Data_Sheet_1_T Regulatory Cells From Non-obese Diabetic Mice Show Low Responsiveness to IL-2 Stimulation and Exhibit Differential Expression of Anergy-Related and Ubiquitination Factors.pdf

Foxp3+ Regulatory T cells (Tregs) are pivotal for the maintenance of tolerance. Alterations in their number and/or function have been proposed to occur in the autoimmune-prone non-obese diabetic (NOD) mouse. Comparing the frequencies and absolute numbers of CD4+Foxp3+CD25+ Tregs among 4 to 6-week old NOD, B6, and BALB/c mice, we observed differences in counts and Foxp3 expression in Tregs from secondary lymphoid organs, but not in the thymus. Upon TCR and IL-2 stimulation, NOD Tregs showed lower responses than Tregs from B6 and BALB/c mice. Indeed, NOD Tregs responded with less proliferation and with smaller increments in the expression of CD25, LAP-1, CD39, PD-1, PD-L1, and LAG-3, when in vitro cultured for 3 days with anti-CD3/CD28 in the absence or presence of IL-2, Tregs from NOD mice showed to be highly dependent on IL-2 to maintain Foxp3 expression. Moreover, NOD Tregs become producers of IL-17 and INF-gamma more easily than Tregs from the other strains. In addition, NOD Tregs showed lower responsiveness to IL-2, with significantly reduced levels of pSTAT5, even at high IL-2 doses, with respect to B6 and BALB/c Tregs. Interestingly, NOD Tregs exhibit differences in the expression of SOCS3, GRAIL, and OTUB1 when compared with Tregs from B6 and BALB/c mice. Both, at steady state conditions and also after activation, Tregs from NOD mice showed increased levels of OTUB1 and low levels of GRAIL. In addition, NOD Tregs had differences in the expression of ubiquitin related molecules that play a role in the maintenance of Foxp3 cellular pools. Indeed, significantly higher STUB1/USP7 ratios were detected in NOD Tregs, both at basal conditions and after stimulation, compared to in B6 and BALB/c Tregs. Moreover, the addition of a proteasome inhibitor to cell cultures, conferred NOD Tregs the ability to retain Foxp3 expression. Herein, we provide evidence indicating a differential expression of SOCS3, GRAIL, and STUB1/USP7 in Tregs from NOD mice, factors known to be involved in IL-2R signaling and to affect Foxp3 stability. These findings add to the current knowledge of the immunobiology of Tregs and may be related to the known insufficiency of Tregs from NOD mice to maintain self-tolerance.

Foxp3阳性调节性T细胞（Foxp3+ Regulatory T cells, Tregs）在免疫耐受的维持中发挥关键作用。已有研究提出，易感自身免疫的非肥胖糖尿病小鼠（non-obese diabetic, NOD）体内，这类细胞的数量和/或功能会出现异常改变。本研究对比了4至6周龄NOD、B6及BALB/c小鼠的CD4+Foxp3+CD25+ Tregs的细胞频率与绝对计数，观察到次级淋巴器官来源的Tregs的细胞计数及Foxp3表达存在品系间差异，但胸腺来源的Tregs未出现此类差异。在经T细胞受体（TCR）与白细胞介素2（IL-2）刺激后，NOD小鼠来源的Tregs应答反应弱于B6与BALB/c小鼠的Tregs。具体而言，当在体外使用抗CD3/抗CD28单克隆抗体，联合或不联合IL-2培养3天后，NOD Tregs的增殖程度更低，且CD25、LAP-1、CD39、PD-1、PD-L1及LAG-3的表达上调幅度也更小。此外，NOD小鼠Tregs高度依赖IL-2以维持Foxp3的表达。除此之外，相较于其他品系小鼠的Tregs，NOD Tregs更易分化为白细胞介素17（IL-17）与干扰素γ（IFN-γ）的分泌细胞。同时，相较于B6及BALB/c小鼠的Tregs，NOD Tregs对IL-2的应答能力更弱，即使在高剂量IL-2处理下，其磷酸化信号转导与转录激活因子5（pSTAT5）的水平也显著降低。有趣的是，相较于B6与BALB/c小鼠来源的Tregs，NOD Tregs的SOCS3、GRAIL及OTUB1表达存在显著差异。无论是在稳态条件下还是活化后，NOD小鼠的Tregs均表现出OTUB1水平升高、GRAIL水平降低的特征。此外，NOD Tregs在参与维持Foxp3细胞库的泛素相关分子表达上存在差异：相较于B6及BALB/c小鼠的Tregs，无论在基础状态还是刺激后，NOD Tregs的STUB1/USP7比值均显著升高。向细胞培养液中加入蛋白酶体抑制剂后，NOD Tregs可维持Foxp3的表达。本研究证实，NOD小鼠Tregs中SOCS3、GRAIL及STUB1/USP7的表达存在差异，而这些因子已被证实参与白细胞介素2受体（IL-2R）信号通路调控并影响Foxp3的稳定性。上述发现丰富了当前对Tregs免疫生物学的认知，或与已知的NOD小鼠Tregs无法有效维持自身耐受的现象密切相关。