免疫性检查点抑制剂相关性心肌炎模型预警

Myocarditis has emerged as an immune-related adverse event of immune checkpoint inhibitor (ICI) cancer therapy associated with significant mortality. To ensure patients continue to safely benefit from life-saving cancer therapy, an understanding of fundamental immunological phenomena underlying ICI myocarditis is essential. We recently developed the NOD-cMHCI/II-/-.DQ8 mouse model that spontaneously develops myocarditis with lower mortality than observed in previous HLA-DQ8 NOD mouse strains. Our strain was rendered murine MHC class I and II deficient using CRISPR/Cas9 technology, making it a genetically clean platform for dissecting CD4+ T cell-mediated myocarditis in the absence of classically selected CD8+ T cells. These mice are highly susceptible to myocarditis and acute heart failure following anti-PD-1 ICI-induced treatment. Additionally, anti-PD-1 administration accelerates skeletal muscle myositis. Using histology, flow cytometry, adoptive transfers, and RNA sequencing analyses, we performed a thorough characterization of cardiac and skeletal muscle T cells, identifying shared and unique characteristics of both populations. Taken together, this report details a mouse model with features of a rare, but highly lethal clinical presentation of overlapping myocarditis and myositis following ICI therapy. This study sheds light on underlying immunological mechanisms in ICI myocarditis and provides the basis for further detailed analyses of diagnostic and therapeutic strategies.

Cardiac fibroblast-derived ANGPTL2 plays a role in immune checkpoint inhibitors -related autoimmune myocarditis by enhancing chemokine-induced recruitment of T cells. Use of immune checkpoint inhibitors (ICIs) as cancer immunotherapy advances rapidly in the clinic. Despite their therapeutic benefits, ICIs can cause clinically significant immune-related adverse events (irAEs), including myocarditis. However, the cellular and molecular mechanisms regulating irAE remain unclear. Here, we investigate the function of Angiopoietin-like protein 2 (ANGPTL2), a potential inflammatory mediator, in a mouse model of ICI-related autoimmune myocarditis. ANGPTL2 deficiency attenuates autoimmune inflammation in these mice, an outcome associated with decreased numbers of T cells and macrophages. We also show that cardiac fibroblasts express abundant ANGPTL2. Importantly, cardiac myofibroblast-derived ANGPTL2 enhances expression of chemoattractants via the NF-κB pathway, accelerating T cell recruitment into heart tissues. Our findings suggest an immunostimulatory function for ANGPTL2 in the context of ICI-related autoimmune inflammation and highlight the pathophysiological significance of ANGPTL2-mediated cardiac myofibroblast/immune cell crosstalk in enhancing autoimmune responses. These findings overall provide insight into mechanisms regulating irAEs.

In the course of the SARS-CoV-2 pandemic, vaccination safety and risk factors of SARS-CoV-2 mRNA-vaccines were under consideration after case reports of vaccine-related side effects, such as myocarditis, which were mostly described in young men. However, there is almost no data on the risk and safety of vaccination, especially in patients who are already diagnosed with acute/chronic (autoimmune) myocarditis from other causes, such as viral infections, or as a side effect of medication and treatment. Thus, the risk and safety of these vaccines, in combination with other therapies that could induce myocarditis (e.g., immune checkpoint inhibitor (ICI) therapy), are still poorly assessable. Therefore, vaccine safety, with respect to worsening myocardial inflammation and myocardial function, was studied in an animal model of experimentally induced autoimmune myocarditis. Furthermore, it is known that ICI treatment (e.g., antibodies (abs) against PD-1, PD-L1, and CTLA-4, or a combination of those) plays an important role in the treatment of oncological patients. However, it is also known that treatment with ICIs can induce severe, life-threatening myocarditis in some patients. Genetically different A/J (most susceptible strain) and C57BL/6 (resistant strain) mice, with diverse susceptibilities for induction of experimental autoimmune myocarditis (EAM) at various age and gender, were vaccinated twice with SARS-CoV-2 mRNA-vaccine. In an additional A/J group, an autoimmune myocarditis was induced. In regard to ICIs, we tested the safety of SARS-CoV-2 vaccination in PD-1−/− mice alone, and in combination with CTLA-4 abs. Our results showed no adverse effects related to inflammation and heart function after mRNA-vaccination, independent of age, gender, and in different mouse strains susceptible for induction of experimental myocarditis. Moreover, there was no worsening effect on inflammation and cardiac function when EAM in susceptible mice was induced. However, in the experiments with vaccination and ICI treatment, we observed, in some mice, low elevation of cardiac troponins in sera, and low scores of myocardial inflammation. In sum, mRNA-vaccines are safe in a model of experimentally induced autoimmune myocarditis, but patients undergoing ICI therapy should be closely monitored when vaccinated.

Immune checkpoint inhibitors (ICIs) have changed cancer treatment. However, ICIs could lead to undesirable events. Myocarditis is one of the rarest, yet most fatal, with a mortality rate of 60%. Our lab has generated a novel murine model to study the susceptibility to develop ICI-myocarditis. In this study, tumor-bearing cd69-/- mice under anti-PD1 treatment presented exacerbated Th17 responses in peripheral blood, cardiac inflammation and dysfunction. Wild-type mice do not develop the disease under the same settings. Previous work has validated mmu-miR-721 as a specific biomarker for acute myocarditis. In our model, mmu-miR-721 levels are significantly elevated at day 10 of the treatment in plasma of cd69-/- mice. This finding has clinical relevance since the mmu-miR-721 could be a potential biomarker and therapeutic target of ICI-myocarditis. Th17 cells are the main producers of mmu-miR-721, and it controls RORγt expression by inhibiting PPARγ. Our hypothesis is that in vivo inhibition mmu-miR-721 will produce an overexpression of PPARγ, and subsequently lower expression of RORγt. Therefore, tumor-bearing cd69-/- susceptible mice under anti-PD1 treatment will exhibit lower Th17 responses and the development of ICI-myocarditis will be impaired without affecting tumor regression. cd69-/- mice were used since our group has described how these animals develop exacerbated Th17 responses and ICI-myocarditis with anti-PD1 treatment. Mice were treated with anti-mmu-miR-721 (single-stranded DNA molecules with four reverse complementary sequences to the microRNA) or scramble the same day as anti-PD1 treatment begin. We analyzed the subsequent immune response in peripheral blood, lymph nodes and heart by flow cytometry, as well as the cardiac dysfunction by echocardiography. We proposed a novel therapeutic target for ICI-myocarditis. Anti-mmu-miR-721 treatment at the beginning of immunotherapy protects susceptible mice of cardiac inflammation with lower immune infiltration, and lower Th17 and γδ T cells responses in the heart. Additionally, these mice do not generate exacerbated Th17 responses in peripheral blood at day 10 of immunotherapy. Consequently, these animals do not overexpress mmu-miR-721 biomarker levels in plasma 10 days after initiating anti-PD1 treatment, as observed in the scramble and control groups. Echocardiography analysis of mice treated with anti-mmu-miR-721 show preserved cardiac function at the end-point of ICI-myocarditis. Anti-mmu-miR-721 therapy in tumor-bearing cd69-/- susceptible mice under anti-PD1 treatment reduces cardiac inflammation due to a lower immune infiltration and Th17 responses towards the heart. Moreover, biomarker mmu-miR-721 levels are not elevated in plasma at day 10 when treated with anti-mmu-miR-721. This finding has clinical relevance since the mmu-miR-721 could be a specific therapeutic target of ICI-myocarditis.  

Immunotherapy has led to considerable clinical improvement in cancer patients, but is associated with serious side effects such as myocarditis, which is associated with a high mortality risk. Immune checkpoint inhibitor-induced myocarditis (ICI-M) is characterised by T cells and macrophages infiltration within the myocardium, which lead to necrosis of cardiac tissue. The NLRP3 inflammasome pathway, associated with interferon-γ (IFN-γ), is overexpressed within the myocardium of ICI-M patients. To study the pathophysiological mechanisms of ICI-M using a preclinical model to investigate the electrophysiological disorders associated with myocardium immune infiltration, and to unravel the molecular mechanisms in a cellular model using IPSC from ICI-M patient. The preclinical ICI-M model was established by subcutaneous injection of murine melanocytic cells into BALB/c mice, treated with anti-PD1/anti-CTLA-4 combination therapy. IPSC-derived cardiomyocytes (CM) and endothelial cells (EC), generated from ICI-M patient and healthy donor, were exposed to IFN-γ. A decrease in tumour volume was observed in mice exhibiting ECG disturbances, with a reduction in QRS and T wave amplitude, as well as a reduction in repolarisation time. Impaired cardiac function was associated with increased CD3 transcript and overexpression of PD-L1 in the myocardium. In hiPSC-derived CM and EC, genes involved in immune response (PD-L1, MHC-II) and in NLRP3 inflammasome (GBP5, GBP6, NLRC5) were up-regulated by IFN-γ. A large number of dysregulated genes were found in EC compared to CM after stimulation. The nature of the inflammasome activated by IFN-γ was different between the two cell types, NLRP3 in hiPSC-CM and AIM2 in hiPSC-EC. The hiPSC-EC from ICI-M patient showed specific regulations, in the immune response (PD-L2, TLR2), and in secreted pro-inflammatory cytokines (CCL2, CCL5, IL-1b), associated with NLRP3 inflammasome. Mice treated with ICI show ECG disturbances correlated with myocardial inflammation. Our cellular model highlighted the major role played by hiPSC-EC in the response to IFN-γ and the specific regulation of the NLRP3 inflammasome. We would like to further investigate the role of endocardium and microvascularisation in the mechanisms of ICI-M.

Introduction: Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that activate immune cells against tumor cells but can cause immune related adverse events such as myocarditis, a serious side effect with up to 40% case fatality rate. We hypothesize that cardiac fibroblasts and cytotoxic T-Cells work in tandem to direct inflammation in the setting of ICI treatment, resulting in myocarditis. Methods: To study the role of CFBs in ICIM, we first examined fibroblast behavior in vivo through picrosirius red staining for cardiac fibrosis. We then cultured C57BL/6 murine CFBs and treated them with cardiac serum acquired from a genetic mouse model of ICIM and analyzed Cxcl9 expression by qPCR. Additionally, we stimulated CFBs with media or IFNγ for 24 hours (uCFBs and sCFBs respectively) before co-culturing them with CD8 CXCR3hi T-Cells for 6 hours, after which we analyzed Cxcl9 and Cxcl10 CFB expression by qPCR (Fig 1C) and T-Cell cytotoxicity by flow cytometry (Fig 1D/E). We next examined CD8 T-Cells harvested from C57BL/6 mice treated with combination immunotherapy to induce ICIM via single-cell sequencing to investigate potential axes of T-Cell/CFB crosstalk in vivo . Results: Our picrosirius red staining revealed a significant increase in cardiac interstitial and perivascular fibrosis, suggesting that CFBs are inflammatorily active and fibrotic in the context of ICIM (Fig 1A). We demonstrated that treating CFBs with ICIM cardiac serum results in a significant upregulation of CXCL9 by qPCR (Fig 1B). Utilizing our co-culture system, we demonstrated that the presence of CD8 CXCR3hi cytotoxic T-cells significantly increases CXCL9/10 expression compared to both control and IFNγ stimulation, underscoring the importance of T-cell presence in CFB activation (Fig 1C). We also used flow cytometry to confirm the cytotoxic quality of the T-Cells used in our co-culture system (Fig 1D/E). Using single-cell sequencing, we identified a cluster of CXCR3hiVLA4hi CD8 T-Cells from ICI-treated myocarditis mice (Fig 1F.) which are highly cytotoxic compared to other clusters (Fig 1G.), demonstrating that VLA-4:VCAM-1 signaling between T-cells and CFBs may act as a critical driver of cytotoxicity in the heart. Conclusions: Our findings position cardiac fibroblasts as potentially critical drivers of T-Cell migration and increased cytotoxicity in the context of ICIM, rendering this crosstalk an important direction of study in future of understanding and treating ICIM.

BACKGROUND: Immune checkpoint inhibitors (ICIs), antibodies targeting PD-1 (programmed cell death protein 1)/PD-L1 (programmed death-ligand 1) or CTLA4 (cytotoxic T-lymphocyte–associated protein 4), have revolutionized cancer management but are associated with devastating immune-related adverse events including myocarditis. The main risk factor for ICI myocarditis is the use of combination PD-1 and CTLA4 inhibition. ICI myocarditis is often fulminant and is pathologically characterized by myocardial infiltration of T lymphocytes and macrophages. Although much has been learned about the role of T-cells in ICI myocarditis, little is understood about the identity, transcriptional diversity, and functions of infiltrating macrophages. METHODS: We used an established murine ICI myocarditis model (Ctla4+/–Pdcd1–/– mice) to explore the cardiac immune landscape using single-cell RNA-sequencing, immunostaining, flow cytometry, in situ RNA hybridization, molecular imaging, and antibody neutralization studies. RESULTS: We observed marked increases in CCR2 (C-C chemokine receptor type 2)+ monocyte-derived macrophages and CD8+ T-cells in this model. The macrophage compartment was heterogeneous and displayed marked enrichment in an inflammatory CCR2+ subpopulation highly expressing Cxcl9 (chemokine [C-X-C motif] ligand 9), Cxcl10 (chemokine [C-X-C motif] ligand 10), Gbp2b (interferon-induced guanylate-binding protein 2b), and Fcgr4 (Fc receptor, IgG, low affinity IV) that originated from CCR2+ monocytes. It is important that a similar macrophage population expressing CXCL9, CXCL10, and CD16α (human homologue of mouse FcgR4) was expanded in patients with ICI myocarditis. In silico prediction of cell-cell communication suggested interactions between T-cells and Cxcl9+Cxcl10+ macrophages via IFN-γ (interferon gamma) and CXCR3 (CXC chemokine receptor 3) signaling pathways. Depleting CD8+ T-cells or macrophages and blockade of IFN-γ signaling blunted the expansion of Cxcl9+Cxcl10+ macrophages in the heart and attenuated myocarditis, suggesting that this interaction was necessary for disease pathogenesis. CONCLUSIONS: These data demonstrate that ICI myocarditis is associated with the expansion of a specific population of IFN-γ–induced inflammatory macrophages and suggest the possibility that IFN-γ blockade may be considered as a treatment option for this devastating condition.

BACKGROUND Although immune checkpoint inhibitors (ICIs) have made an immense breakthrough in cancer therapeutics, they can exert unique, immune-related adverse events. Among them, myocarditis is less frequent, but it is serious and often follows a lethal course. METHODS To examine the changes in cardiac autoimmunity after ICI administration, we developed a mouse experimental autoimmune myocarditis (EAM) model via intraperitoneal administration of murine α-cardiac myosin heavy chain (MyHC-α) fragment. Thereafter, the mouse anti-PD-1 antibody (mPD1ab) was administered at two time points, subsequent to and concurrent with MyHC-α fragment administration. RESULTS Severe EAM developed in 3 weeks; wide inflammatory lesions were observed in the cardiac sections. Furthermore, inflammatory/fibrotic genes, such as interleukin 1β, interleukin 6, and collagen 1, were upregulated, although the cardiac function was not significantly affected. The subsequent administration of mPD1ab at 2 weeks post administration of the first MyHC-α fragment exacerbated EAM, whereas the administration of mPD1ab concurrent with MyHC-α fragment administration did not exacerbate EAM. The subsequent administration of mPD1ab significantly increased the infiltration of cluster of differentiation (CD)4- and F4/80-positive cells, whereas the concurrent administration of mPD1ab significantly decreased the infiltration of CD4-positive cells, indicating that the concurrent and subsequent administration of mPD1ab had opposite effects on immune/inflammatory cell infiltration. CONCLUSIONS These data suggest that the appearance of ICI-induced autoimmune myocarditis might be related to autoimmune system activity before ICI administration. Although ICIs do not adversely affect patients with normal immune systems, we propose that ICI administration should be avoided in patients with autoimmune disorders.

Background: Immune-checkpoint inhibitor (ICI)-induced myocarditis is the most fatal immune-related adverse event (irAE). Early recognition and treatment of ICI myocarditis is associated with improved outcomes. However, current approaches to the diagnosis of ICI myocarditis have limitations. Open-source large language models (LLMs) are an accessible and scalable method of answering queries from human-generated text, and therefore may assist in the real-time early detection of ICI myocarditis among at-risk patients. Research Question: Can a free, open-source LLM detect cases of ICI myocarditis early in admission? Aims: We investigated the ability of a LLM pipeline to screen for ICI myocarditis within one day of admission using hospital medical records. Methods: Hospital admissions of patients on ICI therapy from November 4th, 2021, to September 5th, 2023 were retrospectively reviewed by a multidisciplinary immunotoxicity team using established, published definitions for the presence of ICI myocarditis. Progress notes written the day before, day of, and day after admission were fed into an open-source LLM pipeline built using Mistral 7B OpenOrca with retrieval augmentation generation (RAG). Performance of the LLM was measured via sensitivity and specificity in comparison to the gold standard of manual adjudication. Results: Of the 1874 hospital admissions of patients on ICI therapy, there were 22 (1.2%) cases of myocarditis. The average time to initiation of treatment was 2.45 days. Using notes written within one day after admission, the LLM detected ICI myocarditis with 95.5±8.7% sensitivity and 95.4±1.0% specificity, spending 2.13 seconds per chart. Conclusion: LLMs serve as a useful tool to screen for ICI myocarditis, detecting 95.5% of cases within one day of admission with 95.4% specificity. Future studies will investigate if integrating this tool into the real-time management of irAEs could lead to earlier diagnosis, faster initiation of treatment, and improved patient outcomes.

