In Vivo Detection And Mechanisms Of Regulatory B Cell Function In Transplantation

 描述(由申请人提供)：现在很清楚，B细胞在调节免疫反应中起着重要作用。小鼠的B细胞缺乏或耗尽会恶化自身免疫力，并通过各种被认为以T细胞为靶点的药物来防止同种异体移植耐受。此外，我们还表明，调节性B细胞(Bregs)可以被诱导，并与抗TIM-1介导的移植物存活时间延长有关。这些研究表明Bregs在同种异体移植耐受中起重要作用。这与在耐受性人类肾移植受者中观察到的“B细胞特征”相呼应。然而，我们对Bregs的理解受到了阻碍，因为它们很罕见，而且它们唯一的特异性标记是IL-10，到目前为止，只有在体外刺激后才能检测到IL-10。转移不同的B细胞亚群可以抑制炎症，并以IL-10依赖的方式防止同种异体移植排斥反应。然而，这些亚群受到抑制，因为它们含有给定模型中IL-10+B细胞的最高比例，而不是代表真正的Breg表型。值得注意的是，这些亚群通常很小(每个占B细胞总数的2%-15%)，而IL-10+细胞在每个亚群中仍然只占5%-15%。因此，目前的研究只占IL-10+B细胞的一小部分。相比之下，1%的滤泡(FO)B细胞表达IL-10，但由于所有B细胞中有50%-70%是FO B细胞，这是IL-10+B细胞的主要来源。这使得通过B细胞亚群转移来研究Bregs具有挑战性，而解决方案在于直接鉴定IL-10+B细胞，而不需要体外培养和模拟。这些同样的问题也导致了对Bregs在体内如何发挥作用的明显缺乏了解。接受Bregs治疗的小鼠在很大程度上被视为“黑匣子”。我们不知道哪些IL-10+B亚群作为Bregs发挥作用，它们在哪里产生，以及它们如何和在哪里影响抑制功能。这样的理解对于开发临床上相关的方法以增强Breg数量和功能并避免它们的无意耗尽是关键。我们现在证明，实际上无需体外模拟就可以确定B细胞表达IL-10，而且大多数IL-10+B 细胞实际上是B1A、MZ和FO B细胞，甚至是浆细胞。此外，IL-10+B细胞可直接在原位可见。我们的铅数据证实，IL-10+B细胞存在于脾的MZ、滤泡和红髓中。基于这些数据，我们现在的目标是确定Bregs在体内如何发挥作用，以防止BREG依赖的同种异体移植模型中的排斥反应。不同的B细胞亚群在次级淋巴器官(SLO)中有不同的作用和定位。因此，在目标1中，我们将确定哪些IL-10+B细胞亚群具有BREG活性，以及不同的亚群是否具有亚特化功能。在目标2中，我们将确定SLO Bregs实际上作用于抑制同种异体免疫反应的位置。在目标3中，我们将直接确定Bregs是否通过直接同源相互作用抑制T细胞。我们利用最先进的成像和可诱导的KO小鼠，以前没有应用于BREG研究。这项工作将极大地提高我们对BREG免疫生物学的理解，并提供与同种异体移植耐受高度相关的治疗见解。

 DESCRIPTION (provided by applicant): It is now clear that B cells play an important role regulating immune responses. B cell deficiency or depletion in mice can worsen autoimmunity and prevent allograft tolerance by various agents thought to target T cells. Moreover, we showed that regulatory B cells (Bregs) can be induced and are responsible for prolonged graft survival mediated by anti-TIM-1. These studies suggest that Bregs play an important role in allograft tolerance. This is echoed by the "B cell profile" observed in tolerant human renal allograft recipients. However, our understanding of Bregs is hampered because they are rare and their only specific marker is IL-10, which thus far, has only been detected after stimulation ex vivo. Transfer of various B cell subsets can inhibit inflammation and prevent allograft rejection in an IL-10 dependent fashion. However, these subsets suppress because they contain the highest proportion of IL-10+ B cells in a given model, rather than representing a true Breg phenotype. Of note, these subsets are generally small (each making up 2-15% of total B cells), and IL-10+ cells are still only 5-15% in each subset. Thus, current studies only account for a minority of IL-10+ B cells. In contrast, <1% of follicular (FO) B cells express IL-10, but because of 50-70% of all B cells are FO B cells, these are a major source of IL-10+ B cells. This has made study of Bregs by transfer of B cell subpopulations challenging, and the solution lies in identifying IL-10+ B cells directly without the need for ex vivo culture and simulation. These same issues have contributed to a remarkable lack of understanding of how Bregs function in vivo. Mice receiving Bregs have largely been treated as a "black box". We do not know which IL-10 + B subsets function as Bregs, where they arise, and how and where they effect suppressor function. Such understanding is key to developing clinically relevant approaches to enhance Breg number and function, and to avoid their inadvertent depletion. We now show that IL-10 expression by B cells can in fact be identified without in vitro simulation, and that most IL-10+ B cells are actually, B1a, MZ, and FO B cells, and even Plasma Cells. Moreover, IL-10+ B cells can be directly visualized in situ. Our lead data confirm that IL-10+ B cells populate the splenic MZ, follicle, and red pulp. Based on these data we now aim to determine how Bregs actually function in vivo to prevent rejection in Breg-dependent allograft models. Different subsets of B cells have different roles and localization within the secondary lymphoid organs (SLO). Therefore in Aim 1 we will determine which IL-10+ B cell subpopulations exhibit the Breg activity and whether different subsets have subspecialized functions. In Aim 2, we will determine where within the SLO Bregs actually act to inhibit the alloimmune response. In Aim 3, we will directly determine whether Bregs suppress T cells through direct cognate interactions. We utilize state of the art imaging and inducible KO mice not previously applied to Breg studies. This work will greatly enhance our understanding of Breg immunobiology and provide therapeutic insights highly relevant to allograft tolerance.