B cell lymphoma (BCL)6 and Bcl-xL are expressed in germinal center (GC) B cells and enable them to endure the proliferative and mutagenic environment of the GC. By introducing these genes into peripheral blood memory B cells and culturing these cells with factors produced by follicular helper T cells, CD40L and IL-21, we convert them to highly proliferating, cell surface BCR positive, Ig-secreting B cells with features of GC B cells including expression of activation-induced cytidine deaminase. We generated cloned lines of B cells specific for respiratory syncytial virus and used these cells as a source of antibodies that effectively neutralized this virus in vivo. This method provides a new tool to study GC B cell biology, signal transduction through antigen-specific B cell receptors, and for the rapid generation of high affinity human monoclonal antibodies.
STAT family members have been implicated in regulating the balance between B cell lymphoma (BCL)6 and B lymphocyte induced maturation protein (BLIMP)1 to control plasma cell differentiation. We previously showed that STAT5 induces BCL6 to block plasma cell differentiation and extend the life span of human B cells. The heterogeneity in STAT activation by cytokines and their effects on B cell differentiation prompted us to investigate the effect of STAT3 activation in plasma cell differentiation. First stimulation with IL-21, which promotes plasma cell differentiation, induced robust and prolonged STAT3 activation in primary human B cells. We then investigated effects of direct STAT3 activation on regulation of plasma cell genes, cellular phenotype, and Ig production. Activation of a tamoxifen-regulated STAT3-estrogen receptor fusion protein triggered BLIMP1 mRNA and protein up-regulation, plasma cell phenotypic features, and Ig secretion. When STAT3 was activated by IL-21 in B cells ectopically expressing BCL6, BLIMP1 was up-regulated, but only partial plasma cell differentiation was achieved. Lastly, through coexpression of BCL6 and STAT3-ER, we verified that STAT3 activation functionally mimicked IL-21 treatment and that STAT3-mediated BLIMP1 up-regulation occurred despite high BCL6 expression levels indicating that BCL6 is not the dominant repressor of BLIMP1. Thus, up-regulation of BLIMP1 alone is not sufficient for differentiation of primary human B cells into plasma cells; concomitant down-regulation of BCL6 is absolutely required for completion of the plasma cell differentiation program.
Using multivalent protein probes, an evolutionarily conserved endogenous ligand for EMR2, a human myeloid cell-restricted EGF-TM7 receptor, was identified on the surface of a number of adherent cell lines. In addition, in situ staining of the ligand has revealed specific in vivo patterns consistent with a connective tissue distribution. The interaction is conserved across species and mediated exclusively by the largest EMR2 isoform containing 5 epidermal growth factor (EGF)-like modules. Antibody-blocking studies subsequently revealed that the fourth EGF-like module constitutes the major ligandbinding site. The largest isoform of CD97, a related EGF-TM7 molecule containing an identical EGF-like module, also binds to the putative EMR2 ligand. Through the use of mutant Chinese hamster ovary (CHO) cell lines defective in glycosaminoglycans (GAGs) biosynthesis as well as the enzymatic removal of specific cell surface GAGs, the molecular identity of the EMR2 ligand was identified as chondroitin sulfate (CS). Thus, exogenous CS GAGs blocked the EMR2-ligand interaction in a dose-dependent manner. EMR2-CS interaction is Ca 2؉ -and sulphation-dependent and results in cell attachment. This is the first report of a GAG ligand for the TM7 receptors extending the already vast repertoire of stimuli of the GPCR superfamily.
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