226 Checkpoint blockade hastens a switch from an NKT dominant, TNF-alpha-driven to a CD4+/CD8+ IFN-gamma-driven immune response within MC-38 tumor-infiltrating lymphocytes

BackgroundIt is incompletely understood which populations of tumor-infiltrating lymphocytes (TIL) respond to checkpoint blockade (CB) and when. Recent studies in murine MC-38 colon carcinoma demonstrate CD4+ T cells are among the most prominent responders,1 but these studies were undertaken late in tumor growth, weeks after CB blockade was initiated. Here, we profile how the landscape of CB-responding TIL change between early and late MC-38 tumor growth, and uncover a novel switch that occurs between natural killer T (NKT) and conventional CD4/CD8 T cell responses.MethodsWe treated C57BL/6 mice bearing subcutaneous MC-38 tumors with anti-PD-1 and/or anti-CTLA-4 antibodies, and analyzed TIL 11 or 21 days later using a 23-paramter flow cytometry panel that includes three markers of effector function: TNF-alpha, IFN-gamma, and CD107a. We then investigated major populations, including NKT TIL, in ex vivo cytotoxicity assays and in vivo tumor growth studies using CD1d overexpressin MC-38 cells.ResultsOur analysis identified 37 TIL populations in MC-38 tumors, representing CD4+ or CD8+ T cells, natural killer (NK), and NKT cells. The distribution and effector function among TIL shift dramatically between early and late MC-38 growth. At 11 days, the immune response is dominated by TNF-alpha-producing NKT, which represent 53.5 ± 3.7% of all TIL. These are accompanied by modest frequencies of CD4+ and CD8+ TIL, producing low levels of IFN-gamma. After 21 days, NKT populations are reduced to 15.2 ± 1.5%, giving way to increased NK, CD4+, and CD8+ TIL, with increased IFN-gamma production. CB hastens this switch, markedly reducing NKT to less than 20% of all TIL, downregulating TNF-alpha production across NKT and CD4+ T cell subpopulations, increasing CD4+ and CD8+ TIL frequencies, and significantly up-regulating IFN-gamma production at 11 days. CD107a expression patterns suggest degranulation is most associated with NK and NKT TIL (figure 1). NKT displayed no CD1d-restricted cytotoxicity against MC-38 ex vivo. However, CD1d overexpression on MC-38 significantly delayed tumor growth in vivo, suggesting early NKT activity may indirectly suppress tumor progression, but by what precise mechanism(s) is currently unknown.Abstract 226 Figure 1t-SNE analysis of effector TIL populations identifies distinct, IFN-gamma and TNF-alpha-producing cells at early (day 11) and late (day 21) time points of subcutaneous MC38 growth. (a) Combined pseudocolored density plot of t-SNE param