Cbl-b对Tfh的调控在狼疮肾炎中的作用及机制研究

Activation of polyclonal CD4+ T cells and B cells is a hallmark of systemic lupus nephritis (LN), but the mechanism(s) of activation of T and B cells responsive to self-peptides in LN is poorly defined. T follicular helper (Tfh) cells are critical for providing help to B cells allowing the formation of germinal center and the subsequent long-lived plasma cells differentiation. There is a growing body of evidence by far pointing toward the crucial roles of Tfh cells in the overproduction of pathogenic autoantibodies and tissue damages in SLE. However, how Tfh cells are deregulated in LN in mice and humans are currently unknown. Our preliminary results showed that introducing a point mutation within RING finger domain of E3 ubiquitin ligase Cbl-b into lpr mice (carrying a mutation in Fas gene) on a C57BL/6 background (B6-lpr.CblbC373A), exacerbates the disease. This exacerbation is associated with expanded Tfh cells in the spleens and heightened anti-dsDNA titers in the sera. Based upon these data, we hypothesize that Cbl-b controls Tfh development in B6-lpr, which provides strong help for GC B cells to produce autoantibodies, leading to the development of lupus. To test this hypothesis, we will determine 1. Whether and how Cbl-b regulates Tfh cell development in mouse LN model. In this aim, we will first confirm the LN phenotype in B6-lpr.CblbC373A mice, and verify whether Cbl-b indeed regulates Tfh differentiation in vitro and in vivo. We will then determine the potential molecular targets of Cbl-b in Tfh cells. We will further characterize the cellular compositions of B6-lpr.CblbC373A mice, with the particular focus on Tfh cells. Dr. Jian Zhang’s group in the Department of Microbial Infection and Immunity will be responsible for the experiments proposed in this aim. 2. Whether Cbl-b regulates Tfh cells in LN patients. In this aim, We will verify whether knocking down Cbl-b by Cbl-b siRNA affects the ability of human Tfh subsets identified in LN patients on GC B cell function. Then we will verify whether Cbl-b regulates human Tfh development by using the same molecular mechanism as proposed in Aim-1. Dr. Wael Jarjour’s group in the Division of Rheumatology will be responsible for the experiments proposed in this aim.

肾脏是SLE最常受累的器官，LN导致的肾衰竭是SLE最主要的死亡原因之一，因此寻找LN的防治靶点具有重要意义。辅助性T细胞参与激活B细胞是狼疮肾炎发展的重要环节。虽然目前认为Tfh增殖与LN的发病密切相关，但其机制尚不清楚。我们前期研究发现B6-lpr.CblbC373A较B6-lpr小鼠出现更明显的Tfh增多、GC增加、并有更严重的LN症状；LN患者的Tfh细胞增多但表达Cbl-b的Tfh细胞减少；Cbl-b特异性结合Bcl-6蛋白，Cbl-b表达下调可以使Bcl-6蛋白降解减少。因此我们假设Cbl-b可能靶向调控Bcl-6的泛素化，影响Tfh增殖，参与LN的发病。本研究拟在以往工作的基础上，通过体外诱导Tfh、已建立的B6-lpr.CblbC373A LN小鼠模型和LN患者三方面实验，进一步研究Cblb对Tfh的调控在LN中的作用及其可能的分子机制，为临床上LN的治疗提供新的理论依据。

肾脏是SLE最常受累的器官，LN导致的肾衰竭是SLE最主要的死亡原因之一。辅助性T细胞参与激活B细胞是狼疮肾炎发展的重要环节。虽然目前认为Tfh增殖与LN的发病密切相关，但其机制尚不清楚。. 我们构建了B6-lpr.Cblb-/-小鼠模型，监测B6-lpr.Cblb-/-与B6-lpr小鼠LN疾病进展；观察Tfh细胞在小鼠外周血和肾组织中的改变。体外刺激诱导小鼠CD4+T细胞分化Tfh细胞，寻找Cblb作用的靶蛋白，通过调控Tfh细胞中Cblb的表达诱导靶蛋白泛素化水平。进一步验证在LN患者外周血中Tfh细胞和Tfh细胞中Cblb表达水平改变;体外调控人CD4+T细胞中Cblb表达，观察是否同小鼠试验中的结果一致，证明LN患者中，Cblb对Tfh增殖的调节可能通过泛素化Bcl-6来实现。我们进一步给予小鼠Bcl-6抑制剂皮下注射治疗，检测各组小鼠外周血及肾脏Tfh细胞增殖情况。. 结果表明lpr.Cblb-/-小鼠较B6-lpr小鼠LN表现更为严重，Tfh细胞增殖更为显著。LN肾炎患者较健康对照组外周血Tfh细胞数量明显增多，但表达Cblb的Tfh细胞数减少。体外调控Tfh细胞中Cblb的表达，增强Tfh细胞中Cblb表达，细胞上清液中的IgG的表达水平升高；减弱Tfh细胞Cblb的表达，细胞上清液中的IgG的表达水平降低。同时我们发现Cblb特异性结合Bcl-6，Cblb表达减弱可使Bcl-6泛素化水平降低，Bcl-6蛋白降解减少。接受Bcl-6抑制剂治疗的LN小鼠与对照组相比LN表现明显减轻，外周血及肾脏中Tfh细胞数量显著减少。. 综上所述，Cblb是Tfh细胞的负性调控因子，Cblb的表达下调诱导LN患者外周血中 Tfh细胞增多，加重LN症状；同时研究证明Cblb通过泛素化Bcl-6调节Tfh。因此，Bcl-6可能是LN的一个新的潜在治疗靶点。