Interaction of TAPP adapters with the phosphoinositide PI(3,4)P2 regulates B cell activation and differentiation
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Phosphoinositide 3-kinase is a family of lipid kinases that function by phosphorylating the D3 position of phosphoinositide (PI) lipids generating PI(3)P, PI(3,4)P2 and PI(3,4,5)P3. These D3 phosphoinositides regulate various cellular processes through the recruitment of effector proteins containing lipid specific pleckstrin homology (PH) domains. PI phosphatases such as PTEN and SHIP function to restrain PI3K signaling by limiting the amount of D3 PI available for binding. Deletion of either PTEN or SHIP significantly alters B cell function and humoral immune responses. TAPP1 and TAPP2 are dual PH domain containing adaptors which selectively bind the phosphoinositide PI(3,4)P2 via their C-terminal PH domains. PI(3,4)P2 is a lipid messenger generated by PI3K and through the inositol phosphatase activity of SHIP. The function of PI(3,4)P2 remains incompletely understood. To identify the functional role of TAPP-PI(3,4)P2 interactions, we utilized a knock-in (KI) mouse bearing mutations within the PI-binding pocket of both TAPPs. Our study assessed the effect of PI3K dependent KI mutation on B lymphocyte development, activation and antibody production. Flow cytometry analyses of lymphoid tissues found that TAPP KI mice develop relatively normal frequencies of mature B cell populations with the exception of peritoneal B1 cells, which are increased by approximately 50%. Strikingly, TAPP KI mice developed substantially elevated serum antibody levels. TAPP KI mice were able to generate high affinity antigen-binding antibodies upon immunization with NP-OVA in alum adjuvant; however, total immunoglobulin production was markedly increased under this immunization condition. We further assessed the germinal centre (GC) response, which are known to require PI3K signaling and a hallmark of T cell dependent (TD) antibody responses. TAPP KI mice generated larger germinal centers (GC) upon immunization, which was associated with increased GC B cell survival. We further assessed whether uncoupling of TAPPs from PI(3,4)P2 alters B cell signaling and functional responses in vitro. B cells purified from TAPP KI mice were found to have altered functional responses in vitro, with significantly increased survival and cell division following antigen receptor cross-linking. Consistent with increased cell survival, TAPP KI B cells show increased Akt phosphorylation on Ser473 and Thr308 after antigen receptor cross-linking. However, reconstitution of B cell deficient mice with either WT or TAPP KI B cells was found to generate similar GC responses, suggesting that activation of other cells may contribute to the enhanced in vivo responses. Consistently, when we examined the CD4+ T follicular helper cells, a subset providing critical cues to GC responses, we found increased expression of ICOS activation marker. Our results indicate the interactions of TAPP adapters with PI(3,4)P2 serve to restrain lymphocyte activation and limit antibody production, providing the first in vivo evidence that this interaction is important for immune function.