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

 描述（由申请人提供）：现在清楚的是，B细胞在调节免疫应答中起重要作用。小鼠中的B细胞缺陷或耗竭可使自身免疫恶化，并通过被认为靶向T细胞的各种试剂防止同种异体移植物耐受。此外，我们发现调节性B细胞（BCRs）可被诱导，并负责抗TIM-1介导的移植物存活延长。这些研究表明，Bcl 4在同种异体移植耐受中起重要作用。这与在耐受的人肾移植受者中观察到的“B细胞谱”相呼应。然而，我们对BclB的理解受到阻碍，因为它们很罕见，并且它们唯一的特异性标志物是IL-10，到目前为止，IL-10仅在离体刺激后检测到。 各种B细胞亚群的转移可以以IL-10依赖的方式抑制炎症并防止同种异体移植物排斥。然而，这些亚群抑制，因为它们在给定模型中含有最高比例的IL-10+ B细胞，而不是代表真正的布雷格表型。值得注意的是，这些亚群通常很小（每个亚群占总B细胞的2-15%），并且IL-10+细胞在每个亚群中仍然仅占5-15%。因此，目前的研究仅考虑了少数IL-10+ B细胞。相比之下，<1%的滤泡（FO）B细胞表达IL-10，但由于所有B细胞中的50-70%是FO B细胞，因此这些细胞是IL-10+ B细胞的主要来源。这使得通过转移B细胞亚群来研究IL-10具有挑战性，解决方案在于直接鉴定IL-10+ B细胞而不需要离体培养和模拟。这些相同的问题导致了对Bceptin如何在体内发挥作用的理解明显缺乏。接受贝伐他汀的小鼠在很大程度上被视为“黑匣子”。我们不知道哪种IL-10 + B亚群起抑制因子的作用，它们在哪里产生，以及它们如何和在哪里影响抑制因子的功能。这种理解是开发临床相关方法以增强布雷格数量和功能并避免其意外消耗的关键。 我们现在表明，B细胞的IL-10表达实际上可以在没有体外模拟的情况下被鉴定，并且大多数IL-10+ B细胞表达IL-10。 细胞实际上是B1 a、MZ和FO B细胞，甚至是浆细胞。此外，IL-10+ B细胞可以直接原位可视化。我们的领先数据证实，IL-10+ B细胞填充脾MZ，滤泡和红髓。基于这些数据，我们现在的目标是确定Breg依赖性同种异体移植模型中Breg在体内如何发挥作用以防止排斥反应。B细胞的不同亚群在次级淋巴器官（SLO）内具有不同的作用和定位。因此，在目的1中，我们将确定哪些IL-10+ B细胞亚群表现出布雷格活性以及不同亚群是否具有亚特化功能。在目标2中，我们将确定Bcl 2在SLO内的何处实际上起作用以抑制同种免疫应答。在目标3中，我们将直接确定Bcl 3是否通过直接同源相互作用抑制T细胞。我们利用了先前未应用于布雷格研究的最先进的成像和诱导型KO小鼠。这项工作将大大提高我们对布雷格免疫生物学的理解，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.