IntroductionImmune checkpoint inhibitors (ICIs) are effective for a growing number of cancer indications. ICI-mediated T cell activation can lead to immune related adverse effects, including ICI myocarditis, which has up to a 50% mortality. To date, only a few cytokines have been reported to be associated with ICI myocarditis. We evaluated an expansive repertoire of cytokines associated with ICI myocarditis and the pathways they regulate. MethodsA total of 173 cardio-oncology patients were enrolled in the biomarker study, including 55 who were on ICI. Blood samples of patients who were on ICI for cytokine profiling were sampled when patients presented with symptoms concerning for ICI myocarditis. 71 different cytokines were evaluated and analyzed using Point Biserial correlation analyses and machine learning (XGboost) and explainable artificial intelligence (SHAP) to identify cytokines associated with ICI myocarditis. Analyses were performed to identify pathways associated with ICI myocarditis. Results28 cytokines were associated with ICI myocarditis and machine learning revealed top features associated with ICI myocarditis in the entire cohort with IL33 being the top feature, Accuracy of 0.895, AUC of 0.902, F1 score of 0.73. SHAP was also used to identify features associated with ICI myocarditis and found IL10, CXCL9, CXCL13, CCL3, were positively associated with ICI myocarditis while CCL22, IL33, TNFSF10, CCL8, and CCL24 were negatively associated with ICI myocarditis. 90% of cytokines identified in the correlation model were also identified in SHAP and XG Boost. Top KEGG and GO pathways associated with ICI myocarditis identified by XGBoost and SHAP features include the cytosolic DNA sensing pathway, response to influenza A, IL17, PI3K-Akt, JAK-STAT and lipid/atherosclerosis pathways ConclusionsIdentifying pathways associated with ICI myocarditis could provide insights into optimization of immunosuppression strategies Table of cytokines associated with ICI myocarditis identified by SHAP Cytokine Direction IL10 Upregulated CXCL9 Upregulated CXCL13 Upregulated IL7 Upregulated CCL3 Upregulated IFNL2 Upregulated KITLG Upregulated IL27 Upregulated FLT3LG Upregulated CCL22 Downregulated IL12 Downregulated CCL2 Downregulated IL33 Downregulated TNFSF10 Downregulated CCL8 Downregulated CCL21 Downregulated FGF2 Downregulated CCL24 Downregulated CX3CL1 Downregulated Citation Format: Rachel Jaber Chehayeb, Dat Hong, Nathan W. Chen, Carlos Matute Martinez, Ritujith Jayakrishnan, Ana Ferrigno Guajardo, Derrick Lin, Yunju Im, Stephanie Halene, Jennifer VanOudenhove, John Hwa, Alokkumar Jha, Jennifer M. Kwan. Cytokine profiles associated with ICI myocarditis using machine learning approaches identifies novel cytokines and implicated pathways [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 3632.

Abstract Introduction Myocarditis is a complex cardiac disorder with multifactorial etiology, characterized by myocardial inflammation, cardiomyocyte damage, and fibrosis. Left untreated, myocarditis may lead to acute and chronic heart failure and life-threatening arrhythmias. Reliable experimental models of myocarditis are required to study the underlying pathological mechanisms of the disease and to develop efficient therapies. Materials and Methods Experimental autoimmune myocarditis (EAM) was induced by immunizing 9-week-old female BALB/c mice with a specific peptide derived from the cardiac α-myosin heavy chain (α-MHC) which is the major autoantigen recognized by heart-specific autoantibodies in myocarditis. The immunization activates an anti-α-MHC immune response, which triggers an inflammatory reaction leading to cardiomyocyte damage and myocardial injury. The mice received two immunizations with 100 μL of an emulsion containing α-MHC peptide emulsified 1:1 in Complete Freund’s Adjuvant (CFA) supplemented with 4 mg/mL inactivated Mycobacterium tuberculosis (M. tuberculosis), administered subcutaneously on Days 0 and 7. Results We highlight the essential steps for the successful preparation of the peptide-adjuvant emulsion, a key factor in the effective implementation of the model, and mouse immunization. We also demonstrate how the inflammatory, fibrotic, and functional consequences on the myocardium should be evaluated. Conclusion This efficient and reproducible murine EAM model allows experimental research on the pathophysiology and treatment of myocarditis.

Introduction: Although immune checkpoint inhibitors (ICIs) have transformed cancer therapy, they can cause immune-related adverse effects, including ICI myocarditis, a potentially fatal form of inflammation-induced cardiac injury. What triggers autoreactive T cell activation in patients treated with ICIs remains unclear. Dendritic cells (DCs), which take up and present antigens to T cells for activation, could play a key role in the development of ICI myocarditis. Hypothesis: We hypothesize that reduced DC lysosomal activity in ICI myocarditis leads to reduced degradation of internalized antigens, preserving antigen epitopes and enhancing antigen presentation, leading to increased T cell activation. Methods: We have developed a mouse model of ICI myocarditis wherein three priming doses (100 mg/kg) of isoproterenol (ISO) followed by a high dose (300 mg/kg) of ISO provokes persistent autoreactive CD8+ T cell-driven myocarditis in mice deficient in the immune checkpoint inhibitor PD-1 (PD-1 KO). PD-1 KO mice have increased mortality and reduced cardiac function compared to wild-type mice. We studied DC lysosomes in this model in vivo (heart, mediastinal lymph nodes) and in vitro (bone marrow-derived DCs) using flow cytometry, biochemical tools, and mass spectrometry. Results: Using our ISO model, we observed that PD-1 KO DCs in the heart and mediastinal lymph nodes showed significantly reduced lysosomal acidification than wild-type in response to ISO priming. PD-1 KO bone marrow-derived DCs had significantly reduced cathepsin B activity and antigen processing capabilities than wild-type. Proteomic analyses of lysosomes isolated from antigen presenting cells in lymph nodes also showed increased cardiac peptide abundance in PD-1 KO DCs versus wild-type. Conclusions: Our findings demonstrate altered lysosomal degradation in PD-1 KO DCs. Future work will investigate how this affects antigen presentation, CD8+ T cell expansion, and cardiac outcomes in ICI myocarditis.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy, providing improved outcomes for numerous patients. However, treatment with ICIs can result in ICI-myocarditis, a rare yet severe complication. While current guidelines recommend high-dose glucocorticoids as the first-line approach, steroid-refractory cases require alternative immunosuppressive treatment, such as mycophenolate mofetil (MMF). Given the role of the inflammasome activation in ICI-myocarditis, the NLRP3 inhibitor MCC950 is of potential interest. We aimed to identify early predictors of steroid resistance in patients with ICI-myocarditis. Additionally, a cell culture model was used to explore NLRP3 inhibition as a potential treatment option. In a retrospective study, 73 patients from March 2019 to November 2024 were included. All patients were either hospitalized or assessed at the outpatient clinic due to suspected ICI-myocarditis, indicated by elevation of cardiac biomarkers, typical symptoms, or abnormalities in electrocardiography or echocardiography. 25 patients received glucocorticoids and did not require further immunosuppressive treatment (GC), 20 needed MMF after initial first-line treatment (GC+MMF). In 28 patients, no immunomodulatory treatment was given. In vitro, neonatal rat ventricular myocytes (NRVMs) were treated with Pembrolizumab (ICI), interferon-gamma (INF-γ), human peripheral blood mononuclear cells (hPBMCs) and MCC950. Baseline characteristics of the GC and GC+MMF groups from the day of presentation or the subsequent three days were analyzed. Initial levels of troponin T (hs-cTnT) and troponin I (cTnI) compared with upper reference limits did not differ significantly. Baseline echocardiographic parameters were comparable except for lower TAPSE (tricuspid annular plane systolic excursion) in the GC group. Anti-PD1 treatment was slightly overrepresented in the GC+MMF group (p=0.062). Based on age, sex, BMI, cardiovascular risk factors (arterial hypertension, hypercholesterolemia, tabacco use), presence of symptoms, time between first dose of ICI and suspicion of cardiotoxicity and cardiac biomarkers (hs-cTnT, cTnI, NT-proBNP and CRP) no parameter identified steroid resistance within the first three days following presentation. In vitro, NRVMs exposed to hPBMCs and INF-γ showed increased troponin release, while INF-γ alone was sufficient to induce NLRP3 expression. So far, no early clinical parameter predicts higher risk of steroid resistance and the need for additional treatment in our cohort. At the cellular level, INF-γ drives inflammation and cardiomyocyte injury. Further investigation is needed to assess NLRP3 inhibitors like MCC950 as a therapeutic option or co-therapy for ICI-Myocarditis.

Immune checkpoint inhibitor (ICI)-related myocarditis is a rare but fatal immune-related adverse event in lung cancer patients, with limited multivariate prognostic analysis. This study aimed to identify risk factors for severity, major adverse cardiac events (MACE) and survival time, and develop a survival prediction model. Data from 70 lung cancer patients with ICI-related myocarditis (training set) and 40 patients (validation set) were analyzed, with ≥ 1.5 years of follow-up. Cox regression was employed to determine factors associated with survival time, and Logistic regression models identified risk factors for severe myocarditis and MACE. Several factors were independently associated with all-cause death: protective factors included combined radiotherapy (HR 0.12, 95%CI: 0.01–0.98, p = 0.047) and longer ICI treatment duration (≥ 132 days, HR 0.93, 95%CI: 0.91–0.98, p = 0.013); risk factors included low-dose glucocorticoid use in patients with severe myocarditis (HR 3.92, 95%CI: 1.16–13.2, p = 0.028). A nomogram model constructed based on these three variables yielded area under the time-ROC curves of 0.832, 0.835, and 0.924 for 0.5-, 1-, and 1.5-year survival in the training set, and 0.821, 0.806, and 0.789 in the validation set, respectively. It also demonstrated good discriminative ability and clinical utility for predicting survival in lung cancer patients with ICI-related myocarditis, as this study established a validated nomogram model that may aid survival prediction in this population. Additionally, we analyzed the risk factors for severe ICI related myocarditis and 90-day MACE. We found that the use of angiogenesis inhibitors was an independent risk factor for severe myocarditis (OR 18.72, 95% CI: 2.52–428.27, p = 0.02); a history of coronary artery disease (OR 10.54, 95% CI: 1.62–210.10, p = 0.037) was an independent risk factor for 90-day MACE; and left ventricular ejection fraction (OR 0.94, 95% CI: 0.88–0.99, p = 0.026) was an independent protective factor against 90-day MACE.

Background Immune checkpoint inhibitor (ICIs)-associated cardiotoxicity is a significant cause of immune-related adverse events and mortality in cancer immunotherapy, lacking effective preventative or therapeutic strategies. Xihuang Pill (XHW), a traditional Chinese medicine with established anti-inflammatory properties and clinical use in cancer treatment and adverse event mitigation, merits investigation for its efficacy against ICIs-induced cardiac toxicity. Purpose To investigate XHW’s therapeutic effects on Immune checkpoint inhibitors (ICIs)-associated cardiotoxicity and its underlying mechanisms. Methods This study utilized mass spectrometry technology to identify the chemical components in XHW. The experimental model for ICIs-associated myocarditis was generated in BALB/c mice by immunizing them with murine cardiac troponin I (cTnI) peptide and administering anti-programmed death 1 (PD-1) antibodies to mice. Mice received varying XHW dosages (0.39, 0.78, and 1.56 mg/kg). Myocardial contractility and plasma cardiac injury markers (CK, CK-MB) were assessed. Metabolomics and transcriptomics identified key signaling pathways modulated by XHW, validated via real-time quantitative PCR (QT-PCR). In addition, a correlation analysis was conducted between key genes and differential metabolites. Results Mass spectrometry identified 171 components in XHW. Pharmacological studies demonstrated that XHW improved cardiac contractility, reduced plasma cardiac injury biomarkers, and attenuated myocardial injury in the myocarditis model. Integrated metabolomic and transcriptomic analyses revealed that XHW primarily modulates the HIF-1 signaling pathway, significantly upregulating HIF-1 mRNA expression and downregulating the mRNA expression of Nppa, Angpt1, Angpt2, and Trf. Correlation analysis identified significant associations between 16 metabolites, including 13-tetradecynoic acid, 1-pentadecanoylglycerol, and arginyl-glycyl-aspartic acid, and these genes. Conclusion These findings suggest that XHW may alleviate ICIs-associated myocarditis via HIF-1 signaling pathway, offering a promising therapeutic approach for ICIs-related cardiotoxicity.

No abstract available

Background Immune checkpoint blockade in monotherapy or combinatorial regimens with chemotherapy or radiotherapy have become an integral part of oncology in recent years. Monoclonal antibodies against CTLA-4 or PD-1 or PDL-1 are the most studied ICIs in randomized clinical trials, however, more recently, an anti-LAG3 (Lymphocyte activation gene-3) antibody, Relatlimab, has been approved by FDA in combination with Nivolumab for metastatic melanoma therapy. Moreover, Atezolizumab is actually under study in association with Ipilimumab for therapy of metastatic lung cancer. Myocarditis, vasculitis and endothelitis are rarely observed in these patients on monotherapy, however new combination therapies could expose patients to more adverse cardiovascular events. Methods Human cardiomyocytes co-cultured with human peripheral blood lymphocytes (hPBMCs) were exposed to monotherapy and combinatorial ICIs (PD-L1 and CTLA-4 or PD-1 and LAG-3 blocking agents, at 100 nM) for 48 h. After treatments, cardiac cell lysis and secretion of biomarkers of cardiotoxicity (H-FABP, troponin-T, BNP, NT-Pro-BNP), NLRP3-inflammasome and Interleukin 1 and 6 were determined through colorimetric and enzymatic assays. Mitochondrial functions were studied in cardiomyocyte cell lysates through quantification of intracellular Ca++, ATP content and NADH:ubiquinone oxidoreductase core subunit S1 (Ndufs1) levels. Histone deacetylases type 4 (HDAC-4) protein levels were also determined in cardiomyocyte cell lysates to study potential epigenetic changes induced by immunotherapy regimens. Results Both combinations of immune checkpoint inhibitors exert more potent cardiotoxic side effects compared to monotherapies against human cardiac cells co-cultured with human lymphocytes. LDH release from cardiac cells was 43% higher in PD-L1/CTLA-4 blocking agents, and 35.7% higher in PD-1/LAG-3 blocking agents compared to monotherapies. HDAC4 and intracellular Ca++ levels were increased, instead ATP content and Ndufs1 were reduced in myocardial cell lysates (p < 0.001 vs. untreated cells). Troponin-T, BNP, NT-Pro-BNP and H-FABP, were also strongly increased in combination therapy compared to monotherapy regimen. NLRP3 expression, IL-6 and IL-1β levels were also increased by PDL-1/CTLA-4 and PD-1/LAG-3 combined blocking agents compared to untreated cells and monotherapies. Conclusions Data of the present study, although in vitro, indicate that combinatorial immune checkpoint blockade, induce a pro- inflammatory phenotype, thus indicating that these therapies should be closely monitored by the multidisciplinary team consisting of oncologists, cardiologists and immunologists.

Introduction: Immune checkpoint inhibitors (ICIs, such as anti-CTLA-4 or anti-PD-1) have especially been effective in cancer therapies. However, ICIs treatment can result in myocarditis, the most serious complication of ICIs, which has a case fatality rate as high as 40%. Here, we investigate CXCR3 blockade for targeted treatment of myocardial inflammation in ICI myocarditis while considering concurrent tumor treatment. We hypothesize that CXCR3 blockade will have a direct effect on reducing CD8+ T-cell mediated cardiomyocyte cardiotoxicity without affecting tumor treatment. Methods: To study the impact of CXCR3 blockade on myocardial inflammation, we established an in vitro cardiomyocyte apoptosis model by co-culturing cardiomyocytes and MRL-WT mice CD8+ T-cells with/without ICI stimulation or MRL- Pdcd1-/- mice CD8+ T-cells. We then blocked CXCR3 to assess the effect on CD8+ T cell-mediated cytotoxicity against cardiomyocytes. To assess CXCR3 blockade in an in vivo model of ICI myocarditis accounting for tumor, we treated MRL mice inoculated with B16F1 melanoma cells with six doses of 400 µ g each of anti-PD1 and anti-CTLA4 and investigated the effects of CXCR3-CXCL9/10 blockade on hearts and tumors. Results: In vitro CD8+ T-cell and cardiomyocyte co-culture demonstrated mitigation of cardiomyocytes apoptosis with CXCR3 blockade ( Fig. 1 A - C) , suggesting decreased CD8+ T-cell cytotoxicity with CXCR3 blockade. In vivo treatment with dual immunotherapy (anti-PD1/anti-CTLA4 monoclonal antibodies) in the tumor MRL mouse model demonstrated significant tumor volume reduction, which was not impacted by concurrent treatment with anti-CXCR3 or anti-CXCL9/10 (Fig. 1 D - F) . Concurrently, myocarditis occurred in the group treated with immunotherapy alone, not in anti-CXCL9/10 or anti-CXCR3 (Fig. 1 G - H). Conclusions: CXCR3 blockade reduces CD8+ T-cell cytotoxicity against cardiomyocytes and mitigates myocarditis in an in vivo tumor ICI myocarditis mouse model without affecting anti-tumor ICI efficacy, positioning CXCR3 as a promising target for therapeutic intervention.

