BCR signaling is induced by antigen engagement and is regulated in both quantitative and qualitative ways during developmental maturation by dynamic alterations in the levels or activities of key regulatory molecules. For example, in the previous funding period, we found that the Lyn/CD22/SHP-1 feedback inhibitory pathway is highly active in mature follicular B cells, but has low activity in "transitional" T1 and T2 immatue B cells of the spleen. This clearly is not the only important regulatory change that accompanies B cell maturation, but understanding of the dynamic changes in how BCR signaling is regulated are rudimentary. In the proposed experiments, we shall characterize two mechanisms by which BCR signaling reactions are dynamically regulated with the goal of achieving a more complete understanding of how B cell tolerance and activation are controlled, which is potentially important for vaccine development and for developing therapies designed to promote B cell tolerance in individuals with autoimmune disease and/or undergoing allogeneic organ transplantation. We have found that mature follicular B cells have greatly attenuated Erk MAP kinase signaling compared to T1 transitional B cells, and that this change correlates with upregulation of mRNA levels of two isoforms of diacylglycerol kinase (DGKa and z), the enzyme that removes diacylglycerol. We hypothesize that DGKa and/or DGKz upregulation sets a threshold for activation of follicular B cells. This hypothesis will be tested in Specific Aims 1 ad 2.
Specific Aims 1 is focused on characterizing the effect of deletion of DGKa or DGKz on BCR-induced signaling to Erk and NF-kB in mature follicular B cells.
Specific Aim 2 will study the effect of these mutations on antibody responses. In contrast to the developmental changes in BCR-induced Erk MAP kinase signaling, BCR-induced PIP3/Akt signaling pathway is weaker and more transient in T1 transitional B cells and is stronger and more prolonged in mature follicular B cells. Compared to T1 cells, follicular B cells express about 2-fold less PTEN, a phosphatase that removes PIP3. In addition, in deeply anergic anti-HEL MD4 Ig transgenic B cells, PTEN is upregulated by 2-fold.
In Specific Aim 3, we shall test the hypothesis that negative regulation of PIP3/Akt signaling by PTEN is an important mechanism to prevent activation of immature and anergic B cells. We have generated mice in which PTEN levels are decreased by 50% in B cells, but are normal in other cell types. These mice will be used in the proposed experiments to determine the extent to which the 2-fold increased levels of PTEN in immature T1 cells and in MD4 anergic B cells determines their attenuated PIP3/Akt signaling. In addition, the effect of decreasing PTEN levels to 50% on maintenance of tolerance of B cells that recognize DNA and other nuclear autoantigens will be assessed to understand the importance of the modulation of PTEN expression levels for receptor editing and clonal anergy of self-reactive B cells.
B lymphocytes make antibodies that protect us from infectious diseases, but in some individuals are also directed against components of our own tissues, in which case an autoimmune disease results. The proposed research will study the mechanisms by which B lymphocytes distinguish between foreign components (antigens) and self-antigens so as to achieve the twin goals of strong responsiveness to infectious agents and limited responsiveness to self-components. In particular, this project proposes to study the mechanisms by which the B lymphocyte responds to the presence of antigen and how the biochemical events generated are translated into the decision of the B lymphocyte to make antibody or to be tolerant to the antigen.
|Proekt, Irina; Miller, Corey N; Jeanne, Marion et al. (2016) LYN- and AIRE-mediated tolerance checkpoint defects synergize to trigger organ-specific autoimmunity. J Clin Invest 126:3758-3771|
|Hua, Zhaolin; Gross, Andrew J; Lamagna, Chrystelle et al. (2014) Requirement for MyD88 signaling in B cells and dendritic cells for germinal center anti-nuclear antibody production in Lyn-deficient mice. J Immunol 192:875-85|
|Wheeler, Matthew L; Dong, Matthew B; Brink, Robert et al. (2013) Diacylglycerol kinase Î¶ limits B cell antigen receptor-dependent activation of ERK signaling to inhibit early antibody responses. Sci Signal 6:ra91|
|Gross, Andrew J; Proekt, Irina; DeFranco, Anthony L (2011) Elevated BCR signaling and decreased survival of Lyn-deficient transitional and follicular B cells. Eur J Immunol 41:3645-55|