired thymic HPC homing, and altered early development of thymocytes. Mechanistically, Sirpa-deficient ECs and Cd47-deficient bone marrow progenitor cells or T lymphocytes demonstrated impaired transendothelial migration (TEM). Specifically, SIRPα intracellular ITIM motif-initiated downstream signaling in ECs was found to be required for TEM in an SHP2- and Src-dependent manner. Furthermore, CD47 signaling from migrating cells and SIRPα intracellular signaling were found to be required for VE-cadherin endocytosis in ECs. Thus, our study reveals a novel role of endothelial SIRPα signaling for thymic HPC homing for T cell development.

Preeclampsia is a pregnancy-specific disorder involving placental abnormalities. Elevated placental Sialic acid immunoglobulin-like lectin (Siglec)-6 expression has been correlated with preeclampsia. Siglec-6 is a transmembrane receptor, expressed predominantly by the trophoblast cells in the human placenta. It interacts with sialyl glycans such as sialyl-TN glycans as well as binds leptin. Siglec-6 overexpression has been shown to influence proliferation, apoptosis, and invasion in the trophoblast (BeWo) cell model. However, there is no direct evidence that Siglec-6 plays a role in preeclampsia pathogenesis and its signaling potential is still largely unexplored. Siglec-6 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like motif in its cytoplasmic tail suggesting a signaling function. Site-directed mutagenesis and transfection were employed to create a series of Siglec-6 expressing HTR-8/SVneo trophoblastic cell lines with mutations in specific functional residues to explore the signaling potential of Siglec-6. Co-immunoprecipitation and inhibitory assays were utilized to investigate the association of Src-kinases and SH-2 domain-containing phosphatases with Siglec-6. In this study, we show that Siglec-6 is phosphorylated at ITIM and ITIM-like domains by Src family kinases. Phosphorylation of both ITIM and ITIM-like motifs is essential for the recruitment of phosphatases like Src homology region 2 containing protein tyrosine phosphatase 2 (SHP-2), which has downstream signaling capabilities. These findings suggest Siglec-6 as a signaling molecule in human trophoblasts. Further investigation is warranted to determine which signaling pathways are activated downstream to SHP-2 recruitment and how overexpression of Siglec-6 in preeclamptic placentas impacts pathogenesis.

No abstract available

e13135 Background: First-line treatment for advanced TNBC with PD-L1 CPS ≥10 is chemotherapy plus pembro. ILT3 is an inhibitory receptor expressed on monocytic myeloid cells. High ILT3 expression is associated with immune tolerance and suppression of T-cell function. MK-0482 is an anti-ILT3 mAb hypothesized to relieve immunosuppression and improve T-cell function within the tumor microenvironment. Safety and efficacy data from a dose expansion cohort of the first-in-human, phase 1 MK-0482-001 study (NCT03918278) evaluating MK-0482 in combination with pembro + paclitaxel in participants (pts) with previously untreated advanced TNBC are presented here. Methods: Pts aged ≥18 y with recurrent inoperable or metastatic TNBC with centrally confirmed PD-L1 CPS ≥1, no prior systemic therapy for metastatic disease, and ECOG PS 0 or 1 received MK-0482 750 mg IV + pembro 200 mg IV Q3W for up to 35 cycles + paclitaxel 90 mg/m 2 IV on days 1, 8, and 15 Q4W until PD or discontinuation. The primary end point was safety. Secondary end points included ORR and DOR per RECIST v1.1 by investigator assessment. Exploratory end points included PFS per RECIST v1.1 by investigator assessment. Results: Of 45 pts enrolled and who received ≥1 dose of study treatment, 38 (84%) discontinued treatment at data cutoff (Oct 28, 2024), most commonly because of PD (76%). Median study follow-up was 27.6 mo (range, 23.8-34.0). Median age was 54 y (range, 26-80); 18 pts (40%) had an ECOG PS of 1, 21 (47%) had metastatic disease, 28 (62%) had tumors with CPS ≥1 to <10, and 17 (38%) had tumors with CPS ≥10. Treatment-related AEs occurred in 44 pts (98%); grade 3 or 4 treatment-related AEs occurred in 21 pts (47%), most commonly (≥10%) decreased neutrophil count (22%) and neutropenia (13%). No grade 5 treatment-related AEs occurred. Treatment-related AEs led to discontinuation in 11 pts (24%); 5 (11%) discontinued MK-0482, 3 (7%) discontinued pembro, and 8 (18%) discontinued paclitaxel. Response data are presented in the table. Exploratory biomarker data will be included in the presentation. Conclusions: In pts with advanced TNBC, MK-0482 + pembro + paclitaxel had a manageable safety profile, but showed no difference in efficacy compared to that historically observed with pembro + paclitaxel or gemcitabine-carboplatin. Therefore, further investigation of this combination will not be pursued for advanced TNBC. Clinical trial information: NCT03918278 . Efficacy outcomes. CPS ≥1 to <10n = 28 CPS ≥10n = 17 Total (CPS ≥1)n = 45 Confirmed ORR, % (95% CI) 50 (31-69) 53 (28-77) 51 (36-66) CR, n (%) 1 (4) 2 (12) 3 (7) PR, n (%) 13 (46) 7 (41) 20 (44) SD, n (%) 9 (32) 6 (35) 15 (33) PD, n (%) 4 (14) 2 (12) 6 (13) No assessment, n (%) 1 (4) 0 1 (2) DOR, median (range), months 6.2 (3.5 to 32.5+) 7.5 (4.2 to 30.4+) 6.5 (3.5 to 32.5+) PFS, median (95% CI), months 6.1 (4-8) 7.6 (4-10) 6.2 (5-8) 6/12-month PFS rates, % 54/21 65/24 58/22

2046 Background: For patients with GBM, treatment options are limited once the disease has recurred and become inoperable after standard first-line therapy. ILT3 is an inhibitory receptor expressed on monocytic myeloid cells, including tolerogenic dendritic cells and myeloid-derived suppressor cells (MDSCs). High expression of ILT3 on these immune cells has been associated with immune tolerance and suppression of T-cell function. Data have shown that GBMs have high ILT3 expression and high levels of MDSCs within the tumor microenvironment (TME). MK-0482 is a novel humanized IgG4 mAb targeting ILT3. Inhibition of ILT3 with MK-0482 may help relieve immunosuppression and improve T-cell function within the TME. In the expansion cohort of the first-in-human phase 1 study (MK-0482-001; NCT03918278), MK-0482 was evaluated in combination with pembro in a cohort of patients with recurrent inoperable GBM. The safety and efficacy data from this cohort are presented herein. Methods: Patients aged ≥18 y with their first recurrent inoperable GBM and a Karnofsky performance status (KPS) ≥80 were enrolled. Patients received MK-0482 750 mg Q3W + pembro 200 mg Q3W IV for up to a maximum of 35 cycles. The primary objective was to determine safety and tolerability of MK-0482 + pembro. Secondary objectives were to evaluate ORR, DCR (CR + PR + SD), and DOR by investigator assessment per Response Assessment in Neuro-Oncology (RANO). Exploratory objectives included evaluation of PFS per RANO and OS. Results: Overall, 25 patients were enrolled and received study treatment. At data cutoff (October 3, 2023), median follow-up duration was 9.3 mo (range, 5.6-14.5); treatment was ongoing in 1 patient. Median age was 56 y (range, 33-76). Most patients were male (72%), had a KPS of 80 (68%), and had IDH1 wild-type tumors (88%). All patients had previously received systemic therapy: 24 of 25 (96%) received prior temozolomide and 3 of 25 (12%) received prior glasdegib. Overall, 22 patients (88%) had an adverse event (AE); 10 patients (40%) had treatment-related AEs, most commonly (≥10%) arthralgia (12%), fatigue (12%), and increased ALT (12%). Grade 3 treatment-related AEs occurred in 2 patients (8%; increased ALT [n = 1] and fatigue [n = 1]). No grade 4 or 5 treatment-related AEs occurred. Four patients (16%) experienced immune-mediated AEs (hypothyroidism, n = 2; hepatitis, n = 1; pancreatitis, n = 1); all were grade 1 or 2. Confirmed ORR was 12% (3/25; 95% CI, 2.5-31.2), DCR was 32% (8/25; 14.9-53.5), and median DOR was 11.1 mo (range, 9.9-16.6+). Median PFS was 1.4 mo (95% CI, 1.3-2.9) and median OS was 9.3 mo (6.3-13.7); 6-/12-mo PFS and OS rates were 24%/16% and 72%/36%, respectively. Biomarker data will be included in the presentation. Conclusions: MK-0482 + pembro had a manageable AE profile with modest antitumor activity in patients with recurrent inoperable GBM. Clinical trial information: NCT03918278 .

