Background CD4 T cells have been implicated in cancer immunity for their helper functions. However, their direct cytotoxic potential remains elusive in cancer patients. Here, we aimed at assessing the presence, rate and cytotoxic function of tumor-specific Th-CTX directly in cancer patients.
Methods We capitalized on published single cell transcriptomic analyses of patient samples, integrated with the direct phenotypic and functional characterization of clonal, tumor-specific CD4 T cell populations, using peptide-MHC class II multimers and a novel high-throughput single-cell cytotoxicity assay in picowell arrays. The direct tumor cell killing by cytolytic tumor-specific CD4 T cells in the arrays was monitored in a high-throughput manner by combining multi-channel time-lapse microscopy with deep neural networks.
Results By mining single-cell RNA-seq datasets of tumor infiltrating lymphocytes, we identified CD4 T cells displaying cytotoxic phenotypes in different human tumors. The cytolytic CD4 T cells formed a distinct cluster and expressed genes related to classical cytotoxic functions, largely resembling CD8 T cell gene profiles. Using the peptide MHC class II multimer technology, we confirmed directly ex vivo the presence of cytolytic tumor antigen-specific CD4 T cells, both in the circulation and in the tumors of patients. We performed an integrated phenotypic and functional characterization of cytolytic tumor-specific CD4 T cells, down to the single cell level, through a high-throughput nanobiochip consisting of massive arrays of picowells with sub-nanoliter volumes and machine learning. We demonstrated a direct, contact-dependent, granzyme-dependent cytotoxic activity against tumor cells, with delayed kinetics compared to classical cytotoxic lymphocytes. Lastly, we discovered that this cytotoxic activity was at least in part dependent on the expression of SLAMF7, a homophylic receptor known to regulate NK cell activity.
Conclusions Our work provides a deep characterization of human Th-CTX in cancer and supports their role in tumor immunity. Moreover, our results showing that agonistic engagement of SLAMF7 enhances the cytolytic capacity of tumor-specific CD4 T cells, suggests that targeting these cells might prove synergistic with the use of other immunotherapies in cancer patients.
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