Identification of the central tolerance checkpoint for autoreactive proteinase 3⁺ B cells in human bone marrow

Major target antigens of ANCA-associated vasculitis (AAV) are myeloperoxidase (MPO) and proteinase 3 (PR3). High-affinity MPO- and PR3-ANCA immunoglobulins are produced by antigen-experienced, class-switched autoreactive B cells. To prevent autoreactivity, B cells are subjected to several self-tolerance checkpoints, from the early immature stages in the bone marrow (BM), collectively called "central tolerance", to late mature stages, collectively called "peripheral tolerance"; the latter was recently elucidated for autoreactive PR3⁺ B cells. Here we investigated central tolerance controlling immature PR3⁺B cells in the BM before their migration into the periphery as transitional B cells. We applied an established flow cytometry-based method using labeled recombinant PR3 (rPR3) to identify the PR3⁺B cells to compare the phenotype of PR3⁺B cells in paired samples of BM mononuclear cells (BMMC) and peripheral blood mononuclear cells (PBMC) of non-vasculitis controls (No-AAV), and PBMC of patients with PR3-ANCA-associated vasculitis (PR3-AAV). We observed that the proportion of PR3⁺B cells within BMMC was higher (median [IQR]; 1.98 % [1.77-2.75]) than within PBMC of No-AAV (0.9 % [0.63-1.44], p < 0.01 by paired comparison) and similar to their proportion within PBMC of patients with PR3-AAV (1.82 % [1.66-3.21]; p > 0.05). Within CD24⁺⁺CD38⁺⁺ B cells, the subset of B cell migrating from BM to the periphery, BMMC contained a greater proportion of PR3⁺B cells as compared to PBMC in No-AAV (3.35 % [1.99-4.92] versus 1.23 % [0.62-1.55], p < 0.01), showing different surface markers of maturation (i.e. higher proportion of CD27^-CD10⁺ and lower expression of CD21, IgD, IgM). Importantly, we observed a significant decline of the PR3⁺ fraction from the immature subset (IgD^-IgM⁺; 2.80 % [1.23-4.02]) to the early transitional subset (IgD⁺IgM⁺; 1.76 % [0.96-2.68], p < 0.01) in BMMC, while no significant reduction was observed between the early transitional of BMMC and the transitional compartment of PBMC in No-AAV (1.26 % [0.62-1.56], p > 0.05). In conclusion, to prevent PR3-related autoimmunity, autoreactive PR3⁺B cells pass a stringent selection in the BM, and their removal by central tolerance checkpoint activity occurs mainly between T1-like/immature to T2-like/early transitional B cells of BMMC.