Ting-ting Zhang

Role of the phosphoinositide 3‐kinase p110δ in generation of type 2 cytokine responses and allergic airway inflammation

Phosphoinositide 3‐kinases (PI3K) regulate immune activation via their roles in signal transduction of multiple classes of receptors. Here, we examined the effect of genetic inactivation of the hemopoietic cell‐restricted PI3K isoform p110δ on systemic cytokine and chemokine responses and allergic airway inflammation. We found that type 2 cytokine responses (IL‐4, IL‐5 and IL‐13) are significantly decreased in p110δ mutants, whereas type 1 cytokine responses (IFN‐γ and CXCL10) were robust. Elevated IFN‐γ production during the primary response to ovalbumin (OVA) was associated with reduced production of the regulatory cytokine IL‐10. IFN‐γ and IL‐10 production normalized after secondary OVA immunization; however, type 2 cytokine production was persistently reduced. Type 2 cytokine‐dependent airway inflammation elicited by intranasal challenge with OVA was dramatically reduced, with reduced levels of eosinophil recruitment and mucus production observed in the lungs. Induction of respiratory hyper‐responsiveness to inhaled methacholine, a hallmark of asthma, was markedly attenuated in p110δ‐inactivated mice. Adoptive transfer of OVA‐primed splenocytes from normal but not p110δ‐inactivated mice could induce airway eosinophilia in naive, airway‐challenged recipient mice. These data demonstrate a novel functional role for p110δ signaling in induction of type 2 responses in vivo and may offer a new therapeutic target for Th2‐mediated airway disease.

The p110δ Isoform of Phosphatidylinositol 3-Kinase Controls Susceptibility to Leishmania major by Regulating Expansion and Tissue Homing of Regulatory T Cells

Resistance to Leishmania major and most intracellular pathogens is usually associated with a strong T cell-mediated immunity, particularly a CD4+ Th1 response. Mice with an inactivating knock-in mutation in the p110δ isoform of PI3K (referred to as p110δD910A) show severely impaired T cell responses. Because a strong T cell response is thought to mediate resistance to intracellular pathogens, we examined the outcome of L. major infection in p110δD910A mice. Paradoxically, p110δD910A mice on “resistant” and “susceptible” genetic backgrounds showed more robust resistance manifested as significantly reduced lesion size and accelerated parasite clearance. This enhanced resistance was associated with dramatically diminished immune responses, including impaired cell proliferation and effector cytokine (IFN-γ and TNF) production. Interestingly, the ability of macrophages and dendritic cells from p110δD910A mice to produce NO and destroy Leishmania parasites was similar to those of wild-type mice. We show that the enhanced resistance of p110δD910A mice was due to impaired expansion and effector functions of regulatory T cells (Tregs). Adoptive transfer studies demonstrated that p110δD910A mice lost their increased resistance when given enriched Tregs from wild-type mice. We suggest on the basis of these and further observations that the lack of this enzyme prominently affects Treg expansion and homing to infection sites, and that in the absence of Tregs, weak Th1 responses are capable of containing parasites and prevent pathology. We also suggest that temporary pharmacological inhibition of this enzyme may be a very effective form of treatment against cutaneous leishmaniasis.