4139 Background: Standard first-line treatment of mPDAC includes Gem/Nab-P doublet therapy, which typically results in an ORR of 23%-35%, a median PFS of 5-6 mo, and a median OS of 7-10 mo. ILT3 is an inhibitory receptor expressed on monocytic myeloid cells, including tolerogenic dendritic cells and myeloid-derived suppressor cells. MK-0482 is a novel humanized IgG4 monoclonal antibody targeting ILT3. ILT3 inhibition with MK-0482 has the potential to relieve immunosuppression within the tumor microenvironment. In the expansion cohort of the first-in-human phase 1 study MK-0482-001 (NCT03918278), the combination of MK-0482 + pembrolizumab + Gem/Nab-P was evaluated in pts with treatment-naive mPDAC. The safety and efficacy data from this cohort are presented. Methods: Pts aged ≥18 y with previously untreated mPDAC, measurable disease per RECIST v1.1, and an ECOG PS of 0 or 1 received MK-0482 750 mg Q3W + pembrolizumab 200 mg Q3W IV for ≤35 cycles + Gem/Nab-P (gemcitabine 1000 mg/m2; nab-paclitaxel 125 mg/m2) on days 1, 8, and 15 Q4W until PD or treatment discontinuation. The primary end point was safety. Secondary end points were ORR, DCR (CR + PR + SD), and DOR by investigator assessment per RECIST v1.1. Exploratory end points included PFS per RECIST v1.1 and OS. Results: A total of 37 pts were enrolled and received study treatment. At data cutoff (Oct 3, 2023), median follow-up was 13 mo (range 8.9-18.9). Median age was 65 y (range, 39-77). Most pts were male (54%), had an ECOG PS 1 (65%), and had ≥3 sites of metastasis (54%). Any-cause adverse events (AEs) occurred in all pts and treatment-related AEs (TRAEs) occurred in 36 pts (97%). Grade 3 or 4 TRAEs occurred in 27 pts (73%); the most frequent (≥10%) were decreased neutrophil count (27%), anemia (19%), and neutropenia (19%). No deaths due to TRAEs occurred. Immune-mediated AEs occurred in 12 pts (32%); the most frequent (≥10%) was hypothyroidism (11%). Grade 3 immune-mediated AEs occurred in 5 pts (14%; hepatitis, n = 2; severe skin reactions, n = 2; colitis, n = 1); no grade 4 or 5 events occurred. Confirmed ORR was 43% (16/37; 95% CI, 27.1-60.5), DCR was 84% (31/37; 68.0-93.8), and median DOR was 8.5 mo (range, 3.7-17.8+). Median PFS was 8.5 mo (95% CI, 5.7-12.3) and median OS was 15.6 mo (10.5-22.1); 12-mo PFS and OS rates were 35% and 60%, respectively. Biomarker data will be included in the presentation. Conclusions: MK-0482 + pembrolizumab + Gem/Nab-P showed a manageable AE profile in patients with treatment-naive mPDAC, which was generally consistent with the combined safety profiles of pembrolizumab-based immunotherapy and Gem/Nab-P, respectively. The 4-drug combination showed increased clinical efficacy compared with that historically observed for chemotherapy regimens. Further evaluation is needed to confirm the clinical activities observed. Clinical trial information: NCT03918278 .

Summary Multiple myeloma (MM) is an incurable malignancy of plasma cells. To identify targets for MM immunotherapy, we develop an integrated pipeline based on mass spectrometry analysis of seven MM cell lines and RNA sequencing (RNA-seq) from 900+ patients. Starting from 4,000+ candidates, we identify the most highly expressed cell surface proteins. We annotate candidate protein expression in many healthy tissues and validate the expression of promising targets in 30+ patient samples with relapsed/refractory MM, as well as in primary healthy hematopoietic stem cells and T cells by flow cytometry. Six candidates (ILT3, SEMA4A, CCR1, LRRC8D, FCRL3, IL12RB1) and B cell maturation antigen (BCMA) present the most favorable profile in malignant and healthy cells. We develop a bispecific T cell engager targeting ILT3 that shows potent killing effects in vitro and decreased tumor burden and prolonged mice survival in vivo, suggesting therapeutic relevance. Our study uncovers MM-associated antigens that hold great promise for immune-based therapies of MM.

No abstract available

Introduction CAR-T cell development for refractory or relapsed (R/R) acute myeloid leukemia (AML) has been limited by the lack of specific antigens with high specificity for AML cells and not present on normal hematopoietic stem cells. LILRB4 (also known as ILT3 or CD85k) is an immunosuppressive receptor and highly expressed on monocytic AML blasts (FAB M4 and M5 AML subtypes), which makes it an ideal therapeutic target for monocytic AML. Methods LILRB4-specific nanobodies were obtained from alpacas immunized with the recombinant LILRB4 extracellular domain. These nanobodies were then fused to IgG1 Fc, expressed and purified from CHO cells. The construct used for the manufacture of LILRB4-specific nanobody-based STAR-T (Synthetic T-Cell Receptor and Antigen Receptor-T) contains a double-chain TCR-based receptor with nanobodies fused to the N termini of modified TCR-Cα or TCR-Cβ and OX40 co-stimulatory domain (named Biparatopic LILRB4 STAR-T). In vitro assays and xenograft models were used to assess the potential of LILRB4 STAR-T cells for elimination of leukemic disease. In the first-in-human, single-arm, open-label phase I investigator-initiated trial (NCT05548088), we investigated the safety and efficacy of LILRB4 STAR-T therapy for treating R/R AML patients. By the Bayesian Optimal Interval (BOIN) design, patients who met the inclusion/exclusion criteria were enrolled to evaluate the safety, efficacy, and dose-limited toxicities (DLTs) by four dose groups (DL: 1E6, 3E6, 6E6, 1E7 STAR-T/kg). Peripheral blood mononuclear cells were obtained from either the patients themselves or the transplant donor post allo-HSCT. T-cells were then purified using CD3/CD28+ magnetic beads. All patients were treated with FC lymphodepletion before STAR-T cell infusion. Results In vitro and in vivo cytotoxicity assays demonstrated that LILRB4 STAR-T cells were highly cytotoxic against LILRB4+ AML cell lines, and also could inhibit the immunosuppressive myeloid cells (such as macrophage, and monocytes). As of July 31th, 2024, a total of 9 patients with LILRB4 -positive R/R AML were enrolled and evaluable for safety and efficacy. Median follow-up was 5 months (range 0.5-13.5months). The median age of enrolled patients was 36 years (range 25-60), and 77.8% (7/9) of patients relapsed post allo-HSCT. Median bone marrow (BM) blast percentage was 60% (12-81%) at enrollment to lymphodepletion. LILRB4 expression level in blasts was 77% (42-97%). The median transduction efficiency of the products was 54.75% (32.4%-75.5%). In the BOIN dose escalation, patients received STAR-T therapy at doses of 1E6/kg (n=1), 3E6/kg (n=3), 6E6/kg (n=4), and 1E7/kg (n=1). Six patients completed the 28-day dose-limiting toxicity (DLT) assessments. Two patients died due to infection before the d28 DLT assessments, and one patient's update will be provided at the ASH meeting. The safety analysis revealed that 83.3% (5 out of 6) of patients developed grade 1-2 cytokine release syndrome (CRS), with no grade 3 CRS or immune effector cell-associated neurotoxicity syndrome (ICANS) observed. The most common adverse event was pancytopenia, and no target-related adverse events were reported. Following infusion, the median peak time for circulating STAR-T cells occurred around Day 7 (range Day 5 to Day 10), as measured by q-PCR. The presence of LILRB4-positive cells in peripheral blood (PB) was negatively correlated with the level of STAR-T cells in PB. In responsive patients, LILRB4-positive AML cells in BM were significantly reduced and nearly disappeared. The efficacy data indicated a best overall response rate (ORR) of 50.0% (3 out of 6), comprising complete remission (n=1), morphologic leukemia-free state (n=1), and partial remission (n=1). Two of the responders underwent a second or third allo-HSCT and survived without minimal residual disease (MRD). The estimated 1-year OS was 62.2 % (95CI%: 35.5-100). Conclusion Our first-in-human study demonstrated that LILRB4 STAR-T therapy is a promising approach in LILRB4 -positive r/r AML patients, even in those who have undergone extensive prior treatments and experienced relapse post allo-HSCT. Further data from the phase II trial and longer follow-up time are essential.

LILRB4, a myeloid inhibitory receptor belonging to the family of leukocyte immunoglobulin-like receptors (LILRs/LIRs), plays a pivotal role in the regulation of immune tolerance. LILRB4 primarily mediates suppressive immune responses by transmitting inhibitory signals through immunoreceptor tyrosine-based inhibitory motifs (ITIMs). This immune checkpoint molecule has gained considerable attention due to its potent regulatory functions. Its ability to induce effector T cell dysfunction and promote T suppressor cell differentiation has been demonstrated, indicating the therapeutic potential of LILRB4 for modulating excessive immune responses, particularly in autoimmune diseases or the induction of transplant tolerance. Additionally, through intervening with LILRB4 molecules, immune system responsiveness can be adjusted, representing significant value in areas such as cancer treatment. Thus, LILRB4 has emerged as a key player in addressing autoimmune diseases, transplant tolerance induction, and other medical issues. In this review, we provide a comprehensive overview of LILRB4, encompassing its structure, expression, and ligand molecules as well as its role as a tolerance receptor. By exploring the involvement of LILRB4 in various diseases, its significance in disease progression is emphasized. Furthermore, we propose that the manipulation of LILRB4 represents a promising immunotherapeutic strategy and highlight its potential in disease prevention, treatment and diagnosis.

Lymph node (LN) colonization in cancer is linked to poor prognosis. Evidence suggests that LN colonization induces systemic immunosuppression, facilitating distant metastasis. We investigated LN-mediated immunosuppression in patients with head-and-neck cancer using spatial proteomics, spatial transcriptomics, and an in vivo model of melanoma LN metastasis. Both primary tumors and paired LNs of nodal-positive patients exhibit enhanced interferon-γ signaling and an enrichment of immunosuppressive myeloid cells and cancer-associated fibroblasts (CAFs). The spatial intersection of these myeloid-CAF-enriched niches with perifollicular T cell zones and LN follicles is linked to enhanced T cell dysfunction and Treg activation therein, thereby driving architectural LN remodeling. These immune suppressive changes extend to adjacent non-tumor-involved LN regions and nearby tumor-free LNs, but were not detected in LNs of non-cancer patients, reflecting a systemic effect that compromises anti-tumor immunity beyond the tumor-involved LN. Hence, our findings establish LN colonization as an active driver of systemic immunosuppression, facilitating metastatic progression.

