Background CD4 T cells help coordinating adaptive immune responses. Increasing evidence shows that antigen-specific CD4 T cells are also key players in anti-tumor immunity. We and others recently reported on the frequent existence of fully active, tumor-specific CD4 T cells with cytolytic properties in cancer patients. However, the molecular determinants regulating CD4 T cell cytotoxicity in cancer are poorly defined.

Methods Using peptide-MHC class II fluorescent multimers, we isolated tumor-specific CD4 T cells from peripheral blood and tumor tissues of cancer patients. These cells were classified as helper or cytolytic according to their ability to directly eliminate MHC class II-matched targets.

Results Next, by integrating ex vivo peripheral blood single cell and in situ spatial transcriptomics, we identified key molecules potentially involved in the cytolytic mechanism. To validate the relevance of these molecules in primary human CD4 T cells, we used either pharmacologic inhibitors or CRISPR/Cas9 technology. Killing capacity and immunological synapse formation of wild-type and inhibited/gene-edited cells are evaluated down to single cell level resolution using live cell imaging techniques. Ultimately, our findings will be validated in vivo by adoptive cell transfer of wild-type or gene-edited CD4 T cells in human tumor-bearing NSG mice.

Conclusions Overall, we will provide clues to exploit cytolytic CD4 T cells in the clinics alone or in combination with other cancer immunotherapies.