Immune checkpoint blockade have become an integral part of oncology in recent years. Monoclonal antibodies against CTLA–4 or PD–1 or PDL–1 are the most studied ICIs in randomized clinical trials, however, more recently, an anti–LAG3 monoclonal antibody, Relatlimab, has been approved by FDA for combinatorial treatment with Nivolumab for metastatic melanoma. Moreover, anti PD–L1 blocking agent Atezolizumab is actually under study in association with Ipilimumab as innovative thearpy for metastatic lung cancer. Myocarditis, vasculitis and endothelitis are rarely observed in these patients on monotherapy, however new combination therapies and the use of new targets in immunotherapy could expose patients to more adverse cardiovascular events. In this study, we highlighted the cardiotoxic properties of new Combinatorial immune checkpoint blockade therapies. Human cardiomyocytes co–cultured with human peripheral blood lymphocytes (hPBMCs) were exposed to monotherapy and combinatorial ICIs (PD–L1 and CTLA–4 or PD–1 and LAG–3 blocking agents, at 100 nM) for 48 h. After treatments, cardiac cell lysis and secretion of biomarkers of cardiotoxicity (H–FABP, NT–Pro–BNP), NLRP3–inflammasome and Interleukin 1 and 6 were determined through colorimetric and enzymatic assays. Both combinations of immune checkpoint inhibitors exert more potent cardiotoxic side effects compared to monotherapies against human cardiac cells co–cultured with human lymphocytes. LDH release from cardiac cells was 43% higher in PD–L1/CTLA–4 blocking agents, and 35.7% higher in PD–1/LAG–3 blocking agents compared to monotherapies. NT–Pro–BNP and H–FABP, were also strongly increased in combination therapy with respect to monotherapies. NLRP3, IL–6 and IL–1β levels were also increased by PDL–1/CTLA–4 and PD–1/LAG–3 combined blocking agents compared to untreated cells and monotherapies. Data of the present study, although in vitro, indicate that combinatorial immune checkpoint blockade, induce a pro– inflammatory phenotype, thus indicating that these therapies should be closely monitored by the multidisciplinary team consisting of oncologists, cardiologists and immunologists.

e24016 Background: Immune checkpoint blockade in monotherapy or combinatorial regimens with chemotherapy or radiotherapy have become an integral part of oncology in recent years. Monoclonal antibodies against CTLA-4 or PD-1 or PDL-1 are the most studied ICIs in randomized clinical trials, however, more recently, an anti-LAG3 (Lymphocyte activation gene-3) antibody, Relatlimab, has been approved by FDA in combination with Nivolumab for metastatic melanoma therapy. Moreover, Atezolizumab is actually under study in association with Ipilimumab for therapy of metastatic lung cancer. Myocarditis, vasculitis and endothelitis are rarely observed in these patients on monotherapy, however new combination therapies could expose patients to more adverse cardiovascular events. Methods: human cardiomyocytes, human peripheral blood lymphocytes (hPBMCs) and combinatorial ICIs (PD-L1 and CTLA-4 or PD-1 and LAG-3 blocking agents, at 100 nM) for 48 h. After treatments, cardiac cell lysis and secretion of biomarkers of cardiotoxicity (H-FABP, troponin-T, BNP, NT-Pro-BNP), NLRP3-inflammasome and Interleukin 1 and 6 were determined through colorimetric and enzymatic assays. Mitochondrial functions were studied in cardiomyocyte cell lysates through quantification of intracellular Ca++, ATP content and NADH:ubiquinone oxidoreductase core subunit S1 (Ndufs1) levels. Histone deacetylases type 4 (HDAC-4) protein levels were also determined in cardiomyocyte cell lysates to study potential epigenetic changes induced by immunotherapy regimens. Results: Both combinations of immune checkpoint inhibitors exert more potent cardiotoxic side effects compared to monotherapies against human cardiac cells co-cultured with human lymphocytes. LDH release from cardiac cells was 43% higher in PD-L1/CTLA-4 blocking agents, and 35.7% higher in PD-1/LAG-3 blocking agents compared to monotherapies. HDAC4 and intracellular Ca++ levels were increased, instead ATP content and Ndufs1 were reduced in myocardial cell lysates (p < 0.001 vs untreated cells). Troponin-T, BNP, NT-Pro-BNP and H-FABP, were also strongly increased in combination therapy compared to monotherapy regimen. NLRP3 expression, IL-6 and IL-1β levels were also increased by PDL-1/CTLA-4 and PD-1/LAG-3 combined blocking agents compared to untreated cells and monotherapies. Conclusions: Data of the present study, although in vitro, indicate that combinatorial immune checkpoint blockade, induce a pro- inflammatory phenotype, thus indicating that these therapies should be closely monitored by the multidisciplinary team consisting of oncologists, cardiologists and immunologists.

Background: Immune checkpoint blockade alone or in combination with chemotherapy or radiotherapy or other immune checkpoint blocking agents have become an integral part of oncology in recent years. Monoclonal antibodies against CTLA-4 or PD-1 or PDL-1 are the most studied ICIs in randomized clinical trials, however, more recently, an anti-LAG3 (Lymphocyte activation gene-3) human monoclonal antibody, Relatlimab, has been approved by FDA for combinatorial treatment with Nivolumab for metastatic melanoma. Moreover, anti PD-L1blocking agent Atezolizumab is actually under study in association with Ipilimumab as innovative thearpy for metastatic lung cancer. Cases of cardiotoxicity such as myocarditis, vasculitis and endothelitis are rarely observed in these patients on monotherapy, however new combination therapies and the use of new targets in immunotherapy could expose patients to more adverse cardiovascular events. Methods: human cardiomyocytes co-cultured with human peripheral blood lymphocytes (hPBMCs) were exposed to monotherapy and combinatorial ICIs (PD-L1 and CTLA-4 or PD-1 and LAG-3 blocking agents, at 100 nM) for 48 h. After treatments, cardiac cell lysis and secretion of biomarkers of cardiotoxicity (H-FABP, NT-Pro-BNP), NLRP3-inflammasome and Interleukin 1 and 6 were determined through colorimetric and enzymatic assays. Results: Both combinations of immune checkpoint inhibitors exert more potent cardiotoxic side effects compared to monotherapies against human cardiac cells co-cultured with human lymphocytes. LDH release from cardiac cells was 43% higher in PD-L1/CTLA-4 blocking agents, and 35.7% higher in PD-1/LAG-3 blocking agents compared to monotherapies. Biomarkers of cardiotoxicity, such as NT-Pro-BNP and H-FABP, were also strongly increased in combination therapy with respect to monotherapies. NLRP3 inflammasome, IL-6 and IL-1β levels were also increased by PDL-1/CTLA-4 and PD-1/LAG-3 combined blocking agents compared to untreated cells and monotherapies. Conclusions: Data of the present study, although in vitro, indicate that combinatorial immune checkpoint blockade, induce a pro- inflammatory phenotype.

PD-1/CTLA-4 dual blockade has shown substantial promise against refractory tumors in some patients. Biomarkers were sought to identify patients early in treatment who are responding positively. Strong production of IFNγ shortly after treatment commenced significantly correlated with survival. Immune checkpoint inhibitors are transforming the way cancer is treated. However, these therapies do not benefit all patients and frequently cause significant immune-related adverse events. Biomarkers that identify patients with a favorable early response to therapy are essential for guiding treatment decisions and improving patient outcomes. In this report of our study, we present evidence that shortly after administration of dual PD-1/CTLA-4 blockade, the proinflammatory capacity of peripheral lymphocytes is predictive of tumor progression and survival outcomes in multiple murine models. Specifically, we observed that the quantity of interferon-γ (IFNγ) produced by peripheral lymphocytes in response to CD3/CD28 stimulation was robustly correlated with subsequent survival outcomes. In the tumor models and early time points assessed in this study, this relationship was considerably more predictive than a host of other potential biomarkers, several of which have been previously reported. Overall, these findings suggest that measuring the capacity of peripheral lymphocytes to produce IFNγ may help identify which patients are benefitting from combination anti–PD-1/anti–CTLA-4 immunotherapy. Cancer Immunol Res; 4(8); 650–7. ©2016 AACR.

Background Immune-related adverse events (irAEs), characterized by targeted inflammation, occur in up to 60% of patients with melanoma treated with immune checkpoint inhibitors (ICIs). Evidence proved that the baseline peripheral blood profiles of patients at risk for severe irAEs development paralleled clinical autoimmunity. Interleukin (IL)-23 blockade with risankizumab is recommended for cases that are suffering from autoimmune disease, such as autoimmune colitis. However, currently, the role of IL-23 in irAEs onset and severity remains poorly understood. Methods The pro-inflammatory cytokines most associated with severe irAEs onset were identified by retrospective analysis based on GSE186143 data set. To investigate the efficacy of prophylactic IL-23 blockade administration to prevent irAEs, refer to a previous study, we constructed two irAEs murine models, including dextran sulfate sodium salt (DSS)-induced colitis murine model and a combined-ICIs-induced irAEs murine model. To further explore the applicability of our findings, murine models with graft-versus-host disease were established, in which Rag2−/−Il2rg−/− mice were transferred with human peripheral blood mononuclear cells and received combined cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) and programmed cell death protein-1 (PD-1) treatment. Human melanoma cells were xenografted into these mice concomitantly. Results Here we show that IL-23 was upregulated in the serum of patients suffering from irAEs after dual anti-CTLA-4 and anti-PD-1 treatment, and increased as a function of irAEs severity. Additionally, Augmented CD4+ Tems may preferentially underlie irAEs onset. Treating mice with anti-mouse IL-23 antibody concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, preserves antitumor efficacy. Moreover, in xenografted murine models with irAEs, prophylactic blockade of human IL-23 using clinically available IL-23 inhibitor (risankizumab) ameliorated colitis, hepatitis and lung inflammation, and moreover, immunotherapeutic control of tumors was retained. Finally, we also provided a novel machine learning-based computational framework based on two blood-based features—IL-23 and CD4+ Tems—that may have predictive potential for severe irAEs and ICIs response. Conclusions Our study not only provides clinically feasible strategies to dissociate efficacy and toxicity in the use of combined ICIs for cancer immunotherapy, but also develops a blood-based biomarker that makes it possible to achieve a straightforward and non-invasive, detection assay for early prediction of irAEs onset.

Immune-checkpoint blockade is able to achieve durable responses in a subset of patients; however, we lack a satisfying comprehension of the underlying mechanisms of anti-CTLA-4- and anti-PD-1-induced tumor rejection. To address these issues, we utilized mass cytometry to comprehensively profile the effects of checkpoint blockade on tumor immune infiltrates in human melanoma and murine tumor models. These analyses reveal a spectrum of tumor-infiltrating T cell populations that are highly similar between tumor models and indicate that checkpoint blockade targets only specific subsets of tumor-infiltrating T cell populations. Anti-PD-1 predominantly induces the expansion of specific tumor-infiltrating exhausted-like CD8 T cell subsets. In contrast, anti-CTLA-4 induces the expansion of an ICOS+ Th1-like CD4 effector population in addition to engaging specific subsets of exhausted-like CD8 T cells. Thus, our findings indicate that anti-CTLA-4 and anti-PD-1 checkpoint-blockade-induced immune responses are driven by distinct cellular mechanisms.

Th1 and Th17 cell-mediated autoimmunity is critical for myocarditis induction. Antigen-presenting cell (APCs)-released interleukin (IL)-12 and IL-23 are implicated in the differentiation of Th1 and Th17 lineages. In this study, we utilized cardiac self-antigen myosin heavy chain alpha (α-MyHC)-pulsed bone marrow-derived dendritic cells (bmDCs) and wild-type, IL-12p35-/-, and IL-23p19-/- mice to investigate the influence of IL-12 and IL-23 on CD4+ T cells in experimental autoimmune myocarditis (EAM). All mice (Mus musculus) receiving α-MyHC-pulsed bmDCs developed acute myocarditis and accumulated interferon (IFN)-γ-positive and IL-17A-positive CD4+ T cells in cardiac tissue. Compared to immunization with wild-type bmDCs, adoptive transfer of α-MyHC-pulsed IL-23p19-/- bmDCs resulted in decreased numbers of IL-17A+CD4+ T cells and in a twofold reduction of infiltrating T lymphocytes in the hearts of recipient mice, despite unaffected infiltration count of CD45+ leukocytes. In contrast, IL-12p35-/- bmDCs induced fewer IFN-γ-producing CD4+ T cells but did not affect T lymphocyte infiltration. Furthermore, IL-23p19-/- recipient mice showed reduced heart-infiltrating CD3+ T cells, but not total CD45+, compared to wild-type mice after adoptive transfer of α-MyHC-pulsed IL-23p19-/- bmDCs. Likewise, in the transgenic TCRM model of EAM, TCRMxIL-23p19-/- mice showed reduced myocarditis severity and fewer T lymphocytes in their hearts pointing to impaired T cell trafficking in absence of IL-23. We validated the pro-migratory effect of IL-23 on activated CD4+ T cells in vitro and demonstrated an essential role of Rho GTPases in this process. Our findings provide new insights into the pro-inflammatory activity of IL-23 in autoimmune myocarditis, highlighting its unique role in promoting T cell migration.

Myocarditis is one of the major causes of heart failure in children and young adults and can lead to dilated cardiomyopathy. Lymphocytic myocarditis could result from autoreactive CD4+ and CD8+ T cells, but defining antigen specificity in disease pathogenesis is challenging. To address this issue, we generated T cell receptor (TCR) transgenic (Tg) C57BL/6J mice specific to cardiac myosin heavy chain (Myhc)-α 334–352 and found that Myhc-α-specific TCRs were expressed in both CD4+ and CD8+ T cells. To investigate if the phenotype is more pronounced in a myocarditis-susceptible genetic background, we backcrossed with A/J mice. At the fourth generation of backcrossing, we observed that Tg T cells from naïve mice responded to Myhc-α 334–352, as evaluated by proliferation assay and carboxyfluorescein succinimidyl ester staining. The T cell responses included significant production of mainly pro-inflammatory cytokines, namely interferon (IFN)-γ, interleukin-17, and granulocyte macrophage-colony stimulating factor. While the naïve Tg mice had isolated myocardial lesions, immunization with Myhc-α 334–352 led to mild myocarditis, suggesting that further backcrossing to increase the percentage of A/J genome close to 99.99% might show a more severe disease phenotype. Further investigations led us to note that CD4+ T cells displayed the phenotype of cytotoxic T cells (CTLs) akin to those of conventional CD8+ CTLs, as determined by the expression of CD107a, IFN-γ, granzyme B natural killer cell receptor (NKG)2A, NKG2D, cytotoxic and regulatory T cell molecules, and eomesodermin. Taken together, the transgenic system described in this report may be a helpful tool to distinguish the roles of cytotoxic cardiac antigen-specific CD4+ T cells vs. those of CD8+ T cells in the pathogenesis of myocarditis.

Myocarditis is a major cause of heart failure in children and young adults that can lead to dilated cardiomyopathy. Lymphocytic myocarditis may result from autoreactive CD4 and CD8 T cells but defining their antigen specificity in disease pathogenesis is challenging. To address this issue, we generated T cell receptor (TCR) transgenic (Tg) C57Bl/6 mice specific to cardiac myosin heavy chain (Myhc)-α 334-352 and found that Myhc-specific TCRs were expressed in both CD4 and CD8 T cells. By backcrossing onto A/J mice for four generations, we noted that Tg T cells from naïve mice respond to Myhc-α 334-352 as evaluated by proliferation assay and carboxyfluorescein succinimidyl ester staining. The T cell responses included significant production of pro-inflammatory cytokines including interferon (IFN)-γ, interleukin-17, granulocyte macrophage-colony stimulating factor, and tumor necrosis factor-α. While the naïve Tg mice had isolated myocardial lesions, immunization with Myhc-α 334-352 led to mild myocarditis, suggesting fully backcrossed mice may show more severe disease. Further studies led us to note that the CD4 T cells displayed the phenotype of cytotoxic T cells (CTLs) akin to those of conventional CD8+ CTLs as determined by the expression of CD107a, IFN-γ and granzyme B. Taken together, the transgenic system described here may be helpful to distinguish the roles of cytotoxic cardiac antigen-specific CD4 T cells vs. those of CD8 T cells in the pathogenesis of myocarditis.

No abstract available

No abstract available

No abstract available

The degree to which processed self-peptides contribute to the stimulation of autoreactive T cells has not been determined. In this study we have analyzed a panel of autoreactive T cell hybridomas from normal C57BL/26 mice produced by fusing peripheral lymph node cells with a variant of the BW5147 thymoma line, which does not express endogenous TCR alpha- and beta-chains. All of the autoreactive hybridomas responded to spleen cells expressing the syngeneic I-Ab allele, but not to allogeneic spleen cells. Although all hybridomas were I-Ab restricted, they demonstrated different patterns of reactivity to a panel of APC expressing I-Ab but derived from different genetic backgrounds. In a panel of APC expressing recombinant I-A molecules, changes in the second half of the first domain, which encodes alpha-helix segments that flank the Ag binding site and directly contact the TCR V regions in proposed models, eliminated reactivity of all hybridomas tested. In addition, most of the autoreactive hybridomas also demonstrated inhibition of reactivity to mutations in the amino half of the first domain of the I-A alpha- and beta-chains, which encodes the beta-pleated sheet of the floor of the Ag-binding groove. To confirm the role of processed peptides in the different patterns of reactivity, APC were incubated with competitor Ag and fixed by glutaraldehyde cross-linking before incubation with the autoreactive T hybridomas. The hybridomas were effectively inhibited by exogenous protein and peptide Ag. These results indicate that processed self-peptides are required to activate the autoreactive T cells.