Phosphoinositide 3-kinase-regulated adapters in lymphocyte activation

Summary:  Signaling via phosphoinositide 3‐kinases (PI3Ks) has emerged as a central component of lymphocyte activation via immunoreceptors, costimulatory receptors, cytokine receptors, and chemokine receptors. The discovery of phosphoinositide‐binding pleckstrin homology (PH) domains has substantially increased understanding of how PI3Ks activate cellular responses. Accumulating evidence indicates that PH‐domain containing adapter molecules provide important links between PI3K and lymphocyte function. Here, we review data on PI3K‐regulated adapter proteins of the Grb‐associated binder (GAB), Src kinase‐associated phosphoprotein (SKAP), and B‐lymphocyte adapter molecule of 32 kDa (Bam32)/ dual‐adapter for phosphotyrosine and 3‐phosphoinositides (DAPP)/TAPP families, with a focus on the latter group. Current data support the model that recruitment of these adapters to the plasma membrane of activated lymphocytes is driven by the phosphoinositides phosphatidylinositol‐3,4,5‐tris‐phosphate and phosphatidylinositol‐3,4‐bisphosphate, generated through the action of PI3Ks and under the regulatory control of lipid phosphatases Src homology 2 domain‐containing inositol phosphatase (SHIP), phosphatase and tensin homolog, and inositol polyphosphate 4‐phosphatase. At the plasma membrane, these adapters serve to assemble distinct protein complexes. Bam32/DAPP1 and SKAPs function to promote activation of monomeric guanosine triphosphatases, including Rac and Rap, and promote integrin activation, lymphocyte adhesion to matrix proteins, and cell:cell interactions between B and T lymphocytes. GABs can provide feedforward amplification or feedback inhibition of PI3K signaling. Current work is further defining the molecular interactions driven by these molecules and identifying the functions of TAPP adapters, which also appear to be involved in lymphocyte adhesion and are specific effectors downstream of the SHIP product phosphatidylinositol‐3,4‐bisphosphate.

Interaction of TAPP adapter proteins with phosphatidylinositol (3,4)-bisphosphate regulates B-cell activation and autoantibody production.

TAPP1 and TAPP2 (where TAPP is tandem PH domain containing protein) are dual PH domain adaptors that selectively bind PI(3,4)P2 (phosphatidylinositol (3,4)‐bisphosphate). PI(3,4)P2 is a lipid messenger generated by phosphoinositide 3‐kinase (PI3K) and SHIP, both of which are critical regulators of B‐cell activation. To determine the functional role of TAPP‐PI(3,4)P2 interactions, we utilized a double knock‐in (KI) mouse bearing mutations within the PI‐binding pocket of both TAPP1 and TAPP2. TAPP KI mice show evidence of altered B‐cell development, but generate phenotypically normal mature B‐cell populations. Total serum immunoglobulin IgM and IgG levels were found to be markedly elevated in TAPP KI mice. B cells purified from TAPP KI mice were hyper‐responsive to antigen receptor cross‐linking, showing increased proliferation, CD86 expression, and Akt phosphorylation on Ser473 and Thr308. Female TAPP KI mice developed elevated levels of anti‐DNA and antinuclear antibodies with age, associated with IgG deposition in kidneys and significant glomerulonephritis pathology. Together our results indicate that interaction of TAPPs with PI(3,4)P2 mediates feedback inhibition impacting on BCR signaling, with functional significance for control of autoreactive B cells.

p110δ Phosphoinositide 3-Kinase Represses IgE Switch by Potentiating BCL6 Expression

PI3Ks are key signaling enzymes required for triggering many immunological functions. In B lymphocytes, PI3K signaling is required for Ag-induced proliferation and robust production of most Ab isotypes. Paradoxically, PI3K was found to have a negatively regulatory function regarding Ab class switch recombination, and blockade of PI3K can strongly potentiate IgE switch. In this article, we explore the mechanisms of this unexpected negative regulatory function of PI3K regarding IgE. We demonstrate that p110δ PI3K selectively regulates IgE switch in a B cell-intrinsic manner by controlling germline transcription of the IgE promoter (εGLT). Although p110δ can regulate transcription of activation-induced cytidine deaminase via Akt, repression of εGLT and IgE switch is not dependent on Akt signaling. Inhibition of p110δ, but not Akt, leads to reduced expression of transcriptional repressor B cell lymphoma 6 (BCL6) and concomitant upregulation of εGLT and other BCL6-target genes. p110δ inhibitor treatment strikingly alters the balance between BCL6 and IRF4 (a transcription factor that antagonizes BCL6), leading to increased IRF4 and decreased BCL6 expression levels in germinal center B cells. Ectopic expression of BCL6 can partially overcome the elevated εGLTs and potentiated IgE switching in p110δ-inhibited B cells. To our knowledge, these results provide the first evidence that p110δ PI3K signaling regulates BCL6 expression and indicate that PI3K promotes the germinal center B cell program and selectively represses IgE switch by maintaining sufficient levels of BCL6.