Background: Acute myeloid leukemia (AML) is one of the most common hematopoietic malignancies. Adoptive cellular immunotherapy like chimeric antigen receptor (CAR) T therapy has demonstrated remarkable antitumor activity against B cell malignancies. However, CAR-T therapy exhibited very limited efficacy in treating AML due to the heterogeneity of AML cells and their similarity to normal hematopoietic cells and lack of leukemic cell-specific targets.LILRB4 (also known as ILT3, CD85k) is an immunosuppressive receptor that is highly expressed on monocyte AML blasts (FAB M4 and M5 AML subtypes), which makes it an ideal therapeutic target for monocytic AML. Here we generated LILRB4-specific nanobody-based STAR-T (Synthetic T-Cell Receptor and Antigen Receptor-T) cells, aiming to develop a new treatment strategy to improve outcomes for monocytic AML. Methods: LILRB4-specific nanobodies were obtained from alpacas immunized with the recombinant LILRB4 extracellular domain. These nanobodies were then fused to IgG1 Fc, expressed and purified from CHO cells and used for measuring the affinity, epitope binning, target binding specificity by SPR, BLI and flow cytometry. The LILRB4 STAR was designed as a double-chain TCR-based receptor with nanobodies fused to the N-terminus of the modified mouse TCR-Cα or TCR-Cβ, the two chains were linked with Furin-P2A sequence. Based on STAR structure, LILRB4 single epitopic STAR was generated by fusing an nanobody to the TCR-Cβ,whereas LILRB4 biparatopic STAR contained two nanobodies fused to the TCR-Cα and TCR-Cβ separately. In vitro assays and xenograft models were used to assess the potential of LILRB4 STAR-T cells for elimination of leukemic disease. Results and Conclusions: Multiple STAR-T cells were generated using a lentiviral-based vector and tested for their tumor lysis activity. Among multiple nanobodies tested, two nanobodies that displayed high antigen binding affinity and specificity were identified for further studies. They recognized two distinctive epitopes of LILRB4, and neither of them had competitive binding with APOE or FN1, known ligands of LILRB4. We generated both single paratopic and bi-paratopic STAR constructs and engineered the corresponding STAR-T cells. In vitro and in vivo cytotoxicity assays demonstrated that all LILRB4 STAR-T cells had high cytotoxic activity against LILRB4+ AML cell lines. In addition, LILRB4 biparatopic STAR-T cells exhibited the highest anti-leukemic activity and the best expansion in vivo. Therefore, the LILRB4 STAR-T cells described here may be further developed for the treatment of AML and represent a new immune cell therapy strategy for LILRB4+ AML patients. Citation Format: Wei Rui, Lei Lei, Zezhong Zhang, Chunyan Wu, Yu Xia, Yingying Liu, Xiaojing Pang, Ruifang Du, Lan Wang, Dengyue Sheng, Jing Guo, Yanyan Zhang, Xueqiang Zhao, James Pan. Development of LILRB4 biparatopic synthetic T-cell receptor and antigen receptor (STAR)-T cells for the treatment of acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3185.

Exosomes may represent an interesting antigenic pulse for new forms of anti-tumor immunotherapy. We evaluated exosomes from serum of patients with acute myeloid leukemia (AML) as an antigenic source for dendritic cells (DC) and the effects upon antitumor cytotoxicity, assessed by the percentage of specific lysis of K562 leukemic cells in co-cultures. Surprisingly, incubation of exosomes with DCs decreased lysis of K562, which may correspond to a mechanism of tumor evasion in vivo. However, when immature DCs were pulsed with exosomes purified from K562 culture supernatants, the lysis of target cells was notably enhanced, associated with a substantial increase in the expression of the maturation marker CD83. Thus, the development of vaccines using patients' exosomes would probably add no benefits to the treatment of AML; alternately, exosomes from cultured cells may represent an effective way for maturing DCs into a cytotoxic phenotype, without the immunosuppression observed with patients' exosomes.

Emerging studies suggest that dominant peripheral tolerance is a major mechanism of immune escape in disseminated leukemia. Using an established murine acute myeloid leukemia (AML) model, we here show that systemic administration of recombinant IL-33 dramatically inhibits the leukemia growth and prolongs the survival of leukemia-bearing mice in a CD8+ T cell dependent manner. Exogenous IL-33 treatment enhanced anti-leukemia activity by increasing the expansion and IFN-γ production of leukemia-reactive CD8+ T cells. Moreover, IL-33 promoted dendritic cell (DC) maturation and activation in favor of its cross presentation ability to evoke a vigorous anti-leukemia immune response. Finally, we found that the combination of PD-1 blockade with IL-33 further prolonged the survival, with half of the mice achieving complete regression. Our data establish a role of exogenous IL-33 in reversing T cell tolerance, and suggest its potential clinical implication into leukemia immunotherapy.

Replacement of failed organs followed by safe withdrawal of immunosuppressive drugs have long been the goals of organ transplantation. We studied changes in the balance of T and myeloid cells in blood of HLA-matched and -mismatched patients given living donor kidney transplants (KTx) followed by total lymphoid irradiation (TLI), anti-thymocyte globulin (ATG) conditioning, and donor hematopoietic cell transplant (HCT) to induce mixed chimerism and immune tolerance. The clinical trials were based on a conditioning regimen used to establish mixed chimerism and tolerance in mice. In pre-clinical murine studies, there was a profound depletion of T cells and an increase in immunosuppressive, polymorphonuclear (pmn), myeloid derived suppressor cells (MDSCs) in the spleen and blood following transplant. Selective depletion of the pmn-MDSCs in mice abrogated mixed chimerism and tolerance. In our clinical trials, patients given an analogous tolerance conditioning regimen developed similar changes including profound depletion of T cells and a marked increase in MDSCs in blood post-transplant. Post-transplant pmn-MDSCs transiently increased expression of lectin-type, oxidized LDL receptor-1 (LOX-1), a marker of immunosuppression, and production of the T cell inhibitor, arginase-1. These post-transplant pmn-MDSCs suppressed the activation, proliferation, and inflammatory cytokine secretion of autologous, TCR microbead-stimulated, pre-transplant T cells when co-cultured in vitro. In conclusion, we elucidated changes in receptors, and function of immunosuppressive myeloid cells in patients enrolled in the tolerance protocol that were nearly identical to the that of MDSCs required for tolerance in mice. The clinical trials are registered in Clinicaltrials.gov under NCT #s 00319657 and 01165762.

Supplemental Digital Content is available in the text. Transplantation is now performed globally as a routine procedure. However, the increased demand for donor organs and consequent expansion of donor criteria has created an imperative to maximize the quality of these gains. The goal is to balance preservation of allograft function against patient quality-of-life, despite exposure to long-term immunosuppression. Elimination of immunosuppressive therapy to avoid drug toxicity, with concurrent acceptance of the allograft—so-called operational tolerance—has proven elusive. The lack of recent advances in immunomodulatory drug development, together with advances in immunotherapy in oncology, has prompted interest in cell-based therapies to control the alloimmune response. Extensive experimental work in animals has characterized regulatory immune cell populations that can induce and maintain tolerance, demonstrating that their adoptive transfer can promote donor-specific tolerance. An extension of this large body of work has resulted in protocols for manufacture, as well as early-phase safety and feasibility trials for many regulatory cell types. Despite the excitement generated by early clinical trials in autoimmune diseases and organ transplantation, there is as yet no clinically validated, approved regulatory cell therapy for transplantation. In this review, we summarize recent advances in this field, with a focus on myeloid and mesenchymal cell therapies, including current understanding of the mechanisms of action of regulatory immune cells, and clinical trials in organ transplantation using these cells as therapeutics.

Current rheumatoid arthritis (RA) treatments do not restore immune tolerance. Investigating dendritic cell (DC) populations in human synovial tissue (ST) may reveal pathways to reinstate tolerance in RA. Using single-cell and spatial transcriptomics of ST biopsies, as well as co-culture systems, we identified condition- and niche-specific DC clusters with distinct functions. Healthy tissue contained tolerogenic AXL+ DC2s in the lining niche. In active RA, the hyperplasic lining niche was populated with inflammatory DC3s that activated CCL5-positive effector memory T cells, promoting synovitis. Lymphoid niches that emerged in the sublining layer were enriched with CCR7+ DC2s, which interacted with naive T cells, potentially driving the local expansion of new effector T cells. Remission saw the resolution of these pathogenic niches but lacked recovery of tolerogenic DC2s and exhibited activation of blood precursors of ST-DC3 clusters prior to flare-ups. Targeting pathogenic DC3s or restoring tolerogenic DC2s may help restore immune homeostasis in RA joints.

The tumor microenvironment plays a key role in progression of tumorigenesis, tumor progression, and metastasis. Accumulating data reveal that dendritic cells (DCs) appear to play a key role in the development and progression of metastatic neoplasia by driving immune system dysfunction and establishing immunosuppression, which is vital for tumor evasion of host immune response. Consequently, in this review, we will discuss the function of tumor-infiltrating DCs in immune cell signaling pathways that lead to treatment resistance, tumor recurrence, and immunosuppression. We will also review DC metabolism, differentiation, and plasticity, which are essential for metastasis and the development of lung tumors. Furthermore, we will take into account the interaction between myeloid cells and DCs in tumor-related immunosuppression. We will specifically look into the molecular immune-related mechanisms in the tumor microenvironment that result in reduced drug sensitivity and tumor relapse, as well as methods for combating drug resistance and focusing on immunosuppressive tumor networks. DCs play a crucial role in modulating the immune response. Especially, as cancer progresses, DCs may switch from playing an immunostimulatory to an inhibitory role. This article’s main emphasis is on tumor-infiltrating DCs. We address how they affect tumor growth and expansion, and we highlight innovative approaches for therapeutic modulation of these immunosuppressive DCs which is necessary for future personalized therapeutic approaches.

