Identification and Phenotypic Characterization of Neoantigen-Specific Cytotoxic CD4⁺ T Cells in Endometrial Cancer

Tumor-reactive CD4⁺ T cells often accumulate in the tumor microenvironment (TME) in human cancer, but their functions and roles in antitumor responses remain elusive. Here, we investigated the immunopeptidome of HLA class II–positive (HLA-II⁺) endometrial cancer with an inflamed TME using a proteogenomic approach. We identified HLA-II neoantigens, one of which induced polyclonal CD4⁺ tumor-infiltrating lymphocyte responses. We then experimentally demonstrated that neoantigen-specific CD4⁺ tumor-infiltrating lymphocytes lyse target cells in an HLA-II–dependent manner. Single-cell transcriptomic analysis of the TME coupled with T-cell receptor sequencing revealed the presence of CD4⁺ T-cell clusters characterized by CXCL13 expression. The CXCL13⁺ clusters contained two subclusters with distinct cytotoxic gene expression patterns. The identified neoantigen-specific CD4⁺ T cells were found exclusively in one of the CXCL13⁺ subclusters characterized by granzyme B and CCL5 expression. These results demonstrate the involvement of tumor-reactive CD4⁺ T cells with cytotoxic function in immune surveillance of endometrial cancer and reveal their transcriptomic signature.