Bam32/DAPP1 Promotes B Cell Adhesion and Formation of Polarized Conjugates with T Cells

B cell Ag receptors function in both signaling activation of Ag-specific cells and in collecting specific Ag for presentation to T lymphocytes. Signaling via PI3K is required for BCR-mediated activation and Ag presentation functions; however, the relevant downstream targets of PI3K in B cells are incompletely defined. In this study, we have investigated the roles of the PI3K effector molecule Bam32/DAPP1 in BCR signaling and BCR-mediated Ag presentation functions. In mouse primary B cells, Bam32 was required for efficient activation of the GTPase Rac1 and downstream signaling to JNK, but not activation of BLNK, phospholipase C γ2, or calcium responses. Consistent with a role of this adaptor in Rac-mediated cytoskeletal rearrangement, Bam32 was required for BCR-induced cell adhesion and spreading responses on ICAM-1 or fibronectin-coated surfaces. The function of Bam32 in promoting Rac activation and adhesion required tyrosine 139, a known site of phosphorylation by Lyn kinase. After BCR crosslinking by Ag, Bam32-deficient B cells are able to carry out the initial steps of Ag endocytosis and processing, but show diminished ability to form Ag-specific conjugates with T cells and polarize F-actin at the B-T interface. As a result, Bam32-deficient B cells were unable to efficiently activate Ag-specific T cells. Together, these results indicate that Bam32 serves to integrate PI3K and Src kinase signaling to promote Rac-dependent B cell adhesive interactions important for Ag presentation function.

The Pleckstrin Homology Domain Adaptor Protein Bam32/DAPP1 Is Required for Germinal Center Progression

Ab affinity maturation within germinal centers (GCs) requires weeks to complete. Several signaling pathways in B cells have been shown to be required for initiation of the GC response; however, the signaling checkpoints controlling progression and eventual dissolution of the GC reaction are poorly understood. The adaptor protein Bam32/DAPP1 was originally isolated from human GCs and functions downstream of phosphoinositide 3-kinase enzymes, which are known to have critical roles in B cell activation and GC responses. In this study we identify a unique role of Bam32/DAPP1 in promoting GC progression. Bam32-deficient mice show normal GC initiation, but premature GC dissolution after immunization with protein Ag in alum or low doses of sheep red blood cells. Adoptive transfer studies confirmed that Bam32-deficient B cells have an intrinsic impairment in the ability to mount sustained GC responses. Bam32 deficiency was also associated with impaired Ab affinity maturation. Proliferation of Bam32-deficient GC B cells was not compromised; however, these cells show impaired switch to IgG1 and increased apoptosis in situ. GCs formed by Bam32-deficient B cells contain fewer T cells, indicating that Bam32 is required for B cell–dependent T cell accumulation within established GCs. Exogenous CD40 ligand restored GC B cell numbers and switch to IgG1, indicating that Bam32-deficient B cells are competent to respond to CD40 stimulation when ligand is available. These 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.

Regulation of B-lymphocyte activation by the PH domain adaptor protein Bam32/DAPP1

PI3Ks (phosphoinositide 3-kinases) play critical roles in BCR (B-cell receptor) signalling via the generation of 3-phosphoinositide second messengers. Recruitment of PH domain (pleckstrin homology domain)-containing signal transduction proteins to the plasma membrane through binding to 3-phosphoinositide second messengers represents a major effector mechanism for PI3Ks. Here, we review data on the PH domain-containing adaptor protein Bam32 (B-cell adaptor molecule of 32 kDa)/DAPP1 (dual adaptor for phosphotyrosine and 3-phosphoinositides 1), focusing on its functions in B-lymphocyte activation. Present results support the view that Bam32/DAPP1 mediates multiple PI3K-dependent responses in B-cells through membrane-proximal mechanisms involving Src kinases, Rac1, F-actin and mitogen-activated protein kinases, resulting in selective effects on BCR-mediated proliferation, antigen presentation and generation of antibody responses.