Hepatitis B virus (HBV) remains a leading cause of liver-related morbidity and mortality through chronic hepatitis that may progress to liver cirrhosis and cancer. The central role played by HBV-specific CD8+ T cells in the clearance of acute HBV infection, and HBV-related liver injury is now well established. Vigorous, multifunctional CD8+ T cell responses are usually induced in most adult-onset HBV infections, while chronic hepatitis B (CHB) is characterized by quantitatively and qualitatively weak HBV-specific CD8+ T cell responses. The molecular basis of this dichotomy is poorly understood. Genomic analysis of dysfunctional HBV-specific CD8+ T cells in CHB patients and various mouse models suggest that multifaceted mechanisms including negative signaling and metabolic abnormalities cooperatively establish CD8+ T cell dysfunction. Immunoregulatory cell populations in the liver, including liver resident dendritic cells (DCs), hepatic stellate cells (HSCs), myeloid-derived suppressor cells (MDSCs), may contribute to intrahepatic CD8+ T cell dysfunction through the production of soluble mediators, such as arginase, indoleamine 2,3-dioxygenase (IDO) and suppressive cytokines and the expression of co-inhibitory molecules. A series of recent studies with mouse models of HBV infection suggest that genetic and epigenetic changes in dysfunctional CD8+ T cells are the manifestation of prolonged antigenic stimulation, as well as the absence of co-stimulatory or cytokine signaling. These new findings may provide potential new targets for immunotherapy aiming at invigorating HBV-specific CD8+ T cells, which hopefully cures CHB.

Introduction Cystic Fibrosis (CF) is the commonest genetically inherited disease (1 in 4,500 newborns) and 70% of people with CF (pwCF) harbour the F508Del mutation, resulting in misfolding and incorrect addressing of the channel CFTR to the epithelial membrane and subsequent dysregulation of fluid homeostasis. Although studies have underscored the importance and over-activation of myeloid cells, and in particular neutrophils in the lungs of people with CF (pwCF), relatively less emphasis has been put on the potential immunological bias in CF blood cells, at homeostasis or following stimulation/infection. Methods Here, we revisited, in an exhaustive fashion, in pwCF with mild disease (median age of 15, median % FEV1 predicted = 87), whether their PBMCs, unprimed or primed with a ‘non specific’ stimulus (PMA+ionomycin mix) and a ‘specific’ one (live P.a =PAO1 strain), were differentially activated, compared to healthy controls (HC) PBMCs. Results 1) we analysed the lymphocytic and myeloid populations present in CF and Control PBMCs (T cells, NKT, Tgd, ILCs) and their production of the signature cytokines IFN-g, IL-13, IL-17, IL-22. 2) By q-PCR, ELISA and Luminex analysis we showed that CF PBMCs have increased background cytokines and mediators production and a partial functional tolerance phenotype, when restimulated. 3) we showed that CF PBMCs low-density neutrophils release higher levels of granule components (S100A8/A9, lactoferrin, MMP-3, MMP-7, MMP-8, MMP-9, NE), demonstrating enhanced exocytosis of potentially harmful mediators. Discussion In conclusion, we demonstrated that functional lymphoid tolerance and enhanced myeloid protease activity are key features of cystic fibrosis PBMCs.

The intestinal microbiota contributes to gut immune homeostasis, where short-chain fatty acids (SCFAs) function as the major mediators. We aimed to elucidate the immunomodulatory effects of acetate, propionate, and butyrate. With that in mind, we sought to characterise the expression of SCFA receptors and transporters as well as SCFAs' impact on the activation of different immune cells. Whereas all three SCFAs decreased tumour necrosis factor (TNF)-α production in activated T cells, only butyrate and propionate inhibited interferon (IFN)-γ, interleukin (IL)-17, IL-13, and IL-10 production. Butyrate and propionate inhibited the expression of the chemokine receptors CCR9 and CCR10 in activated T- and B-cells, respectively. Similarly, butyrate and propionate were effective inhibitors of IL-1β, IL-6, TNF-α, and IL-10 production in myeloid cells upon lipopolysaccharide and R848 stimulation. Acetate was less efficient at inhibiting cytokine production except for IFN-α. Moreover, SCFAs inhibited the production of IL-6 and TNF-α in monocytes, myeloid dendritic cells (mDC), and plasmacytoid dendritic cells (pDC), whereas acetate effects were relatively more prominent in pDCs. In monocytes and mDCs, acetate was a less efficient inhibitor, but it was equally effective in inhibiting pDCs activation. We also studied the ability of SCFAs to induce trained immunity or tolerance. Butyrate and propionate - but not acetate - prevented Toll-like receptor-mediated activation in SCFA-trained cells, as demonstrated by a reduced production of IL-6 and TNF-α. Our findings indicate that butyrate and propionate are equally efficient in inhibiting the adaptive and innate immune response and did not induce trained immunity. The findings may be explained by differential SCFA receptor and transporter expression profiles of the immune cells.

Abstract Leukocyte immunoglobulin-like receptor B4 (LILRB4) is an inhibitory receptor in the LILR family mainly expressed on normal and malignant human cells of myeloid origin. By binding to ligands, LILRB4 is activated and subsequently recruits adaptors to cytoplasmic immunoreceptor tyrosine inhibitory motifs to initiate different signaling cascades, thus playing an important role in physiological and pathological conditions, including autoimmune diseases, microbial infections, and cancers. In normal myeloid cells, LILRB4 regulates intrinsic cell activation and differentiation. In disease-associated or malignant myeloid cells, LILRB4 is significantly correlated with disease severity or patient survival and suppresses T cells, thereby participating in the pathogenesis of various diseases. In summary, LILRB4 functions as an immune checkpoint on myeloid cells and may be a promising therapeutic target for various human immune diseases, especially for cancer immunotherapy.

No abstract available

Although innate immunity is linked to metabolic health, the effect of leptin signaling in cells from the innate immune system on glucose homeostasis has not been thoroughly investigated. We generated two mouse models using Cre-lox methodology to determine the effect of myeloid cell-specific leptin receptor (Lepr) reconstitution and Lepr knockdown on in vivo glucose metabolism. Male mice with myeloid cell-specific Lepr reconstitution (Lyz2Cre+LeprloxTB/loxTB) had better glycemic control as they aged compared to male mice with whole-body transcriptional blockade of Lepr (Lyz2Cre−LeprloxTB/loxTB). In contrast, Lyz2Cre+LeprloxTB/loxTB females only had a trend for diminished hyperglycemia after a prolonged fast. During glucose tolerance tests, Lyz2Cre+LeprloxTB/loxTB males had a mildly improved plasma glucose profile compared to Cre− controls while Lyz2Cre+LeprloxTB/loxTB females had a similar glucose excursion to their Cre− controls. Myeloid cell-specific Lepr knockdown (Lyz2Cre+Leprflox/flox) did not significantly alter body weight, blood glucose, insulin sensitivity, or glucose tolerance in males or females. Expression of the cytokine interleukin 10 (anti-inflammatory) tended to be higher in adipose tissue of male Lyz2Cre+LeprloxTB/loxTB mice (p = 0.0774) while interleukin 6 (pro-inflammatory) was lower in male Lyz2Cre+Leprflox/flox mice (p < 0.05) vs. their respective controls. In conclusion, reconstitution of Lepr in cells of myeloid lineage has beneficial effects on glucose metabolism in male mice.

Key Points Lean Il4raΔmyel mice have a deteriorated insulin sensitivity. Obese Il4raΔmyel mice have a rather improved metabolic phenotype. IL-13 stimulation increases CD11c expression in macrophages. IL-4 receptor signaling is supposed to play a major role in anti-inflammatory polarization and proliferation of adipose tissue macrophages. In this study, we examined the metabolic and inflammatory phenotype of C57BL/6J mice (IIl4ra) with LysM-dependent knockout (IIl4raΔmyel) of the IL-4 receptor α-chain (IL-4Rα), the mandatory signaling component of IL-4 and IL-13, on chow and high-fat diet. Lean IIl4raΔmyel mice showed decreased insulin sensitivity, no divergent adipose tissue macrophage polarization, but an increased percentage of CD8+ T cells in visceral adipose tissue. After 20 wk of a high-fat diet, IIl4raΔmyel mice exhibited higher glucose tolerance, no changes in the lymphocyte compartment and fewer M1 macrophages in visceral adipose tissue. In vivo adipose tissue macrophage proliferation measured by BrdU incorporation was unaffected by Il4ra knockout. Interestingly, we show that IL-4Rα signaling directly augmented Itgax (Cd11c) gene expression in bone marrow–derived macrophages and increased the amount of CD11c+ macrophages in adipose tissue explants. Myeloid cell–specific knockout of Il4ra deteriorated insulin sensitivity in lean mice but improved parameters of glucose homeostasis and partially protected from adipose tissue inflammation in obese mice. Hence, IL-4Rα signaling probably plays a minor role in maintaining the macrophage M2 population and proliferation rates in vivo. Moreover, our data indicate that IL-4 signaling plays a proinflammatory role in adipose tissue inflammation by directly upregulating CD11c on adipose tissue macrophages.

Despite advances in identifying the key immunoregulatory roles of many of the human leukocyte immunoglobulin-like receptor (LILR) family members, the function of the inhibitory molecule LILRB3 (ILT5, CD85a, LIR3) remains unclear. Studies indicate a predominant myeloid expression; however, high homology within the LILR family and a relative paucity of reagents have hindered progress toward identifying the function of this receptor. To investigate its function and potential immunomodulatory capacity, a panel of LILRB3-specific monoclonal antibodies (mAbs) was generated. LILRB3-specific mAbs bound to discrete epitopes in Ig-like domain 2 or 4. LILRB3 ligation on primary human monocytes by an agonistic mAb resulted in phenotypic and functional changes, leading to potent inhibition of immune responses in vitro, including significant reduction in T cell proliferation. Importantly, agonizing LILRB3 in humanized mice induced tolerance and permitted efficient engraftment of allogeneic cells. Our findings reveal powerful immunosuppressive functions of LILRB3 and identify it as an important myeloid checkpoint receptor.

