Increased Phosphorylation of Intracellular Signaling Molecules Indicates Continuous Activation of Human Autoreactive B-Cells

Many human autoimmune diseases (AIDs) are hallmarked by the presence and persistence of autoreactive B-cells. While autoreactive B-cells may frequently encounter antigens, the signals required to balance and maintain their activation and survival are mostly unknown. Understanding such signals may be important for strategies aimed at eliminating human B-cell autoreactivity. Here, we assessed intracellular signaling pathways in B cells targeting citrullinated protein antigens isolated from patients with rheumatoid arthritis (RA), a common and well-characterized AID. Peripheral blood mononuclear cells of 15 RA patients positive for anti-citrullinated protein antibodies (ACPA) were analyzed directly ex vivo using spectral flow cytometry and B-cell differentiation markers, citrullinated antigen-biotin-streptavidin tetramers, and intracellular (phosphoflow) markers. Tetanus toxoid (TT)-specific B cells served as antigen-specific comparators. In absence of any in vitro BCR stimulation, ACPA-expressing memory B cells (MBCs) displayed enhanced expression of Ki-67 and increased SYK-, BTK-, AKT-, and S6-phosphorylation compared with TT-specific MBCs. We demonstrate the simultaneous detection of B cell antigen-specificity and intracellular protein phosphorylation on the single-cell level. The data reveal that autoreactive B-cells in RA, in contrast to B cells against recall antigens, display enhanced phosphorylation of signaling molecules that point toward continuous, presumably antigen-mediated activation of the autoreactive B-cell compartment.