Functionally heterogeneous intratumoral CD4+CD8+ double positive T cells can give rise to single positive T cells [bulkRNA-seq]

Conventional single positive (SP) CD4+ and CD8+ T cells recognize tumor antigens and help mediate clinical responses with cancer immunotherapy. Double positive CD4+CD8+ (DP) T cells have also been described in human cancers, but their role in the tumor microenvironment (TME) remains unclear. By generating a multi-omic single cell atlas of DP and SP T cells, we find that DP T cells possess phenotypic heterogeneity similar to SP T cells that includes multiple clonally expanded populations of cytotoxic DP T cells in human renal cell carcinoma (RCC). These intratumoral DP T cells can mediate by both MHC class I- and class II-dependent killing of autologous tumor cells. In addition, transcriptional profiling of DP TCR-bearing T cells revealed a gene signature enriched for clinical responders to PD-1 blockade in advanced RCC. We confirm prior observations of SP T cells transitioning into DP T cells and more notably, demonstrate that intratumoral T cells are capable of bidirectional differentiation in which DP T cells serve as precursors to SP T cells in vivo. In the latter scenario, intratumoral DP T cells are shown to express Rag2, suggesting that the tumor may act as an extrathymic site of T cell development. These findings reveal the multiple roles that DP T cells can possess in anti-tumor immunity. Overall design: To characterize T-cell heterogeneity in renal cell carcinoma (RCC), we performed single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq) using the 10x Genomics platform. Samples were collected from each patient's tumor tissue, adjacent normal tissue, and peripheral blood mononuclear cells (PBMCs), along with PBMCs from healthy donors. To assign cells to their respective individuals following sequencing, we applied demuxlet, which leverages donor-specific genetic variants derived from bulk RNA-seq to enable genotype-based sample demultiplexing.

传统单阳性（single positive, SP）CD4+及CD8+ T细胞可识别肿瘤抗原，并助力癌症免疫疗法介导临床应答。双阳性CD4+CD8+（double positive, DP）T细胞在人类癌症中已有报道，但其在肿瘤微环境（tumor microenvironment, TME）中的作用仍不明确。
通过构建DP与SP T细胞的多组学单细胞图谱，我们发现DP T细胞具有与SP T细胞相似的表型异质性：在人类肾细胞癌（renal cell carcinoma, RCC）中存在多个克隆扩增的细胞毒性DP T细胞亚群。这些瘤内DP T细胞可通过主要组织相容性复合体I类（Major Histocompatibility Complex, MHC class I）及II类（MHC class II）依赖的途径介导自体肿瘤细胞杀伤。此外，对表达T细胞受体（T cell receptor, TCR）的DP T细胞进行转录组分析发现，在晚期RCC中，该基因特征与PD-1阻断治疗的临床应答者显著富集相关。
我们验证了此前关于SP T细胞可转化为DP T细胞的观察结果，更重要的是，证实了瘤内T细胞具有双向分化能力——其中DP T细胞可作为体内SP T细胞的前体细胞。在后一种情况下，瘤内DP T细胞可表达Rag2，提示肿瘤可作为T细胞发育的胸腺外位点。上述研究结果揭示了DP T细胞在抗肿瘤免疫中可发挥的多重作用。

整体实验设计：为解析肾细胞癌（RCC）中的T细胞异质性，我们借助10x Genomics平台开展了单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）及单细胞TCR测序（single-cell TCR sequencing, scTCR-seq）。实验样本取自每名患者的肿瘤组织、癌旁正常组织及外周血单个核细胞（peripheral blood mononuclear cells, PBMCs），同时收集了健康供者的外周血单个核细胞。为在测序后将细胞分配至对应的供者个体，我们采用了demuxlet工具——该工具利用源自bulk RNA-seq的供者特异性遗传变异，实现基于基因型的样本拆分。