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Momentum grant of the Hungarian Academy of Sciences Immune checkpoint inhibitors (ICI), such as monoclonal antibodies targeting programmed death ligand-1 (PD-1), revolutionized cancer treatment. However, they can lead to several cardiovascular adverse effects, ranging from mild cardiac dysfunction to fulminant, lethal myocarditis. Nevertheless, the mechanisms and risk factors behind the diverse forms of ICI-induced cardiotoxicity are not entirely understood currently. In this study, we hypothesized that a prior cardiac ischemic injury, leading to acute immune cell infiltration and activation, but without subsequent heart failure, can exacerbate the cardiotoxicity and cardiac inflammation caused by anti-PD-1 monoclonal antibodies. Furthermore, we aimed to investigate in our mouse model whether abatacept, an inhibitor of T-cell co-stimulation, can ameliorate ICI-induced cardiac effects. First, we treated 8 weeks-old C57BL/6J mice with isoprenaline (ISOP group, 160 mg/kg, n = 43) or with its solvent (CON group, n = 38), to induce reversible cardiac ischemia. Validation of the ischemic injury was performed in 6 randomly selected animals from each group two days after the treatment with histology and echocardiography. After this, the animals underwent 16 weeks of recovery period, followed by echocardiography to confirm cardiac functional recovery. Here, mice from both groups were randomized to three further treatment groups: isotype control, anti-PD-1 alone, or anti-PD-1 combined with abatacept and were treated for two weeks, with three weekly intraperitoneal injections (immune checkpoint inhibition phase). Echocardiography, qRT-PCR and histology was performed to evaluate cardiac function and inflammation. Two days after the initial ISOP treatment, mice displayed significant reduction in ejection fraction and infiltration of inflammatory cells were seen on histology. During the recovery period, 8 mice from the ISOP group and one mouse from the CON died. After the immune checkpoint inhibition phase, mice with prior ischemic injury and anti-PD-1 treatment (ISOP + anti-PD-1 alone) showed significant cardiac dysfunction on echocardiography, while animals with abatacept treatment (ISOP+anti-PD-1+abatacept) showed normal cardiac function. With qRT-PCR and histology, increased infiltration of T-cells and macrophages was seen in the myocardium of the ISOP+anti-PD-1 treated group compared to CON animals, with increased expression of pro-inflammatory cytokines, including Il17a, Il23 and Ifng. However, no cardiac infiltration was seen in mice without prior ischemic injury and the pro-inflammatory cytokine response was less pronounced as well. Prior cardiac ischemic injury without overt cardiac dysfunction exacerbates cardiac inflammation and cardiotoxicity induced by anti-PD-1 immune checkpoint inhibition therapy. Patients with pre-existing ischemic heart disease may be at greater risk for developing ICI-induced severe cardiac adverse events.Hypothesis

AIMS Cardiac immune-related adverse events (irAEs) from immune checkpoint inhibition (ICI) targeting programmed death 1 (PD1) are of growing concern. Once cardiac irAEs become clinically manifest, fatality rates are high. Cardio-oncology aims to prevent detrimental effects before manifestation of severe complications by targeting early pathological changes. We therefore aimed to investigate early consequences of PD1 inhibition for cardiac integrity to prevent the development of overt cardiac disease. METHODS AND RESULTS We investigated cardiac-specific consequences from anti-PD1 therapy in a combined biochemical and in vivo phenotyping approach. Mouse hearts showed broad expression of the ligand PDL1 on cardiac endothelial cells as a main mediator of immune-crosstalk. Using a novel melanoma mouse model, we assessed that anti-PD1 therapy promoted myocardial infiltration with CD4+ and CD8+ T cells, the latter being markedly activated. Left ventricular (LV) function was impaired during pharmacological stress, as shown by pressure-volume catheterization. This was associated with a dysregulated myocardial metabolism, including the proteome and the lipidome. Analogous to the experimental approach, in patients with metastatic melanoma (n = 7) receiving anti-PD1 therapy, LV function in response to stress was impaired under therapy. Finally, we identified that blockade of tumour necrosis factor alpha (TNFα) preserved LV function without attenuating the anti-cancer efficacy of anti-PD1 therapy. CONCLUSIONS Anti-PD1 therapy induces a disruption of cardiac immune homeostasis leading to early impairment of myocardial functional integrity, with potential prognostic effects on the growing number of treated patients. Blockade of TNFα may serve as an approach to prevent the manifestation of ICI-related cardiotoxicity.

Cancer immunotherapy is a well-established therapeutic approach for various types of cancer. However, its clinical utility is usually limited by cardiovascular adverse events. Immune Checkpoint Inhibitors (ICIs) can induce diverse forms of cardiotoxicity, with myocarditis being the most fatal complication. The underlying mechanism of its occurrence remains elusive. Therefore, this study aims to elucidate the impact of programmed death-1 (PD-1) inhibitor on myocarditis development in mice. Myeloid-epithelial-reproductive tyrosine kinase (MerTK) receptors, located on the surface of macrophages, play a pivotal role in phagocytic regulation. We established a mouse model of autoimmune myocarditis by injecting 6-week-old normal male BALB/c mice with PD-1 inhibitor and cardiac troponin I peptide fragments, which resulted in elevated levels of serum soluble MerTK (SolMer) and reduced numbers of MerTK-CD68 double-positive macrophages, accompanied by cardiac injury in mice. In vitro, PD-1 inhibitor promotes a disintegrin and metalloproteinase17 (ADAM17)-mediated shed of the MerTK, forming SolMer, through MKK3/P38 MAPK pathway, leading to downregulation of MerTK expression on the macrophage surface. This results in the inhibition of efferocytosis and impairment of tissue repair function, ultimately contributing to myocarditis development. TAPI-0 inhibited the activity of ADAM17, while SB203580 inhibited the phosphorylation of P38 MAPK. Both inhibitors effectively restored the inhibition of efferocytosis induced by the PD-1 inhibitor. In vitro, when the PD-1 receptor on the surface of RAW264.7 macrophages was knocked down and then stimulated with a PD-1 inhibitor, no further significant alterations in the pathway were elicited. In conclusion, the PD-1 inhibitor induces the shedding of MerTK in macrophages by binding to the PD-1 receptor on the surface of macrophages and activating the MKK3/P38 MAPK/ADAM17 pathway, leading to impaired efferocytosis. Elucidation of this molecular mechanism holds promise for improved prognosis and therapeutic strategies in cancer patients.

No abstract available

Cardiotoxicity caused by immune checkpoint inhibitors is one of the most severe and potentially fatal side effects. Hence it is crucial from a therapeutic standpoint to understand the underlying processes and devise countermeasures. This study sought to determine whether the SOCS3/JAK/STAT3 signaling pathway, which controls macrophage polarization, contributes to the cardiotoxicity caused by PD-1/PD-L1 inhibitors. The PD-1/PD-L1 inhibitor BMS-1 (10 mg/kg) was used to create a mouse model of immune checkpoint inhibitor-related cardiotoxicity, and hematoxylin and Masson’s trichome tests were used to measure cardiomyocyte apoptosis and cardiotoxicity. The production of M1 factors (tumor necrosis factor α [TNF-α] and interleukin [IL]-1 b), as well as the blood levels of myocardial enzymes (creatine kinase, aspartate transaminase, creatine kinase-MB, and lactate dehydrogenase), were evaluated by ELISA. Echocardiography was used to assess the heart’s health. The processes were investigated using flow cytometric analysis, real-time PCR, Western blot, and chromatin immunoprecipitation. We found that the PD-1/PD-L1 inhibitor BMS-1 dramatically reduced tumor weight while considerably impairing cardiac function in melanoma-induced tumor-bearing mice. At the gene and protein levels, it was found that levels of SOCS3, JAK, STAT3, and the inflammatory mediators IL-6 and TNF-α had all significantly decreased. Immune checkpoint inhibitor-induced cardiotoxicity may be linked to major changes in the SOCS3/JAK/STAT3 signaling pathway, as indicated by the knockdown of SOCS3, JAK, and STAT3. Finally, immune checkpoint inhibitor intervention demonstrated a large elevation of CD86+ and MHCII+ as well as a considerable increase in macrophages. These data suggest that the SOCS3/JAK/STAT3 signaling pathway, which controls macrophage polarization, may be linked to cardiotoxicity caused by PD-1/PD-L1 inhibitor therapy.

AIMS The role of cardiac programmed cell death ligand 1 (PDL1) in immune checkpoint inhibitor (ICI) - related myocarditis (irMyocarditis) remains unclear. We aimed to investigate whether ligand PDL1 could serve as an early indicator and a potential therapeutic target for irMyocarditis. METHODS Cardiac PDL1 expression was assessed using single-nucleus RNA sequencing and multiplex immunohistochemistry in human patients and mouse models of irMyocarditis. A PDL1-targeted magnetic resonance imaging (MRI) nanoprobe was developed for noninvasive imaging of irMyocarditis. Additionally, an adeno-associated virus 9 (AAV9) vector was employed to deliver the PDL1 gene to cardiomyocytes, and its therapeutic effects on irMyocarditis were evaluated in a mouse model. RESULTS PDL1 expression was significantly elevated in the myocardium of irMyocarditis patients and mouse models. The PDL1-targeted MRI nanoprobe successfully detected myocarditis in vivo, with enhanced cardiac signals observed in affected mice compared to isotype controls. Therapeutic intervention using AAV9-mediated PDL1 gene delivery significantly reduced immune cell infiltration and cardiomyocyte apoptosis, improving left ventricular ejection fraction over a 2-month follow-up period. CONCLUSIONS This study identifies PDL1 as a critical biomarker and therapeutic target for irMyocarditis. PDL1-targeted MRI nanoprobe enables early, noninvasive diagnosis, while AAV9-mediated PDL1 gene therapy offers a promising strategy to mitigate irMyocarditis and restore cardiac function in ICI therapy recipients.

Despite accumulating data on underlying mechanisms, the influence of sex and prevalent cardio-metabolic co-morbidities on the manifestation and severity of immune checkpoint inhibitor (ICI)-induced cardiotoxicity has not been well defined. To elucidate whether sex and prevalent cardio-metabolic co-morbidities affect ICI-induced cardiotoxicity, we randomized 17-month-old male and female mice to receive control diet (CON) or high-fat diet (HFD) + L-NAME—a well-established mouse model of cardio-metabolic co-morbidities—for 17 weeks (n = 5–7), and evaluated markers of T-cell function in the spleen. As expected, HFD + L-NAME significantly increased body- and heart weight, and serum cholesterol levels, and caused no systolic dysfunction, however, led to diastolic dysfunction, cardiomyocyte hypertrophy, and increased fibrosis only in males compared to corresponding CON. Western blot analyses of splenic immune checkpoint protein levels showed differential expression depending on sex and prevalent cardio-metabolic co-morbidities, suggesting T-cell exhaustion in both sexes on HFD + L-NAME, but more pronounced in males. In a sub-study with a similar setup, we tested cardiotoxic manifestations of ICI by treating mice with anti-PD-1 monoclonal antibody (ICI) for the last 2 weeks of diet administration (n = 5–7). After 2 weeks of ICI treatment, cardiac systolic functions significantly decreased in CON, but not in HFD + L-NAME groups of both sexes compared to baseline (before ICI administration). In conclusion, in this exploratory study using aged mice, we describe for the first time that ICI-related systolic dysfunction is diminished in both sexes when obesity and hypercholesterolemia are present, possibly due to obesity-related T-cell exhaustion.

Immune checkpoint inhibitor-related cardiotoxicity is one of the most lethal adverse effects, and thus, the identification of underlying mechanisms for developing strategies to overcome it has clinical importance. This study aimed to investigate whether microbiota-host interactions contribute to PD-1/PD-L1 inhibitor-related cardiotoxicity. A mouse model of immune checkpoint inhibitor-related cardiotoxicity was constructed by PD-1/PD-L1 inhibitor BMS-1 (5 and 10 mg/kg), and cardiomyocyte apoptosis and cardiotoxicity were determined by hematoxylin and eosin, Masson’s trichome and TUNEL assays. 16S rRNA sequencing was used to define the gut microbiota composition. Gut microbiota metabolites short-chain fatty acids (SCFAs) were determined by HPLC. The serum levels of myocardial enzymes (creatine kinase, aspartate transaminase, creatine kinase-MB and lactate dehydrogenase) and the production of M1 factors (TNF-α and IL-1β) were measured by ELISA. The colonic macrophage phenotype was measured by mmunofluorescence and qPCR. The expression of Claudin-1, Occludin, ZO-1 and p-p65 was measured by western blot. The gene expression of peroxisome proliferator-activated receptor α (PPARα) and cytochrome P450 (CYP) 4X1 was determined using qPCR. Statistical analyses were performed using Student’s t-test for two-group comparisons, and one-way ANOVA followed by Student–Newman–Keul test for multiple-group comparisons. We observed intestinal barrier injury and gut microbiota dysbiosis characterized by Prevotellaceae and Rikenellaceae genus depletion and Escherichia-Shigella and Ruminococcaceae genus enrichment, accompanied by low butyrate production and M1-like polarization of colonic macrophages in BMS-1 (5 and 10 mg/kg)-induced cardiotoxicity. Fecal microbiota transplantation mirrored the effect of BMS-1 on cardiomyocyte apoptosis and cardiotoxicity, while macrophage depletion and neutralization of TNF-α and IL-1β greatly attenuated BMS-1-induced cardiotoxicity. Importantly, Prevotella loescheii recolonization and butyrate supplementation alleviated PD-1/PD-L1 inhibitor-related cardiotoxicity. Mechanistically, gut microbiota dysbiosis promoted M1-like polarization of colonic macrophages and the production of proinflammatory factors TNF-α and IL-1β through downregulation of PPARα-CYP4X1 axis. Intestinal barrier dysfunction amplifies PD-1/PD-L1 inhibitor-related cardiotoxicity by upregulating proinflammatory factors TNF-α and IL-1β in colonic macrophages via downregulation of butyrate-PPARα-CYP4X1 axis. Thus, targeting gut microbiota to polarize colonic macrophages away from the M1-like phenotype could provide a potential therapeutic strategy for PD-1/PD-L1 inhibitor-related cardiotoxicity.

Background Serious but rare side effects associated with immunotherapy pose a difficult problem for regulators and practitioners. Immune checkpoint inhibitors (ICIs) have come into widespread use in oncology in recent years and are associated with rare cardiotoxicity, including potentially fatal myocarditis. To date, no comprehensive model of myocarditis progression and outcomes integrating time-series based laboratory and clinical signals has been constructed. In this paper, we describe a time-series neural net (NN) model of ICI-related myocarditis derived using supervised machine learning. Methods We extracted and modeled data from electronic medical records of ICI-treated patients who had an elevation in their troponin. All data collection was performed using an electronic case report form, with approximately 300 variables collected on as many occasions as available, yielding 6000 data elements per patient over their clinical course. Key variables were scored 0–5 and sequential assessments were used to construct the model. The NN model was developed in MatLab and applied to analyze the time course and outcomes of treatments. Results We identified 23 patients who had troponin elevations related to their ICI therapy, 15 of whom had ICI-related myocarditis, while the remaining 8 patients on ICIs had other causes for troponin elevation, such as myocardial infarction. Our model showed that troponin was the most predictive biomarker of myocarditis, in line with prior studies. Our model also identified early and aggressive use of steroid treatment as a major determinant of survival for cases of grade 3 or 4 ICI-related myocarditis. Conclusion Our study shows that a supervised learning NN can be used to model rare events such as ICI-related myocarditis and thus provide clinical insight into drivers of progression and treatment outcomes. These findings direct attention to early detection biomarkers and clinical symptoms as the best means of implementing early and potentially life-saving steroid treatment.

Immune checkpoint inhibitors (ICIs) have revolutionized treatment for advanced lung cancer, yet their cardiotoxicity, particularly immune checkpoint inhibitor-related myocarditis, poses significant clinical challenges. This study aims to create a predictive model using cardiac biomarkers to identify patients prone to myocarditis during treatment, thereby enhancing clinical decision-making and patient outcomes. In this retrospective cohort study, 1,838 patients with locally advanced and metastatic lung cancer and abnormal baseline cardiac parameters receiving immunotherapy from June 2018 to August 2024 were analyzed, with a follow-up date cutoff of September 20, 2024. Patients were randomly divided into training (70%) and validation (30%) cohorts. Logistic regression analysis was conducted on demographic information, clinical characteristics, treatments, and cardiac parameters of these patients prior to immunotherapy. A nomogram was constructed via multivariable logistic regression, and AUC and Hosmer-Lemeshow tests were performed to verify the accuracy of the model. Among 1,838 patients, 89 (4.84%) developed myocarditis. Independent predictors included α-HBDH > 910 U/L (OR = 10.57, 95%CI: 2.47–45.22, P = 0.001), CK-MB > 15 ng/mL (OR = 3.87, 95%CI: 1.06–14.11, P = 0.040), hs-cTnT elevation (14–28 pg/mL: OR = 4.19; 28–42 pg/mL: OR = 13.10; >42 pg/mL: OR = 25.43, P < 0.001), NT-proBNP > 3× age-adjusted upper limit (OR = 9.72, 95%CI: 1.09–86.73, P = 0.042), and Caprini score ≥ 4 (OR = 4.49, 95%CI: 2.26–8.90, P < 0.001). The nomogram demonstrated strong discrimination ability, with an AUC of 0.831 in the training cohort (sensitivity: 0.842, specificity: 0.717) and an AUC of 0.844 in the validation cohort. This study establishes a validated risk assessment model integrating cardiac biomarkers (α-HBDH, CK-MB, hs-cTnT, NT-proBNP) and Caprini risk score to predict ICI-related myocarditis in lung cancer patients with cardiac abnormalities. The tool facilitates early identification of high-risk patients, enabling tailored monitoring and preemptive management. These findings underscore the critical role of baseline cardiac profiling in optimizing immunotherapy safety.

Immune checkpoint inhibitor (ICI) therapy represents a ground-breaking paradigm in cancer treatment, harnessing the immune system to combat malignancies by targeting checkpoints such as cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1). The use of ICI therapy generates distinctive immune-related adverse events (irAEs) including cardiovascular toxicity, necessitating targeted research efforts. This comprehensive review explores preclinical models dedicated to ICI-mediated cardiovascular complications including myocarditis. Tailored preclinical models of ICI-mediated myocardial toxicities highlight the key role of CD8+ T cells, emphasizing the profound impact of immune checkpoints on maintaining cardiac integrity. Cytokines and macrophages were identified as possible driving factors in disease progression, and at the same time, initial data on possible cardiac antigens responsible are emerging. The implications of contributing factors including thoracic radiation, autoimmune disorder, and the presence of cancer itself are increasingly understood. Besides myocarditis, mouse models unveiled an accelerated progression of atherosclerosis, adding another layer for a thorough understanding of the diverse processes involving cardiovascular immune checkpoint signalling. This review aims to discuss current preclinical models of ICI cardiotoxicity and their potential for improving enhanced risk assessment and diagnostics, offering potential targets for innovative cardioprotective strategies. Lessons from ICI therapy can drive novel approaches in cardiovascular research, extending insights to areas such as myocardial infarction and heart failure.

