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

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 In unpublished studies we explored B cell immunity in the arterial wall vs secondary lymphoid organs (SLOs) in mice.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.