调控性T细胞（Treg）传统上被认为是内源性和治疗诱导的抗肿瘤免疫的抑制剂，然而，它们在调节免疫检查点阻断（ICB）反应中的作用尚不清楚。本研究将单细胞RNA测序/T细胞受体测序（TCRseq）与治疗前和抗PD-1治疗的非小细胞肺癌（NSCLC）中的>73,000肿瘤浸润性调节性T细胞（TIL-Treg）相结合，同时进行了基于小鼠肿瘤模型的肿瘤相关抗原（TAA）特异Treg的单细胞分析。我们发现10个人类TIL-Treg亚群，其中大多数与小鼠TIL-Treg亚群高度一致。只有一个亚群选择性地表达高水平的TNFRSF4（OX40）和TNFRSF18（GITR），其通过对应配体的结合介导了增殖程序和NF-κB活化，以及调节Treg抑制相关的多个基因，包括LAG3。功能上，OX40hiGITRhi亚群在体外具有最高的抑制作用，其在总TIL-Treg中的比例与对PD-1阻断的抵抗力相关。令人意外的是，在小鼠肿瘤模型中，我们发现几乎所有TIL-Treg表达的特异性TAA的T细胞受体，在进入肿瘤后经过2周时间内完全发展出明显的类似TH1的特征，下调FoxP3，上调TBX21（Tbet），IFNG和某些促炎性颗粒酶的表达。将小鼠TAA特异性TH1-like Treg亚群的基因评分应用于人类单细胞数据集的传输学习结果显示，这个亚群在抗PD-1治疗应答性肿瘤中富集。这些发现表明TIL-Treg分为多个不同的转录亚群，对ICB诱导的抗肿瘤免疫可能产生相互对立的影响，并且表明TAA特异性TIL-Treg可能积极促进抗肿瘤反应。

Regulatory T cells (Treg) are conventionally viewed as suppressors of endogenous and therapy-induced antitumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNA-seq/T cell receptor sequencing (TCRseq) of >73,000 tumor-infiltrating Treg (TIL-Treg) from anti-PD-1-treated and treatment-naive non-small cell lung cancers (NSCLC) with single-cell analysis of tumor-associated antigen (TAA)-specific Treg derived from a murine tumor model. We identified 10 subsets of human TIL-Treg, most of which have high concordance with murine TIL-Treg subsets. Only one subset selectively expresses high levels of TNFRSF4 (OX40) and TNFRSF18 (GITR), whose engangement by cognate ligand mediated proliferative programs and NF-κB activation, as well as multiple genes involved in Treg suppression, including LAG3. Functionally, the OX40hiGITRhi subset is the most highly suppressive ex vivo, and its higher representation among total TIL-Treg correlated with resistance to PD-1 blockade. Unexpectedly, in the murine tumor model, we found that virtually all TIL-Treg-expressing T cell receptors that are specific for TAA fully develop a distinct TH1-like signature over a 2-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFNG, and certain proinflammatory granzymes. Transfer learning of a gene score from the murine TAA-specific TH1-like Treg subset to the human single-cell dataset revealed a highly analogous subcluster that was enriched in anti-PD-1-responding tumors. These findings demonstrate that TIL-Treg partition into multiple distinct transcriptionally defined subsets with potentially opposing effects on ICB-induced antitumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to antitumor responses.