ZFP36-family RNA-binding proteins in regulatory T cells reinforce immune homeostasis [single cell RNA-seq]

RNA binding proteins (RBP) of the ZFP36 family limit the differentiation and effector functions of CD4 and CD8 T cells, but little is known of their expression or function in regulatory T cells (Treg). By Treg-restricted deletion of Zfp36 family members we identify the essential role of Zfp36l1 and Zfp36l2 in Treg to maintain immune homeostasis. Mice with Tregs deficient in these RBP display an inflammatory phenotype with an expansion in the numbers of type-2 conventional dendritic cells, T effector cells, T follicular helper and germinal center B cells and elevated serum cytokines and immunoglobulins. In the absence of Zfp36l1 and Zfp36l2, the pool of cycling CTLA-4 in naïve Treg was reduced, Tregs were less sensitive to IL-2 and IL-7 but were more sensitive to IFNγ. In mice lacking both RBP in Treg, the deletion of a single allele of Ifng is sufficient to ameliorate the pathology. Thus, ZFP36L1 and ZFP36L2 regulate multiple pathways that enable Tregs to enforce immune homeostasis. Single cell RNA-sequencing of control and Zfp36l1/Zfp36l2-deficient regulatory T cells (Treg) from 14-week-old mice, with one replicate per genotype. Deletion was driven by the Foxp3-YFP-Cre allele. Treg from the two genotypes were labelled using Totalseq oligo-conjugated antibodies (BioLegend), before pooling, such that library generation and sequencing was performed within a single sample.

ZFP36家族的RNA结合蛋白（RNA binding proteins, RBP）会抑制CD4和CD8 T细胞的分化及效应功能，但目前对其在调节性T细胞（regulatory T cells, Treg）中的表达与功能仍知之甚少。通过对Treg进行Zfp36家族基因的特异性敲除，我们明确了Zfp36l1与Zfp36l2在Treg维持免疫稳态过程中的关键作用。缺失这类RBP的Treg小鼠会表现出炎症表型，伴随2型常规树突状细胞、效应T细胞、滤泡辅助T细胞及生发中心B细胞的数量扩增，同时血清细胞因子与免疫球蛋白水平升高。在缺失Zfp36l1与Zfp36l2的情况下，初始Treg中循环型CTLA-4的表达库减少，Treg对IL-2与IL-7的敏感性降低，但对IFNγ的敏感性升高。在Treg中同时缺失这两种RBP的小鼠中，仅敲除单等位基因Ifng即可缓解其病理表型。综上，ZFP36L1与ZFP36L2可调控多条通路，使Treg能够维持免疫稳态。本研究对14周龄小鼠的野生型与Zfp36l1/Zfp36l2缺陷型调节性T细胞（Treg）开展单细胞RNA测序，每个基因型设置一个生物学重复。基因敲除由Foxp3-YFP-Cre等位基因介导。两种基因型的Treg分别使用Totalseq寡核苷酸偶联抗体（BioLegend）进行标记，随后将样本混合，确保文库构建与测序在同一批次样本中完成。