Single-Cell Analysis Identifies Distinct Populations of Cytotoxic CD4 T Cells Linked to the Therapeutic Efficacy of Immune Checkpoint Inhibitors in Metastatic Renal Cell Carcinoma.

BACKGROUND

The involvement of cytotoxic CD4 T cells (CD4 CTLs) and their potential role in dictating the response to immune checkpoint inhibitors (ICIs) in patients with metastatic renal cell carcinoma (mRCC) remains an unexplored area of research.

METHODS

Utilizing single-cell RNA sequencing, we analyzed the immunophenotype and expression patterns of CD4 T lymphocyte subtypes in mRCC patients, followed by preliminary validation via multi-immunofluorescent staining. In addition, we obtained a comprehensive immunotherapy dataset encompassing single-cell RNA sequencing datasets and bulk RNA-seq cohorts from the European Genome-Phenome Archive and ArrayExpress database. Utilizing the CIBERSORTx deconvolution algorithms, we derived a signature score for CD4 CTLs from the bulk-RNA-seq datasets of the CheckMate 009/025 clinical trials.

RESULTS

Single-cell analysis of CD4 T lymphocytes in mRCC reveals several cancer-specific states, including diverse phenotypes of regulatory T cells. Remarkably, we observe that CD4 CTLs cells constitute a substantial proportion of all CD4 T lymphocyte sub-clusters in mRCC patients, highlighting their potential significance in the disease. Furthermore, within mRCC patients, we identify two distinct cytotoxic states of CD4 T cells: CD4GZMK T cells, which exhibit a weaker cytotoxic potential, and CD4GZMB T cells, which demonstrate robust cytotoxic activity. Both regulatory T cells and CD4 CTLs originate from proliferating CD4 T cells within mRCC tissues. Intriguingly, our trajectory analysis indicates that the weakly cytotoxic CD4GZMK T cells differentiate from their more cytotoxic CD4GZMB counterparts. In comparing patients with lower CD4 CTLs levels to those with higher CD4 CTLs abundance in the CheckMate 009 and 25 immunotherapy cohorts, the latter group exhibited significantly improved OS and PFS probability.

CONCLUSION

Our study underscores the pivotal role that intratumoral CD4 CTLs may play in bolstering anti-tumor immunity, suggesting their potential as a promising biomarker for predicting response to ICIs in patients with mRCC.