Loss of Bcl3 influences gene expression in Tregs and Treg subsets [Bcl3-WT_Treg_bulkRNAseq]

Peripherally induced regulatory T cells (pTregs) expressing the retinoic acid receptor-related orphan-receptor gamma t (RORgammat) are indispensable for intestinal immune homeostasis. Previous studies have determined the role of the atypical NFkappaB inhibitor Bcl3 in the development of colitis. In this study we analyzed the influence of Bcl3 on pTreg development and functionality in healthy mice and under colitogenic conditions. Our findings reveal that expression of Bcl3, in health, limits not just the development of pTregs in a T cell intrinsic manner but also the formation of a RORgammat+Helios+ double positive subset (DPTreg) expressing an activated transcriptional profile. Consequently, loss of Bcl3 in Tregs does not impede their suppressive capacity in a model of T cell transfer colitis but moreover leads to increased production of anti-inflammatory cytokines IL-10 and TGFbeta. We further demonstrate that lack of Bcl3 in Tregs results in trans-differentiation towards Th17-like cells. Finally, we provide a Bcl3 dependent gene signature in pTregs including altered signaling of the cytokines IL-2, IL-6 and TNFα. We therefore conclude that expression of Bcl3 regulates the sensitivity of Tregs towards homeostatic cytokines and consequently limits the formation of an unphysiological number of RORgammat+ Tregs. Cells from mesenteric lymph nodes (mLN) and spleen were isolated from wildtype mice (WT), Bcl3-/- Bcl3 deficient mice (KO) and mice conditionally overexpressing Bcl3 in Tregs (OE). Via fluorescence-activated cell sorting (FACS) we isolated CD45+CD3+CD4+Foxp3+ Tregs and CD45+CD3+CD4+Foxp3- Thelper cells (Th). We FACS sorted Foxp3+ T cells (CD45+, CD3+, CD4+) from mLN, spleen and small intestine lamina propria (SI) of Bcl3 sufficient and Bcl3 deficient mice, both with reporters for Foxp3-RFP and RORgt-GFP. We sorted peripherally induced Foxp3+RORgt+ pTregs and thymically induced Foxp3+RORgt- tTregs.表达视黄酸受体相关孤儿受体γt（retinoic acid receptor-related orphan-receptor gamma t，RORgammat）的外周诱导性调节性T细胞（peripherally induced regulatory T cells，pTregs）对肠道免疫稳态不可或缺。既往研究已阐明非典型核因子κB（NFκB）抑制剂Bcl3在结肠炎发生发展中的功能。本研究分析了Bcl3对健康小鼠及结肠炎造模条件下pTreg发育与功能的影响。本研究结果显示，生理状态下Bcl3的表达不仅以T细胞固有方式限制pTreg的发育，还抑制表达活化转录特征的RORgammat+Helios+双阳性亚群（double positive subset，DPTreg）的形成。因此，在T细胞过继转移结肠炎模型中，Treg特异性缺失Bcl3并不会削弱其免疫抑制功能，反而会促进抗炎细胞因子IL-10与转化生长因子β（transforming growth factor beta，TGFβ）的分泌。本研究进一步证实，Treg中Bcl3的缺失会导致其向辅助性T细胞17（Th17）样细胞转分化。最后，本研究明确了pTreg中Bcl3依赖的基因特征谱，其中涉及IL-2、IL-6及肿瘤坏死因子α（tumor necrosis factor alpha，TNFα）的信号通路异常。据此我们认为，Bcl3的表达可调节Treg对稳态细胞因子的敏感性，进而限制病理性过量的RORgammat+ Treg的形成。本研究从野生型小鼠（wildtype mice，WT）、Bcl3基因敲除小鼠（Bcl3 deficient mice，KO）以及Treg特异性条件性过表达Bcl3的小鼠（OE）中，分离得到肠系膜淋巴结（mesenteric lymph nodes，mLN）与脾脏中的细胞。通过荧光激活细胞分选术（fluorescence-activated cell sorting，FACS），我们分选出CD45+CD3+CD4+Foxp3+ Treg以及CD45+CD3+CD4+Foxp3- 辅助性T细胞（Thelper cells，Th）。我们还通过FACS分取了Bcl3表达充足与Bcl3缺失小鼠的肠系膜淋巴结、脾脏及小肠固有层（small intestine lamina propria，SI）中的Foxp3+ T细胞（表型为CD45+、CD3+、CD4+），这些小鼠均携带Foxp3-RFP与RORgt-GFP报告基因。我们进一步分选得到外周诱导性Foxp3+RORgt+ pTreg，以及胸腺源性诱导的Foxp3+RORgt- 调节性T细胞（thymically induced regulatory T cells，tTregs）。