Exhausted intratumoral Vδ2- γδ T cells in human kidney cancer retain effector function [bulk RNA-seq]

Gamma delta (γδ) T cells reside within human tissues including tumors, but their role in mediating anti-tumor response with immune checkpoint inhibition is unknown. Using single-cell approaches, we found that kidney cancers are infiltrated by diverse Vδ2- γδ T cells, with equivalent representation of Vδ1+ and Vδ1- cells, that are distinct from γδ T cells found in normal human tissues. These tumor-resident Vδ2- T cells can express the transcriptional program of exhausted alpha beta (ab) CD8+ T cells as well as canonical markers of terminal T cell exhaustion including PD-1, TIGIT and TIM-3. While Vδ2- γδ T cells have blunted IL-2 production, they retain expression of cytolytic effector molecules and costimulatory receptors like 4-1BB. Exhausted Vδ2- γδ T cells are comprised of three distinct populations that lack TCF-7, are clonally expanded, express of cytotoxic molecules, and possess multiple Vδ2- TCRs. Human tumor-derived Vδ2- γδ T cells maintain cytotoxic function and pro-inflammatory cytokine secretion in vitro. The transcriptional program of Vδ2- T cells in pre-treatment tumor biopsies predicted subsequent clinical responses to PD-1 blockade in vivo in cancer patients. Thus, Vδ2- γδ T cells within the tumor microenvironment can contribute to anti-tumor efficacy. Tumor samples were obtained from resection surgery from 6 adult patients with histologically confirmed renal cell carcinoma (RCC). Samples were minced and digested, then FACS-sorted for live gd, CD4, and CD8 T cells. Bulk RNA libraries were constructed using the Smart-seq2 protocol and sequenced on an Illumina HiSeq 4000. The sequenced data was used to generate a variant call format (VCF) file containing single-nucleotide polymorphism (SNP) data for each patient (used later by Demuxlet to demultiplex pooled scRNA data generated from the same tumor samples).

γδ T细胞（Gamma delta T cells）定植于包括肿瘤在内的人体组织中，但其在联合免疫检查点阻断介导抗肿瘤应答过程中发挥的作用尚不明确。本研究通过单细胞技术发现，肾癌组织中浸润着多种Vδ2阴性γδ T细胞，其中Vδ1阳性与Vδ1阴性细胞的占比相当，且这群细胞与正常人体组织中的γδ T细胞存在显著差异。这群肿瘤驻留性Vδ2阴性T细胞可表达耗竭型αβ CD8阳性T细胞（alpha beta CD8+ T cells）的转录程序，同时也表达终末T细胞耗竭的经典标志物，包括PD-1、TIGIT及TIM-3。尽管Vδ2阴性γδ T细胞的白细胞介素2（IL-2）产生能力受损，但它们仍可表达溶细胞效应分子与共刺激受体（如4-1BB）。耗竭型Vδ2阴性γδ T细胞可分为三个互不相同的亚群，均不表达转录因子7（TCF-7），处于克隆扩增状态，可表达细胞毒性分子，并携带多种Vδ2阴性T细胞受体（TCR）。从人体肿瘤中分离的Vδ2阴性γδ T细胞在体外仍可维持细胞毒性功能与促炎性细胞因子分泌能力。治疗前肿瘤活检样本中Vδ2阴性T细胞的转录程序，可预测癌症患者体内后续接受PD-1阻断治疗的临床应答情况。由此可见，肿瘤微环境中的Vδ2阴性γδ T细胞可参与抗肿瘤效应的发挥。本研究的肿瘤样本来自6名经组织学确诊的肾细胞癌（Renal Cell Carcinoma, RCC）成人患者的手术切除标本。样本经切碎、消化处理后，通过荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）分选出活的γδ T细胞、CD4阳性T细胞与CD8阳性T细胞。采用Smart-seq2技术构建批量RNA文库，并在Illumina HiSeq 4000测序平台上完成测序。将测序所得数据用于生成变异调用格式（Variant Call Format, VCF）文件，其中包含每名患者的单核苷酸多态性（Single Nucleotide Polymorphism, SNP）数据，后续将通过Demuxlet工具对同一肿瘤样本的混合单细胞RNA测序数据进行解多重分析。