Single-cell analysis identifies distinct populations of cytotoxic CD4+ T cells (CD4+ CTLs) linked to the therapeutic efficacy of immune checkpoint inhibitors in metastatic renal cell carcinoma.

Purpose: In this study, we systematically evaluated CD4 + T cell subtypes and investigated the correlation between tumor-specific cytotoxic CD4 + T cells (CD4 + CTLs) and the therapeutic efficacy in metastatic renal cell carcinoma (mRCC) patients. Methods The expression patterns of CD4 + T lymphocytes subtypes in RCC patients were systematically characterized using CD4 + T lymphocytes single-cell RNA sequencing, and verification via immunofluorescent staining techniques. Datasets of mRCC patients undergoing immunotherapy were analyzed to assess the synergistic role of CD4 + CTLs in predicting responses to immune checkpoint inhibitors (ICIs). The CD4 + CTLs signature score was derived from the CheckMate 009, 010, and 025 bulk RNA-seq datasets using CIBERSORTx algorithms. Results We found that cytotoxic CD4 + T cells accounted for a significant proportion among all CD4 + T lymphocyte sub-clusters in mRCC patients. Additionally, two distinct cytotoxic states, CD4 + GZMK + T cells with weak cytotoxic activity and CD4 + GZMB + T cells with strong cytotoxic activity, were identified in mRCC patients. Both regulatory T cells and CD4 + CTLs were derived from proliferating CD4 + T cells in mRCC tissues. Moreover, trajectory analysis showed that weak cytotoxic CD4 + GZMK + T cells differentiated from higher cytotoxic CD4 + GZMB + T cells. Higher CD4 + CTLs abundance group showed better prognosis in the CheckMate 009, 010, and 025 immunotherapy cohorts. Conclusions Our study suggests that intratumoral CD4 + CTLs may play a crucial role in anti-tumor immunity and could serve as a potential marker for predicting the therapeutic efficacy of immune checkpoint inhibitors in mRCC patients.