In Vivo Detection And Mechanisms of Regulatory B cell Function in Transplantation

B细胞在免疫反应中起着重要的调节作用。小鼠的B细胞缺乏或耗尽会恶化 自身免疫和防止同种异体移植耐受。我们发现TIM-1是IL-10+调节性B细胞的广泛标志物 Bregs和抗TIM-1通过诱导IL-10+Bregs诱导耐受。基于这一发现， 我们的合作者Kuchroo博士刚刚证明TIM-1调节B细胞上的各种抑制分子 除了IL-10之外，缺乏B细胞-TIM-1的小鼠会产生自发的系统性自身免疫。 这毫不含糊地表明了布雷格在保持宽容方面的关键作用。大体上同意， 耐受性人肾移植受者表现出“Breg图谱”和移植围术期B细胞的耗竭 肾移植和心脏移植后急性排斥反应和血管病变分别增加。尽管如此 尽管如此，我们对Bregs的理解仍处于初级阶段。首先，甚至对于哪一个B都没有达成一致意见 细胞执行Breg功能，为什么不同研究中的Bregs属于不同的亚集，甚至是否 实际上是浆细胞(PC)而不是B细胞本身执行Breg功能。其次，人们对此知之甚少。 Bregs在体内受到调节，或者什么信号可以用来扩大它们。基于我们和我们的合作者的 这项提议将直接解决这两个主要差距。这一领域一直受到阻碍，因为 Bregs很少见，其最好的标记物IL-10只有在体外刺激B细胞后才能检测到。 转移不同的B细胞亚群可以抑制移植和自身免疫模型中的炎症。然而， 这些亚群是抑制的，因为它们在给定的 模型，而不是代表真正的Breg表型。这些亚群中只有5%-15%的B细胞表达IL-10， 每个亚群占所有IL-10+B细胞的20%。因此，该领域已经确定了无数不同的 Breg“亚群”(通常是较小的或不成熟的)，对于它们的功能还没有达成共识。 10-GFP报告小鼠，我们最近证明无需体外培养即可直接鉴定IL-10+B细胞 文化。此外，在蛋白质水平上，规范卵泡B细胞(FOB)、边缘区B细胞(MZB)和PC 每种细胞约占所有B系IL-10的30%。我们假设Bregs属于这些典范子集 在SLO中有明显定位的，具有不同的功能。在AIM 1中，使用纯化的IL-10+或TIM-1+ Bregs属于这三个子集，我们现在将直接确定他们监管的相同或不同 免疫反应的各个方面(例如体液与细胞)。而据报道，个人电脑对Breg来说是必不可少的 功能，我们发现由于BLIMP-1的B细胞特异性缺失而不能产生PC的小鼠表现出 同种异体移植物存活率和不那么严重的EAE的调节表型，两者均显著增加 IL-10+和Tim-1+Bregs的频率和数量。因此，在目标2中，我们将确定 BLIMP-1、PC和微生物区系相互作用，调节体内的Breg数量。这项工作将极大地提高我们的 了解B细胞和BREG生物学，并提供与同种异体移植耐受高度相关的治疗见解。

B cells play an important role regulating immune responses. B cell deficiency or depletion in mice can worsen autoimmunity and prevent allograft tolerance. We showed that TIM-1 is a broad marker for IL-10+ regulatory B cells (Bregs) and that anti-TIM-1 induces tolerance through induction of IL-10+ Bregs. Based on this discovery, our collaborator Dr. Kuchroo, has just shown that TIM-1 regulates various inhibitory molecules on B cells in addition to IL-10, and that mice specifically lacking B cell-TIM-1 develop spontaneous systemic autoimmunity. This unequivocally demonstrates a critical role for Bregs in maintaining tolerance. In general agreement, tolerant human renal allograft recipients demonstrate a “Breg profile” and peri-transplant depletion of B cells increased acute rejection and vasculopathy after renal and cardiac transplantation, respectively. Despite these advances, our understanding of Bregs remains in its infancy. First, there is no agreement even about which B cells carry out Breg function, why Bregs in different studies belong to different subsets, or even whether plasma cells (PCs), and not B cells per se, actually carry out Breg function. Second, little is known about how Bregs are regulated in vivo, or what signals can be used to expand them. Based on our and our collaborator’s advances, this proposal, will directly address both of these major gaps. The field has been hampered because Bregs are rare and their prior best marker, IL-10, was only detected after stimulation of B cells ex vivo. Transfer of various B cell subsets can inhibit inflammation in transplant and autoimmune models. However, these subsets are suppressive because they contain a relatively high proportion of IL-10+ B cells in a given model, rather than representing a true Breg phenotype. Only 5-15% of B cells in such subsets express IL-10, and each subset comprises <20% of all IL-10+ B cells. As a result, the field has identified a myriad of different Breg "subsets” (often minor or immature), and there is no consensus as to their function. However, using IL- 10-GFP reporter mice, we recently demonstrated that IL-10+ B cells can be directly identified without in vitro culture. Moreover, on a protein level, canonical Follicular B cells (FOB), Marginal Zone B cells (MZB) and PCs each account for ~30% of all B-lineage IL-10. We hypothesize that Bregs belonging to these canonical subsets that have distinct localization in the SLO, have different functions. In AIM 1, using purified IL-10+ or TIM-1+ Bregs belonging to these 3 subsets, we will now directly determine whether they regulate the same or different aspects of the immune response (e.g. humoral vs. cellular). While PCs were reported to be essential for Breg function, we found that mice unable to generate PCs due to B cell-specific deletion of BLIMP-1 exhibit a regulated phenotype with improved allograft survival and less severe EAE, and a marked increase in both frequency and number of IL-10+ and TIM-1+ Bregs. Therefore, in Aim 2, we will identify how changes in BLIMP-1, PCs, and the microbiota interact to regulate Breg numbers in vivo. This work will greatly enhance our understanding of B cell and Breg biology and provide therapeutic insights highly relevant to allograft tolerance.