Regulation of VH replacement in human immature B cells by B cell receptor (BCR)-mediated signaling.

摘要

VH replacement occurs through RAG-mediated secondary recombination to change unwanted IgH genes. In this dissertation, I focused on studying the molecular mechanism that regulates VH replacement in human immature B cells.;In part I, our results show that VH replacement is regulated by B cell antigen receptor (BCR) mediated signaling. Using the human EU12 muHC + cells as an experimental model system, crosslinking BCR with F(ab') 2 anti-IgM antibodies results in BCR internalization, cell proliferation arrest, and induction of VH replacement. Pretreatment of human EU12 muHC+ cells with the protein tyrosine kinase inhibitor Genistein, Syk kinase inhibitors, and a Src kinase inhibitor blocks BCR-mediated signaling events and inhibits VH replacement. Inhibition of PI3K kinase enhances VH replacement, conversely, activation of PI3K by anti-CD19 antibodies inhibits BCR signaling induced VH replacement. Furthermore, analyses of large numbers of IgH sequences reveal that the VH replacement products are highly enriched in different autoimmune diseases and anti-viral responses.;In part II, we further dissect BCR-mediated signaling events in EU12 muHC + immature B cells compare to that in human Daudi and Ramos mature B cells. EU12 muHC+ cells have features of human bone marrow immature B cells. After 30 minutes of treatment with anti-IgM antibodies, almost 100% of EU12 muHC+ cells lost their surface BCR, in contrast, Daudi and Ramos cells only partially lost their surface BCR. After BCR internalization, restimulation of EU12 muHC+ cells with anti-IgM antibodies results in normal level of Erk1/2 activation, but with delayed Syk phosphorylation, reduced Ca2+ mobilization, and decreased FoxO1 phosphorylation. Thus, internalization of BCR on EU12 muHC + immature B cells attenuates BCR signaling with selective effects on downstream signaling events, which may favor receptor editing.;Taken together, the results presented in this dissertation provide the first evidence that VH gene replacement is regulated by BCR signaling on immature B cells. Complete internalization of BCR receptor specifically changes BCR-mediated signaling events in immature B cells.