Neoantigen specific CD4+ T cells in human melanoma have diverse differentiation states and correlate with CD8+ T cell, macrophage, and B cell function

Tumor antigen-specific CD4+ T cells are required for the efficacy of immune checkpoint inhibitors in murine models but their contributions in human cancer are less understood. We used targeted single cell RNA sequencing and matching of T cell receptor sequences to identify signatures and functional correlates of tumor antigen-specific CD4+ T cells infiltrating human melanoma tumors. CD4+ T cells that recognize tumor-specific neoantigens express CXCL13 and are subdivided into clusters expressing memory and T follicular helper markers, and those expressing cytolytic markers, exhaustion markers and IFN-. In a cohort of melanoma patients, the frequency of CXCL13+ CD4+ T cells in the tumor correlated with the transcriptional states of CD8+ T cells and macrophages, maturation of B cells, and patient survival. Similar correlations were observed in a breast cancer cohort. These results identify distinct phenotypes and functional correlates of tumor antigen-specific CD4+ T cells in melanoma and suggest the possibility of using such cells to modify the tumor microenvironment. Cryopreserved single cell suspensions from melanoma tumors from 20 patients were stained with oligonucleotide labelled antibodies and sorted for either lymphocytes or non-lymphocyte CD45+ hematopoietic cells and subjected to single cell sequencing targeted to TCR and VDJ recombination events and a panel of immune response genes using the BD Rhapsody platform.