Immune checkpoint inhibitors (ICIs)have significantly improved overall survival rates in many aggressive cancers. Despite the recent clinical success, a rapidly increasing number of patients suffer from ICI-induced cardiotoxicity with often fatal outcomes, nonspecific symptoms and uninvestigated underlying pathological mechanisms. Therefore, this study explored metabolic, muscle wasting, and autophagic pathways and their roles in ICI-induced cardiac remodeling and dysfunction. Female C57BL/6 wildtype and LC3 transgenic (autophagy reporter) mice were randomly assigned to control (CON) and ICI-treated (ICI) groups. Mice underwent 4 weeks of ICI treatment (200 µg/mouse, intraperitoneally, twice/week). Echocardiography assessed ICI-induced changes in cardiac structure and function. At euthanasia, cardiac tissue was collected for Western Blot analysis of metabolic and muscle wasting signaling pathways and confocal fluorescent microscopy of autophagic flux. ICI treatment reduced tumor burden (-48% mass, P < 0.05) and led to significantly decreased cardiac function (-20%) and remodeling, including left ventricular dilation (+ 50%) and thinning of posterior cardiac walls (P < 0.05), indicative of dilated cardiomyopathy. Exploratory protein level analysis revealed dysfunctional muscle wasting (Atrogin1, MuRF1) disrupted AKT and FoxO1 signaling, and altered autophagic flux (P < 0.05). In this model, ICI-induced cardiotoxicity was characterized by severe cardiac remodeling and dysfunction, associated with dysfunctional metabolism, muscle wasting, and autophagy. To our knowledge, this is one of the first studies to explore underlying pathological mechanisms, adding novel and impactful insight to the still unclear characteristics of ICI-induced cardiotoxicity and supporting the critical need to further investigate side effects of immunotherapies to optimize clinical treatment of cancers.

Inflammatory biomarkers, including CRP, the neutrophil‐to‐lymphocyte ratio (NLR), and the neutrophil‐to‐eosinophil ratio (NER), may predict outcomes in cancer. However, their value in immune checkpoint inhibitor (ICI) therapy‐associated cardiotoxicity remains elusive. We aimed to characterize the relationship of inflammatory markers with severity of ICI‐related cardiotoxicities (iRCs) and prognosis among patients with iRCs.

Immune checkpoint inhibitors (ICI), such as anti‐PD‐1 monoclonal antibodies, have revolutionized cancer therapy by enhancing the cytotoxic effects of T‐cells against tumours. However, enhanced T‐cell activity also may cause myocarditis and cardiotoxicity. Our understanding of the mechanisms of ICI‐induced cardiotoxicity is limited. Here, we aimed to investigate the effect of PD‐1 inhibition on cardiac function and explore the molecular mechanisms of ICI‐induced cardiotoxicity.

Immuno-checkpoint inhibitors (ICI) are frequently used as a major cancer therapy, frequently combined with radiotherapy, particularly among patients with lung cancer. There is an increasing awareness of potential cardiotoxicities from either therapy, thus raising concerns when both therapies are combined. It is likely that radiation-induced cardiac inflammation becomes unchecked when ICIs are administered, contributing to myocarditis although the underlying specific mechanisms have not been characterized. We developed a mouse model of ICI and cardiac irradiation-induced myocarditis through an image-guided small animal irradiator in the A/J mice, which is prone to ICI-induced myocarditis. Our results showed that 20 % of ICI treated mice developed mild myocarditis, whereas 40 to 50% of mice with ICI plus irradiation treatment showed severe myocarditis, characterized by lots of infiltration of CD4+ T cells, CD8+ T cells, B cells and macrophages in hearts. Flow cytometry analysis showed that the number of infiltrated CD4+ T cells is 2 to 3 folds of CD8+ T cells in the heart tissue with disease. Severe fibrosis in hearts was associated with severe myocarditis. The levels of cardiomyocyte-specific protein troponin I in serum was correlated well with the severity of myocarditis, implicating that ICI and irradiation caused many cardiomyocyte damages. ELISA assay showed that serum IL-17 was dramatically increased in mice treated with ICI alone or ICI plus radiation, implying Th17 cells may play a pathogenic role in ICI & irradiation-induced myocarditis. In addition, we compared the myocarditis induced by ICI and irradiation in less susceptible prone of C57 mice with the more susceptible strain of A/J mice. The incidence of myocarditis was significantly lower in C57 mice compared with A/J Mice. Overall, our study indicates that irradiation promotes the pathogenesis of ICI-induced myocarditis, and individuals with autoimmune tendency have a high risk to develop severe myocarditis after administration of combined ICIs and irradiation. (This work is supported by NIH grant R01) Citation Format: Shiguang Yu, Nhan T Vu, Tyler Verburg, Bo Lu. Autoimmunity mechanisms in combined radiotherapy and immune-checkpoint inhibitor therapy-induced myocarditis. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr B013.

Immune checkpoint inhibitors (ICI) targeting CTLA-4 or PD-1/PD-L1 have transformed cancer therapy but are associated with immune-related adverse events (irAEs), including myocarditis. Here, we report a robust preclinical mouse model of ICI-associated myocarditis in which mono-allelic loss of Ctla4 in the context of complete genetic absence of Pdcd1 leads to premature death in approximately half of mice. Premature death results from myocardial infiltration by T cells and macrophages and severe electrocardiographic abnormalities, closely recapitulating the clinical and pathological hallmarks of ICI-associated myocarditis observed in patients. Using this model, we show that Ctla4 and Pdcd1 functionally interact in a gene dosage-dependent manner, providing a mechanism by which myocarditis arises with increased frequency in the setting of combination ICI therapy. We demonstrate that intervention with CTLA-4-Ig (abatacept) is sufficient to ameliorate disease progression and additionally provide a case series of patients in which abatacept mitigates the fulminant course of ICI-myocarditis.

Advances in oncology have led to the discovery of immune checkpoint inhibitors (ICIs) that exhibit remarkable results in cancer patients’ survival and tumour regression. Despite their antitumor potential, some life-threatening immune-related adverse effects (AEs) including cardiotoxicity have been reported, while the pathomechanism of the cardiac AEs is obscure. We herein investigated the cardiotoxic effects of ipilimumab (IPI, anti-CTLA-4), pembrolizumab (PEM, anti-PD-1) and avelumab (AVE, anti-PD-L1) in primary murine ventricular cardiomyocytes (mAVCs) and spleenocytes in vitro and established the mechanism and progression of Pem’s cardiotoxicity in an in vivo murine model. Primary mAVCs and spleenocytes were isolated and incubated with IPI, PEM and AVE (0-100 μg/ml) for 24h. Cell viability was assessed by MTT assay. Subsequently conditioned media from spleenocytes treated with the aforementioned ICIs was transferred onto the mAVCs for 24h. The experiments were repeated for molecular analyses. For the confirmation of PEM binding on the murine PD-1, human and murine PD-1 extracellular domains (ED) were biotechnologically produced and PEM binding was confirmed by circular dichroism (CD). C57BL6/J male mice were randomized into i. IgG4 and ii. Pem groups and treated for 2 (n = 5/group) and 5 weeks (n = 9/group). IgG4 and Pem were administered intraperitoneally once weekly at 2mg/ml. Pem dose was directly translated from humans. Mice underwent weekly echocardiography analysis, while at the endpoint, mice were sacrificed for myocardial sampling and histology analyses. IPI, PEM and AVE did not induce direct cytotoxicity on primary mAVCs, whereas IPI was excluded from the study due to unexpected spleenocyte cytotoxicity. Incubation of mAVCs with PEM and AVE conditioned media, revealed that only PEM could induce Immune cell (IC)-dependent cytotoxicity at 50 and 100 μg/ml and establishment of inflammation and autophagy in vitro. CD experiments showed that PEM binds on the murine PD-1 ED, which supports for the first time the use of the murine model for the extraction of translational results. In vivo Pem led to %Fractional shortening (FS%) decline, which was significantly reduced versus baseline at 2 weeks and further depressed at 5 weeks. At two weeks, Pem increased ICAM-1 expression as well as iNOS and beclin-1 expression in the myocardium, which was accompanied by intra-cardiac IC infiltration. At five weeks, Pem further increased vascular permeability, as shown by e-selectin and ICAM-1 upregulation and led to increase of cardiac autophagy and inflammation markers. Herein we established for the first time a murine in vivo model of PEM cardiotoxicity. PEM induces an IC-mediated mAVCs cytotoxicity, via induction of inflammation, autophagy and ER-stress. Increased vascular permeability and dysregulated endothelial homeostasis seem to orchestrate early-on Pem’s cardiotoxicity manifestation in vivo.

Background Immune checkpoint inhibitor-associated myocarditis (ICI myocarditis) is a rare but highly fatal immune-related adverse reaction. This study aimed to develop nomogram prognostic models for both short-term and long-term survival outcomes in patients with ICI myocarditis based on key biomarkers in peripheral blood. Methods In this single-center retrospective study, we included 90 patients with ICI myocarditis at the Fourth Hospital of Hebei Medical University. Critical peripheral biomarkers associated with 40-day and 1-year overall survival (OS) were identified. Two prognostic models were developed and evaluation of the models were performed with receiver operating characteristic (ROC) curves, C-index, calibration curves, and decision curve analysis (DCA). Results A total of 24 patients (26.7%) succumbed within 40 days, while 40 patients (44.4%) died within one year. Cardiac troponin-I (cTnI), N-terminal pro-brain natriuretic peptide (NTBNP) and lactic dehydrogenase-to-albumin ratio (LAR) were identified as critical prognostic factors for 40-day OS in patients with ICI myocarditis and utilized to develop a nomogram model. The model demonstrates an area under the curve (AUC) of 0.867 [95% confidence interval (CI): 0.774−0.960] and a C-index of 0.824. Another predictive model for the 1-year OS was developed based on cTnI, NTBNP, LAR and systemic inflammatory response index (SIRI) with an AUC of 0.765 (95% CI: 0.664−0.866) and a C index of 0.742. The calibration curve demonstrates that both models exhibit strong consistency. The results of the DCA further indicate that both nomograms possess substantial clinical utility. Conclusions These two prediction models will enable clinicians to more effectively utilize readily available peripheral blood biomarkers for the convenient and efficient identification of high-risk patients with poor prognoses, thereby facilitating early intervention.

BACKGROUND Esophageal cancer is a serious global health concern with poor prognosis in advanced stages. Immune checkpoint inhibitors (ICIs) have shown promise in enhancing survival, but they are associated with immune-related adverse events, including potentially life-threatening myocarditis. Moreover, ICI-induced myocarditis can be asymptomatic, necessitating early diagnosis. Specific risk factors and biomarkers for esophageal cancer remain poorly characterized. AIM To investigate the determinants of ICI-associated asymptomatic myocarditis in patients with esophageal cancer and explore potential early biomarkers. METHODS A retrospective analysis was conducted on 202 cancer patients who received treatment at Shanxi Province Cancer Hospital from July 2019 to July 2024. RESULTS Older age, male gender, and elevated creatine kinase isoenzymes (CK-MB) and CK levels were found to be significant risk factors for asymptomatic myocarditis. The myocarditis occurrence group had higher CK-MB (3.05 ng/mL vs 5.02 ng/mL; P < 0.001) and CK levels (187.29 U/L vs 212.25 U/L; P = 0.005), and the predictive value of age, gender, CK, and CK-MB was low [are under the receiver operating characteristic curve (AUC) = 0.579-0.608]. However, their combination in a predictive model showed improved diagnostic capability, with an AUC of 0.808. CONCLUSION Age, gender, and cardiac biomarker levels considerably contribute to the risk of ICI-related myocarditis in patients with esophageal cancer. The integration of these factors into a predictive model enhances early diagnosis, facilitating personalized risk management.

The introduction of programmed cell death protein 1 (PD-1) inhibitors has revolutionized cancer immunotherapy, offering new treatment options for patients with refractory malignancies. However, immune-related myocarditis (IRM), occurring in approximately 1.14% of patients receiving immune checkpoint inhibitors (ICIs), presents a significant challenge, with mortality rates of 25-50%, necessitating early detection and effective management. Glucocorticoids, the standard treatment for IRM, complicate management by suppressing T-cell activity and diminishing PD-1 efficacy. Diagnosing IRM remains difficult, as endomyocardial biopsy, the gold standard, is often impractical, while cardiac MRI, commonly used as an alternative, has limitations (sensitivity 0.66, specificity 0.73). To improve IRM management, integrating routine cardiac MRI with biomarker assessments, supported by AI-driven diagnostic tools, can enhance early detection and reduce diagnostic uncertainty. Flexible clinical trial protocols should allow PD-1 therapy resumption post-IRM, independent of glucocorticoid duration. Additionally, creating separate trial arms for patients recovering from long-term glucocorticoid use can help isolate its impact on treatment response, while advanced statistical models can account for glucocorticoid duration, ensuring robust efficacy assessments. Finally, educating trial stakeholders on these strategies is essential for optimizing patient safety and generating reliable clinical outcomes in PD-1 inhibitor trials. Implementing these approaches will enhance the management of IRM while preserving the therapeutic benefits of PD-1 inhibitors.

Immune checkpoint inhibitors (ICIs) are monoclonal antibodies that block immune checkpoints and therefore activate immune cells, allowing them to recognize and attack cancer cells. ICIs have revolutionized oncology practice, but their use has been complicated by immune-related adverse events (irAEs). Of cardiovascular (CV) irAEs, ICI-related myocarditis has received significant attention due to high mortality rates, ranging from 25% to 50%, despite its overall low incidence. Establishing the early diagnosis of ICI-myocarditis is important for early initiation of steroids and consideration of hospitalization in patients who are at risk for hemodynamic compromise and need high acuity care in a tertiary setting. In this review, we summarize the diagnostic and prognostic tools for ICI-myocarditis, including electrocardiography, echocardiography, cardiac magnetic resonance imaging, with emphasis on circulating biomarkers. Cardiac troponins (cTns) are an essential component of the diagnosis of ICI-myocarditis, and we provide a summary of the recent studies that utilized different assays (cTnI vs. cTnT) and outcomes (diagnosis vs. prognosis including major adverse cardiac outcomes). With the exponential increase in ICI use across different oncology indications, there is a major need to include biomarkers in risk stratification to guide diagnosis and treatment. Our review proposes a framework for future multisite registries, including cTn evaluation at baseline and at the time of irAE suspicion, with development of central biobanking to allow head-to-head evaluation and clinical validation of different biomarker assays in ICI-myocarditis. This approach, with the inclusion of CV biomarkers into clinical and pragmatic oncology trials, holds promise to improve the early recognition and management of ICI-myocarditis and CV irAEs, thus leading to better outcomes.

e14698 Background: Immune checkpoint inhibitor (ICI)-related myocarditis is a rare but severe side effect that requires attention to distinguish it from other cardiovascular emergencies. Therefore, new biomarkers are needed for the early diagnosis of ICI-related myocarditis, especially to distinguish it from other cardiovascular emergencies. Our study aims to explore plasma biomarkers with high specificity and sensitivity using proteomic analysis for the early diagnosis of ICI-related myocarditis and to distinguish it from acute myocardial infarction (AMI). Methods: Patients with ICI-related myocarditis and AMI were selected and matched for potential confounding effects such as age, gender, smoking history, and chronic heart diseases. A label-free liquid chromatography-mass spectrometry (LC-MS) proteomics approach was used to analyze plasma samples and screen sensitive and specific myocarditis protein biomarkers. Further validation will be performed at the level of patients and animal models of ICI-related myocarditis and AMI. Results: We performed plasma proteomics on 15 plasma samples from 5 pairs of samples with PD-1 inhibitor-associated myocarditis at baseline (control group) and diagnosis (myocarditis group) and 5 cases of acute myocardial infarction (MI group) confirmed by coronary angiography in the Second Affiliated Hospital of Nanchang University. In total, we identified a total of 1521 plasma proteins and 1325 quantifiable plasma proteins across all 15 plasma samples. Further analysis of the differential proteins showed that 34 proteins with significantly differentially expressed in myocarditis group samples compared with other groups. Among them, several proteins were significantly up-regulated (MYOM3, TNNC1, MYL1, MYOM2, CA3, MYLPF, ECI1, LGALS1, PSMA7, PSMB3, PSMA4, PSMB6, CKM, ABCB6, UGP2, ALDOA, TPM2) or down-regulated (MAN1A1, SPTB, YTHDC2, CLEC11A, GSN, APOA4) in myocarditis samples. Compared with the other groups, the ICI-related myocarditis group showed molecular changes in myocardial contraction, proteasome, immunoregulation, NF-κB signaling pathway, arginine and proline metabolism, and cysteine and methionine metabolism. We then performed ELISA on additional ICI-related myocarditis and AMI patients and corresponding animal models. The Troponin C, Myom 3, UGP2, and Galectin-1 proteins related to immune or inflammation were significantly increased. Conclusions: Our study has revealed that proteins related to the immune system or inflammation may be sensitive and specific plasma biomarkers for identifying ICI-related myocarditis. However, further large sample cohorts are needed to confirm these findings.

