Background/Purpose : We recently identified a human B cell population, which is naturally autoreactive and tolerized by functional anergy. This population was naïve, fully mature, negative for surface IgM and expressing only IgD BCR (BND cells: naïve, D-only). The BND cells have antibody variable region genes in germline configuration and a proportion of anergic cells exhibit natural reactivity to antinuclear antigens and dsDNA. Break of B cell tolerance leading to presence of autoantibodies in rheumatic diseases may cause the initiation and perpetuation of various autoimmune processes. The aim of this study was therefore to investigate if anergy in BND cells could be overcome by T cell mediated signals as has been reported in murine systems, and furthermore, study the molecular mechanisms underlying this process.

Methods : Untouched naive and BND human B cells from healthy blood donors were purified and cultured in the presence of T cell help (e.g. IL-4 and CD40L) followed by various functional assays such as Ca2+-flux, phospho-flow (pSyk, pLyn) and standard flow cytometry

measurements of intracellular and surface B cell markers. Finally, CD45 phosphatase activity was measured utilizing fluorescent peptide-based assay.

Results : Stimulation mimicking T cell help broke anergy and forced BND cells to fully respond to antigenic stimulation by restoring normal signaling through the BCR. This was dependent on de novo protein synthesis as opposed to direct crosstalk between the BCR, IL-4 and CD40 signaling pathways. We traced the restoration of BCR signaling from downstream signaling stages such as intracellular calcium release to phosphorylation of Syk and Lyn kinase, which is located proximal to the BCR. We observed an inability of activation of Lyn in BND cells upon BCR-stimulation, while subsequent to IL-4/CD40L treatment, the activation state of Lyn was restored. Lyn kinase activity is principally regulated by CD45 phosphatase (activating) and Csk kinase (inactivating). By inhibition studies, we identified CD45 phosphatase as a key component in the process of restoration of BCR signaling by T cell mediated signals. As a next step, we therefore investigated if T helper signals regulate CD45 expression in anergic B cells. A modest upregulation of CD45 surface expression on B

cells stimulated with T cell mimicking signals was observed. Since CD45 is a complex molecule with many possible isoforms exhibiting different levels of phosphatase activity, quantitation of CD45 phosphatase activity of B cells upon T cell stimulation was performed. These analyses showed enhanced CD45 phosphatase activity upon T cell help in BND cells as well as in naive B cells, pinpointing CD45 as a key regulator of BCR signaling thresholds mediated by T cell help.

Conclusion : Our findings reveal a novel connection between T cell help, increased CD45 phosphatase activity and BCR signaling in human primary anergic and naïve B cells, and further implies that anergic BND cells may represent the precursors of autoantibody secreting plasma cells in B cell driven autoimmune conditions. Our data provide new insight in the break of humoral immune tolerance with possible diagnostic and therapeutic implications in patients with systemic autoimmune and rheumatic diseases.