Lung tumor-infiltrating Treg have divergent transcriptional profiles and function linked to checkpoint blockade response [mouse]

Regulatory T cells (Treg) are conventionally viewed to suppress endogenous and therapy-induced anti-tumor immunity; however, their role in modulating responses to immune checkpoint blockade (ICB) is unclear. In this study, we integrated single-cell RNAseq/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. Notably, only one subset selectively expresses high levels of OX40 and GITR, whose engangement by cognate ligand mediated proliferative programs and NF-kB 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. Surprisingly, 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 two-week period after entry into the tumor, down-regulating FoxP3 and up-regulating expression of TBX21 (Tbet), IFN and certain pro-inflammatory 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 anti-tumor immunity and suggest that TAA-specific TIL-Treg may positively contribute to anti-tumor responses. Libraries were prepared accordingn to 10X Genomics protocols for 5' barcoded GEX, feature barcode, and V(D)J TCR (Version 1)

调节性T细胞（Regulatory T cells, Treg）传统上被认为可抑制内源性及治疗诱导的抗肿瘤免疫，但其在调控免疫检查点阻断（immune checkpoint blockade, ICB）应答中的作用尚不明确。本研究整合了来自抗PD-1治疗及未接受治疗的非小细胞肺癌（non-small cell lung cancer, NSCLC）中超过73000个肿瘤浸润性调节性T细胞（tumor-infiltrating Treg, TIL-Treg）的单细胞RNA测序/单细胞T细胞受体测序（single-cell RNAseq/TCRseq）数据，同时结合了小鼠肿瘤模型中肿瘤相关抗原（tumor-associated antigen, TAA）特异性调节性T细胞的单细胞分析结果。本研究鉴定出10个人类TIL-Treg亚群，其中大多数与小鼠TIL-Treg亚群具有高度一致性。值得注意的是，仅一个亚群选择性高表达OX40与GITR；该亚群通过同源配体结合可介导增殖程序与核因子κB（NF-κB）激活，并上调多种参与Treg抑制功能的基因（包括LAG3）。功能实验显示，OX40高表达且GITR高表达（OX40hiGITRhi）的亚群是体外抑制活性最强的亚群，且该亚群在总TIL-Treg中的占比越高，与PD-1阻断治疗的耐药性相关性越强。令人意外的是，在小鼠肿瘤模型中，本研究发现几乎所有表达肿瘤相关抗原特异性T细胞受体的TIL-Treg在进入肿瘤后的两周内，会完全形成独特的Th1样特征：下调FoxP3的表达，同时上调TBX21（T-bet）、干扰素γ（IFN-γ）以及多种促炎症颗粒酶的表达。将小鼠TAA特异性Th1样Treg亚群的基因评分迁移学习至人类单细胞数据集后，发现了一个高度相似的亚簇，该亚簇在抗PD-1治疗应答的肿瘤中显著富集。上述研究结果表明，TIL-Treg可分为多个基于转录特征定义的独特亚群，这些亚群对免疫检查点阻断诱导的抗肿瘤免疫可能存在相反的调控作用；同时提示TAA特异性TIL-Treg或可对抗肿瘤应答产生积极贡献。文库构建参照10X Genomics的5'端条形码基因表达（5' barcoded GEX）、特征条形码及V(D)J T细胞受体（V(D)J TCR，版本1）实验流程进行制备。