2904 - Radiation Enhances PD-L1 in Cardiomyocytes: Implications for Combined Radiation and Anti-Programmed Death-1 (PD-1) Antibody-Induced Myocardial Injury

Y. Zhou¹, Y. Wu², N. Zhang³, Q. Li⁴, and J. Wang²; ¹Hebei General Hospital, Shijiazhuang, China, ²Department of Radiation Oncology, the Fourth Hospital of Hebei Medical University, Shijiazhuang, China, ³Shijiazhuang People’s Hospital, Shijiazhuang, China, ⁴General Hospital of Hebei, Shijiazhuang, Hebei, China, China

Purpose/Objective(s): Radiation and immunotherapy combined-induced myocardial injury has potentially related to CD8⁺ T cells, but the mechanism is unclear. This study aims to explore the effects of on the expression of PD-L1 in cardiomyocytes and its role in the occurrence of myocardial injury induced by radiation and anti-programmed death-1 (PD-1) antibody combined. Materials/

Methods: Human ventricular cardiomyocytes (AC16) were irradiated at varying doses and time points, and PD-L1 levels were assessed using flow cytometry, real-time qPCR, Western blot, and immunofluorescence staining. A cell counting kit cell viability assay, flow cytometry apoptosis, and ELISA detection were performed on AC16 cells before and after radiation. Subsequently, siRNA knockdown of PD-L1 was used to verify its role in radiation-induced myocardial injury. Then, simulation of radiation and PD-1 antibody-induced myocardial injury was conducted in vitro. Activated and inactivated human CD8+ T cells were prepared and co-cultured with cardiomyocytes, with or without PD-L1 knockdown, before and after radiation with PD-1 antibody, the vitality of cardiomyocytes in each group was detected by a cell counting kit, and the expression of PD-1, IFN-?, and granzyme B of co-culture CD8+ T cells were detected by flow cytometry and ELISA, respectively.
Results: 10Gy radiation significantly upregulated PD-L1 expression in AC16 cells in a time-dependent manner (p<0.001), while PD-L2 expression was not significantly expressed. and PD-L1 was enriched in the nucleus after 72h of radiation. Following radiation, AC16 cells viability decreased, apoptosis increased, and the expression of IL-6, CCL5, and CXCL10 increased, while IL-10 expression decreased. siRNA knockdown of PD-L1 had no significant effect on the viability and apoptosis (P>0.05), but significantly upregulated the expression of IL-6, CCL5, CXCL10 (p<0.0001), suggesting the chemotaxis of CD8⁺ T cells had enhanced. Co-culture of activated and inactivated CD8⁺ T cells with AC16 cells before and after radiation therapy showed that the cytotoxicity of the activated group was significantly higher than that of the inactivated group. The co-culture of activated CD8+ T cells with AC16 cells demonstrated that AC16 cell vitality was lowest in the combined group, followed by the single radiation therapy group, then the single PD-1 antibody group, and all were lower than the normal co-culture group (p < 0.01). SiPD-L1 co-culture revealed that the PD-L1 knockdown group exhibited lower vitality than the control group, regardless of radiation (p < 0.01). Additionally, PD-1, IFN-?, and granzyme B expression in CD8+ T cells was higher in the co-culture radiation group compared to the non-radiation and control CD8+ T cell groups.
Conclusion: Radiation increases PD-L1 expression in ventricular cardiomyocytes AC16, enhancing CD8+ T cell chemotaxis. The exacerbation of myocardial injury in combined radiation and PD-1 antibody treatment may be due to PD-1/PD-L1 pathway blockade and upregulation of CD8+ T cell function.