Immune checkpoint inhibitors-related myocarditis (ICI-M) is one of the immune-related adverse events (irAEs), which is rare and highly lethal. This study aimed to establish nomograms based on ratio biomarkers to predict the severity and prognosis of ICI-M. We retrospectively examined patients with advanced cancers who were also diagnosed with ICI-M at the Fourth Hospital of Hebei Medical University. The patients of ICI-M were divided into mild and severe groups and a 40-day following up was carried out. The major adverse cardiovascular events(MACEs) were regarded as the endpoint. Nomogram-based models were established and validated. Seventy-seven patients were involved, including 31 severe cases(40.3%). Lactate dehydrogenase-to-albumin ratio(LAR) combined with the change rate from baseline to onset of LAR(▵\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\triangle$$\end{document}LAR) which performed best to diagnose the severe ICI-M was identified to establish the nomogram-based model. The bootstrap-corrected concordance index [0.752 95% confidence interval (CI): 0.635-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$-$$\end{document}0.866] and calibration plot with good degree of fitting confirmed this diagnostic model. Neutrophil-to-high-density lipoprotein cholesterol ratio(NHR) and LAR were also screened into the nomogram-based model for 40-day MACEs after ICI-M, which performed well by validating for concordance index(0.779 95% CI: 0.677-\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$-$$\end{document}0.865)and calibration plots after being bootstrap-corrected. Moreover, a ≥\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\ge$$\end{document}101% increase in LAR significantly separated patients in MACE-free survival. Ratio indexes at onset and their change rates from baseline showed good diagnostic value for the severity of ICI-M and prognostic value for subsequent MACEs, particularly LAR, NHR and their change rates. The nomogram-based models of ratio indexes could provide a potential choice for early detection and monitor of the severe ICI-M and subsequent MACEs.

Background Immune checkpoint inhibitor (ICI)-related myocarditis is a rare but potentially fatal adverse event that can occur following ICI exposure. Early diagnosis and treatment are key to improve patient outcomes. Somatostatin receptor-based positron emission tomography–CT (PET/CT) showed promising results for the assessment of myocardial inflammation, yet information regarding its value for the diagnosis of ICI-related myocarditis, especially at the early stage, is limited. Thus, we investigated the value of 68Ga-DOTA(0)-Phe(1)-Tyr(3)-octreotide (68Ga-DOTATOC) PET/CT for the early detection and diagnosis of ICI-related myocarditis. Methods Consecutive patients with clinically suspected ICI-related myocarditis from July 2018 to February 2021 were retrospectively evaluated in this single-center study. All patients underwent imaging for the detection of ICI-related myocarditis using either cardiac magnetic resonance (CMR) imaging or 68Ga-DOTATOC PET/CT. PET/CT images were acquired 90 min after the injection of 2 MBq/kg 68Ga-DOTATOC with pathological myocardial uptake in the left ventricle (LV) suggestive of myocarditis defined using a myocardium-to-background ratio of peak standard uptake value to mean intracavitary LV standard uptake (MBRpeak) value above 1.6. Patients had a full cardiological work-up including ECG, echocardiography, serum cardiac troponin I (cTnI), cardiac troponin T and creatine kinase (CK), CK-MB. Endomyocardial biopsy and inflammatory cytokine markers were also analyzed. The detection rate of ICI-related myocarditis using 68Ga-DOTATOC PET/CT and CMR was assessed. Results A total of 11 patients had clinically suspected ICI-related myocarditis; 9 underwent 68Ga -DOTATOC PET/CT. All nine (100%) patients with 68Ga-DOTATOC PET/CT presented with pathological myocardial uptake in the LV that was suggestive of myocarditis (MBRpeak of 3.2±0.8, range 2.2–4.4). Eight patients had CMR imaging and 3/8 (38%) patients had lesions evocative of myocarditis. All PET-positive patients were previously treated with a high dose of steroids and intravenous immunoglobulin prior to PET/CT had elevated serum cTnI except for one patient for whom PET/CT was delayed several days. Interestingly, in 5/6 (83%) patients who presented with concomitant myositis, pathological uptake was seen on whole-body 68Ga-DOTATOC PET/CT images in the skeletal muscles, suggesting an additional advantage of this method to assess the full extent of the disease. In contrast, four patients with CMR imaging had negative findings despite having elevated serum cTnI levels (range 20.5–5896.1 ng/mL), thus defining possible myocarditis. Newly identified immune correlates could provide specific biomarkers for the diagnosis of ICI-related myocarditis. Most tested patients (six of seven patients) had serum increases in the inflammatory cytokine interleukin (IL)-6 and in the chemokines CXCL9, CXCL10, and CXCL13, and the mass cytometry phenotypes of immune cell populations in the blood also showed correlations with myocardial inflammation. Four of five patients with myocarditis exhibited a Th1/Th2 imbalance favoring a pronounced inflammatory Th1, Th1/Th17, and Th17 CD4 memory T-cell response. The high proportion of non-classical monocytes and significantly reduced levels of CD31 in four to five patients was also consistent with an inflammatory disease. Conclusion The use of 68Ga-DOTATOC PET/CT along with immune correlates is a highly sensitive method to detect ICI-related myocarditis especially in the early stage of myocardial inflammation, as patients with elevated cTnI may present normal CMR imaging results. 68Ga-DOTATOC PET/CT is also useful for detecting concomitant myositis. These results need to be confirmed in a larger population of patients and validated against a histological gold standard if available.

No abstract available

Simple Summary Immune checkpoint inhibitors are revolutionizing cancer treatment, but lead to the occurrence of immune related adverse events including ICI-associated myocarditis (ICIM). To date, transcriptional alterations of this rare phenomenon with a high mortality rate are not characterized. 19 ICIM patients at the University Hospitals Heidelberg and Kiel showed diverse clinical presentations. Comparative transcriptomics was able to distinguish ICIM patients from patients with dilated cardiomyopathy or virus-induced myocarditis in the upregulation of 3784 genes. The RNA-based analyses and immunohistology revealed a potential role of an inflammasome-regulating protein, GBP5, as a potential pathomechanism in cardiomyocytes. These alterations may help to diagnose ICIM and potentially enable to identify patients at risk in an early stage. Abstract Immune checkpoint inhibitors (ICIs) are revolutionizing cancer treatment. Nevertheless, their increasing use leads to an increase of immune-related adverse events (irAEs). Among them, ICI-associated myocarditis (ICIM) is a rare irAE with a high mortality rate. We aimed to characterize the transcriptional changes of ICIM myocardial biopsies and their possible implications. Patients suspected for ICIM were assessed in the cardio-oncology units of University Hospitals Heidelberg and Kiel. Via RNA sequencing of myocardial biopsies, we compared transcriptional changes of ICIM (n = 9) with samples from dilated cardiomyopathy (DCM, n = 11), virus-induced myocarditis (VIM, n = 5), and with samples of patients receiving ICIs without any evidence of myocarditis (n = 4). Patients with ICIM (n = 19) showed an inconsistent clinical presentation, e.g., asymptomatic elevation of cardiac biomarkers (hs-cTnT, NT-proBNP, CK), a drop in left ventricular ejection fraction, or late gadolinium enhancement in cMRI. We found 3784 upregulated genes in ICIM (FDR < 0.05). In the overrepresented pathway ‘response to interferon-gamma’, we found guanylate binding protein 5 and 6 (compared with VIM: GBP5 (log2 fc 3.21), GBP6 (log2 fc 5.37)) to be significantly increased in ICIM on RNA- and protein-level. We conclude that interferon-gamma and inflammasome-regulating proteins, such as GBP5, may be of unrecognized significance in the pathophysiology of ICIM.

Immune checkpoint inhibitors (ICIs)-associated myocarditis was a rare yet severe complication observed in individuals undergoing immunotherapy. This study investigated the immune status and characteristics of patients diagnosed with ICIs- associated myocarditis. A total of seven patients diagnosed with ICIs-associated myocarditis were included in the study, while five tumor patients without myocarditis were recruited as reference controls. Additionally, 30 healthy individuals were recruited as blank controls. Biochemical indices, electrocardiogram, and echocardiography measurements were obtained both prior to and following the occurrence of myocarditis. High-throughput sequencing of T cell receptor (TCR) was employed to assess the diversity and distribution characteristics of TCR CDR3 length, as well as the diversity of variable (V) and joining (J) genes of T lymphocytes in peripheral blood. In the seven patients with ICIs-associated myocarditis, Troponin T (TNT) levels exhibited a significant increase following myocarditis, while other parameters such as brain natriuretic peptide (BNP), QTc interval, and left ventricular ejection fraction (LVEF) did not show any significant differences. Through sequencing, it was observed that the diversity and uniformity of CDR3 in the ICIs-associated myocarditis patients were significantly diminished. Additionally, the distribution of CDR3 nucleotides deviated from normality, and variations in the utilization of V and J gene segments. The reconstitution of the TCR immune repertoire may play a pivotal role in the recognition of antigens in patients with ICIs-associated myocarditis.

An 80‐year‐old man, who developed multiple lymph node and skin metastasis of malignant melanoma, received nivolumab monotherapy. Two weeks after the first dose, he experienced anorexia and fatigue, and suffered from progressive, severe dyspnea and muscle weakness. We diagnosed him with myocarditis, myositis, and myasthenic crisis induced by nivolumab. We commenced steroid therapy, immune absorption therapy, plasma exchange therapy, and i.v. immunoglobulin therapy, and succeeded in saving his life. Because his serum level of anti‐acetylcholine receptor antibodies in a sample collected before nivolumab treatment were positive and were elevated significantly after nivolumab, we suspected that nivolumab triggered a severe autoimmune response, which progressed subclinical myasthenia gravis to myasthenic crisis. We carried out T cell receptor repertoire analysis using next‐generation sequencing technologies and identified infiltration of clonally expanded T cell populations in the skeletal muscle after nivolumab treatment, implying a very strong T cell immune response against muscular cells. To avoid severe immune‐related adverse events, the exclusion of patients with subclinical autoimmune disease is very important for treatment with immune checkpoint inhibitors.

The T cell receptor (TCR) determines specificity and affinity for both foreign and self-peptides presented by the major histocompatibility complex (MHC). Although the strength of TCR interactions with self-pMHC impacts T cell function, it has been challenging to identify TCR sequence features that predict T cell fate. To discern patterns distinguishing TCRs from naive CD4+ T cells with low versus high self-reactivity, we used data from 42 mice to train a machine learning (ML) algorithm that identifies population-level differences between TCRβ sequence sets. This approach revealed that weakly self-reactive T cell populations were enriched for longer CDR3β regions and acidic amino acids. We tested our ML predictions of self-reactivity using retrogenic mice with fixed TCRβ sequences. Extrapolating our analyses to independent datasets, we predicted high self-reactivity for regulatory T cells and slightly reduced self-reactivity for T cells responding to chronic infections. Our analyses suggest a potential trade-off between TCR repertoire diversity and self-reactivity. A record of this paper's transparent peer review process is included in the supplemental information.

Many experimental and bioinformatics approaches have been developed to characterize the human T cell receptor (TCR) repertoire. However, the unknown functional relevance of TCR profiling hinders unbiased interpretation of the biology of T cells. To address this inadequacy, we developed tessa, a tool to integrate TCRs with gene expression of T cells to estimate the effect that TCRs confer on the phenotypes of T cells. Tessa leveraged techniques combining single-cell RNA-sequencing with TCR sequencing. We validated tessa and showed its superiority over existing approaches that investigate only the TCR sequences. With tessa, we demonstrated that TCR similarity constrains the phenotypes of T cells to be similar and dictates a gradient in antigen targeting efficiency of T cell clonotypes with convergent TCRs. We showed this constraint could predict a functional dichotomization of T cells postimmunotherapy treatment and is weakened in tumor contexts. Tessa, a Bayesian model, integrates TCR sequencing data with gene expression data to analyze the effect of the TCR repertoire on T cell functionality.

Immune checkpoint modulators have revolutionized tumor therapy with significant therapeutic benefits. However, immune-related adverse events, especially cardiovascular sequelae have been documented in clinical use. Current studies have focused on conventional immune checkpoint inhibitors (anti-PD-1/PD-L1, anti-CTLA-4), which are associated with severe myocarditis in ~1-2% of patients. There is an increasing need to understand whether emerging immune checkpoint modulators contribute to cardiovascular risk and the progression of chronic cardiovascular diseases. Such agents target different mechanisms and are often used in combination with conventional immune checkpoint inhibitors. For example, combination of OX40 agonist and PD-1 inhibitor demonstrates promising anti-tumor effects in pre-clinical and early clinical studies. Here, we employed single-cell RNA-sequencing, lineage tracing, immunostaining, flow cytometry and antibody neutralization studies to explore the cardiac effects of combined OX40 activation and PD-1 inhibition. We observed that combined PD1 inhibition and OX40 activation increased the recruitment of monocytes and CCR2+ macrophages into the heart, and the frequency of cardiac effector memory T-cells along with elevated pro-inflammatory chemokines and cytokines in both naïve and tumor bearing mice. These changes persisted at chronic time points highlighting the long-term effects of combined OX40-PD1 therapy on cardiac immune niches. Unlike conventional immune checkpoint inhibition, these changes were independent of CD8+ T-cell mediated IFN-γ signaling. Instead, monocyte recruitment depended on signals generated by CD4 + T-cells and the effector memory phenotype of T-cells induced by OX40 agonist relied on TNF-α signaling. From a clinical perspective, prior OX40 agonism aggravated ischemia reperfusion injury and angiotensin II and phenylephrine infusion induced cardiac hypertrophy, fibrosis and inflammation in Pdcd1 -/- mice establishing the functional relevance of shifts in the cardiac immune landscape induced by combined PD-1 inhibition and OX40 agonism. Collectively, these findings indicate that combined OX40 activation and PD1 inhibition reshape the cardiac immune environment, promote myocardial inflammation, and vulnerability.

Background: Numerous studies indicate an association between cardiac dilation and autoimmune processes. Still, a deeper understanding of the mechanisms linking immune regulation and pathophysiological processes in the heart is required. Programmed cell death protein 1 (PD-1) is an immune checkpoint regulator. PD-1 knockout (PD-1 KO) in mice leads to heart dilation in only some mice. Purpose: This study investigated differences between PD-1 KO mice with and without a cardiac phenotype. Methods: Female and male mice aged 3-14 weeks were included, n=12 PD-1 KO mice showing cardiac dilation (PD-1 dil), n=16 with non-dilated hearts (PD-1 ctrl) and n=11 BALB/c mice (ctrl). B cell activating factor (BAFF) levels were measured in serum via ELISA. Heart weights were normalized. Proteome analysis and gene expression profiling were conducted using the apical third of the heart, significant alterations (fold change of ≥|1.5|, adjusted p-value <0.05) were analysed. Results: Cardiac dilation occurred in 6% of PD-1 KO mice. Heart weights and BAFF levels of PD-1 dil were significantly higher compared to PD-1 ctrl and ctrl (heart weight: 12.9/6.9/6.8 mg/mm; BAFF: 25.9/19.7/15.2 ng/ml). Proteomics analysis revealed more than 1,100 significantly altered proteins (PD-1 dil vs. PD-1 ctrl: 504↓, 623↑; PD-1 dil vs. ctrl: 676↓, 702↑). Only 19 proteins were differentially abundant comparing both ctrls (4↓, 15↑). Principal component analysis clearly distinguished PD-1 dil from both ctrl groups, which in turn strongly overlapped. Activation z-score analysis showed an inhibition of mainly mitochondrial functions (z≤-2) in PD-1 dil vs. PD-1 ctrl and in PD-1 dil vs. ctrl. In these comparison groups, some of the most significantly enriched terms associated with cardiovascular diseases were cardiac fibrosis, dilation and inflammation. A regulation of proteins associated with immunological diseases was observed in both comparisons. Moderate correlation between datasets for proteome and transcriptome was observed (PD-1 dil vs. PD-1 ctrl: r=0.5, p<0.0001; PD-1 dil vs. ctrl: r=0.41, p<0.0001). Conclusion: PD-1 KO mice show an inconsistent phenotype. Thereby PD-1 dil and PD-1 ctrl display significant differences in immune regulation and cardiac pathways, underscoring immunological influences on the development of cardiac pathologies. Future studies will focus on identifying molecular mechanisms that lead to the described phenotype.

Immune checkpoints, such as PD-1 and CTLA-4, are crucial regulators of immune responses, acting as gatekeepers to balance immunity against foreign antigens and self-tolerance. These checkpoints play a key role in maintaining cardiac homeostasis by preventing immune-mediated damage to critical organs like the heart. In this study, we explored the involvement of PD-1 and CTLA-4 in cardiovascular complications, particularly atherosclerosis and myocarditis, which can lead to heart failure. We conducted a comprehensive analysis using animal models and clinical data to assess the effects of immune checkpoint inhibition on cardiac function. Our findings indicate that disruption of PD-1 and CTLA-4 pathways exacerbates myocardial inflammation, accelerates atherosclerotic plaque formation, and promotes the development of heart failure. Additionally, we observed that immune checkpoint inhibition in these models led to increased infiltration of T lymphocytes, higher levels of pro-inflammatory cytokines, and enhanced tissue damage. These results suggest that PD-1 and CTLA-4 are critical in preserving cardiac health, and their inhibition can result in severe cardiovascular toxicity. Our study emphasizes the need for careful monitoring of cardiovascular health in patients undergoing immune checkpoint inhibitor therapies.

No abstract available

Background: Immunotherapy, particularly PD-1/PD-L1 blockers, is effective in cancer treatment but can cause rare, but potentially fatal heart issues. Despite the significant impact of these adverse events, there is limited research into the underlying mechanisms of immune checkpoint inhibitor (ICI)-mediated cardiotoxicity. PD-L1, which is abundantly expressed in cardiac tissue, particularly exhibits high expression levels over myeloid cells. However, the precise role of myeloid-specific PD-L1 signaling in mediating autoimmune cardiac disease pathology remains unclear. Methods and Results: To study the role of myeloid cell-specific PD-L1 (myeloid-PD-L1) in cardiac pathophysiology, we generated myeloid-specific PD-L1 KO mice, using Lyz2tm1(Cre) Ifo systems. As expected, PD-L1 expression was significantly down-regulated in Mφs from myeloid-PD-L1 KOs. The cardiac function was followed by echocardiography. Controls and myeloid-PD-L1-KO hearts had comparable ventricular function at 1 month of age. Interestingly, as early as 2 months of age, myeloid-PD-L1-KOs displayed marked cardiac dysfunction as reflected by reduced LVEF, LVFS, and HF markers. Immune profiling was conducted whether chronic inflammation contributes to cardiac dysfunction and remodeling at a later age as observed at 2 and 3 months of age. Interestingly, excessive systemic inflammation and substantial immune infiltration (innate and adoptive), activation, and a pro-inflammatory cytokine storm were detected in the KO hearts. Furthermore, myeloid-PD-L1 KO hearts showed increased recruitment of pro-inflammatory CCR2+ monocytes and macrophages. Conclusion: Our data with myeloid-PD-L1 KOs suggest that deleting PD-L1 in myeloid lineage promotes myocardial and systemic inflammation through innate and adaptive immune cell activation and recruitment of CCR2+ MΦs. Subsequently, this leads to impaired cardiac pathophysiology. Thus, these translational studies will directly evaluate the potential of targeting myeloid-PD-L1 signaling to alleviate cardiac damage.

