PD-1 restrains thymic IL-2 production and regulatory T cell development. PD-1 restrains thymic IL-2 production and regulatory T cell development

Inhibitory proteins, such as programmed cell death protein 1 (PD-1), have been extensively studied in peripheral T cell responses to foreign, self, and neoantigens. Notably, these proteins are first expressed during T cell development in the thymus. Reports suggest that PD-1 limits regulatory T cell (Treg) development, but the mechanism by which PD-1 exerts this function remains unknown. The present study expands the evaluation of PD-1 and its ligands in the thymus, demonstrating that some of the highest expressers of PD-1 and PD-L1 are agonist selected cells. Surprisingly, we reveal a selective role for PD-1 in regulating the developmental niche only for Tregs as other agonist selected cell populations, such as natural killer T cells, remain unchanged. We also ruled out PD-1 as a regulator of proliferation or cell death of agonist selected Tregs and further demonstrated that PD-1 deficient Tregs have reduced TCR signaling. Unexpectedly, the data suggests that PD-1 deficient thymocytes produce elevated levels of IL-2, a Treg niche limiting cytokine. Collectively, these data suggest a novel role for PD-1 in regulating IL-2 production and the concurrent agonist selection of thymic Tregs. This observation has implications for the use of checkpoint blockade in the context of cancer and infection. Overall design: Thymus from 3-week-old WT or PD-1 knockout animals (C57BL/6J) were processed for flourescence-activated cell sorting (FACS) to sort CD4+ HSA+ CD73- CD25- (CD4 single positive thymocytes) and CD4+ HSA+ CD73- CD25+ (regulatory T cells and CD25+ regulatory T cell progenitors). We sought to examine differences between the WT and PD-1 knockout cells using scRNAseq.

程序性细胞死亡蛋白1（programmed cell death protein 1, PD-1）等抑制性蛋白，在外周T细胞针对外源抗原、自身抗原以及新抗原的免疫应答中已被广泛研究。值得注意的是，这类蛋白在胸腺内的T细胞发育阶段便已首次表达。有研究指出PD-1会限制调节性T细胞（regulatory T cell, Treg）的发育，但PD-1发挥该功能的具体分子机制仍未阐明。本研究拓展了PD-1及其配体在胸腺中的表达与功能评估，结果显示PD-1与PD-L1的部分高表达群体为激动剂选择细胞。令人意外的是，我们发现PD-1仅在调控调节性T细胞的发育微环境中发挥选择性作用，而其他激动剂选择的细胞群体（如自然杀伤T细胞（natural killer T cell））则无明显变化。我们还排除了PD-1作为激动剂选择的调节性T细胞增殖或细胞死亡调控因子的可能性，并进一步证实PD-1缺陷的调节性T细胞的T细胞受体（T cell receptor, TCR）信号通路活性降低。出乎意料的是，数据显示PD-1缺陷的胸腺细胞会产生更高水平的IL-2——一种限制调节性T细胞发育微环境的细胞因子。综上，本研究数据表明PD-1在调控IL-2产生以及胸腺调节性T细胞的同步激动剂选择过程中具有全新的生物学功能。这一发现对于癌症与感染场景下的免疫检查点阻断治疗具有重要参考价值。
整体实验设计：选取3周龄野生型（wild type, WT）或PD-1基因敲除（knockout, KO）的C57BL/6J品系小鼠，获取其胸腺组织并进行处理，通过荧光激活细胞分选（flourescence-activated cell sorting, FACS）分选出CD4+ HSA+ CD73- CD25- 细胞（CD4单阳性胸腺细胞）以及CD4+ HSA+ CD73- CD25+ 细胞（调节性T细胞与CD25+调节性T细胞前体）。我们旨在通过单细胞RNA测序（single-cell RNA sequencing, scRNAseq）分析野生型与PD-1基因敲除细胞之间的转录组差异。