The novel role of the immunoproteasome subunit LMP7 in allo-immunity and T cell exhaustion

摘要 T细胞衰竭的特征是炎性细胞因子的进行性丢失 产生，随后是功能和增殖缺陷，导致细胞凋亡。虽然这 最近在同种异体移植的背景下研究了这一现象，但仍不清楚 明白识别和靶向诱导同种免疫T细胞的关键途径 疲劳将促进同种异体移植物的接受。 我们最近发现，免疫蛋白酶体（i-20 S）取代了宪法的 蛋白酶体（c-20 S），并在同种异体移植后在人和鼠T细胞中高度表达。 活化使其成为同种免疫的理想靶点。因此，在与林刚博士的合作中， 我们设计并合成了一种高度选择性的i-20 S LMP 7亚基抑制剂， 揭示了LMP 7在T细胞耗竭中的意想不到的作用，通过特异性降解LMP 7， JAK-STAT 5通路和促进小鼠心脏移植物存活的主要机制 组织相容性复合物（Esilida et al，PNAS，Dec 2016）。虽然瞄准c-20 S受到阻碍， 显著的脱靶副作用，i-20 S是同种免疫的理想靶点，因为它富含 免疫细胞激活。 我们的假设是，在同种免疫T细胞活化过程中，免疫蛋白酶体 LMP 7亚基被上调，通过以下途径促进磷酸化STAT 5（pSTAT 5）的降解： 其保护抗原反应效应T细胞免受免疫调节的蛋白酶活性， 疲惫不堪因此，抑制免疫蛋白酶体亚基LMP 7增加pSTAT 5，诱导 T细胞耗竭和Fas/FasL凋亡导致克隆性缺失，促进同种异体移植 没有毒性脱靶效应的生存。 为了验证我们的假设，我们将使用与Dr. 林刚在抗原特异性移植模型（OTI、OTII、OVA）中将追踪同种异体反应性 使用一种新的非侵入性荧光素酶体内技术的T细胞克隆。我们还产生 在OTI和OTII背景上敲除LMP 7和条件性敲除LMP 7 选择性地在CD 4和CD 8 T细胞中。我们将使用分子技术来鉴定LMP 7 如果STAT 5的活化导致T细胞凋亡和克隆缺失，最后为 生成临床前数据，我们将在人类免疫系统上测试我们的治疗策略 使用移植了人类胰岛的人源化小鼠模型。

Abstract T cell exhaustion is characterized by a progressive loss of inflammatory cytokines production followed by a functional and proliferation defect leading to apoptosis. While this phenomenon was studied recently in the context of allo-transplantation, it remains not well understood. Identifying and targeting key pathways involved in the induction of allo-immune T cell exhaustion will promote allograft acceptance. We recently showed that the immunoproteasome (i-20S) replaces the constitutional proteasome (c-20S) and becomes highly expressed in the human and murine T cells upon allo- activation making it an ideal target for allo-immunity. Hence, in collaboration with Dr. Gang Lin, we designed and synthesized a highly selective inhibitor of the LMP7 subunit of the i-20S that revealed an unexpected role for the LMP7 in T cell exhaustion through specific degradation of the JAK-STAT5 pathway and in promoting murine heart allograft survival across major histocompatibility complex (Esilida et al, PNAS, Dec 2016). While targeting c-20S is hindered by significant off target side effects, i-20S is an ideal target for allo-immunity as it's enriched in immunocytes upon activation. Our hypothesis is that during alloimmune T cell activation, the immunoproteasome subunit LMP7 is upregulated, promoting degradation of phosphorylated STAT5 (pSTAT5) through its protease activity to protect the antigen-reacting effector T cells from immune regulation and exhaustion. Hence, inhibiting the immunoproteasome subunit LMP7 increases pSTAT5, induces T cell exhaustion and Fas/FasL apoptosis leading to clonal deletion, and promotes allograft survival without toxic off target effects. To test our hypothesis, we will use selective inhibitors generated in collaboration with Dr. Gang Lin in an antigen specific transplant model (OTI, OTII, OVA) and will track the allo-reactive T cell clones using a novel non-invasive luciferase technique in vivo. We also generated knockouts of LMP7 on the OTI and OTII backgrounds and a conditional knockout of LMP7 selectively in CD4 and CD8 T cells. We will use molecular techniques to identify how the LMP7 degrade STAT5 and if STAT5 activation leads to T cell apoptosis and clonal deletion. Finally, to generate preclinical data, we will test our therapeutic strategy on the human immune system using a humanized mouse model transplanted with human islets.