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Summary Intestinal disequilibrium leads to inflammatory bowel disease (IBD), and chronic inflammation predisposes to oncogenesis. Antigen-presenting dendritic cells (DCs) and macrophages can tip the equilibrium toward tolerance or pathology. Here we show that delta-9-tetrahydrocannabinol (THC) attenuates colitis-associated colon cancer and colitis induced by anti-CD40. Working through cannabinoid receptor 2 (CB2), THC increases CD103 expression on DCs and macrophages and upregulates TGF-β1 to increase T regulatory cells (Tregs). THC-induced Tregs are necessary to remedy systemic IFNγ and TNFα caused by anti-CD40, but CB2-mediated suppression of APCs by THC quenches pathogenic release of IL-22 and IL-17A in the colon. By examining tissues from multiple sites, we confirmed that THC affects DCs, especially in mucosal barrier sites in the colon and lungs, to reduce DC CD86. Using models of colitis and systemic inflammation we show that THC, through CB2, is a potent suppressor of aberrant immune responses by provoking coordination between APCs and Tregs.

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Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid cells, are characterized by their immunosuppressive abilities through the secretion of various cytokines such as inducible nitric oxide synthase, nitric oxide, reactive oxygen species, transforming growth factor-β, and arginase-1. Accumulating evidence highlights its potential role in maintaining immune tolerance in solid organ and hematopoietic stem cell transplantation. Mechanistically, MDSCs-induced transplant tolerance is mainly dependent on direct suppression of allogeneic reaction or strengthened cross-talk between MDSCs and Treg or NKT cells. Adopted transfer of in vitro– or in vivo–induced MDSCs by special drugs therefore becomes a potential strategy for maintaining transplantation tolerance. In this review, we will summarize the previously published data about the role of MDSCs in the biology of transplantation tolerance and gain insights into the possible molecular mechanism governing this process.

The gastrointestinal tract is continuously exposed to foreign antigens in food and commensal microorganisms with potential to induce adaptive immune responses. Peripherally induced T regulatory (pTreg) cells are essential for mitigating inflammatory responses to these agents1, 2, 3–4. Although RORγt+ antigen-presenting cells (APCs) have been shown to programme gut microbiota-specific pTreg cells5, 6–7, their definition remains incomplete, and the APC responsible for food tolerance has remained unknown. Here we identify an APC subset that is required for differentiation of both food- and microbiota-specific pTreg cells and for establishment of oral tolerance. Development and function of these APCs require expression of the transcription factors PRDM16 and RORγt, as well as a unique Rorc(t)cis-regulatory element. Gene expression, chromatin accessibility, and surface marker analysis establish the pTreg-inducing APCs as myeloid in origin, distinct from type 3 innate lymphoid cells, and sharing epigenetic profiles with classical dendritic cells, and designate them PRDM16+RORγt+ tolerizing dendritic cells (tolDCs). Upon genetic perturbation of tolDCs, we observe a substantial increase in food antigen-specific T helper 2 cells in lieu of pTreg cells, leading to compromised tolerance in mouse models of asthma and food allergy. Single-cell analyses of freshly resected mesenteric lymph nodes from a human organ donor, as well as multiple specimens of human intestine and tonsil, reveal candidate tolDCs with co-expression of PRDM16 and RORC and an extensive transcriptome shared with tolDCs from mice, highlighting an evolutionarily conserved role across species. Our findings suggest that a better understanding of how tolDCs develop and how they regulate T cell responses to food and microbial antigens could offer new insights into developing therapeutic strategies for autoimmune and allergic diseases as well as organ transplant tolerance. A subset of antigen-presenting cells of myeloid origin that require PRDM16 and RORγt regulates T cell function to enable immune tolerance of food and commensal gut microorganisms.

Summary Loss of p53 function contributes to the development of many cancers. While cell-autonomous consequences of p53 mutation have been studied extensively, the role of p53 in regulating the anti-tumor immune response is still poorly understood. Here, we show that loss of p53 in cancer cells modulates the tumor-immune landscape to circumvent immune destruction. Deletion of p53 promotes the recruitment and instruction of suppressive myeloid CD11b+ cells, in part through increased expression of CXCR3/CCR2-associated chemokines and macrophage colony-stimulating factor (M-CSF), and attenuates the CD4+ T helper 1 (Th1) and CD8+ T cell responses in vivo. p53-null tumors also show an accumulation of suppressive regulatory T (Treg) cells. Finally, we show that two key drivers of tumorigenesis, activation of KRAS and deletion of p53, cooperate to promote immune tolerance.

Innate immune responses are rapid, dynamic and highly regulated to avoid overt reactions. This regulation is executed by innate immune tolerance mechanisms that remain obscure. Wnt5a is a signalling protein mainly involved in developmental processes and cancer. The effect of Wnt5a on inflammatory myeloid cells is controversial. Here, we combine primary cell cultures, in vitro binding studies, mass spectrometry and Drosophila protein modelling to show that Wnt5a is a direct ligand of toll-like receptor (TLR) 2 and 4. The binding promotes a MyD88-non-canonical nuclear factor of kappa B (NFκB) and AP-1 signalling cascade, with contradictory profiles in mouse (pro-inflammatory) and human (anti-inflammatory) myeloid immune cells. These data reveal that the true nature of Wnt5a in inflammatory cells, is to regulate TLR signals, and in human myeloid cells it acts as an endogenous, tolerance-associated molecular pattern (TAMP), inducing IL-10 and innate immune tolerance. Meliha Mehmeti et al. show that Wnt5a is a direct ligand of toll-like receptor 2 and 4 to act as an endogenous tolerance-associated molecular pattern, triggering innate immune tolerance in human myeloid cells. This study expands the functional repertoire of Wnt5a that is mainly known as a signalling protein involved in development and cancer.

Thymic myeloid cells regulate T cell tolerance. Here, we describe their heterogeneity. We found that thymic “DC2” comprise four distinct lineages, including monocyte-derived DCs and macrophages, and conventional DC2. Among DC2, a novel population of transitional DCs, representing thymus immigrating cells, was identified.

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The immunotherapy targeting tumor immune escape mechanisms has become a critical strategy in anticancer treatment; however, the challenge of immune resistance remains significant. Autophagy, a cellular response to various stressors, involves the degradation of damaged proteins and organelles via lysosomal pathways, maintaining cellular homeostasis. This process not only supports tumor cell survival but also profoundly impacts the efficacy of cancer immunotherapies. The modulation of autophagy in tumor cells or immune cells exerts dual effects on tumor immune escape and immunotherapy. However, the mechanistic details of how autophagy influences the immune system and therapy remain inadequately understood. Given this complexity, a deeper understanding of the role of autophagy in the tumor-immune landscape could reveal novel therapeutic avenues. By manipulating autophagy appropriately, it may be possible to overcome immune resistance and enhance the effectiveness of immunotherapeutic strategies. This article summarizes the role of autophagy in tumor immunity, its relationship with immunotherapy, and the potential therapeutic benefits of targeting autophagy to strengthen antitumor immune responses and optimize the outcomes of immunotherapy.

Tumor immunotherapy, an innovative anti-cancer therapy, has showcased encouraging outcomes across diverse tumor types. Among these, the PD-1/PD-L1 signaling pathway is a well-known immunological checkpoint, which is significant in the regulation of immune evasion by tumors. Nevertheless, a considerable number of patients develop resistance to anti-PD-1/PD-L1 immunotherapy, rendering it ineffective in the long run. This research focuses on exploring the factors of PD-1/PD-L1-mediated resistance in tumor immunotherapy. Initially, the PD-1/PD-L1 pathway is characterized by its role in facilitating tumor immune evasion, emphasizing its role in autoimmune homeostasis. Next, the primary mechanisms of resistance to PD-1/PD-L1-based immunotherapy are analyzed, including tumor antigen deletion, T cell dysfunction, increased immunosuppressive cells, and alterations in the expression of PD-L1 within tumor cells. The possible ramifications of altered metabolism, microbiota, and DNA methylation on resistance is also described. Finally, possible resolution strategies for dealing with anti-PD-1/PD-L1 immunotherapy resistance are discussed, placing particular emphasis on personalized therapeutic approaches and the exploration of more potent immunotherapy regimens.

Metabolic reprogramming is a k`ey hallmark of tumors, developed in response to hypoxia and nutrient deficiency during tumor progression. In both cancer and immune cells, there is a metabolic shift from oxidative phosphorylation (OXPHOS) to aerobic glycolysis, also known as the Warburg effect, which then leads to lactate acidification, increased lipid synthesis, and glutaminolysis. This reprogramming facilitates tumor immune evasion and, within the tumor microenvironment (TME), cancer and immune cells collaborate to create a suppressive tumor immune microenvironment (TIME). The growing interest in the metabolic reprogramming of the TME, particularly its significance in colorectal cancer (CRC)—one of the most prevalent cancers—has prompted us to explore this topic. CRC exhibits abnormal glycolysis, glutaminolysis, and increased lipid synthesis. Acidosis in CRC cells hampers the activity of anti-tumor immune cells and inhibits the phagocytosis of tumor-associated macrophages (TAMs), while nutrient deficiency promotes the development of regulatory T cells (Tregs) and M2-like macrophages. In CRC cells, activation of G-protein coupled receptor 81 (GPR81) signaling leads to overexpression of programmed death-ligand 1 (PD-L1) and reduces the antigen presentation capability of dendritic cells. Moreover, the genetic and epigenetic cell phenotype, along with the microbiota, significantly influence CRC metabolic reprogramming. Activating RAS mutations and overexpression of epidermal growth factor receptor (EGFR) occur in approximately 50% and 80% of patients, respectively, stimulating glycolysis and increasing levels of hypoxia-inducible factor 1 alpha (HIF-1α) and MYC proteins. Certain bacteria produce short-chain fatty acids (SCFAs), which activate CD8+ cells and genes involved in antigen processing and presentation, while other mechanisms support pro-tumor activities. The use of immune checkpoint inhibitors (ICIs) in selected CRC patients has shown promise, and the combination of these with drugs that inhibit aerobic glycolysis is currently being intensively researched to enhance the efficacy of immunotherapy.

