Upregulation of the PD-1 pathway in non-ICI myocarditis

Immune checkpoint (IC) inhibitors are a potent line of anti-cancer treatment that disable the brake system of T-cells, inducing their anti-tumor effect. However, a notorious complication of their application is IC inhibitor-mediated myocarditis. This suggests that ICs play a key immunoregulatory role within the heart, however its mechanism remains unknown. Therefore, we investigated the role of the IC programmed cell death protein 1 (PD-1) and its ligand, PD-L1, signaling in the heart in non-ICI myocarditis.

In a comparative analysis, we assessed 1) a T1L murine myocarditis model via single-cell RNA sequencing (scRNAseq) data; 2) a CVB3 and 3) a MYH6-specific T-cell receptor transgenic mice (TCRM) murine myocarditis model both via single-nucleus RNA sequencing (snRNAseq). All three murine models revealed that myocarditis induced a significant increase in PD-L1 gene expression in endothelial cells (ECs) and cardiac fibroblasts (CFs). In cardiomyocytes (CMs), this response was only observed in the CVB3 model. Additionally, T-cells displayed elevated PD-1 levels, rising and falling over time in line with the amount of myocardial inflammation. A translational validation in a snRNAseq dataset of human endomyocardial biopsies of acute myocarditis was performed, revealing elevated PD-L1 and PD-1 expression in all the respective cell types. Cell-cell communication analyses of the murine models identified predominant communication between PD-L1 on ECs and PD-1 on T-cells. Functionally, gene ontology pathway analyses revealed that PD-L1 expressing ECs and CFs exhibited increased immune interactions and nonprofessional antigen presenting cell (APC) properties. Finally, we performed in vitro experiments where stimulation with tumor necrosis factor alpha (TNFα), a cytokine known for its pro-inflammatory effects in myocarditis, significantly increased de novo gene and protein PD-L1 expression on human ECs and CFs, but not in CMs. These cells also revealed APC properties, further corroborating the findings in the various sequencing datasets.

These data illustrate a robust and consistent activation of the PD-1/PD-L1 pathway in non-ICI myocarditis. Specifically, ECs and CFs exhibit nonprofessional APC properties upon presentation of PD-L1, peaking at the same time as PD-1 on T-cells, which presents an important defense mechanism by these cells. Therefore, ICs have global importance in myocarditis and may serve as a therapeutic target.