Regulatory roles of PI3Ks and PH domain-containing adaptor protein Bam32 in humoral immune responses
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PI3Ks (phosphoinositide 3-kinases), a family of enzymes expressed in immune cells, are activated in response to a wide variety of stimuli by generating second lipid messengers. A subset of singnaling molecules containing lipid-binding pleckstrin homology (PH) domains are downstream molecules of PI3K signaling pathway, essential to mediate the functional outcomes of PI3Ks. Bam32 / DAPP1 is a PH domain-containing adaptor protein, which was discovered from human tonsil germinal centers (GCs); however, its biological function related to GCs, where efficient T-cell-dependent (TD) antibody responses are generated, is unknown. This thesis is focused on the effect of genetic or pharmacological blockade of PI3K p110delta activity on T and B cells, and the role of Bam32 in GC responses. Type 2 cytokine responses are significantly decreased in p110delta-inactivated mice, whereas Type 1 cytokine responses are increased or comparable after primary and secondary immunization. Hallmarks of asthma, airway inflammation and respiratory hyper-responsiveness are dramatically reduced in those mice. Adoptive transfer of OVA-primed splenocytes from normal, but not p110delta-inactivated mice could induce airway eosinophilia in naïve, airway-challenged recipient mice. These data demonstrate a novel functional role for p110delta signaling in induction of Type 2 responses in vivo and may offer a new therapeutic target for Th2-mediated airway disease. Paradoxically, serum IgE levels are markedly increased in OVA-immunized p110delta-inactivated mice despite lower level of swich factor IL-4. In vitro studies showed that p110delta is required to restrain IgE class switch recombination in a B-cell intrinsic manner. Blockade of PI3K activity using broad-spectrum PI3K inhibitors PIK-90 and PI-103 generates similar results. In vivo administration of p110delta-selective inhibitor IC87114 into OVA-immunized mice results in selective elevation of antigen-specific IgE production. Disruption of p110delta signaling leads to increased germline transcription at the epsilon locus (epsilon GLT) and increased induction of activation induced cytidine deaminase (AID) enzyme, suggesting deregulation at the level of the isotype switch process. Moreover, p110delta signaling selectively regulates the expression level of transcription factor Bcl6 and IRF4, which may be responsible for the regulation of AID and epsilon GLT. PI3K signaling regulates multiple steps of GC development, and Bam32 may be involved. GCs dissipate prematurely in Bam32-deficient mice after immunization with OVA/alum. In vitro, Bam32-deficient B cells are functional competent in proliferation, chemotaxis, isotype switching and plasma cell differentiation in response to signals present in GCs. In vivo, Bam32-deficient GC B cells proliferate normally; however, they are more apoptotic. Adoptive transfer studies indicated that intrinsic defect of Bam32-/- B cells leads to premature GC dissolution. Additionally, GCs formed by Bam32-/- B cells contain fewer T cells, implying that Bam32 is required for B cell-dependant T cell accumulation within established GCs. Treatment of Bam32-/- mice with agonistic anti-CD40 fully restored GC persistence and IgG1 isotype switching, demonstrating that Bam32-deficient GC B cells are functionally competent when access to cognate signals is not limiting. Collectively, those data demonstrate that Bam32 is not required for GC initiation, but rather functions in a late checkpoint of GC progression associated with T cell recruitment and GC B cell survival. In general, by focusing on PI3K p110delta and its downstream adaptor protein Bam32, my studies clearly indicate that p110delta is a potential therapeutic target for the treatment of Th2-induced airway inflammation. The unexpected immunomodulatory acitivity on IgE switching associated with multiple PI3K inhibitor compounds is first discovered in this thesis, suggesting that more need to be investigated in this aspect before those inhibitor compounds are widely used in the clinic. Furthermore, the specific regulatory role of Bam32 in GCs represents a unique model for us to study the late GC checkpoint in regarding to in vivo GC B cell and T cell interaction, which is an important issue need to be clarified in order to fully understand GC responses.