Cellular senescence, a hallmark of aging, has emerged as a captivating area of research in tumor immunology with profound implications for cancer prevention and treatment. In the tumor microenvironment, senescent cells exhibit a dual role, simultaneously hindering tumor development through collaboration with immune cells and evading immune cell attacks by upregulating immunoinhibitory proteins. However, the intricate immune escape mechanism of cellular senescence in the tumor microenvironment remains a subject of intense investigation. Chronic inflammation is exacerbated by cellular senescence through the upregulation of pro-inflammatory factors such as interleukin-1β, thereby augmenting the risk of tumorigenesis. Additionally, the interplay between autophagy and cellular senescence adds another layer of complexity. Autophagy, known to slow down the aging process by reducing p53/p21 levels, may be downregulated by cellular senescence. To harness the therapeutic potential of cellular senescence, targeting its immunological aspects has gained significant attention. Strategies such as immune checkpoint inhibitors and T-cell senescence inhibition are being explored in the context of cellular senescence immunotherapy. In this comprehensive review, we provide a compelling overview of the regulation of cellular senescence and delve into the influencing factors, including chronic inflammation, autophagy, and circadian rhythms, associated with senescence in the tumor microenvironment. We specifically focus on unraveling the enigmatic dual role of cellular senescence in tumor immune escape. By deciphering the intricate nature of cellular senescence in the tumor microenvironment, this review aims to advance our understanding and pave the way for leveraging senescence as a promising target for tumor immunotherapy applications.

BACKGROUND Diffuse large B-cell lymphoma (DLBCL) is a prevalent and aggressive form of non-Hodgkin's lymphoma with a complex etiology. NOP2/Sun domain 2 (NSUN2) is an RNA methyltransferase that has been linked to the regulation of gene expression in various cancers. However, the function of NSUN2 in DLBCL, specifically its contribution to exosome-driven tumor progression, remains to be thoroughly elucidated. METHODS Quantitative real-time polymerase chain reaction was used to analyze the expression of NSUN2 and programmed death ligand 1 variant (PDL1). Western blotting assay was performed to detect the protein levels of NSUN2, PDL1 and Y-box binding protein 1 (YBX1). Cell proliferation was analyzed by cell counting kit-8 and 5-Ethynyl-2'-deoxyuridine assays. Cell apoptosis and CD206-positive cells were quantified by flow cytometry. The levels of tumor necrosis factor-alpha and interferon-γ in cell supernatant were analyzed by enzyme-linked immunosorbent assays. m6A RNA immunoprecipitation and RNA pull-down assays were performed to determine the association between NSUN2 and PDL1. An RNA immunoprecipitation assay was used to analyze the association of YBX1 and PDL1. In vitro findings were validated in a mouse model. RESULTS NSUN2 was overexpressed in DLBCL tissues and cells. DLBCL cell-derived exosomes facilitated the transfer of NSUN2 to DLBCL cells, which in turn promoted tumor cell proliferation, M2 macrophage polarization, and immune escape and inhibited cell apoptosis. In addition, NSUN2 stabilized PDL1 mRNA through an m5C-dependent mechanism and a YBX1-dependent pathway. Moreover, the suppression of PDL1 significantly mitigated the effects induced by NSUN2 within DLBCL cell-derived exosomes on cellular proliferation, apoptosis, M2 macrophage polarization, and immune evasion. Further, DLBCL cell-derived exosomal NSUN2 promoted tumor growth by regulating PDL1. CONCLUSION NSUN2 in DLBCL cell-derived exosomes stabilized PDL1 in a YBX1-dependent manner and thus promoted tumor immune escape and M2 macrophage polarization. These findings highlight the potential of targeting the NSUN2-PDL1 axis as a novel therapeutic strategy for DLBCL.

Background Tumor cells employ various mechanisms to evade detection and attack by the immune system, a phenomenon known as tumor immune escape, which represents a significant target for immunotherapy. Both primary and secondary immune escape mechanisms pose substantial challenges that hinder the efficacy of immunotherapy. This study aims to systematically examine the knowledge structure, hotspot frontiers, emerging trends, and future directions in the field of tumor immune escape through the application of bibliometric methods and knowledge mapping analysis. Methods A comprehensive search of the Web of Science Core Collection (WoSCC) was conducted for publications pertaining to tumor immune escape from January 1, 2015, to November 30, 2024. The annual publication data retrieved from the WoSCC were analyzed utilizing Microsoft Office Excel 2019. Furthermore, bibliometric analysis and visualization were executed using VOSviewer, Biblioshiny, and CiteSpace. Results This study encompassed a total of 11,128 articles published across 1,612 journals, authored by 71,684 individuals affiliated with 9,254 institutions in 121 countries. The United States, China, and Germany emerged as the leading contributors to this field, collectively accounting for 79.99% of all publications. Notable international collaboration was observed between the United States and China. Frontiers in Immunology, Nature Communications, the Journal for ImmunoTherapy of Cancer, and Nature were identified as the four most influential journals in tumor immune escape research. Zhang Wei was noted for the highest publication output, while Freeman Gordon J achieved the highest citation rate. Fudan University was recognized as the most productive institution, whereas Harvard Medical School was acknowledged as the most cited institution. Current hotspot frontiers in tumor immune escape research include immunotherapy, the tumor microenvironment, PD-L1, and PD-1. Additionally, emerging frontiers in recent years encompass immune checkpoint inhibitors, immune infiltration, natural killer cells, extracellular vesicles, immunogenic cell death, metabolism, ferroptosis, melanoma, lung adenocarcinoma, and prognosis. Conclusion A comprehensive investigation into the mechanisms of tumor immune escape is essential for overcoming the existing challenges in immunotherapy. This study systematically analyzes the current state, research frontiers, and future directions, identifying the most prolific and highly cited documents, journals, authors, institutions, and countries in the field of tumor immune escape.

Background Immune escape, a process by which tumor cells evade immune surveillance, remains a challenge for cancer therapy. Tumor cells produce extracellular vesicles (EVs) that participate in immune escape by transferring bioactive molecules between cells. The main body of the abstract EVs refer to heterogeneous vesicles that participate in intercellular communication. EVs from tumor cells usually carry tumor antigens and have been considered a source of tumor antigens to induce anti-tumor immunity. However, evidence also suggests that these EVs can accelerate immune escape by carrying heat shock proteins (HSPs), programmed death-ligand 1 (PD-L1), etc. to immune cells, suppressing function and exhausting the immune cells pool. EVs are progressively being evaluated for therapeutic implementation in cancer therapies. EVs-based immunotherapies involve inhibiting EVs generation, using natural EVs, and harnessing engineering EVs. All approaches are associated with advantages and disadvantages. The EVs heterogeneity and diverse physicochemical properties are the main challenges to their clinical applications. Short conclusion Although EVs are criminal; they can be useful for overcoming immune escape. This review discusses the latest knowledge on EVs population and sheds light on the function of tumor-derived EVs in immune escape. It also describes EVs-based immunotherapies with a focus on engineered EVs, followed by challenges that hinder the clinical translation of EVs that are essential to be addressed in future investigations. Video Abstract

Lactic acid, a key metabolic byproduct within the tumor microenvironment, has garnered significant attention for its role in immune evasion mechanisms. Tumor cells produce and release large amounts of lactic acid into the tumor microenvironment through aberrant glycolysis via the Warburg effect, leading to a drop in pH. Elevated lactic acid levels profoundly suppress proliferation capacity, cytotoxic functions, and migratory abilities of immune effector cells such as macrophages and natural killer cells at the tumor site. Moreover, lactic acid can modulate the expression of surface molecules on immune cells, interfering with their recognition and attack of tumor cells, and it regulates signaling pathways that promote the expansion and enhanced function of immunosuppressive cells like regulatory T cells, thereby fostering immune tolerance within the tumor microenvironment. Current research is actively exploring strategies targeting lactic acid metabolism to ameliorate tumor immune evasion. Key approaches under investigation include inhibiting the activity of critical enzymes in lactic acid production to reduce its synthesis or blocking lactate transporters to alter intracellular and extracellular lactate distribution. These methods hold promise when combined with existing immunotherapies such as immune checkpoint inhibitors and chimeric antigen receptor T-cell therapies to enhance the immune system’s ability to eliminate tumor cells. This could pave the way for novel combinatorial treatment strategies in clinical cancer therapy, effectively overcoming tumor immune evasion phenomena, and ultimately improving overall treatment efficacy.

