Immune tolerance established during the development of B lymphocytes can be subverted in mature cells and lead to autoimmunity. This study focuses on the recently discovered subset of CD19+CD27−IgD+IgMlow/− B cells that recognize self-antigens and have the capacity to produce autoantibodies, but under normal conditions do not generate autoimmune response due to intrinsic signaling inhibition (a condition known as clonal anergy and characterized by impaired antigen receptor signaling). Phosphorylation of intracellular signaling proteins and Ca2+ responses in anergic B cells were measured by multicolor flow cytometry. Our results demonstrate a distinct phosphoryation pattern for major signal transduction proteins, which distinguishes anergic B cells. Comparison of B cell signaling properties in Rheumatoid Arthritis patients and healthy controls revealed a reversal of pTyr and Ca2+ anergic signaling features in patients, accompanied by phosphorylation decreases of Blnk, Syk, SHP2, CD19. We identified BCR signaling pathway alterations associated with the loss of anergic B cell tolerance in Rheumatoid Arthritis.
B lymphocytes exhibit phenotypic differences that correlate with their developmental or functional stages and affect humoral immune responses. One recently described subset of naturally occurring immature transitional type 3 (T3) B lymphocytes is believed to consist of potentially autoimmune cells whose signaling properties have not been studied in detail. This study characterizes intracellular signaling in T3 B cells in wildtype C57BL/6 mice. Protein phosphorylation and Ca2+ responses upon B cell antigen receptor (BCR) engagement were measured by multicolor flow cytometry. We observed high baseline signaling activity and reduced BCR-mediated responses in T3 cells, which confirmed their anergy – a functional state in which lymphocytes recognize chronically present self-antigens but cannot produce immune response due to intrinsic signaling inhibition. Our results also revealed a previously unknown T3-specific phosphorylation pattern of 24 key signaling molecules involved in BCR signal transduction. These characteristics reflect the balance between stimulatory and inhibitory BCR signaling pathways in anergy. Results obtained in the collagen induced arthritis model demonstrate the loss of anergy in T3 B cells during the onset of the disease. Our findings provide rationale for further investigating alterations in B cell signaling patterns as earliest functional biomarkers of changes in the immune tolerance of autoreactive B cells.
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