Checkpoint blockade accelerates a novel switch from an NKT-driven TNFα response toward a T cell driven IFN-γ response within the tumor microenvironment

BACKGROUND: The temporal response to checkpoint blockade (CB) is incompletely understood. Here, we profiled the tumor infiltrating lymphocyte (TIL) landscape in response to combination checkpoint blockade at two distinct timepoints of solid tumor growth. METHODS: C57BL/6 mice bearing subcutaneous MC38 tumors were treated with anti-PD-1 and/or anti-CTLA-4 antibodies. At 11 or 21 days, TIL phenotype and effector function were analyzed in excised tumor digests using high parameter flow cytometry. The contributions of major TIL populations toward overall response were then assessed using ex vivo cytotoxicity and in vivo tumor growth assays. RESULTS: The distribution and effector function among 37 distinct TIL populations shifted dramatically between early and late MC38 growth. At 11 days, the immune response was dominated by Tumor necrosis factor alpha (TNFα)-producing NKT, representing over half of all TIL. These were accompanied by modest frequencies of natural killer (NK), CD4(+), or CD8(+) T cells, producing low levels of IFN-γ. At 21 days, NKT populations were reduced to a combined 20% of TIL, giving way to increased NK, CD4(+), and CD8(+) T cells, with increased IFN-γ production. Treatment with CB accelerated this switch. At day 11, CB reduced NKT to less than 20% of all TIL, downregulated TNFα across NKT and CD4(+) T cell populations, increased CD4(+) and CD8(+) TIL frequencies, and significantly upregulated IFN-γ production. Degranulation was largely associated with NK and NKT TIL. Blockade of H-2kb and/or CD1d during ex vivo cytotoxicity assays revealed NKT has limited direct cytotoxicity against parent MC38. However, forced CD1d overexpression in MC38 cells significantly diminished tumor growth, suggesting NKT TIL exerts indirect control over MC38 growth. CONCLUSIONS: Despite an indirect benefit of early NKT activity, CB accelerates a switch from TNFα, NKT-driven immune response toward an IFN-γ driven CD4(+)/CD8(+) T cell response in MC38 tumors. These results uncover a novel NKT/T cell switch that may be a key feature of CB response in CD1d(+) tumors.