Background The antitumor activity of natural killer (NK) cells can be enhanced by specific targeting with therapeutic antibodies that trigger antibody-dependent cell-mediated cytotoxicity (ADCC) or by genetic engineering to express chimeric antigen receptors (CARs). Despite antibody or CAR targeting, some tumors remain resistant towards NK cell attack. While the importance of ICAM-1/LFA-1 interaction for natural cytotoxicity of NK cells is known, its impact on ADCC induced by the ErbB2 (HER2)-specific antibody trastuzumab and ErbB2-CAR-mediated NK cell cytotoxicity against breast cancer cells has not been investigated. Methods Here we used NK-92 cells expressing high-affinity Fc receptor FcγRIIIa in combination with trastuzumab or ErbB2-CAR engineered NK-92 cells (NK-92/5.28.z) as well as primary human NK cells combined with trastuzumab or modified with the ErbB2-CAR and tested cytotoxicity against cancer cells varying in ICAM-1 expression or alternatively blocked LFA-1 on NK cells. Furthermore, we specifically stimulated Fc receptor, CAR and/or LFA-1 to study their crosstalk at the immunological synapse and their contribution to degranulation and intracellular signaling in antibody-targeted or CAR-targeted NK cells. Results Blockade of LFA-1 or absence of ICAM-1 significantly reduced cell killing and cytokine release during trastuzumab-mediated ADCC against ErbB2-positive breast cancer cells, but not so in CAR-targeted NK cells. Pretreatment with 5-aza-2'-deoxycytidine induced ICAM-1 upregulation and reversed NK cell resistance in ADCC. Trastuzumab alone did not sufficiently activate NK cells and required additional LFA-1 co-stimulation, while activation of the ErbB2-CAR in CAR-NK cells induced efficient degranulation independent of LFA-1. Total internal reflection fluorescence single molecule imaging revealed that CAR-NK cells formed an irregular immunological synapse with tumor cells that excluded ICAM-1, while trastuzumab formed typical peripheral supramolecular activation cluster (pSMAC) structures. Mechanistically, the absence of ICAM-1 did not affect cell–cell adhesion during ADCC, but rather resulted in decreased signaling via Pyk2 and ERK1/2, which was intrinsically provided by CAR-mediated targeting. Furthermore, while stimulation of the inhibitory NK cell checkpoint molecule NKG2A markedly reduced FcγRIIIa/LFA-1-mediated degranulation, retargeting by CAR was only marginally affected. Conclusions Downregulation of ICAM-1 on breast cancer cells is a critical escape mechanism from trastuzumab-triggered ADCC. In contrast, CAR-NK cells are able to overcome cancer cell resistance caused by ICAM-1 reduction, highlighting the potential of CAR-NK cells in cancer immunotherapy.

Adenosine monophosphate-activated protein kinase (AMPK) is associated with the development of liver hepatocellular carcinoma (LIHC). AMPKα2, an α2 subunit of AMPK, is encoded by PRKAA2, and functions as the catalytic core of AMPK. However, the role of AMPKα2 in the LIHC tumor immune environment is unclear. RNA-seq data were obtained from the Cancer Genome Atlas and Genotype-Tissue Expression databases. Using the single-cell RNA-sequencing dataset for LIHC obtained from the China National Genebank Database, the communication between malignant cells and T cells in response to different PRKAA2 expression patterns was evaluated. In addition, the association between PRKAA2 expression and T-cell evolution during tumor progression was explored using Pseudotime analysis, and the role of PRKAA2 in metabolic reprogramming was explored using the R “scMetabolis” package. Functional experiments were performed in LIHC HepG2 cells. AMPK subunits were expressed in tissue-specific and substrate-specific patterns. PRKAA2 was highly expressed in LIHC tissues and was associated with poor patient prognosis. Tumors with high PRKAA2 expression displayed an immune cold phenotype. High PRKAA2 expression significantly promoted LIHC immune escape. This result is supported by the following evidence: 1) the inhibition of major histocompatibility complex class I (MHC-I) expression through the regulation of interferon-gamma activity in malignant cells; 2) the promotion of CD8+ T-cell exhaustion and the formation of CD4+ Treg cells in T cells; 3) altered interactions between malignant cells and T cells in the tumor immune environment; and 4) induction of metabolic reprogramming in malignant cells. Our study indicate that PRKAA2 may contribute to LIHC progression by promoting metabolic reprogramming and tumor immune escape through theoretical analysis, which offers a theoretical foundation for developing PRKAA2-based strategies for personalized LIHC treatment.

Immunotherapy with checkpoint inhibitors (ICIs) has radically changed the landscape of therapeutic opportunities in oncology, but much still needs to be understood from a mechanistic point of view. There is space for further improving tumors’ response to ICIs, as supported by a strong biological rationale. For this achievement a detailed analysis of tumor cell phenotype with functional dissection of the molecular interactions occurring in the TME is required. Galectin-3 is a pleiotropic tumor relevant molecule, which deserves particular attention in immuno-oncology. Due to its ability to finely modulate immune response in vivo, Galectin-3 is a potential target molecule to be considered for overcoming tumor immune escape.

Cholesterol metabolism reprogramming plays essential roles in hepatocellular carcinoma (HCC). However, precisely how cholesterol metabolism is dysregulated is not clear. Here, we show that the palmitoyltransferase ZDHHC3 and depalmitoylase ABHD17A regulate HCC cell cholesterol biosynthesis by dynamically S-acylating SREBP cleavage-activating protein (SCAP). SCAP S-acylation by ZDHHC3 at C264 antagonizes HACE1-mediated SCAP ubiquitination. Intriguingly, SREBP2 transcriptionally upregulates ZDHHC3 to form a positive feedback loop, which explains why negative feedback regulation of SCAP/SREBP2 signaling fails in HCC. Increased cholesterol in the tumor microenvironment (TME) restrains CD4+ T cell cytotoxicity. Hence, the cholesterol metabolism reprogramming and cholesterol level alternation in the TME cooperate to promote HCC development. We identified a small-molecule inhibitor of ZDHHC3 that, combined with anti-PD-1 immunotherapy, inhibited diethyl nitrosamine (DEN)/CCl4-induced HCC growth in mice. ZDHHC3-mediated SCAP S-acylation reprograms cholesterol metabolism and promotes HCC immune escape. ZDHHC3 is thus identified as a rational chemotherapy target for HCC.

Immune cell therapy based on chimeric antigen receptor (CAR) technology platform has been greatly developed. The types of CAR immune cell therapy have expanded from T cells to innate immune cells such as NK cells and macrophages, and the diseases treated have expanded from hematological malignancies to non-tumor fields such as infectious diseases and autoimmune diseases. Among them, CAR-T and CAR-NK therapy have observed examples of rapid remission in approved clinical trials, but the efficacy is unstable and plagued by tumor resistance. Trogocytosis is a special phenomenon of intercellular molecular transfer that is common in the immune system and is achieved by recipient cells through acquisition and internalization of donor cell-derived molecules and mediates immune effects. Recently, a novel short-term drug resistance mechanism based on trogocytosis has been proposed, and the bidirectional molecular exchange between CAR immune cells and tumor cells triggered by trogocytosis partially explains the long-term relapse phenomenon after treatment with CAR immune cells. In this review, we summarize the research progress of trogocytosis in CAR immunotherapy, discuss the influencing factors of trogocytosis and its direct and indirect interference with CAR immune cells and emphasize that the interference of trogocytosis can further release the potential of CAR immune cell therapy.

OBJECTIVE This study aims to dissect impacts of exosomes-delivered PD-L1 and CTLA-4 siRNAs on colorectal cancer (CRC) progression and immune responses. METHODS Exosomes containing PD-L1 siRNA and CTLA-4 siRNA were prepared and utilized to treat CRC cells to evaluate their effects. A tumor-bearing mouse model was established for verification. RESULTS Exosomes containing PD-L1 siRNA and CTLA-4 siRNA repressed malignant features of CRC cells and restrained tumor growth and activated tumor immune responses in vivo. Co-culture of CRC cells treated with exosomes containing PD-L1 siRNA and CTLA-4 siRNA with human CD8+ T cells increased the percentage of CD8+ T cells, decreased the apoptotic rate of CD8+ T cells, elevated IL-2, IFN-γ, and TNF-α expression in cell supernatants, reduced adherent density of CRC cells, augmented the positive rate of CRC cells, and subdued tumor immune escape. CONCLUSION Exosomes containing PD-L1 siRNA and CTLA-4 siRNA suppressed CRC progression and enhanced tumor immune responses.

Hypoxia, a common feature of the tumor microenvironment in various types of cancers, weakens cytotoxic T cell function and causes recruitment of regulatory T cells, thereby reducing tumoral immunogenicity. Studies have demonstrated that hypoxia and hypoxia-inducible factors (HIFs) 1 and 2 alpha (HIF1A and HIF2A) are involved in tumor immune escape. Under hypoxia, activation of HIF1A induces a series of signaling events, including through programmed death receptor-1/programmed death ligand-1. Moreover, hypoxia triggers shedding of complex class I chain-associated molecules through nitric oxide signaling impairment to disrupt immune surveillance by natural killer cells. The HIF-1-galactose-3-O-sulfotransferase 1-sulfatide axis enhances tumor immune escape via increased tumor cell-platelet binding. HIF2A upregulates stem cell factor expression to recruit tumor-infiltrating mast cells and increase levels of cytokines interleukin-10 and transforming growth factor-β, resulting in an immunosuppressive tumor microenvironment. Additionally, HIF1A upregulates expression of tumor-associated long noncoding RNAs and suppresses immune cell function, enabling tumor immune escape. Overall, elucidating the underlying mechanisms by which HIFs promote evasion of tumor immune surveillance will allow for targeting HIF in tumor treatment. This review discusses the current knowledge of how hypoxia and HIFs facilitate tumor immune escape, with evidence to date implicating HIF1A as a molecular target in such immune escape. This review provides further insight into the mechanism of tumor immune escape, and strategies for tumor immunotherapy are suggested.

An immunosuppressive state is a typical feature of the tumor microenvironment. Despite the dramatic success of immune checkpoint inhibitor (ICI) therapy in preventing tumor cell escape from immune surveillance, primary and acquired resistance have limited its clinical use. Notably, recent clinical trials have shown that epigenetic drugs can significantly improve the outcome of ICI therapy in various cancers, indicating the importance of epigenetic modifications in immune regulation of tumors. Recently, RNA modifications (N6-methyladenosine [m6A], N1-methyladenosine [m1A], 5-methylcytosine [m5C], etc.), novel hotspot areas of epigenetic research, have been shown to play crucial roles in protumor and antitumor immunity. In this review, we provide a comprehensive understanding of how m6A, m1A, and m5C function in tumor immunity by directly regulating different immune cells as well as indirectly regulating tumor cells through different mechanisms, including modulating the expression of immune checkpoints, inducing metabolic reprogramming, and affecting the secretion of immune-related factors. Finally, we discuss the current status of strategies targeting RNA modifications to prevent tumor immune escape, highlighting their potential.