Mitochondria were critical for pathogenesis of PD-1 inhibitors-induced carditis, which was demonstrated to be the mechanism for Ganoderma atrum polysaccharide (PSG) against cardiomyopathy. Hence, the present study aimed to determine the role of PSG in controlling mitochondrial homeostasis in PD-1 inhibitors-induced carditis of Lewis lung carcinoma mice. Results showed that PSG significantly alleviated PD-1 inhibitors-induced cardiotoxicity without compromising their anti-tumor effects, as evidenced by inhibiting cardiac histopathological disorders, creatine kinase (CK) release, and tumor growth. PSG administration significantly ameliorated inflammation by reducing pro-inflammatory cytokine IL-1β release and NLRP3 expression. Meanwhile, the reduction of pyroptosis was demonstrated to be implicated in PSG-inhibited carditis evidenced by the decrease in Caspase-1, gasdermin D (GSDMD). Mechanistically, mitochondria were sites of ROS generation and NLRP3 inflammasome activation. Our results showed that PSG suppressed NLRP3- induced pyroptosis, which was associated with inhibition of ROS attack and mitochondrial protection by maintaining mitochondrial membrane potential, reversing a deficiency in mitochondrial fission, suppressing mitochondrial hyper-fusion, suggesting that ROS/NLRP3/pyroptosis axis was a vital process in avoiding mitochondrial dysfunction during PSG-mediated cardioprotection. Additionally, the modification of the redox system was also shown in the context of cardioprotection of PSG, by elevating antioxidant enzyme activities and suppressing lipid oxidation.

Background: Immune checkpoint inhibitors (ICIs) have revolutionized cancer treatment by enhancing the immune system’s ability to recognize and attack tumor cells. However, despite their efficacy, ICIs can trigger immune-related adverse events (jrAEs), including severe cardiotoxicities such as myocarditis, heart failure, and arrhythmias. While previous studies have primarily focused on T-cell-mediated mechanisms, the role of myeloid-specific PD-L1 signaling in ICI-induced myocarditis remains poorly understood. Methods and Results: In this study, we investigated the impact of myeloid-specific PD-L1 deletion using a Lyz2tm1 (Cre)Ifo mouse model. Surprisingly, as early as 1 month of age, myeloid-PD-L1 KOs displayed marked cardiac dysfunction as reflected by reduced LVEF, LVFS, and elevated HF markers. Immune profiling was conducted whether chronic inflammation contributes to cardiac dysfunction and remodeling. Interestingly, KO hearts demonstrated excessive systemic inflammation, substantial myocardial immune infiltration (innate and adaptive), and activation. Of note, the cardiac and systemic inflammation phenotype precedes the onset of cardiac dysfunction and adverse remodeling. This implies that excessive inflammation is critical to driving impaired cardiac pathophysiology. Notably, we observed increased recruitment of pro-inflammatory CCR2+ macrophages in KO hearts, suggesting a crucial role of myeloid PD-L1 in modulating immune response and cardiac pathology. Additionally, fibroblasts from wild-type mice co-cultured with bone marrow-derived macrophages (BMDMs) from KO mice exhibited significant upregulation of fibrosis-related genes and inflammatory cytokines. Furthermore, BMDMs from KO co-cultured with T-cells from wild-type mice lead to increased T-cell frequency and activation, as demonstrated by the upregulation of TCRαβ+, CD4+ and CD8+ T-cell populations, with a pro-inflammatory Th1 polarization. Conclusion: Overall, our findings demonstrate that myeloid-derived immunosuppressive PD-L1 signaling is essential to maintaining adult heart homeostasis. The absence of myeloid PD-L1 leads to progressive cardiac dysfunction and adverse remodeling, likely driven by excessive inflammation. Nonetheless, we also identified CCR2+ macrophages as a targetable population in myeloid-PD-L1-mediated myocarditis. Future studies should explore whether interventions targeting pathogenic CCR2+ macrophages could be leveraged to halt or reverse ICI-induced cardiac dysfunction.

e14631 Background: Radiation in combination with programmed cell death 1 (PD-1) antibodies greatly enhanced the anti-tumor activity by reactivating T lymphocytes. Whether it will produce the superposition of toxic side effects and the underlying mechanism remains elusive. Pyroptosis is a novel type of programmed cell death associated with the pathogenesis of many inflammatory diseases. We hypothesized that GSDMD plays a pathogenic role in PD-1 inhibitor combined with radiation-induced myocardial injury (RIMI) and that GSDMD gene knockout (KO) or blockade the caspase-1/GSDMD pathway will alleviate RIMI. Methods: GSDMD-KO mice and their wildtype (WT) C57BL/6J were used to investigate whether PD-1 inhibitor aggravated RIMI through pyroptosis pathway. Myocardial contractile functions, myocardial inflammation and fibrosis, and myocardial injury were assessed. Pyroptosis-related proteins and inflammatory cytokines were evaluated. Flow cytometry was used to detect the level of lymphocyte in myocardium. To further confirm the presence of pyroptosis in cardiomyocyte, double-immunostaining was performed. We verified the effect of radiotherapy combined with PD-1 inhibitor on pyroptosis in cell experiments. Results: Compared with irradiation or anti-PD-1 alone, PD-1 inhibitor could aggravate RIMI, increase the infiltration of CD8+T cells in the myocardium, and aggravate inflammatory reactions. The expressions of pyroptosis-related proteins and inflammatory cytokines were upregulated. Immunofluorescence also indicated that pyroptosis cells were increasing. While GSDMD-KO or caspase-1GSDMD inhibitor, myocardial injury and pyroptosis-related proteins were significantly reduced. Conclusions: We speculate that PD-1 inhibitors activate CD8+T cells and mediate cardiomyocyte pyroptosis. GSDMD-triggered immune-inflammatory response plays a key role in the aggravation of myocardial injury.

Introduction: Immune checkpoint inhibitors (ICI), such as anti-PD-1 monoclonal antibodies are increasingly used in anti-cancer therapy. However, several cardiovascular adverse effects can occur with the use of ICIs, including new-onset heart failure. Hypothesis: We hypothesized that prior myocardial ischemic injury could exacerbate cardiac dysfunction and inflammation caused by anti-PD-1 treatment. Moreover, we investigated whether abatacept, a T-cell co-stimulation blocker, can prevent ICI-induced cardiac effects. Methods: To induce reversible cardiac ischemia, C57Bl/6J mice were treated with isoprenaline (ISOP group) or with PBS (CON group), followed by 16 weeks of recovery period. Following this, mice from both groups were divided into three further treatment groups: isotype control, anti-PD-1, or anti-PD-1 combined with abatacept, and were treated for two weeks, with three weekly intraperitoneal injections. Echocardiography was performed to evaluate cardiac function while myocardial inflammation was assessed by qRT-PCR and immunohistochemistry. Flow cytometry and Western blot were used to investigate changes occurring in the thymus. Results: Mice with normal heart function but with prior ischemic injury and anti-PD-1 treatment (ISOP + anti-PD-1) showed significantly decreased fractional shortening and cardiac index on echocardiography, while in animals with abatacept co-treatment (ISOP+anti-PD-1+abatacept) cardiac function was not altered. Increased immune cell infiltration was seen in the myocardium of the ISOP+anti-PD-1 treated group compared to CON animals, including T-cells and macrophages, with increased expression of pro-inflammatory cytokines, while co-treatment with abatacept ameliorated the inflammatory response. In the thymus, increased expression of PD-1 was found after abatacept co-treatment. Conclusion: Prior myocardial ischemic injury was associated with cardiac dysfunction and inflammation after anti-PD-1 treatment, which was ameliorated by abatacept co-treatment. Patients with prior cardiac ischemic events may be at greater risk for developing ICI-induced cardiotoxicity, including new-onset HF.

Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ricerca Corrente, Ministero della Salute. Immune checkpoint inhibitors (ICIs) have significantly changed the oncology clinic in recent years, improving survival expectations in cancer patients. ICI therapy have a broad spectrum of side effects from endocrinopathies to cardiovascular diseases. In this study, pro-inflammatory and pro-fibrotic effects of short-term ICIs therapy in preclinical models were analyzed. Firstly, in a human in vitro model, human cardiomyocytes co-cultured with hPBMC were exposed to ICIs (with CTLA-4 or PD-1 blocking agents, at 200 nM) for 72h. After treatment, production of DAMPs and 12 cytokines were analyzed in the supernatant through colorimetric and enzymatic assays. C57/Bl6 mice were treated with CTLA-4 or PD-1 blocking agents (15 mg/kg) for 10 days. Before (T0), after three days (T3) and after treatments (T10), ejection fraction, fractional shortening, radial and longitudinal strain were calculated by using bidimensional echocardiography (Vevo 2100,Fujfilm). Fibrosis, necrosis, hypertrophy and vascular NF-kB expression were analyzed through Immunohistochemistry. Myocardial expression of DAMPs (S100- Calgranulin, Fibronectin and Galectine-3), MyD88, NLRP3 and twelve cytokines have been analyzed. Systemic levels of SDF-1, IL-1β and IL-6 were analyzed before, during and after ICIs therapy. Radial and longitudinal strain were decreased after 10 days of ICIs therapy. Histological analysis of NF-kB expression shows that short-term anti-CTLA-4 or anti-PD-1 treatment increased vascular and myocardial inflammation. No myocardial hypertrophy was seen with the exception of the pembrolizumab group. Myocardial fibrosis and expression of galectin-3, pro-collagen 1-α and MMP-9 were increased after treatment with all ICIs. Both anti-CTLA-4 or anti-PD-1 treatments increased the expression of DAMPs, NLRP3 inflammasome and MyD88 and induced both in vitro and in vivo the secretion of IL-1β, TNF-α and IL-6. Systemic levels of SDF-1, IL-1β and IL-6 were increased during and after treatment with ICIs. Short therapy with PD-1 and CTLA-4 blocking agents increases vascular expression of NF-kB, systemic SDF-1, IL-1β, IL-6 levels and myocardial NLRP3, MyD88 and DAMPs expression in preclinical models. A pro-inflammatory cytokine storm was induced in myocardial tissues and in cultured cardiac cells after ICIs therapy. The overall picture of the study suggests new putative biomarkers of ICIs-mediated systemic and myocardial damages potentially useful in clinical cardioncology.

Immune checkpoint inhibitors (ICIs) have significantly changed the oncology clinic in recent years, improving survival expectations in cancer patients. ICI therapy have a broad spectrum of side effects from endocrinopathies to cardiovascular diseases. In this study, pro–inflammatory and pro–fibrotic effects of short–term ICIs therapy in preclinical models were analyzed. Firstly, in a human in vitro model, human cardiomyocytes co–cultured with hPBMC were exposed to ICIs (with CTLA–4 or PD–1 blocking agents, at 200 nM) for 72h. After treatment, production of DAMPs and 12 cytokines were analyzed in the supernatant through colorimetric and enzymatic assays. C57/Bl6 mice were treated with CTLA–4 or PD–1 blocking agents (15 mg/kg) for 10 days. Before (T0), after three days (T3) and after treatments (T10), ejection fraction, fractional shortening, radial and longitudinal strain were calculated by using bidimensional echocardiography (Vevo 2100,Fujfilm). Fibrosis, necrosis, hypertrophy and vascular NF–kB expression were analyzed through Immunohistochemistry. Myocardial expression of DAMPs (S100– Calgranulin, Fibronectin and Galectine–3), MyD88, NLRP3 and twelve cytokines have been analyzed. Systemic levels of SDF–1, IL–1β and IL–6 were analyzed before, during and after ICIs therapy. Radial and longitudinal strain were decreased after 10 days of ICIs therapy. Histological analysis of NF–kB expression shows that short–term anti–CTLA–4 or anti–PD–1 treatment increased vascular and myocardial inflammation. No myocardial hypertrophy was seen with the exception of the pembrolizumab group. Myocardial fibrosis and expression of galectin–3, pro–collagen 1–α and MMP–9 were increased after treatment with all ICIs. Both anti–CTLA–4 or anti–PD–1 treatments increased the expression of DAMPs, NLRP3 inflammasome and MyD88 and induced both in vitro and in vivo the secretion of IL–1β, TNF–α and IL–6. Systemic levels of SDF–1, IL–1β and IL–6 were increased during and after treatment with ICIs. Short therapy with PD–1 and CTLA–4 blocking agents increases vascular expression of NF–kB, systemic SDF–1, IL–1β, IL–6 levels and myocardial NLRP3, MyD88 and DAMPs expression in preclinical models. A pro–inflammatory cytokine storm was induced in myocardial tissues and in cultured cardiac cells after ICIs therapy.

Myocardial fibrosis is a common pathological feature that affects the outcome of various heart diseases, typically in the condition of myocardial infarction (MI), and hence the extent of fibrosis becomes a major risk factor for predicting the prognosis of heart failure. On the other hand, while the roles of T cells in heart failure have been increasingly recognized, however, it remains not fully known whether PD1+ T cells modulate the cardiac fibrosis during the post-MI pathological remodeling process. Single cell RNA sequencing and mass cytometry were conducted to identify the proportion of PD1+ T cells both in human ischemic diseases and mouse MI model. Bulk RNA sequencing and cytokine array were utilized to investigate the function of PD1+ T cells. Cardiac function and histology analysis were evaluated in non-human primates and rodents post MI. We observed significantly increased PD1+ T cells in the MI heart, the amount of which was positively associated with cardiac fibroblast activation and hence collagen secretion. Unlike in tumor, PD1+ T cells in MI heart demonstrated activated characteristics. PD-1 knockout (PD1-/-) mice exhibited reduced cardiac fibrosis, resulting in an increase in cardiac performance following MI. Mechanistically, the activated PD1+ T cells were shown to induce fibrosis via modulating the CXCL9/CXCR3 axis via interacting with cardiac fibroblasts, and this was independent of PD1/PD-L1 pathway. Importantly, the anti-PD1 therapy by novolumab administration in both mouse and non-human primate (NHP) MI models exerted the anti-fibrosis effects and resulted in increased cardiac function, without any noticeable side effects on other organ-tissues including the liver, the kidney and the thyroid. The present study revealed the pro-fibrotic role of PD1+ T cells during the post-MI cardiac remodeling process, which was independent of its correlation with PD-L1. Our data strongly supported that PD-1 inhibition can serve as an effective immunotherapy for preventing post-MI cardiac fibrosis.

No abstract available

Background Immune checkpoint inhibitor-associated myocarditis (ICI myocarditis) is an infrequent but potentially fatal immune-related adverse event. This study aimed to identify valuable indicators for risk prediction and evaluation of disease severity and outcomes. Methods A total of 79 patients with severe or mild ICI myocarditis and 158 controls without post-ICI immune-related adverse events were enrolled in this retrospective study. The clinical application value of a series of simple biomarkers were tested. Results Higher levels of the systemic immune-inflammation index (SII), neutrophil-to-eosinophil ratio (NER), aspartate transferase-to-albumin ratio (AAR), and lactic dehydrogenase-to-albumin ratio (LAR) at myocarditis onset were associated with severe disease conditions. In the receiver operating characteristic analysis, biomarkers areas under the curve (AUC) close to or greater than 0.8 were LAR (AUC: 0.810) and AAR (AUC: 0.806). Patients with higher SII, AAR, and LAR also exhibited poorer overall survival. The SII, NER, AAR, and LAR before the last ICI treatment increased relative to baseline in patients with ICI myocarditis, whereas no significant changes in the tested biomarkers were observed in the control group. For SII, AAR, and LAR, high ratios of the biomarker levels before the last ICI to baseline was associated with the incidence of myocarditis. Conclusions Surveillance of these economical biomarkers during ICI therapy might contribute to the risk prediction of ICI myocarditis, as well as the assessment of disease severity and prognosis.

BACKGROUND AND AIMS Immune checkpoint inhibitors (ICI) are associated with life-threatening myocarditis but milder presentations are increasingly recognized. The same autoimmune process that causes ICI myocarditis can manifest concurrent generalized myositis, myasthenia-like syndrome, and respiratory muscle failure. Prognostic factors for this 'cardiomyotoxicity' are lacking. The main aim of this study was to determine predictors and construct a risk score associated with negative outcomes in patients admitted for ICI myocarditis. METHODS A multicentre registry collected data retrospectively from 17 countries between 2014 and 2023. A multivariable Cox regression model was used to determine risk factors for the primary composite outcome: time to severe arrhythmia, heart failure, respiratory muscle failure, and/or cardiomyotoxicity-related death. Covariates included demographics, comorbidities, cardiomuscular symptoms, diagnostics, and treatments. Time-dependent covariates were used, and missing data were imputed. A point-based prognostic risk score was derived and externally validated. RESULTS In 748 patients (67% male, age 23-94 years), 30-day incidence of the primary composite outcome, cardiomyotoxic death, and overall death were 33%, 13%, and 17%, respectively. By multivariable analysis, the primary composite outcome was associated with active thymoma (hazard ratio [HR] 3.6, 95% confidence interval [CI] 1.7-7.7), presence of cardiomuscular symptoms (HR 2.6 [1.5-4.2]), low QRS voltage on presenting electrocardiogram (HR for ≤0.5 mV vs >1 mV 1.9 [1.1-3.1]), left ventricular ejection fraction (LVEF) < 50% (HR 1.7 [1.1-2.6]), and incremental troponin elevation (HR 1.8 [1.4-2.4], 2.9 [1.8-4.7], and 4.6 [2.3-9.3], for 20, 200, and 2000-fold above upper reference limit, respectively). A prognostic risk score developed using these parameters showed good performance; 30-day primary outcome incidence increased gradually from 4% (risk score = 0) to 81% (risk score ≥ 4). This risk score was externally validated in two independent French and US cohorts. This risk score was used prospectively in the external French cohort to identify low-risk patients who were managed with no immunosuppression resulting in no cardiomyotoxic events. CONCLUSIONS ICI-associated myocarditis can manifest with high morbidity and mortality. Myocarditis severity is associated with magnitude of troponin, thymoma, low QRS voltage, depressed LVEF, and cardiomuscular symptoms. A risk score incorporating these features performed well. CLINICAL TRIAL REGISTRATION NCT04294771 and NCT05454527.

