Mechanisms of action, optimization and application of Bregs

项目总结/摘要 有人认为，医学的未来取决于细胞和基因治疗。虽然这一断言可能为时过早， 很明显，这些创新疗法具有非凡的，范式转变的潜力。对于基于细胞的疗法， 嵌合抗原受体（CAR）T细胞在治疗血液病中的强大影响是最好的例证。 目前标准治疗难治性恶性肿瘤（5）。在移植领域，MSC，干细胞衍生的胰岛， 促进细胞促进骨髓对肾移植物的耐受已经显示出令人鼓舞的结果， 治疗学（6-8）。此外，许多早期临床试验正在进行中，以探索调节T细胞的潜力。 细胞（THEORY），以减轻肝和肾同种异体移植物的排斥反应（9，10）。最近，CAR技术已经被应用于 以产生CAR T细胞作为更有效的现成的供体抗原特异性调节群体（11）。 在目前的建议中，我们研究了适应性免疫的另一个分支中的调节特性，以关注 B细胞的调节活性。在发现B细胞是各种实验移植耐受所必需的之后， 而且从对胰岛耐受的小鼠中分离的B细胞（或BCRs）可以过继性地将耐受转移到其他小鼠中。 未处理的B细胞缺陷型宿主中，我们试图离体扩增致耐受性布雷格群体。在这样做的过程中， 出乎意料的观察是，即使是用Toll样受体（TLR）离体刺激的幼稚B细胞也表现出有效的 在MLR中的抑制活性和在体内防止移植物排斥（12）。随着进一步的研究，我们证明，各种 B细胞激活信号的激活可诱导布雷格抑制，并且依赖于激活触发， 体内抑制不同。我们的总体假设是，布雷格抑制是通过抗原呈递 在局部免疫调节细胞因子如TGF-β和IL-10的情况下。为了支持这一观点，我们发现 供体的B细胞特异性对抑制功能至关重要，可能表明同源Breg-T细胞/Treg 通信（13，14）。同样值得注意的是，我们发现对供体抗原具有特异性的克隆布雷格群体， 在体外和体内表现出最大的抑制效力（13）。根据这些发现，在目标3中，我们将开展 创新的研究，以确定是否bepaly在线网投，充满了捐助者的特异性，通过暂时或永久 供体特异性CAR的表达，防止同种异体移植排斥。此外，最近的研究表明，CAR可以 使用修饰有抗体的脂质纳米颗粒（LNP）在体内有效递送以靶向选择的细胞群， 例如宿主B细胞（15）。为提供疫苗而开发的LNP方法也适用于平行转移， 编码抑制性分子（IL-10、TGF-β等）的mRNA有效载荷的表达增加效力（16）。总的来说， 我们的工作将进一步了解布雷格的分化和抑制机制，优化布雷格的表达， 功能和评估作为细胞治疗剂的BCR 4和CAR BCR 4的潜力。

PROJECT SUMMARY / ABSTRACT It has been suggested that the future of medicine rests in cell and gene therapy. While this assertion may be premature, it seems clear that these innovative therapies have extraordinary, paradigm shifting potential. For cell-based therapies, this is best exemplified by the powerful impact of chimeric antigen receptor (CAR) T cells in the treatment of hematological malignancies refractory to current standard of care (5). In the transplant realm, MSC’s, stem cell derived islets, and facilitator cells to promote bone marrow tolerance to renal grafts have shown encouraging results as cell-based therapeutics (6-8). In addition, numerous early phase clinical trials are underway exploring the potential of regulatory T cells (Tregs) to mitigate rejection of liver and kidney allografts (9, 10). More recently, CAR technology has been employed to generate CAR Tregs as more potent, off-the-shelf, donor antigen-specific regulatory populations (11). In the current proposal, we investigate the regulatory properties in the other arm of adaptive immunity to focus on the regulatory activities of B cells. After finding that B cells were required for tolerance in varied experimental transplant models and that B cells (or Bregs) isolated from mice tolerant to islets could adoptively transfer tolerance to otherwise untreated B cell deficient hosts, we sought to expand the tolerogenic Breg population ex vivo. In so doing, we made the unexpected observation that even naïve B cells stimulated ex vivo by Toll-like receptors (TLRs) manifested potent suppressive activity in MLRs and prevented graft rejection in vivo (12). With further study, we demonstrate that a variety of B cell activating signals induce Breg suppression and that, depending on the activating trigger, the mechanism of suppression in vivo varies. Our overarching hypothesis is that Breg suppression is executed through antigen presentation in the context of local immunoregulatory cytokine elaboration, such as TGF-ß and IL-10. Supporting this notion, we found that B cell specificity for the donor was essential to suppressive function, perhaps indicating cognate Breg-T cell/Treg communication (13, 14). Also noteworthy is our finding that clonal Breg populations with specificity for donor antigens exhibited the greatest suppressive potency in vitro and in vivo (13). Based on these findings, in Aim 3, we will conduct innovative studies to determine whether Bregs, imbued with donor specificity, through either transient or permanent expression of a donor-specific CAR, prevent allograft rejection. Furthermore, recent studies indicate that CAR can be delivered effectively in vivo using lipid nanoparticles (LNP) decorated with antibodies to target select cell populations, such as host B cells (15). The LNP approach developed for vaccine delivery also lends itself to parallel transfer and expression of mRNA payloads encoding suppressive molecules (IL-10, TGF-ß, etc.) to augment potency (16). Collectively, our proposed work will advance understanding of Breg mechanisms of differentiation and suppression, optimize Breg function and evaluate the potential of Bregs and CAR Bregs as cellular therapeutics.