Regulatory B-cells Limit CNS Inflammation and Infarct in Experimental Stroke

描述(申请人提供)：最近的研究已经确定了一小部分但功能强大的产生IL-10的CD1dhiCD5+调节性B淋巴细胞(B10)，可以在啮齿动物模型中限制中枢神经系统炎症和神经疾病的临床症状。在这种多PI应用中，我们假设内源性调节性B细胞，包括B10亚群，通过控制局灶性卒中在中枢和外周免疫器官触发的免疫介导性炎症来限制MCAO的梗塞范围。此外，我们预测，无论是选择性地诱导或被动转移B10细胞，以增加B10细胞的频率，都将在野生型(WT)小鼠中提供额外的调节作用，并导致较小的梗塞和改善的功能性卒中结果。本研究的目的是：1)研究B淋巴细胞耗竭及调节性B10细胞修复和治疗对MCAO后大鼠脾、脑半球梗死面积、免疫细胞组成和炎症反应的影响。假说是B细胞KO小鼠比WT小鼠遭受更大的组织损伤和更差的功能结局。我们将确定负责的B细胞亚群，包括分泌IL-10的B10亚群。2)探讨PD-1/PD-L负共激活通路对小鼠脾、脑梗塞面积、免疫细胞组成及炎症反应的影响。假设B淋巴细胞，特别是CD1dhiCD5+B细胞中这种受体-配体通路的激活改变了MCAO的结果。本研究的目的是评估PD-1、PD-L1和PD-L2在B细胞上的表达，证实我们关于PD-1KO小鼠梗塞面积增加的初步发现，并将分析扩展到PD-L1和PD-L2KO小鼠，通过将WT B10-细胞重组PD-L KO小鼠，评估B10-细胞在卒中后PD-1/PD-L负共激活中的作用。3)探讨B细胞抑制小胶质细胞(MG)激活和释放神经毒性因子的机制。假说是B细胞，特别是CD1dhiCD5+B调节细胞，通过抑制小胶质细胞的激活来改善缺血后的结果，无论是通过分泌的因子还是直接的细胞-细胞接触，从而实现了PD-1负的共激活机制。具体地说，这个目标将评估PD-L+CD19+B细胞或产生IL-10的CD1dhiCD5+B-reg细胞与激活的PD-1+MG之间的相互作用。拟议的研究对卒中领域产生了很大的影响，因为它们：1)完全新颖，2)专注于选定的B细胞亚群的炎症抑制调节能力，3)为急性卒中的调节性B细胞治疗奠定翻译基础。

DESCRIPTION (provided by applicant): Recent studies have identified a small but powerful subset of IL-10-producing CD1dhiCD5+ regulatory B lymphocytes (B10) that can limit CNS inflammation and clinical signs of neurological disease in rodent models. In this multi-PI application, we hypothesize that endogenous regulatory B-cells, including the B10 subset, limit infarct size in MCAO by controlling immune-mediated inflammation triggered both in the CNS and in peripheral immune organs by focal stroke. Further, we predict that either selective induction or passive transfer of B10 cells so as to augment B10 cell frequency will provide additional regulatory effects in wild type (WT) mice, and result in smaller infarct and improved functional stroke outcome. The aims of this application are: 1) Determine the effects of B lymphocyte depletion and regulatory B10 cell restoration and treatment on infarct size, immune cellular composition and inflammatory signature in both spleen and brain hemispheres after MCAO. The hypothesis is that B cell KO mice sustain larger histological damage and poor functional outcome than do WT mice. We will determine the responsible B-cell subpopulations, including the IL-10-secreting B10 subset. 2) Evaluate the contribution of the PD-1/PD-L negative co-activation pathway to infarct size, immune cell composition and inflammatory signature in spleen and brain. The hypothesis is that activation of this receptor-ligand pathway in B lymphocytes, particularly CD1dhiCD5+ B-cells, alters outcomes from MCAO. This aim will evaluate PD-1, PD-L1 and PD-L2 expression on B-cells, confirm our preliminary findings of increased infarct size in PD-1 KO mice, extend the analysis to PD-L1 and PD-L2 KO mice, and evaluate the contribution of B10- cells to the PD-1/PD-L negative co-activation after stroke by reconstituting PD-L KO mice with WT B10-cells. 3) Evaluate the mechanisms through which B-cells inhibit microglial (MG) activation and release of neurotoxic factors. The hypothesis is that B cells, particularly CD1dhiCD5+ B regulatory cells, improve post-ischemic outcomes by suppressing microglial activation, either via secreted factors or direct cell-cell contact that enables a PD-1 negative co-activation mechanism. Specifically, this aim will evaluate interaction between PD-L+ CD19+ B-cells or IL-10-producing CD1dhiCD5+ B-reg cells and activated PD-1+ MG. The proposed studies offer high impact for the stroke field by virtue of their: 1) complete novelty, 2) focus on the inflamma-suppressive regulatory capacity of select B-cell subsets and 3) ability to lay translational groundwork for regulatory B-cell therapy in acute stroke.