Background Adrenocortical carcinoma is rare and aggressive endocrine cancer of the adrenal gland. Within adrenocortical carcinoma, a recently described subtype characterized by a CpG island methylator phenotype (CIMP) has been associated with an especially poor prognosis. However, the drivers of CIMP remain unknown. Furthermore, the functional relation between CIMP and poor clinical outcomes of patients with adrenocortical carcinoma stays elusive. Results Here, we show that CIMP in adrenocortical carcinoma is linked to the increased expression of DNA methyltransferases DNMT1 and DNMT3A driven by a gain of gene copy number and cell hyperproliferation. Importantly, we demonstrate that CIMP contributes to tumor aggressiveness by favoring tumor immune escape. This effect could be at least partially reversed by treatment with the demethylating agent 5-azacytidine. Conclusions In sum, our findings suggest that co-treatment with demethylating agents might enhance the efficacy of immunotherapy and could represent a novel therapeutic approach for patients with high CIMP adrenocortical carcinoma.

Blockade of the programmed death 1 (PD‐1)/ programmed death ligand 1 (PD‐L1) immune checkpoint could increase antitumor immunotherapy for multiple types of cancer, but the response rate of patients is about 10%–40%. Peroxisome proliferator activated receptor γ (PPARγ) plays an important role in regulating cell metabolism, inflammation, immunity, and cancer progression, while the mechanism of PPARγ on cancer cell immune escape is still unclear. Here we found that PPARγ expression exhibits a positive correlation with activation of T cells in non‐small‐cell lung cancer (NSCLC) by clinical analysis. Deficiency of PPARγ promoted immune escape of NSCLC by inhibiting T‐cell activity, which was associated with increased PD‐L1 protein level. Further analysis showed that PPARγ reduced PD‐L1 expression independent of its transcriptional activity. PPARγ contains the microtubule‐associated protein 1A/1B‐light chain 3 (LC3) interacting region motif, which acts as an autophagy receptor for PPARγ binding to LC3, leading to degradation of PD‐L1 in lysosomes, which in turn suppresses NSCLC tumor growth by increasing T‐cell activity. These findings suggest that PPARγ inhibits the tumor immune escape of NSCLC by inducing PD‐L1 autophagic degradation.

No abstract available

No abstract available

Tumors develop strategies to evade immunity by suppressing antigen presentation. In this work, we show that prosaposin (pSAP) drives CD8 T cell–mediated tumor immunity and that its hyperglycosylation in tumor dendritic cells (DCs) leads to cancer immune escape. We found that lysosomal pSAP and its single-saposin cognates mediated disintegration of tumor cell–derived apoptotic bodies to facilitate presentation of membrane-associated antigen and T cell activation. In the tumor microenvironment, transforming growth factor–β (TGF-β) induced hyperglycosylation of pSAP and its subsequent secretion, which ultimately caused depletion of lysosomal saposins. pSAP hyperglycosylation was also observed in tumor-associated DCs from melanoma patients, and reconstitution with pSAP rescued activation of tumor-infiltrating T cells. Targeting DCs with recombinant pSAP triggered tumor protection and enhanced immune checkpoint therapy. Our studies demonstrate a critical function of pSAP in tumor immunity and may support its role in immunotherapy. Editor’s summary Cancers develop mechanisms to evade detection and subsequent killing by the immune system. One such strategy involves suppressing the presentation of tumor-associated antigens. Sharma et al. report that a protein called prosaposin is key for triggering immunity against tumors. Immune sentinels called dendritic cells (DCs) engulf dead cancer cells and use prosaposin to process tumor-derived antigens for subsequent display and activation of protective T lymphocytes. Tumor growth results in the addition of complex sugar chains to prosaposin molecules in DCs, leading to prosaposin secretion and the depletion of lysosomal saposins. Targeting tumor DCs with an engineered form of prosaposin enhances the antitumor immune response and improves tumor control. —Priscilla N. Kelly Prosaposin facilitates antigen cross-presentation and tumor immunity, and its hyperglycosylation leads to immune evasion.

Graphical abstract

Tumor immune escape is an important strategy of tumor survival. There are many mechanisms of tumor immune escape, including immunosuppression, which has become a research hotspot in recent years. The programmed death ligand-1/programmed death-1 (PD-L1/PD-1) signaling pathway is an important component of tumor immunosuppression, which can inhibit the activation of T lymphocytes and enhance the immune tolerance of tumor cells, thereby achieving tumor immune escape. Therefore, targeting the PD-L1/PD-1 pathway is an attractive strategy for cancer treatment; however, the therapeutic effectiveness of PD-L1/PD-1 remains poor. This situation requires gaining a deeper understanding of the complex and varied molecular mechanisms and factors driving the expression and activation of the PD-L1/PD-1 signaling pathway. In this review, we summarize the regulation mechanisms of the PD-L1/PD-1 signaling pathway in the tumor microenvironment and their roles in mediating tumor escape. Overall, the evidence accumulated to date suggests that induction of PD-L1 by inflammatory factors in the tumor microenvironment may be one of the most important factors affecting the therapeutic efficiency of PD-L1/PD-1 blocking.

Tumor immune escape is a critical step in the malignant progression of tumors and one of the major barriers to immunotherapy, making immunotherapy the most promising therapeutic approach against tumors today. Tumor cells evade immune surveillance by altering the structure of their own, or by causing abnormal gene and protein expression, allowing for unrestricted development and invasion. These genetic or epigenetic changes have been linked to microRNAs (miRNAs), which are important determinants of post-transcriptional regulation. Tumor cells perform tumor immune escape by abnormally expressing related miRNAs, which reduce the killing effect of immune cells, disrupt the immune response, and disrupt apoptotic pathways. Consequently, there is a strong trend toward thoroughly investigating the role of miRNAs in tumor immune escape and utilizing them in tumor treatment. However, because of the properties of miRNAs, there is an urgent need for a safe, targeted and easily crossed biofilm vehicle to protect and deliver them in vivo, and exosomes, with their excellent biological properties, have successfully beaten traditional vehicles to provide strong support for miRNA therapy. This review summarizes the multiple roles of miRNAs in tumor immune escape and discusses their potential applications as an anti-tumor therapy. Also, this work proposes exosomes as a new opportunity for miRNA therapy, to provide novel ideas for the development of more effective tumor-fighting therapeutic approaches based on miRNAs.

Liver cancer, which most commonly manifests as hepatocellular carcinoma (HCC), is the sixth most common cancer in the world. In HCC, the immune system plays a crucial role in the growth and proliferation of tumor cells. HCC achieve immune escape through the tumor microenvironment, which significantly promotes the development of this cancer. Here, this article introduces and summarizes the functions and effects of regulatory T cells (Tregs) in the tumor microenvironment, highlighting how Tregs inhibit and regulate the functions of immune and tumor cells, cytokines, ligands and receptors, etc, thereby promoting tumor immune escape. In addition, it discusses the mechanism of CAR-T therapy for HCC and elaborate on the relationship between CAR-T and Tregs.

Tumor-associated macrophages (TAMs) are important components of the tumor microenvironment (TME) and strongly associated with poor prognosis and drug resistance, including checkpoint blockade immunotherapy in solid tumor patients. However, the mechanism by which TAM affects immune metabolism reprogramming and immune checkpoint signalling pathway in the TME remains elusive. In this study we found that transforming growth factor-beta (TGF-β) secreted by M2-TAMs increased the level of glycolysis in bladder cancer (BLCA) and played important role in PD-L1-mediated immune evasion through pyruvate kinase isoenzymes M2 (PKM2). Mechanistically, TGF-β promoted high expression of PKM2 by promoting the nuclear translocation of PKM2 dimer in conjunction with phosphorylated signal transducer and activator of transcription (p-STAT3), which then exerted its kinase activity to promote PD-L1 expression in BLCA. Moreover, SB-431542 (TGF-β blocker) and shikonin (PKM2 inhibitor) significantly reduced PD-L1 expression and inhibited BLCA growth and organoids by enhancing anti-tumour immune responses. In conclusion, M2-TAM-derived TGF-β promotes PD-L1-mediated immune evasion in BLCA by increasing the PKM2 dimer-STAT3 complex nuclear translocation. Combined blockade of the TGF-β receptor and inhibition of PKM2 effectively prevent BLCA progression and immunosuppression, providing a potential targeted therapeutic strategy for BLCA.

Extrachromosomal DNA (ecDNA) is a type of circular and tumor specific genetic element. EcDNA has been reported to display open chromatin structure, facilitate oncogene amplification and genetic material unequal segregation, and is associated with poor cancer patients’ prognosis. The ability of immune evasion is a typical feature for cancer progression, however the tumor intrinsic factors that determine immune evasion remain poorly understood. Here we show that the presence of ecDNA is associated with markers of tumor immune evasion, and obtaining ecDNA could be one of the mechanisms employed by tumor cells to escape immune surveillance. Tumors with ecDNA usually have comparable TMB and neoantigen load, however they have lower immune cell infiltration and lower cytotoxic T cell activity. The microenvironment of tumors with ecDNA shows increased immune-depleted, decreased immune-enriched fibrotic types. Both MHC class I and class II antigen presentation genes’ expression are decreased in tumors with ecDNA, and this could be the underlying mechanism for ecDNA associated immune evasion. This study provides evidence that ecDNA formation is an immune escape mechanism for cancer cells.

No abstract available