Single cell RNAseq of Wild Type and IL27ra KO Tumor infiltrating lymphocytes isolated from B16F10 melanoma batch 5

Expression of co-inhibitory receptors, such as CTLA-4 and PD-1, on effector T cells is a key mechanism for ensuring immune homeostasis. Dysregulated co-inhibitory receptor expression on CD4+ T cells promotes autoimmunity while sustained overexpression on CD8+ T cells promotes T cell dysfunction or exhaustion, leading to impaired ability to clear chronic viral infections and cancer. Here, we used RNA and protein expression profiling at single-cell resolution to identify a module of co-inhibitory receptors that includes not only several known co-inhibitory receptors (PD-1, Tim-3, Lag-3, and TIGIT), but also a number of novel surface receptors. We functionally validated two novel co-inhibitory receptors, Activated protein C receptor (Procr) and Podoplanin (Pdpn). The module of co-inhibitory receptors is co-expressed in both CD4+ and CD8+ T cells and is part of a larger co-inhibitory gene program that is shared by non-responsive T cells in multiple physiological contexts and is driven by the immunoregulatory cytokine IL-27. Computational analysis identified the transcription factors Prdm1 and c-Maf as cooperative regulators of the co-inhibitory module, which we validated experimentally. This molecular circuit underlies the co-expression of co-inhibitory receptors in T cells and identifies novel regulators of T cell function with the potential to regulate autoimmunity and tumor immunity. Single cell RNAseq analysis of IL-27 induced T cell gene signature in the tumor

效应T细胞上的共抑制受体（co-inhibitory receptors），如CTLA-4与PD-1的表达，是维持免疫稳态的关键机制。CD4+ T细胞上共抑制受体的表达失调会诱发自身免疫病，而CD8+ T细胞上共抑制受体的持续过表达则会导致T细胞功能异常或耗竭，使其清除慢性病毒感染与肿瘤的能力受损。本研究采用单细胞分辨率的RNA与蛋白质表达谱分析，鉴定出一组共抑制受体模块，该模块不仅包含数种已报道的共抑制受体（PD-1、Tim-3、Lag-3及TIGIT），还涵盖了多种全新的表面受体。我们通过功能实验验证了两种全新的共抑制受体：活化蛋白C受体（Activated protein C receptor，Procr）与Podoplanin（Pdpn）。该共抑制受体模块在CD4+与CD8+ T细胞中均存在共表达现象，且属于更大范围的共抑制基因程序的一部分；该基因程序在多种生理环境下的无应答T细胞中均存在，并由免疫调节性细胞因子IL-27所驱动。通过计算分析，我们鉴定出转录因子（transcription factors）Prdm1与c-Maf是该共抑制受体模块的协同调控因子，并通过实验进行了验证。该分子环路是T细胞中共抑制受体共表达的分子基础，同时也发现了全新的T细胞功能调控因子，其有望用于调控自身免疫与肿瘤免疫。针对肿瘤中IL-27诱导的T细胞基因特征的单细胞RNA测序（single cell RNAseq）分析。