Transcriptional profiling of tumor infiltrating and ciculating T lymphocytes of mice harboring MOC1 tumors

Combination TGF-β neutralization and PD-L1 immune checkpoint blockade promotes anti-tumoral systemic immunity by enhancing tumor egress of CD8+ TILs. This enhanced egress associated with reduced expression of the tissue residency marker Itgae and its upstream regulator Znf683, with the circulating CD8+ T cells expressing higher Cxcr3, an observation made as well in samples from patients treated with dual TGF- neutralization and ICB. Tumor tissues from five individual mice were processed and cell suspensions were stained with anti-mouse TCR-β and T cells were sorted to >99% purity. Two captures per treatment of 10,000 cells each were performed. Cells were mixed with barcoded gel beads and 5’ GEM Kit v3 reagents and single cell capture was performed (10X Genomics). Following reverse transcription, cDNA and murine TCRs were amplified, and gene expression and TCR sequencing libraries were re-constructed. Each DNA library was loaded into a sequencing lane on a NovaSeq system. Spleens and tumor draining lymph nodes from individual mice were processed and T cells were isolated using the EasySep Mouse T cell Isolation Kit from StemCell Technologies. RNA was extracted from all sorted samples using the RNEasy Mini Kit and deep TCR sequencing was performed using the SMARTer Mouse TCR Profiling Kit from MiLaboratories.

联合转化生长因子β（TGF-β）中和与程序性死亡受体配体1（PD-L1）免疫检查点阻断，可通过增强CD8+肿瘤浸润淋巴细胞（CD8+ Tumor-Infiltrating Lymphocytes, TILs）的肿瘤迁出能力，促进抗肿瘤系统性免疫应答。该增强的迁出能力与组织驻留标志物Itgae及其上游调控因子Znf683的表达下调相关，同时循环CD8+ T细胞的CXC趋化因子受体3（Cxcr3）表达水平升高；这一观测结果在接受双重TGF-β中和与免疫检查点阻断（ICB）治疗的患者样本中同样存在。本研究对五只个体小鼠的肿瘤组织进行处理，制备细胞悬液后使用抗小鼠TCR-β抗体进行染色，随后将T细胞分选至纯度大于99%。每个处理组设置两次单细胞捕获，每次捕获10000个细胞。将细胞与带条形码的凝胶微珠及5’ GEM Kit v3试剂盒试剂混合后，依托10X Genomics平台完成单细胞捕获。逆转录反应完成后，扩增互补脱氧核糖核酸（cDNA）与小鼠T细胞受体（TCR），并重建基因表达文库与TCR测序文库。将每个DNA文库加载至NovaSeq测序系统的测序泳道中进行测序。此外，本研究对个体小鼠的脾脏及肿瘤引流淋巴结进行处理，采用StemCell Technologies公司的EasySep小鼠T细胞分离试剂盒分离T细胞。使用RNEasy Mini试剂盒从所有分选后的样本中提取核糖核酸（RNA），并使用MiLaboratories公司的SMARTer小鼠TCR分型试剂盒完成深度TCR测序。