Abstract 4475: Site-specific profiling of TCR signaling

Abstract

Abstract Background: T cell exhaustion is a hyporesponsive state commonly found in cancer and is characterized by overexpression of inhibitory receptors and secretion of immunosuppressive cytokines. Immune checkpoint inhibitors (ICIs) target inhibitory receptors such as PD-1 and CTLA-4 to reinvigorate exhausted T cells and promote anti-tumor responses. While ICIs have improved clinical outcomes, no biomarker to date reliably determines the extent of T cell exhaustion. Since robust T cell receptor (TCR) signaling is critical for effective antitumor immunity, we hypothesized that interrogating the phospho-signaling states within the T cells along with their functional states will enhance our understanding of T cell exhaustion and yield potential biomarkers. Methods: In this study, we developed a validated mass cytometry panel of 24 markers to quantify phosphorylation states of 8 intracellular kinases involved in the TCR signaling within multiple CD8+ T cell subtypes. To identify potential differences between the local and peripheral immunological responses, we profiled CD8+ T cells derived from four sites (tumor, tumor draining lymph node, spleen and peripheral blood) in mice bearing MC38-induced flank tumors. We performed a clustering analysis of the dataset in aggregate using FlowSOM and visualized the results with UMAP, a dimensionality reduction algorithm, to compare the phospho-profiles of CD8+ T cell subtypes in all four sites. Results: Bulk analysis of CD8+ T cells demonstrates site-specific concordance between phosphoprotein markers and T cell functional markers (i.e. Ki67 and Granzyme B) with the highest expression in tumor followed by tumor draining lymph node, spleen and peripheral blood. Based on the results from the clustering analysis, site-specific breakdown of CD8+ T cells revealed high prevalence of effector, memory, and exhausted memory T cells in tumors compared to other sites where naïve T cells predominated. Despite these variations in CD8+ T cell immunophenotypic distribution, TCR signaling profiles correlated strongly with specific T cell subtypes across all sites. Conclusion: These data demonstrate that subtype-specific TCR signaling is preserved systemically and that phospho-immune subtyping of CD8+ T cells in the peripheral blood may be used to identify T cell exhaustion states that are found in the tumor microenvironment. Further studies will be conducted to investigate the changes in the TCR phospho-profiles in response to ICI treatment and their associated functions. Citation Format: Zaw Phyo, Rohan Verma, Won J. Ho, Elizabeth M. Jaffee. Site-specific profiling of TCR signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4475.