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 PR3B 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 PR3B cells to compare the phenotype of PR3B 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 PR3B 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 CD24CD38 B cells, the subset of B cell migrating from BM to the periphery, BMMC contained a greater proportion of PR3B 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 CD27CD10 and lower expression of CD21, IgD, IgM). Importantly, we observed a significant decline of the PR3 fraction from the immature subset (IgDIgM; 2.80 % [1.23-4.02]) to the early transitional subset (IgDIgM; 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 PR3B 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.