Background: Immune checkpoint inhibitors have transformed the treatment of several cancers by releasing restrained antitumor immune responses. Ipilimumab, an anti–cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) antibody, and nivolumab, an anti–programmed death-1 (PD-1) antibody, have individually improved survival in patients with melanoma, and their combination further enhances antitumor activity and survival. More recently, an anti-LAG3 human monoclonal antibody, Relatlimab, has been approved by FDA for combinatorial treatment with Nivolumab for metastatic melanoma and an anti-PD-L1 mAb,Atezolizumab, is undergoing clinical trials evaluation in combination with Ipilimumab for metastatic lung cancer.However,adverse events associated with these agents include dermatitis, endocrinopathies, colitis, hepatitis, and pneumonitis, which are all thought to arise from aberrant activation of autoreactive T cells. These toxic effects are more frequent and severe when immunomodulatory mAbs are used in combination. Methods: human cardiomyocytes co-cultured with hPBMC were exposed to monotherapy and combinatorial ICIs (PD-L1 and CTLA-4 or PD-1 and LAG-3 blocking agents, at 200 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 exertmore 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 PDL-1/CTLA-4 blocking agents, and 35.7% higher in PD-1/LAG-3 blocking agents compared to monotherapies. Biomarkers of cardiotoxicity, such as H-FABP and NT-Pro-BNP were also strongly increased in combination therapy than monotherapies. NLRP3 inflammasome, IL-1β and IL-6 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 on co-cultures of hPBMCs and cardiac cells, including for the first time the anti LAG-3 monoclonal antibody, induce a pro- inflammatory phenotype, thus indicating that these therapies should be closely monitored by the multidisciplinary team consisting of oncologists, cardiologists and immunologists.