TCR sequencing of Treg and Tconv subsets from murine spleen

While unique subsets of Treg cells have been described in some non-lymphoid tissues, their relationship to Treg cells in secondary lymphoid organs and circulation remains unclear. We have identified a recirculating and highly suppressive effector Treg cell subset that expresses the a2 integrin, CD49b, and exhibits a unique tissue distribution. We used TCR sequencing to ask whether CD49b+ activated Treg cells and CD49b- activated Treg cells represent stable states of Treg cell differentiation instructed by distinct TCR repertoires. We did not find a pattern of clonotypes that was specific to either subset, suggesting that essentially all CD49b- Treg cells could give rise to CD49b+ cells. These results shed light on the identity and development of a functionally potent subset of mature effector Treg cells that recirculate through and survey peripheral tissues. Overall design: Tcra cDNA libraries were prepared from FACS-purified splenic Tconv and Treg cells from Foxp3GFP Tcra+/- DO11.10tg mice. In these mice, the presence of the DO11.10 TCRß transgene inhibits the recombination of the endogenous Tcrb loci, thus restricting TCR repertoire variability to TCRa chains. Further limitation of the TCR repertoire was achieved by the presence of only one functional Tcra gene. 2 CD4+ T-cell lineages x 3 activation states x 4 replicates = 24 samples.尽管部分非淋巴组织中已报道存在独特的调节性T细胞（Treg）亚群，但它们与次级淋巴器官及循环系统中Treg细胞的关联仍未明确。本研究鉴定出一种可循环且具有高度抑制活性的效应性Treg细胞亚群，该亚群表达α2整合素（α2 integrin）CD49b，并呈现独特的组织分布特征。我们借助T细胞受体（TCR）测序技术，探究CD49b阳性活化Treg细胞与CD49b阴性活化Treg细胞是否代表由不同TCR库调控的Treg细胞分化稳定状态。研究未发现任一亚群具有特异性的克隆型模式，这表明几乎所有CD49b阴性Treg细胞均可分化为CD49b阳性细胞。这些结果阐明了一类功能强效的成熟效应性Treg细胞亚群的身份与发育过程，该亚群可循环并监测外周组织。 总体实验设计：从Foxp3GFP Tcra+/- DO11.10tg小鼠的荧光激活细胞分选（FACS）纯化的脾脏Tconv细胞与Treg细胞中构建Tcra cDNA文库。在该类小鼠中，DO11.10 TCRβ转基因的存在会抑制内源性Tcrb基因座的重组，从而将TCR库的变异限制仅存在于TCRα链。进一步限制TCR库变异的手段为仅保留一个功能性Tcra基因。实验分组为2种CD4+ T细胞谱系 × 3种激活状态 × 4次生物学重复，共计24个样本。