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. (10th December 2020)

• Record Type: Journal Article
• Authors: Aoyama, Shota; Nakagawa, Ryosuke; Nemoto, Satoshi; Villarroel, Patricio Perez; Mulé, James; Mailloux, Adam
• Is Part Of: Journal for immunotherapy of cancer. Volume 8(2020)Supplement 3
• Page Start: A134
• Page End: A135
• Publication Date: 2020-12-10
• Subjects: Cancer -- Immunotherapy -- Periodicals; Cancer -- Immunological aspects -- Periodicals; Tumors -- Immunological aspects -- Periodicals; Immunotherapy -- Periodicals; 616.99406105
• DOI: 10.1136/jitc-2020-SITC2020.0226
• Languages: English
• ISSNs: 2051-1426

Abstract

Abstract : Background: It 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. Methods: We 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. Results: Our 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. Conclusions: Despite evidence of an indirect benefit of early NKT activity, CB hastens a switch from TNF-alpha-driven NKT involvement toward a IFN-gamma-driven CD4+ and CD8+ T cell response in subcutaneous MC-38 tumors. These results corroborate earlier findings that CD4+ TIL are a major CB-responding population, and introduce a NKT/T cell switch that may be a key feature of the CB response in certain tumors. Ethics Approval: Animal experiments in this study were performed according to protocols approved by the University of South Florida's institutional animal care and use committee (IACUC) committee, number R IS00005710. Reference: Wei S, Levine J, Coghill A, Zhao Y, Anang N, Andrews M, Sharma P, Wang J, Wargo J, Pe'er D, Allison J. Distinct cellular mechanisms underlie anti-CTLA-4 and anti-PD-1 checkpoint blockade. Cell 2017 Sep 7; 170(6): 1120–1133.e17.