Miller Stav, Eizenberg-Magar Inbal, Reich-Zeliger Shlomit, Rimer Jacob, Zaretsky Irina, Reshef Dan, Kopitman Ekaterina, Friedman Nir, Antebi Yaron E
Department of Immunology and Regenerative Biology, Weizmann Institute of Science, Rehovot, Israel.
Department of Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot, Israel.
Front Immunol. 2025 Apr 28;16:1462045. doi: 10.3389/fimmu.2025.1462045. eCollection 2025.
T helper 17 and Regulatory T cells (Th17 and Treg, respectively) are two well-described lymphocyte subsets with opposing actions. The divergent fates of Th17 and Treg cells are accounted for, at least in part, by molecular antagonism that occurs between their respective specific transcription factors, RORγt and Foxp3. An imbalance between Th17 and Treg cells may lead to tissue inflammation and is associated with certain types of autoimmunity. In order to understand the heterogeneity and dynamics of the differentiation process, we studied Th17/Treg cell differentiation of naïve cells , using RORγtFoxp3 dual-reporter mouse. Flow cytometry revealed the consistent emergence of a population of double positive RORγtFoxp3 (DP) cells during the early stages of Th17 cell differentiation. These DP cells are closely related to RORγt single positive (SPR) cells in terms of global gene expression. Nevertheless, for some genes, DP cells share an expression pattern with Foxp3 single positive (SPF) Treg cells, most importantly by reducing IL17 levels. Using time-lapse microscopy, we could delineate the expression dynamics of RORγt and Foxp3 at a clonal level. While the RORγt expression level elevates early during differentiation, Foxp3 rises later and is more stable upon environmental changes. These distinct expression profiles are independent of each other. During differentiation and proliferation, individual cells transit between SPR, DP, and SPF states. Nevertheless, the differentiation of sister cells within a clone progeny was highly correlated. We further demonstrated that sorted SPR and DP populations were not significantly affected by changes in their environment, suggesting that the correlated fate decision emerged at early time points before the first division. Overall, this study provides the first quantitative analysis of differentiation dynamics during the generation of DP RORγtFoxp3 cells. Characterizing these dynamics and the differentiation trajectory could provide a profound understanding and be used to better define the distinct fates of T cells, critical mediators of the immune response.
辅助性T细胞17和调节性T细胞（分别为Th17和Treg）是两种作用相反且已被充分描述的淋巴细胞亚群。Th17和Treg细胞的不同命运至少部分是由它们各自特异性转录因子RORγt和Foxp3之间发生的分子拮抗作用所导致的。Th17和Treg细胞之间的失衡可能会导致组织炎症，并与某些类型的自身免疫相关。为了了解分化过程的异质性和动态变化，我们使用RORγtFoxp3双报告基因小鼠研究了初始细胞的Th17/Treg细胞分化。流式细胞术显示在Th17细胞分化的早期阶段持续出现一群双阳性RORγtFoxp3（DP）细胞。就整体基因表达而言，这些DP细胞与RORγt单阳性（SPR）细胞密切相关。然而，对于某些基因，DP细胞与Foxp3单阳性（SPF）Treg细胞共享一种表达模式，最重要的是通过降低IL17水平。使用延时显微镜，我们能够在克隆水平上描绘RORγt和Foxp3的表达动态。虽然RORγt表达水平在分化早期升高，但Foxp3稍后升高且在环境变化时更稳定。这些不同的表达谱彼此独立。在分化和增殖过程中，单个细胞在SPR、DP和SPF状态之间转变。然而，克隆后代中姐妹细胞的分化高度相关。我们进一步证明，分选的SPR和DP群体不受其环境变化的显著影响，这表明相关的命运决定在第一次分裂之前的早期时间点就已出现。总体而言，这项研究首次对DP RORγtFoxp3细胞生成过程中的分化动态进行了定量分析。表征这些动态变化和分化轨迹可以提供深刻的理解，并用于更好地定义T细胞的不同命运，T细胞是免疫反应的关键介质。
