How GRAIL controls Treg function to maintain self-tolerance.
How GRAIL controls Treg function to maintain self-tolerance.
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GRAIL如何控制Treg功能以维持自身耐受性。
DOI：
10.3389/fimmu.2022.1046631
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发表时间：
2022
影响因子：
7.3
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中科院分区：
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调节性T细胞（Tcells）通常维持自身耐受性。免疫系统识别“自我”，因此当它们不能正常工作时，例如在自身免疫中，免疫系统可以攻击和破坏自己的组织。目前用于自身免疫的疗法依赖于相对无效且通常有毒的疗法来“治疗”破坏性炎症。恢复有缺陷的内源性免疫调节（自身耐受性）将代表这些疾病治疗的范式转变。一种最近的恢复自身耐受性的方法是使用“低剂量IL-2”作为增加循环T细胞的数量的疗法。然而，迄今为止的研究尚未证明低剂量IL-2疗法可以恢复伴随的Treg功能，并且在低剂量IL-2治疗的自身免疫性疾病患者中的2期研究未能证明显著的临床益处。我们假设，在自身免疫中看到的自身耐受性缺陷不是由于可用Treg的数量不足，而是由于通常控制Treg功能和稳定性的IL-2 R下游的第二信使的缺陷。我们实验室和其他人的先前研究已经证明GRAIL（一种泛素E3连接酶）在Treg功能中很重要。GRAIL表达在来自患有自身免疫性疾病和过敏性哮喘的患者的TCR 4中显著减少，并且在被认为是自身免疫性倾向的小鼠的TCR 4中也减少。在T细胞中的相关通路中，GRAIL通常阻断cullin环连接酶活性，其抑制T细胞中的IL-2 R脱敏，从而促进Treg功能。由于GRAIL表达中的这种缺陷，患有自身免疫性疾病和过敏性哮喘的患者的T细胞在用低剂量IL-2激活后降解IL-2 R相关的pJAK 1，因此不能维持pSTAT 5表达。pSTAT 5控制Treg功能所需基因的转录。此外，GRAIL介导的缺陷也可能允许mTOR抑制剂，含DEP结构域的mTOR相互作用蛋白（Deptor）的降解。这可导致自身免疫患者中mTOR的IL-2 R活化和Treg稳定性的丧失。使用胰蛋白酶消化后的泛素化蛋白质上的残余双甘氨酸标签的单克隆抗体，我们鉴定了在Treg功能中重要的GRAIL泛素化的蛋白质cullin 5。我们的数据表明，GRAIL通过泛素化cullin 5上的赖氨酸来发挥IL-2 R脱敏的负调节作用，所述赖氨酸必须被neddylated以允许cullin 5 cullin环连接酶活性。我们假设neddylation抑制剂与低剂量IL-2激活的组合可用于替代GRAIL，并恢复自身免疫性和过敏性哮喘患者T细胞中Treg的功能和稳定性。然而，当全身给予时，neddylation活化酶抑制剂（NAEi）是有毒的。通过产生由NAEi通过可切割接头结合到鼠IL-2的融合蛋白（以将药物靶向TcR）组成的蛋白质药物缀合物（PDC），我们能够使用比治疗有效的全身递送所需的量少1000倍的neddylation抑制剂药物。PDC在几种自身免疫小鼠模型（1型糖尿病、系统性红斑狼疮和多发性硬化症）和过敏性哮喘小鼠模型中有效阻断疾病的发作或进展，且无可检测的毒性。这种PDC策略代表了最佳的靶向药物递送，其中确定了导致疾病的缺陷，设计和开发了药物来纠正缺陷，并且药物仅靶向并递送到需要它的细胞，从而最大限度地提高安全性和有效性。
Regulatory T cells (Tregs) normally maintain self-tolerance. Tregs recognize “self” such that when they are not working properly, such as in autoimmunity, the immune system can attack and destroy one’s own tissues. Current therapies for autoimmunity rely on relatively ineffective and too often toxic therapies to “treat” the destructive inflammation. Restoring defective endogenous immune regulation (self-tolerance) would represent a paradigm shift in the therapy of these diseases. One recent approach to restore self-tolerance is to use “low dose IL-2” as a therapy to increase the number of circulating Tregs. However, studies to-date have not demonstrated that low-dose IL-2 therapy can restore concomitant Treg function, and phase 2 studies in low dose IL-2 treated patients with autoimmune diseases have failed to demonstrate significant clinical benefit. We hypothesize that the defect in self-tolerance seen in autoimmunity is not due to an insufficient number of available Tregs, but rather, due to defects in second messengers downstream of the IL-2R that normally control Treg function and stability. Previous studies from our lab and others have demonstrated that GRAIL (a ubiquitin E3 ligase) is important in Treg function. GRAIL expression is markedly diminished in Tregs from patients with autoimmune diseases and allergic asthma and is also diminished in Tregs of mice that are considered autoimmune prone. In the relevant pathway in Tregs, GRAIL normally blocks cullin ring ligase activity, which inhibits IL-2R desensitization in Tregs and consequently promotes Treg function. As a result of this defect in GRAIL expression, the Tregs of patients with autoimmune diseases and allergic asthma degrade IL-2R-associated pJAK1 following activation with low dose IL-2, and thus cannot maintain pSTAT5 expression. pSTAT5 controls the transcription of genes required for Treg function. Additionally, the GRAIL-mediated defect may also allow the degradation of the mTOR inhibitor, DEP domain-containing mTOR interacting protein (Deptor). This can lead to IL-2R activation of mTOR and loss of Treg stability in autoimmune patients. Using a monoclonal antibody to the remnant di-glycine tag on ubiquitinated proteins after trypsin digestion, we identified a protein that was ubiquitinated by GRAIL that is important in Treg function, cullin5. Our data demonstrate that GRAIL acts a negative regulator of IL-2R desensitization by ubiquitinating a lysine on cullin5 that must be neddylated to allow cullin5 cullin ring ligase activity. We hypothesize that a neddylation inhibitor in combination with low dose IL-2 activation could be used to substitute for GRAIL and restore Treg function and stability in the Tregs of autoimmune and allergic asthma patients. However, the neddylation activating enzyme inhibitors (NAEi) are toxic when given systemically. By generating a protein drug conjugate (PDC) consisting of a NAEi bound, via cleavable linkers, to a fusion protein of murine IL-2 (to target the drug to Tregs), we were able to use 1000-fold less of the neddylation inhibitor drug than the amount required for therapeutically effective systemic delivery. The PDC was effective in blocking the onset or the progression of disease in several mouse models of autoimmunity (type 1 diabetes, systemic lupus erythematosus, and multiple sclerosis) and a mouse model of allergic asthma in the absence of detectable toxicity. This PDC strategy represents targeted drug delivery at its best where the defect causing the disease was identified, a drug was designed and developed to correct the defect, and the drug was targeted and delivered only to cells that needed it, maximizing safety and efficacy.