Novel CD28 antagonist mPEG PV1-Fab’ mitigates experimental autoimmune uveitis by suppressing CD4+ T lymphocyte activation and IFN-γ production

Autoimmune Uveitis is an important chronic inflammatory disease and a leading cause of impaired vision and blindness. This ocular autoimmune disorder is mainly mediated by T CD4+ lymphocytes poising a TH1 phenotype. Costimulatory molecules are known to play an important role on T cell activation and therefore represent interesting therapeutical targets for autoimmune disorders. CD28 is the prototypical costimulatory molecule for T lymphocytes, and plays a crucial role in the initiation, and maintenance of immune responses. However, previous attempts to use this molecule in clinical practice achieved no success. Thus, we evaluated the efficacy of mPEG PV1-Fab’ (PV1), a novel selective CD28 antagonist monovalent Fab fragment in the treatment of Experimental Autoimmune Uveitis (EAU). Here, we showed that PV1 treatment decreases both average disease score and incidence of EAU. A decrease in the activation profile of both T CD4+ and T CD8+ eye-infiltrating lymphocytes was evidenced. In the periphery, T CD4+ cells from PV1-treated mice also showed a decrease in their activation status, with reduced expression of CD69, CD25, and PD-1 molecules. This suppression was not dependent on Treg cells, as both their frequency and absolute number were lower in PV1-treated mice. In addition, frequency of CD4+IFN-γ+ T cells was significantly lower in PV1-treated group, but not of IL-17-producing T cells. Moreover, after specific restimulation, PV1 blockade selectively blocked IFN-γ production by CD4+ lymphocytes Taken together, our data suggest that mPEG PV1-Fab’ acts mainly on IFN-γ-producing CD4+ T cells and emphasize that this specific CD28 blockade strategy is a potential specific and alternative tool for the treatment of autoimmune disorders in the eye.

自身免疫性葡萄膜炎（Autoimmune Uveitis）是一类重要的慢性炎症性疾病，也是视力受损与失明的主要诱因之一。该眼部自身免疫紊乱主要由呈现T辅助细胞1（TH1）表型的CD4阳性T淋巴细胞介导。已知共刺激分子（Costimulatory molecules）在T细胞活化过程中发挥关键作用，因此成为自身免疫性疾病极具潜力的治疗靶点。CD28是T淋巴细胞的经典共刺激分子，在免疫应答的启动与维持中至关重要。然而，既往将该分子应用于临床实践的尝试均未取得成功。为此，我们评估了mPEG PV1-Fab’（PV1）——一种新型选择性CD28阻断剂单价Fab片段——在治疗实验性自身免疫性葡萄膜炎（Experimental Autoimmune Uveitis, EAU）中的疗效。本研究证实，PV1治疗可同时降低EAU的平均疾病评分与发病率。研究还观察到，眼内浸润的CD4阳性与CD8阳性T淋巴细胞的活化水平均有所下降。在外周免疫环境中，经PV1处理的小鼠体内的CD4阳性T细胞活化状态也有所减弱，其CD69、CD25及程序性死亡蛋白1（PD-1）分子的表达水平均降低。该抑制效应并不依赖于调节性T细胞（Treg），因为经PV1处理的小鼠体内调节性T细胞的频率与绝对计数均更低。此外，PV1处理组小鼠体内CD4阳性干扰素-γ（IFN-γ）阳性T细胞的占比显著降低，但产生白细胞介素-17（IL-17）的T细胞占比无明显变化。进一步的特异性再刺激实验显示，PV1阻断可选择性抑制CD4阳性淋巴细胞产生干扰素-γ。综上，我们的研究结果表明，mPEG PV1-Fab’主要通过作用于产生干扰素-γ的CD4阳性T细胞发挥功效，同时证实了这种特异性CD28阻断策略有望成为眼部自身免疫性疾病治疗的特异性替代方案。