CD4 T cells have been implicated in cancer immunity for their helper functions. Moreover, their direct cytotoxic potential has been shown in some patients with cancer. Here, by mining single-cell RNA-seq datasets, we identified CD4 T cell clusters displaying cytotoxic phenotypes in different human cancers, resembling CD8 T cell profiles. Using the peptide-MHCII-multimer technology, we confirmed ex vivo the presence of cytolytic tumor-specific CD4 T cells. We performed an integrated phenotypic and functional characterization of these cells, down to the single-cell level, through a high-throughput nanobiochip consisting of massive arrays of picowells and machine learning. We demonstrated a direct, contact-, and granzyme-dependent cytotoxic activity against tumors, with delayed kinetics compared to classical cytotoxic lymphocytes. Last, we found that this cytotoxic activity was in part dependent on SLAMF7. Agonistic engagement of SLAMF7 enhanced cytotoxicity of tumor-specific CD4 T cells, suggesting that targeting these cells might prove synergistic with other cancer immunotherapies.

CD4 T 细胞因其辅助功能而与癌症免疫有关。此外，它们的直接细胞毒性潜力已在一些癌症患者中显示出来。在这里，通过挖掘单细胞 RNA-seq 数据集，我们确定了 CD4 T 细胞簇在不同的人类癌症中表现出细胞毒性表型，类似于 CD8 T 细胞谱。使用肽-MHCII-多聚体技术，我们在体外证实了溶细胞性肿瘤特异性 CD4 T 细胞的存在。我们通过由大量皮孔阵列和机器学习组成的高通量纳米生物芯片对这些细胞进行了综合表型和功能表征，直至单细胞水平。我们展示了对肿瘤的直接、接触和颗粒酶依赖性细胞毒活性，与经典细胞毒淋巴细胞相比具有延迟的动力学。最后的，我们发现这种细胞毒活性部分依赖于 SLAMF7。SLAMF7 的激动作用增强了肿瘤特异性 CD4 T 细胞的细胞毒性，这表明靶向这些细胞可能与其他癌症免疫疗法具有协同作用。