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

B淋巴细胞亚群在动脉粥样硬化中扮演着相反的角色，但目前仍不清楚动脉粥样硬化B细胞免疫是在哪里以及如何组织的。在未发表的研究中，我们探讨了小鼠动脉壁和次级淋巴器官(SLO)中的B细胞免疫。转录图谱、流式细胞仪、细胞转移和免疫球蛋白ELISPOT分析表明，动脉粥样硬化的B细胞免疫在衰老过程中在动脉第三淋巴器官(ATLO)中组织起来。在ATLOS、内膜斑块和SLO中发现了大量外膜B细胞相关的转录本。ATLO B-2B细胞亚型包括幼稚细胞、过渡性细胞、滤泡性细胞、生发中心细胞、转换型Ig+、Ig G1+、Ig A+和Ig E+记忆细胞、B10+B调节细胞、短存活期和长存期浆细胞。在构成上，分泌IgM和免疫球蛋白的浆细胞在ATLOS中丰富，而在SLO中较低。标记数量的B-1细胞聚集在ATLO内，并且这个B细胞隔室强烈地向B-1b细胞倾斜，而不是B-1a细胞。我们得出结论，在衰老过程中，ATLO可协调多层动脉粥样硬化B-1和B-2B细胞的反应。B-2细胞亚型的范围表明，自身抗原触发的生发中心反应，Ig类转换，以及病变动脉壁节段内结构性的IgM和IgG分泌。我们在过去2-3年的研究表明，我们建立了用NGS检测BCR谱带的方法；ii)建立了从单个B细胞中获得成对的H+L链的方法。利用这些技术，我们的初步数据表明，在ApoE-/-小鼠动脉粥样硬化发展的不同阶段，可能会发生自身免疫B细胞反应。分离生发中心B细胞，对其BCR进行测序和克隆，并在体外表达相应的免疫球蛋白并获得相应的抗体。这些抗体的第一次测试显示，它们与外膜和邻近斑块中的未知结构具有特异性结合，尽管它们与平滑肌细胞、内皮细胞和脂肪细胞的反应性仍为阴性。在目前的计划中，我们旨在利用各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，并开始检测抗体在体内对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 In unpublished studies we explored B cell immunity in the arterial wall vs secondary lymphoid organs (SLOs) in mice.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.