Laboratory of Medical Immunology, Department of Laboratory Medicine, Radboud University Medical Center, Nijmegen, Netherlands.
College of Computer Science, Qinghai Normal University, Xining, China.
Front Immunol. 2018 Mar 21;9:573. doi: 10.3389/fimmu.2018.00573. eCollection 2018.
A crucial issue for Treg-based immunotherapy is to maintain a Treg phenotype as well as suppressive function during and after expansion. Several strategies have been applied to harness Treg lineage stability. For instance, CD28 superagonist stimulation , in the absence of CD3 ligation, is more efficient in promoting Treg proliferation, and prevention of pro-inflammatory cytokine expression, such as IL-17, as compared to CD3/CD28-stimulated Treg. Addition of the mTOR inhibitor rapamycin to Treg cultures enhances FOXP3 expression and Treg stability, but does impair proliferative capacity. A tumor necrosis factor receptor 2 (TNFR2) agonist antibody was recently shown to favor homogenous expansion of Treg . Combined stimulation with rapamycin and TNFR2 agonist antibody enhanced hypo-methylation of the gene, and thus promoting Treg stability. To further explore the underlying mechanisms of rapamycin and TNFR2 agonist-mediated Treg stability, we here stimulated FACS-sorted human Treg with a CD28 superagonist, in the presence of rapamycin and a TNFR2 agonist. Phenotypic analysis of expanded Treg revealed an autocrine loop of TNFα-TNFR2 underlying the maintenance of Treg stability . Addition of rapamycin to CD28 superagonist-stimulated Treg led to a high expression of TNFR2, the main TNFR expressed on Treg, and additional stimulation with a TNFR2 agonist enhanced the production of soluble as well as membrane-bound TNFα. Moreover, our data showed that the expression of histone methyltransferase EZH2, a crucial epigenetic modulator for potent Treg suppressor function, was enhanced upon stimulation with CD28 superagonist. Interestingly, rapamycin seemed to downregulate CD28 superagonist-induced EZH2 expression, which could be rescued by the additional addition of TNFR2 agonist antibody. This process appeared TNFα-dependent manner, since depletion of TNFα using Etanercept inhibited EZH2 expression. To summarize, we propose that an autocrine TNFα-TNFR2 loop plays an important role in endorsing Treg stability.
基于 Treg 的免疫疗法的一个关键问题是在扩增过程中和扩增后维持 Treg 表型和抑制功能。已经应用了几种策略来利用 Treg 谱系稳定性。例如，与 CD3/CD28 刺激的 Treg 相比，在缺乏 CD3 结合的情况下，CD28 超激动剂刺激更有效地促进 Treg 增殖，并防止促炎细胞因子如 IL-17 的表达。在 Treg 培养物中添加 mTOR 抑制剂雷帕霉素可增强 FOXP3 表达和 Treg 稳定性，但会损害增殖能力。最近显示，肿瘤坏死因子受体 2（TNFR2）激动剂抗体有利于 Treg 的同质扩增。雷帕霉素和 TNFR2 激动剂抗体的联合刺激增强了 基因的低甲基化，从而促进 Treg 稳定性。为了进一步探索雷帕霉素和 TNFR2 激动剂介导的 Treg 稳定性的潜在机制，我们在这里用 CD28 超激动剂刺激 FACS 分选的人 Treg，同时存在雷帕霉素和 TNFR2 激动剂。扩增的 Treg 的表型分析揭示了 TNFα-TNFR2 的自分泌环是维持 Treg 稳定性的基础。将雷帕霉素添加到 CD28 超激动剂刺激的 Treg 中导致 TNFR2 的高表达，TNFR2 是 Treg 上主要表达的 TNFR，另外用 TNFR2 激动剂刺激增强了可溶性和膜结合 TNFα的产生。此外，我们的数据表明，组蛋白甲基转移酶 EZH2 的表达增强，EZH2 是强效 Treg 抑制功能的关键表观遗传调节剂，用 CD28 超激动剂刺激后表达增强。有趣的是，雷帕霉素似乎下调了 CD28 超激动剂诱导的 EZH2 表达，这可以通过添加 TNFR2 激动剂抗体来挽救。这个过程似乎是 TNFα 依赖的方式，因为使用依那西普耗尽 TNFα 抑制了 EZH2 的表达。总之，我们提出自分泌 TNFα-TNFR2 环在维持 Treg 稳定性方面起着重要作用。
