Decoding T cell clonotypes, biology and predictive biomarkers associated with immune checkpoint Myocarditis - The success of immune checkpoint inhibitor (ICI) therapy is often limited by immune-related adverse events (irAEs) that are accompanied by significant morbidity, often leading to treatment termination and in some cases resulting in death. This proposal focuses on immune-related (ir)Myocarditis, which is the most fatal irAE. As the incidence of irMyocarditis continues to rise through the increasing use of ICI combination regimens, thousands of cancer patients will face the morbidity and mortality of irMyocarditis. Despite its high morbidity, little is known about the mechanistic underpinnings of irMyocarditis, resulting in limited targeted treatment solutions that currently involves general immunosuppression that may inhibit anti-tumor response and interruption of their life- saving ICI therapy. Together, this emphasizes the urgent need for our patient-centric project to decipher the mechanisms leading to irMyocarditis and identify better mitigation strategies. Our preliminary single-cell RNAseq (scRNA-seq) data from heart tissue (n=84,576 cells) and blood (n= 366,066 cells) across 15 cases and 8 controls showed that irMyocarditis heart tissue is enriched with T cell, myeloid cell, dendritic cell (DC), and fibroblast populations. Associated cytotoxic T lymphocytes (CTL) spanned a spectrum defined by markers of cytotoxicity (GZMK) and exhaustion/activation (PDCD1, LAG3). T cell receptor sequencing (TCR-seq) showed clonal expansion of these heart CTLs that were detectable in paired blood. TCR-seq of 4 paired tumor, irMyocarditis heart and control heart showed distinct expanded TCR clones in paired tumor and irMyocarditis heart, suggesting that the irMyocarditis expanded TCR clones may recognize a self-antigen. We showed that the 52 most expanded heart TCRs do not recognize alpha-myosin, previously associated with irMyocarditis, suggesting the involvement of other cardiac autoantigen(s). DCs were downregulated in blood and upregulated in irMyorcarditis heart, supporting the role for DC in disease pathogenesis. These preliminary results were the genesis our central hypothesis that ICI therapy prompt self-antigen loaded DC to prime and amplify quiescent autoreactive cytotoxic CD8+ T cells targeting cardiac proteins, leading to profound alteration of the heart microenvironment and harmful inflammatory responses. To test this hypothesis, we will seek to identify and validate novel cardiac autoantigens recognized by the top 30 heart-expanded TCRs we defined, using complementary T-Scan unbiased approach (Aim 1.1) and ex vivo T cell assays (Aim 1.2), and then probe for the presence of autoreactive T cells in circulation (Aim 1.3). Aim 2 will study the spatial interactions of expanded CTLs populations with DCs, other immune cells, cardiomyocytes, and stromal cells using spatial transcriptomics analysis to gain mechanistic insights into cell- cell interactions and signaling pathways driving fatal and non-fatal irMyocarditis (Aims 2.1-2.2), and define the biology conserved in the myocarditis Pdcd1-/-Ctla4+/- model (Aim 2.3). We will assess the impact of therapeutic interventions in reshaping the cellular ecosystem sustaining irMyocarditis in Pdcd1-/-Ctla4+/- mice (Aim 2.4). These results will provide new mechanistic insights and nominate therapeutic targets to mitigate irMyocarditis.