Myocarditis is a known complication of immune checkpoint inhibitor (ICI) therapy. Viral myocarditis often follows seasonal patterns, raising the question of whether ICI-associated myocarditis may also exhibit temporal variation or periods of heightened susceptibility. Identifying temporal clustering may guide targeted monitoring strategies during higher-risk periods. Given the modest sample size, the analyses were designed as exploratory and hypothesis-generating. We identified 54 high-suspicion cases of ICI-associated myocarditis through inpatient consults, outpatient follow-up, or troponin monitoring. Six patients were excluded: two without confirmed ICI exposure, two without documented troponin elevation, one with inaccessible records, and one with a missing ICI start date, yielding a final cohort of 48 patients. Cases were grouped by peak year as pre-COVID (before 2020), during COVID (2020-2021), or post-COVID (2022-2025). Seasonality was assessed by comparing January-June versus July-December, a half-year grouping chosen to ensure adequate statistical stability with limited sample size. Statistical analyses included Fisher’s exact Test and binomial testing. Among 48 high-suspicion ICI-myocarditis cases, ICIs were most commonly initiated for gastrointestinal (17%), lung (15%), head and neck (13%), and melanoma (13%) cancers. Pembrolizumab was the predominant regimen (54%), followed by ipilimumab + nivolumab (25%), cemiplimab (8%), nivolumab (6%), durvalumab (4%), and envafolimab (2%). Most patients (77%, 37/48) experienced symptom onset within the first two months of ICI initiation. A greater proportion of ICI-myocarditis events occurred between July and December (31/48, 64.6%) compared with January-June (17/48, 35.4%), although this did not reach statistical significance (binomial test, p = 0.059). This seasonal pattern persisted across troponin thresholds: 70% (16/23) of cases >100 ng/L, 70% (23/33) >500 ng/L, and 63% (25/40) >1000 ng/L occurred between July and December. Although elevated troponin cases were numerically higher post-COVID, the difference was not statistically significant (Fisher’s exact p = 0.496). Most ICI-myocarditis cases occurred within two months of treatment initiation, with a skew toward the latter half of the year suggesting a potential seasonal pattern in early-onset cardiotoxicity. This trend may reflect interactions between increased ICI use, improved surveillance, or immunologic priming related to COVID-19 infection or vaccination. While preliminary, these findings support further investigation into temporal and immunologic risk factors that may inform risk-adapted monitoring strategies. Crystal Griffith, Elizabeth Park, Evaline Cheng, Stephen McCroskey, Tamiko Katsumoto, Han Zhu, Pauline Funchain. Seasons of risk: Investigating temporal trends in ICI-associated Myocarditis [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 B069.

Endomyocardial biopsy (EMB) is the gold standard for myocarditis diagnosis. However, the Dallas pathological criteria(1) prevail over multimodal cardiovascular imaging and focus on viral myocarditis. Immune checkpoint inhibitors myocarditis(2) are easier to diagnose in severe cases, but non-severe, asymptomatic forms have emerged. The value of EMB has not been prospectively assessed(3). The lack of predictors for disease severity highlights the need to identify low-risk populations. To assess the correlation between clinical features and histology characteristics in patients with suspected ICI-myocarditis. This single-centre prospective sub-study of a larger cohort included adults for suspected cardiovascular ICI-related IRAEs who underwent EMB performed when deemed necessary by the MDT to rule in or out ICI-myocarditis. EMBs were reviewed by few experienced pathologists using current guidelines, including CD3/CD68 immunohistochemistry.The primary endpoint was the correlation between EMB findings and clinical features. Among 175 consecutive patients referred, we performed a total of 48 biopsies. Of these, 4 were normal, 4 showed definite myocarditis (i.e., >7 T cells/mm² and myocardial loss), 37 showed significant myocardial inflammation (>7 T cells/mm²) but no cell loss, and 1 showed cardiac metastasis.The median number of lymphocytes in the definite myocarditis group was 114/mm² versus 16/mm² in the borderline pattern (Dallas criteria). The clinical characteristics of patients with definite myocarditis and the histological/clinical/radiological parameters of EMB population are depicted in Figure 1. Patients with a definite myocarditis pattern were more likely to: Be thymoma patients [2 (50%) versus 1 (2.7%); p=0.001], have non-lung/non-breast cancers [3 (75%) versus 3 (16.2%); p=0.028], stage IV cancer [4 (100%) versus 10 (27%); p=0.003], higher median CPK levels [741 U/L (IQR 122-1648) versus 61 (IQR 40-100); p=0.035], have received ICIs without other cancer drugs [2 (50%) versus 3 (8.1%); p=0.015] and have undergone fewer cycles of ICIs [2 (IQR 1.25-2) versus 7 (IQR 3-9); p=0.002] and were more frequently hospitalized [4 (100%) versus 8 (21.6%); p=0.001] . Breast cancer patients [28 (78.4%) versus 0 (0%); p=0.001] treated with a combination of ICIs and other chemotherapies [32 (86.5%) versus 1 (25%)p=0.003] and anthracyclines [22 (64.7%) versus 0 (0%); p=0.029] were more likely to present with a borderline pattern(Figure 2). N° of T-cells were significantly correlated with the levels of troponin normalized to the upper normal limit (R=0.3, P=0.028) (Figure 2). ICI-myocarditis shows a complex biopsy spectrum and is not a binary phenomenon. T-cell infiltration without necrosis or symptoms remains of unclear significance. We found that some clinical features correlate with histological Dallas criteria. Further research is needed to establish a gold standard for ICI-myocarditis.  

11177 Background: Immune checkpoint inhibitor (ICI)-associated myocarditis is a rare but life-threatening immune-related adverse event (irAE), with a mortality rate of 30–50%. Most available evidence comes from case series and adverse event reporting databases, leaving its risk factors and clinical outcomes largely undefined. Methods: We performed a retrospective investigation using the TriNetX Global Research Network. Patients were included if they had been diagnosed with solid tumors after 2016 for which ICIs are approved. We calculated myocarditis incidence among ICI users and compared myocarditis risk between ICI and non-ICI cohorts using propensity score matching (accounting for demographics, comorbidities, cancer types, and treatments). Univariate and multivariate models were applied to assess risk factors. Risks of hospitalization, ICU admission, and major adverse cardiovascular events (MACE) were compared in ICI-treated patients with and without myocarditis using propensity score matching. Results: Among 130,234 ICI-treated patients, 643 (0.49%) developed myocarditis. Most cases (82.0%) occurred within the first year of ICI initiation. The risk of myocarditis was 27 times higher in the ICI group compared to the non-ICI group (Relative Risk [RR]=27.78, 95% CI: [17.36-44.45]). Univariate analysis identified older age, male sex, White race, hypertension, hyperlipidemia, ischemic heart disease, diabetes, chronic kidney disease, heart failure, cardiomyopathy, urothelial cancer, melanoma, ipilimumab, nivolumab, pembrolizumab, radiation therapy, inflammatory arthritis, lupus, and systemic connective tissue disease as potential risk factors. In multivariate analysis, only age over 65 (Hazard Ratio [HR] = 1.34, 95% CI: [1.11–1.63], p < 0.01), hypertension (HR=1.31, 95% CI: [1.06-1.62], p=0.01), hyperlipidemia (HR=1.34, 95% CI: [1.09-1.65], p<0.01), melanoma (HR=1.52, 95% CI: [1.25-1.86], p<0.01), and ipilimumab (HR= 2.44, 95% CI: [1.87-3.19], p<0.01) were significantly associated with myocarditis. Patients who developed ICI-associated myocarditis had higher risks of hospitalization (RR=1.39, 95% CI: [1.28-1.52]), ICU admission (RR=2.35, 95% CI: [1.86-2.98]), myocardial infarction (RR=5.58, 95% CI: [3.73-8.34]), heart failure (RR=1.59, 95% CI: [1.38-1.85]), stroke (RR=2.94, 95% CI: [1.75-4.97]), MACE (RR=3.92, 95% CI: [2.91-5.27]), and all-cause mortality (RR=1.32, 95% CI: [1.13-1.55]) within one year compared to those who received ICIs but did not develop myocarditis. Conclusions: ICI-associated myocarditis is a rare irAE but carries an increased risk of cardiovascular complications and death. Advanced age, hypertension, hyperlipidemia, melanoma, and ipilimumab use appear to be risk factors. Clinical vigilance is warranted to facilitate early detection and management of this potentially fatal complication.

This article pertains to the study by Liu et al. Myocarditis caused by immune checkpoint inhibitors in patients with esophageal cancer is a concerning issue. This was found to be linked by increased levels of creatine kinase (CK) and CK isoenzymes, as well as older age and male gender. All these risk factors behind this phenomenon, which could be incorporated into a unified prediction model, have been briefly discussed. Several recommendations have been made to validate this prediction model for use in different clinical scenarios.

Clonal hematopoiesis of indeterminate potential (CHIP) has been shown to increase all-cause mortality and risk of cardiomyopathy in patients with solid malignancies. CHIP has also been shown to increase T cell activation in heart failure patients. It is unclear whether CHIP can affect the risk of immune checkpoint inhibitor (ICI) myocarditis in patients with cancer treated with immunotherapy. We enrolled patients with solid tumors in a prospective study, determined CHIP status at time of enrollment through blood whole exome sequencing, and assessed incidence of ICI myocarditis from time of enrollment through December 1st, 2023. We performed a competing risk cox regression to evaluate the role of CHIP in ICI myocarditis, accounting for patient demographics, cardiac comorbidities, cardiotoxic cancer therapy, and dual ICI use in our covariates. We also generated cumulative incidence curves using subdistribution hazards to evaluate development of ICI myocarditis stratified by CHIP vs no CHIP. Chart review was performed to evaluate patient co-morbidities, lab values, imaging findings and outcomes. Among the 88 patients receiving ICI therapy, average age was 67 ± 14 years, of which 50% harbored CHIP variants. Among all comorbidities, including diabetes, heart failure and obstructive coronary artery disease, only coronary artery calcifications were significantly increased in patients with CHIP. There were no statistically significant differences in cancer therapy or cardiovascular drugs between patients with and without CHIP. Among examined outcomes, patients with CHIP had a statistically higher rate of ICI myocarditis (overall: 57%, CHIP: 73% (32/44), no CHIP: 41% (18/44), p = 0.003) and death (CHIP: 60%, no CHIP 31%, p = 0.011). In a multivariate competing risk analysis, CHIP status doubled the risk of developing ICI myocarditis, similar to the risk of dual ICI use (CHIP status HR 2.74, 95% CI: 1.44–5.22, p = 0.002 vs dual ICI use HR 2.39, 95% CI: 1.11–5.14, p = 0.026). This study is the first to show that CHIP independently increases risk of ICI myocarditis, with implications for risk stratification of patients prior to ICI initiation and frequency of cardiac monitoring.

e24026 Background: ICI-associated myocarditis carries a high morbidity and mortality, with close to half of patients experiencing major adverse cardiac events (MACE). Risk factors previously associated with short-term MACE include troponin T (TnT), neutrophil/lymphocyte ratio (NLR), and absolute lymphocyte count (ALC). In this study, we aimed to analyze the predictors of long-term survival. Methods: This single-center retrospective study included 37 patients diagnosed with ICI-associated myocarditis at Massachusetts General Hospital between March 2016 and March 2022. We collected demographics, diagnostic results, and myocarditis treatments from the electronic medical record. Patients were grouped by survival time: short-term ( < 30 day), intermediate (30 to 360 day), or long-term ( > 360 days) survival. We used chi-square and Kruskal-Wallis tests for descriptive statistics. Cut-off points of continuous variables were determined by ROC curve. Predictors of survival were analyzed by logistic regression models. Results: The cohort consisted of 29 males and 8 females with a median age of 75 years (IQR 68-79), and median follow-up of 5.6 months (IQR 0.5-14.9). The median time from ICI start to myocarditis onset was 50 days (IQR 36-91). Ten, 14, and 13 patients were in the short-term, intermediate, and long-term survival groups, respectively. By univariate analysis, predictors of long-term survival included initial NT-proBNP ≤ 450, NLR ≤ 4.44, ALC ≥ 1122, normal EKG QTc interval (≤ 450 ms in male or ≤ 460 ms in female), and TnT decrement ≥ 42% by day 8 of immunosuppression (Table 1). All patients with long-term survival presented with a normal left ventricular ejection fraction (LVEF ≥ 55%), which made it uninterpretable using logistic regression models. Normal LVEF was significantly associated with long-term survival by using chi-square test (p = 0.002). While TnT elevation > 75x the upper limit of normal (ULN) was strongly associated with short-term survival (p = 0.001), it was not associated with long-term survival (p = 0.23). The use of second-line immunosuppression in addition to corticosteroids did not have a significant impact on long-term survival (p = 0.69). Conclusions: NLR, ALC, NT-proBNP, QTc interval, and a substantial decrease in TnT at day 8 after immunosuppression are all associated with long-term survival in ICI myocarditis. [Table: see text]

Background: Immune-checkpoint inhibitors (ICI) are associated with life-threatening myocarditis but milder presentations are increasingly recognized. The same autoimmune process that causes ICI-myocarditis can manifest concurrent generalized myositis, myasthenia-like syndrome, and respiratory muscle failure. Prognostic factors for this 'cardiomyotoxicity' are lacking. Methods: A multicenter registry collected data retrospectively from 17 countries between 2014-2023. A multivariable cox regression model (hazard-ratio(HR), [95%confidence-interval]) was used to determine risk factors for the primary composite outcome: severe arrhythmia, heart failure, respiratory muscle failure, and/or cardiomyotoxicity-related death. Covariates included demographics, comorbidities, cardio-muscular symptoms, diagnostics, and treatments. Time-dependent covariates were used and missing data were imputed. A point-based prognostic risk score was derived and externally validated. Results: In 748 patients (67% male, age 23-94), 30-days incidence of the primary composite outcome, cardiomyotoxic death, and overall death were 33%, 13%, and 17% respectively. By multivariable analysis, the primary composite outcome was associated with active thymoma (HR=3.60[1.93-6.72]), presence of cardio-muscular symptoms (HR=2.60 [1.58-4.28]), low QRS-voltage on presenting electrocardiogram (HR for [≤]0.5mV versus >1mV=2.08[1.31-3.30]), left ventricular ejection fraction (LVEF) <50% (HR=1.78[1.22-2.60]), and incremental troponin elevation (HR=1.86 [1.44-2.39], 2.99[1.91-4.65], 4.80[2.54-9.08], for 20, 200 and 2000-fold above upper reference limit, respectively). A prognostic risk score developed using these parameters showed good performance; 30-days primary outcome incidence increased gradually from 3.9%(risk-score=0) to 81.3%(risk-score=4). This risk-score was externally validated in two independent French and US cohorts. This risk score was used prospectively in the external French cohort to identify low risk patients who were managed with no immunosuppression resulting in no cardio-myotoxic events. Conclusions: ICI-myocarditis can manifest with high morbidity and mortality. Myocarditis severity is associated with magnitude of troponin, thymoma, low-QRS voltage, depressed LVEF, and cardio-muscular symptoms. A risk-score incorporating these features performed well.

Background Immune checkpoint inhibitor (ICI) therapy has dramatically improved the prognosis for some cancers but can be associated with myocarditis, adverse cardiovascular events, and mortality. Objectives The aim of this study was to develop an artificial intelligence (AI) model to predict the increased likelihood for the development of ICI-related myocarditis and adverse cardiovascular events. Methods Cancer patients treated with ICI at a tertiary institution from 2011 to 2022 were reviewed. Baseline characteristics, laboratory values, electrocardiograms, and cardiovascular clinical outcomes were extracted. A composite outcome of ICI-related myocarditis and major adverse cardiovascular events (transient ischemic attack/stroke, new diagnosis of heart failure, myocardial infarction, and cardiac death) was used to develop a multimodal joint fusion AI model by combining baseline tabular data with electrocardiogram (ECG) in a single end-to-end model. ECG data were parsed using 1-D convolution and tubular data using multilayer perceptron. Results Of 2,258 cancer patients who had ICI therapy and troponin measurement (mean age 68.5 ± 11.5 years, 59.7% male), the composite of cardiovascular clinical adverse events, including ICI-related myocarditis and major adverse cardiovascular events, occurred in 264 (11.7%) unique patients, with 428 events overall (including 59 [3%] ICI-related myocarditis events and 59 [3%] cardiac deaths). The proposed joint fusion model outperformed individual ECG and baseline electronic medical record data and laboratory value models with an area under the operating characteristics curve of 0.72 (0.64 true positive rate and 0.98 negative predictive value). Conclusion A multimodal fusion AI model to predict myocarditis and adverse cardiovascular events in cancer patients starting ICI therapy had good prognostic performance. It may have clinical utility in identifying at-risk patients who may benefit from closer surveillance.