B Cell Autoimmunity in ApoE-deficient (ApoE-/-) Mice

B淋巴细胞亚群在动脉粥样硬化中起相反的作用，但动脉粥样硬化B细胞免疫在何处以及如何组织尚不清楚。在未发表的研究中，我们探讨了小鼠动脉壁B细胞免疫与次级淋巴器官（slo）的关系。转录图谱、流式细胞术、细胞转移和免疫球蛋白ELISPOT检测显示，动脉粥样硬化B细胞免疫在衰老过程中在动脉三级淋巴器官（ATLOs）中组织。在ATLOs与内膜斑块和slo中发现了大的外基质B细胞相关转录组。ATLO B-2 B细胞亚型包括naïve，移行，滤泡，生发中心，切换Ig+， IgG1+， IgA+和IgE+记忆细胞，B10+ B调节细胞，以及短寿命和长寿命浆细胞。组成性IgM和igg分泌浆细胞在ATLOs中大量存在，而在slo中含量较低。ATLOs中积累了大量的B-1细胞，这种B细胞区室强烈偏向于B-1b细胞而不是B-1a细胞。我们得出结论，ATLOs在衰老过程中协调多层动脉粥样硬化的B-1和B-2细胞反应。B-2细胞亚型的范围表明了自身抗原触发的生发中心反应、Ig类转换以及病变动脉壁段内IgM和IgG的组成性分泌。我们在过去2-3年中获得的研究表明，我们i)建立了用NGS检测BCR谱的方法；ii)建立了从单个B细胞中获得配对H + L链的方法。利用这些技术，我们的初步数据表明，自身免疫B细胞反应可能发生在ApoE-/-小鼠动脉粥样硬化发展的不同阶段。分离生发中心B细胞，对其bcr进行测序和克隆，体外表达相应的igg并获得相应的抗体。这些抗体的第一次测试显示特异性结合外膜和邻近斑块的未知结构，但它们与平滑肌细胞、内皮细胞和脂肪细胞的反应性仍为阴性。在目前的项目中，我们的目标是使用多种技术分离自身抗原，测试atlo衍生的自身免疫抗体的反应性，并开始研究抗体对体内动脉粥样硬化进展的影响。

B lymphocyte subsets play opposing roles in atherosclerosis, yet it remains unclear where and how atherosclerosis B cell immunity is organized. In unpublished studies we explored B cell immunity in the arterial wall vs secondary lymphoid organs (SLOs) in mice. Transcript maps, flow cytometry, cell transfers, and immunoglobulin ELISPOT assays revealed that atherosclerosis B cell immunity is organized in artery tertiary lymphoid organs (ATLOs) during aging. Large adventitial B cell-related transcriptomes were identified in ATLOs vs intima plaques and SLOs. ATLO B-2 B cell subtypes included naïve, transitional, follicular, germinal centre, switched Ig+, IgG1+, IgA+, and IgE+ memory cells, B10+ B regulatory cells, and short- and long-lived plasma cells. Constitutively IgM- and IgG-secreting plasma cells were abundant in ATLOs though they were low in SLOs. Marked numbers of B-1 cells accumulated in ATLOs and this B cell compartment was strongly skewed towards B-1b versus B-1a cells. We conclude that ATLOs orchestrate multi-layered atherosclerosis B-1 and B-2 B cell responses during aging. The range of B-2 cell subtypes indicates autoantigen-triggered germinal center reactions, Ig class switching, and constitutive IgM and IgG secretion within diseased arterial wall segments. Our studies obtained during the past 2-3 years showed that we i) established methods to examine the BCR repertoires by NGS; ii) established the methods to obtain the paired H + L chains from single B cells. Using these technologies, our preliminary data suggest that autoimmune B cell responses may occur at different stages of atherosclerosis development in ApoE-/- mice. Germinal center B cells were isolated, their BCRs sequenced and cloned, and the respective Igs were in vitro expressed and the corresponding antibodies obtained. The first test of these antibodies shows specific binding to unknown structures in the adventitia and in the adjacent plaques though their reactivity with smooth muscle cells, endothelia cells, and adipocytes remained negative. In the current program, we aim at the isolation of the autoantigens using a variety of techniques, test the reactivity of ATLO-derived autoimmune antibodies, and begin to examine the impact of the antibodies for atherosclerosis progression in vivo.