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. 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.抑制性蛋白如程序性死亡蛋白1（programmed cell death protein 1, PD-1），已在外周T细胞针对外来抗原、自身抗原以及新抗原的应答过程中得到广泛研究。值得注意的是，这类蛋白最早在胸腺内的T细胞发育阶段即开始表达。已有研究表明PD-1可限制调节性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明确。本研究拓展了对PD-1及其配体在胸腺中表达与功能的分析，结果显示，部分表达PD-1与PD-L1水平最高的细胞群体为经激动剂选择的细胞。令人意外的是，本研究揭示PD-1仅在调控调节性T细胞（Treg）的发育微环境中发挥选择性作用，而其他经激动剂选择的细胞群体（如自然杀伤T细胞（natural killer T cells））的发育状态未受影响。本研究同时排除了PD-1对经激动剂选择的Treg的增殖或细胞死亡的调控功能，并进一步证实PD-1缺陷型Treg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缺陷型胸腺细胞会产生水平升高的白细胞介素2（interleukin-2, IL-2）——一种可限制Treg发育微环境的细胞因子。综上，上述结果表明PD-1在调控IL-2生成以及胸腺Treg的同步激动剂选择过程中发挥了全新的功能。该发现对于癌症与感染场景下的免疫检查点阻断疗法的应用具有重要参考价值。本研究选取3周龄野生型（wild type, WT）或PD-1基因敲除的C57BL/6J小鼠胸腺，通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）分选出CD4+ HSA+ CD73- CD25-（CD4单阳性胸腺细胞）以及CD4+ HSA+ CD73- CD25+（调节性T细胞及CD25+调节性T细胞前体细胞），并拟通过单细胞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表达差异。