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.

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.

Contagious bovine pleuropneumonia (CBPP) is a severe infectious disease caused by Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) and is a vast problem in Africa. Current CBPP prevention is based on attenuated live strain vaccines, but these are limited by factors such as short-term immunity, cold-chain dependence and retained virulence. CBPP can be diagnosed using post-mortem examination, identification of the agent using culture and PCR based methods as well as serological diagnostic methods, but the latter are generally not sensitive enough and there is also demand for an inexpensive, pen side field test.The research presented in this thesis was focused on using recombinantly expressed surface proteins from M. mycoides SC to characterize humoral immune responses to CBPP. Thereby candidate proteins to be used in development of serological diagnostic methods and possibly subunit vaccines could be identified. As a first step, five putative variable surface proteins of M. mycoides SC were expressed and purified from E. coli in Paper I. These proteins were analyzed using immunoblotting techniques and results showed that one protein, MSC_0364, was variably expressed on the surface of M. mycoides SC in vitro. Paper II presents expanded efforts including cloning and expression of 64 recombinant surface proteins and an assay for high throughput analysis of protein-specific IgG, IgA and IgM titers in hundreds of sera using a bead-based screening assay. The assay was evaluated by protein-specific inhibition experiments, comparisons to Western blotting and monitoring of immune responses over time in a study with sera taken from eight animals over 293 days from a previous vaccine trial.Papers III and IV present applications using the recombinant proteins and bead-based screening assay wherein proteins for diagnostic and vaccine development were identified. In Paper III, the assay was used to screen 61 proteins using well-characterized serum samples from cattle with CBPP and healthy controls, resulting in selection of eight proteins suitable for diagnostic use. These proteins were combined and evaluated in a proof-of-concept ELISA with a discriminative power that enabled 96% correct classification of sera from CBPP-affected and CBPP-free bovines. Paper IV reports the results and protein-specific analyses of a vaccine trial using the recombinant putative variable surface proteins presented in Paper I as a subunit vaccine. The vaccine conferred no protection, but a weak vaccine response could not be excluded as the cause of failure. In an effort to identity other protein candidates to be used in a subunit vaccine, protein-specific analysis of humoral immune responses elicited by the currently approved live strain vaccine, T1/44, were investigated. Here, five proteins with high IgG titers associated to immunity were identified: LppQ, MSC_02714, MSC_0136, MSC_0079 and MSC_0431. These proteins may be important in the development of a novel subunit vaccine against CBPP. / QC 20100719