IL-33 signaling alters regulatory T cell diversity in support of tumor development

Regulatory T cells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct from those of peripheral Tregs, but their contribution to tumor development remains unknown. Here, we use single cell RNA-Seq to longitudinally profile dynamic shifts in the distribution of Tregs in a genetically-engineered mouse model of lung adenocarcinoma. In this model, interferon-responsive Tregs are more prevalent early in tumor development, while a specialized effector phenotype characterized by enhanced expression of the interleukin 33 receptor ST2 is predominant in advanced disease. Treg-specific deletion of ST2 alters the evolution of effector Treg diversity, increases infiltration of CD8+ T cells into tumors, and decreases tumor burden. Our study shows that ST2 plays a critical role in Treg-mediated immunosuppression in cancer, highlighting potential paths for therapeutic intervention. Overall design: Tconv (DAPIneg, i.v.neg, Thy1.2+CD4+Foxp3-GFPneg) and Treg (DAPIneg, i.v.neg, Thy1.2+CD4+Foxp3-GFPpos) cells were single-cell sorted into Buffer TCL (Qiagen) plus 1% ß-mercaptoethanol in 96-well plates using a MoFlo Astrios cell sorter. Each plate had 30-100 cell population well and an empty well as controls. Following sorting, plates were spun down for 1” at 2000 RPM and frozen immediately at -80C. Plates were thawed and RNA was purified using 2.2X RNAclean SPRI beads (Beckman Coulter) without final elution (Shalek et al., 2013). SMART-seq2 and Nextera library preparation was performed as previously described (Picelli et al., 2013), with some modifications as described in a previous study (Singer et al., 2017). Plates were pooled into 384 single-cell libraries, and sequenced 50 x 25 paired end reads using a single kit on the NextSeq500 5 instrument.

调节性T细胞（Regulatory T cells, Tregs）可抑制抗肿瘤免疫应答，且与多种癌症的不良预后密切相关。人类肿瘤中的Tregs存在与外周血Tregs截然不同的多样化转录状态，但其对肿瘤发生发展的具体贡献仍未明确。本研究借助单细胞RNA测序（single cell RNA-Seq），对基因工程改造的肺腺癌小鼠模型中Tregs分布的动态变化进行了纵向表征。在该模型中，干扰素应答型Tregs在肿瘤发生早期更为富集，而以白介素33受体ST2（interleukin 33 receptor, ST2）高表达为特征的特异性效应表型Tregs则在疾病晚期占据主导地位。
特异性敲除Tregs中的ST2可改变效应Tregs多样性的演化进程，增加CD8+T细胞向肿瘤组织的浸润，并降低肿瘤负荷。本研究证实ST2在癌症中Tregs介导的免疫抑制过程中发挥关键作用，为肿瘤治疗干预提供了潜在方向。
总体实验设计：将常规T细胞（conventional T cells, Tconv）（DAPI阴性、静脉注射染色阴性、Thy1.2+CD4+Foxp3-GFP阴性）与Treg细胞（DAPI阴性、静脉注射染色阴性、Thy1.2+CD4+Foxp3-GFP阳性）通过MoFlo Astrios细胞分选仪，以单细胞形式分选至添加了1% β-巯基乙醇的Qiagen Buffer TCL缓冲液中，置于96孔板内。每块板设置30-100个细胞的群体孔与空白孔作为对照。分选完成后，将板以2000转/分钟离心1分钟，随后立即置于-80℃冰箱冷冻保存。后续将冻存板解冻，使用2.2X RNAclean SPRI磁珠（Beckman Coulter）纯化RNA，无需最终洗脱（参考Shalek等，2013年研究）。参照既往研究（Picelli等，2013年）的方法开展SMART-seq2与Nextera文库构建，并参照另一项前期研究（Singer等，2017年）进行部分参数优化。将所有样本混合为384个单细胞文库，使用NextSeq500测序仪配套单试剂盒完成50×25双端读长测序。