Antigen-specific peripheral tolerance is crucial to prevent the development of organ-specific autoimmunity. However, its function decoupled from thymic tolerance remains unclear. We used desmoglein 3 (Dsg3), a pemphigus antigen expressed in keratinocytes, to analyze peripheral tolerance under physiological antigen-expression conditions. Dsg3-deficient thymi were transplanted into athymic mice to create a unique condition in which Dsg3 was expressed only in peripheral tissue but not in the thymus. When bone marrow transfer was conducted from high-avidity Dsg3-specific T cell receptor–transgenic mice to thymus-transplanted mice, Dsg3-specific CD4⁺ T cells developed in the transplanted thymus but subsequently disappeared in the periphery. Additionally, when Dsg3-specific T cells developed in Dsg3^−/− mice were adoptively transferred into Dsg3-sufficient recipients, the T cells disappeared in an antigen-specific manner without inducing autoimmune dermatitis. However, Dsg3-specific T cells overcame this disappearance and thus induced autoimmune dermatitis in Treg-ablated recipients but not in Foxp3-mutant recipients with dysfunctional Tregs. The molecules involved in disappearance were sought by screening the transcriptomes of wild-type and Foxp3-mutant Tregs. OX40 of Tregs was suggested to be responsible. Consistently, when OX40 expression of Tregs was constrained, Dsg3-specific T cells did not disappear. Furthermore, Tregs obtained OX40L from dendritic cells in an OX40-dependent manner in vitro and then suppressed OX40L expression in dendritic cells and Birc5 expression in Dsg3-specific T cells in vivo. Lastly, CRISPR/Cas9-mediated knockout of OX40 signaling in Dsg3-specific T cells restored their disappearance in Treg-ablated recipients. Thus, Treg-mediated peripheral deletion of autoreactive T cells operates as an OX40-dependent regulatory mechanism to avoid undesired autoimmunity besides thymic tolerance.

抗原特异性外周耐受对于防止器官特异性自身免疫的发展至关重要。然而，其与胸腺耐受脱钩的功能仍不清楚。我们使用桥粒芯蛋白 3 (Dsg3)，一种在角质形成细胞中表达的天疱疮抗原，来分析生理抗原表达条件下的外周耐受性。将 Dsg3 缺陷的胸腺移植到无胸腺小鼠中，以创造一种独特的条件，其中 Dsg3 仅在外周组织中表达，而不在胸腺中表达。当从高亲和力 Dsg3 特异性 T 细胞受体转基因小鼠向胸腺移植小鼠进行骨髓移植时，Dsg3 特异性 CD4 ⁺ T 细胞在移植的胸腺中发育，但随后在外周消失。此外，当 Dsg3 特异性 T 细胞在Dsg3^-/-小鼠被过继转移到 Dsg3 充足的受体中，T 细胞以抗原特异性方式消失，而不会诱发自身免疫性皮炎。然而，Dsg3 特异性 T 细胞克服了这种消失，因此在 Treg 消融受体中诱发了自身免疫性皮炎，但在具有功能失调 Treg的Foxp3突变受体中却没有。通过筛选野生型和Foxp3突变 Treg 的转录组来寻找与消失有关的分子。建议Tregs的OX40负责。一致地，当Tregs 的OX40表达受到限制时，Dsg3 特异性 T 细胞并没有消失。此外，Tregs 从OX40中的树突细胞中获得 OX40L体外依赖性方式，然后在体内抑制树突状细胞中的 OX40L 表达和 Dsg3 特异性 T 细胞中的 Birc5 表达。最后，CRISPR/Cas9 介导的 Dsg3 特异性 T 细胞中OX40信号的敲除恢复了它们在 Treg 消融受体中的消失。因此，Treg 介导的自身反应性 T 细胞的外周缺失作为OX40依赖性调节机制起作用，以避免除胸腺耐受之外的不希望的自身免疫。