B Cell Receptor Signaling in Human B Cells

B cells play an important role in the early phase of the immune response particularly in polysaccharide-encapsulated bacteria-induced responses, in which B and T cell cooperation is interfered. The mechanisms of these T cell-independent (TI) -antigen-induced B cell responses have been studied mainly in mice, but the responses and the role of BCR-mediated activation in human B cells are not known. The purpose of this study was to analyze the function and regulation of antigen-specific BCR signaling in human B cells. The role of BCR signaling and a separate second signal was analyzed in an experimental model mimicking TI B cell responses caused by polysaccharide-encapsulated bacteria. It was shown that human macrophage (Mφ)-derived cytokines, as a second signal, were important enhancers of BCR stimulation-induced class switch recombination and cytokine production in B cells. In addition, it was demonstrated that B cells and Mφ function in close cooperation in TI responses as soluble mediators from activated B cells significantly enhanced cytokine production in Mφ. The regulation of BCR signaling by CD45 isoforms was studied in human GC-derived follicular lymphoma B cell lines. Novel human B cell lines expressing distinct CD45 isoforms (RA and R0) were established, and the CD45 isoform expression was shown to play a role in fine-tuning of the basal, BCRand cytokine-induced proliferation, and BCR-mediated cytokine production, and BCR-induced intracellular signaling. In addition, CD45R0 was shown to be a positive regulator of BCR-induced cellular events, whereas the CD45RA isoform was shown to function as a negative regulator. BCR-induced apoptosis is one of the most important ways to eliminate self-reactive B cells during development or GC reaction. The apoptotic process has classically been measured by detecting morphological changes or by biochemical methods such as the detection of DNA degradation. However, these methods have limited sensitivity and ability to detect apoptotic sub-populations. Therefore, a multi-parametric Annexin V-FITC, PI and SYTO 17 staining method for flow cytometric detection of apoptosis was established and evaluated. It was found that this assay increased the sensitivity to detect early apoptotic cells. As a model for B cell targeted and specific adenoviral gene therapy, a novel fusion gene, hCAR-EGFP, was constructed. It was successfully introduced into hCAR negative human follicular B cell lymphoma cells with a lentiviral gene transfer. In this experimental model it was indirectly shown that adenovirus retargeting made adenovirus resistant cells to sensitive ones, suggesting that adenoviral gene therapy of B cell-specific cancers cells is a feasible method, but further development of appropriately targeted adenovirus vectors is still required to increase the cell-type specificity and efficacy. National Library of Medicine Classification: QU 375, QW 568, QY 95, QZ 52, WH 200 Medical Subject Headings: Adaptor Proteins, Signal Transducing; Adenoviridae/genetics; Antigens, CD45; Antigens, T-Independent; Apoptosis; Apoptosis Regulatory Proteins; BLymphocytes; Cell Line; Cells, Cultured; Cytokines; Flow Cytometry; Gene Therapy; Gene Transfer Techniques; Human; Lymphoma, B-Cell; Lymphoma, Follicular; Receptors, Antigen, B-Cell; Receptors, Virus To Anna, Roosa and Elsa