Unbiased Multiparameter Analyses Reveal Mechanisms of Variable Restimulation-Induced Cell Death Sensitivity across Human T cell Populations 2251586

Abstract Introduction Restimulation-Induced Cell Death (RICD) is a crucial self-regulatory apoptosis pathway that constrains effector T cell expansion during an adaptive immune response. Our lab and others have implicated multiple genes involved in regulating RICD sensitivity, but molecular mechanisms that explain highly hetergeneous RICD susceptibility across healthy human donors remain nebulous. We hypothesized that relative RICD sensitivity could be explained by specific gene expression programs that correspond to functional differentiation states of multiple, distinct effector T cell subsets. Methods We took an unbiased approach to this question by amassing extensive phenotypic data on T cells collected from >50 healthy human donors, employing spectral flow cytometry and scRNA-seq on effector T cells from the most highly resistant vs. sensitive individuals. Results Our results unveiled enrichment of CD8+ T cells expressing senescence (CD57, NKG2D) and exhaustion (TIGIT) markers in highly sensitive donors; indeed, FACS-based purification confirmed CD8+CD57+ T cells were markedly more susceptible to RICD compared to CD57- counterparts. Moreover, scRNA-seq revealed upregulation of PI-3K pathway genes in RICD sensitive donors, consistent with enhanced PI-3K signaling in senescent T cells. RICD sensitive donors also harbored more “aged” CD4+ T cells expressing ZEB2 and cytotoxicity genes (PRF1, GZMB/K, GNLY, NKG7). Conversely, RICD-resistant CD4+ and CD8+ effector T cells were enriched for genes/markers corresponding to more stem-like properties (IL7R, SELL, CD27, LRRN3, LEF1, BACH2). Indeed, more naïve/central memory T cells were observed in initial PBMC collected from RICD-resistant donors. Conclusion Collectively, our work provides the first “atlas” of human RICD variability that can be used to elucidate and predict relative RICD susceptibility across humans donors, informing new approaches for optimizing adoptive T cell immunotherapies and understanding capricious T cell responses to infection/immunization. Funding Source NIH R35GM139619 Topic Categories Lymphocyte Differentiation and Peripheral Maintenance (LYM)