Elisa Bjørgo

PI3K p110δ regulates T cell cytokine production during primary and secondary immune responses in mice and humans

We have previously described critical and nonredundant roles for the phosphoinositide 3-kinase p110delta during the activation and differentiation of naive T cells, and p110delta inhibitors are currently being developed for clinical use. However, to effectively treat established inflammatory or autoimmune diseases, it is important to be able to inhibit previously activated or memory T cells. In this study, using the isoform-selective inhibitor IC87114, we show that sustained p110delta activity is required for interferon-gamma production. Moreover, acute inhibition of p110delta inhibits cytokine production and reduces hypersensitivity responses in mice. Whether p110delta played a similar role in human T cells was unknown. Here we show that IC87114 potently blocked T-cell receptor-induced phosphoinositide 3-kinase signaling by both naive and effector/memory human T cells. Importantly, IC87114 reduced cytokine production by memory T cells from healthy and allergic donors and from inflammatory arthritis patients. These studies establish that previously activated memory T cells are at least as sensitive to p110delta inhibition as naive T cells and show that mouse models accurately predict p110delta function in human T cells. There is therefore a strong rationale for p110delta inhibitors to be considered for therapeutic use in T-cell-mediated autoimmune and inflammatory diseases.

Cross Talk between Phosphatidylinositol 3-Kinase and Cyclic AMP (cAMP)-Protein Kinase A Signaling Pathways at the Level of a Protein Kinase B/β-Arrestin/cAMP Phosphodiesterase 4 Complex

ABSTRACT Engagement of the T-cell receptor (TCR) in human primary T cells activates a cyclic AMP (cAMP)-protein kinase A (PKA)-Csk inhibitory pathway that prevents full T-cell activation in the absence of a coreceptor stimulus. Here, we demonstrate that stimulation of CD28 leads to recruitment to lipid rafts of a β-arrestin/phosphodiesterase 4 (PDE4) complex that serves to degrade cAMP locally. Redistribution of the complex from the cytosol depends on Lck and phosphatidylinositol 3-kinase (PI3K) activity. Protein kinase B (PKB) interacts directly with β-arrestin to form part of the supramolecular complex together with sequestered PDE4. Translocation is mediated by the PKB plextrin homology (PH) domain, thus revealing a new role for PKB as an adaptor coupling PI3K and cAMP signaling. Functionally, PI3K activation and phosphatidylinositol-(3,4,5)-triphosphate (PIP3) production, leading to recruitment of the supramolecular PKB/β-arrestin/PDE4 complex to the membrane via the PKB PH domain, results in degradation of the TCR-induced cAMP pool located in lipid rafts, thereby allowing full T-cell activation to proceed.

Novel mechanism of signaling by CD28

Ligation of both the T cell receptor (TCR) and the CD28 receptor is required for full T cell activation to occur. Engagement of the TCR in primary T cells is followed by rapid cAMP production in lipid rafts and activation of the cAMP-protein kinase A (PKA)-Csk pathway inhibiting proximal T cell signaling. However, CD28 stimulation leads to recruitment of a beta-arrestin/phosphodiesterase-4 (PDE4) complex to rafts, resulting in down-regulation of cAMP levels. Thus, the activities of both PKA and PDE4 seem to be important for regulation of TCR-induced signaling and T cell function. This review will focus on the novel mechanism whereby CD28 through PI3K regulates recruitment of a PKB/beta-arrestin/PDE4 complex thereby allowing a complete T cell activation to proceed.

Phosphodiesterases as Targets for Modulating T-Cell Responses

The cAMP-protein kinase A (PKA) signaling pathway is strongly involved in the regulation and modulation of immune responses, and cAMP is the most potent and acute inhibitor of T-cell activation. Thus, cAMP levels in the cell must be tightly regulated. Cyclic AMP-specific phosphodiesterases (PDEs) provide the only mechanism for degrading cAMP in cells, thereby functioning as key regulators of signaling. To obtain a complete immune response with optimal cytokine production and T-cell proliferation, ligation of both the T-cell receptor (TCR) and the CD28 receptor is required. However, engagement of the TCR in primary T cells is followed by rapid cAMP production in lipid rafts and activation of the cAMP- PKA-Csk pathway inhibiting proximal T-cell signaling. In contrast, TCR/CD28 costimulation leads to the recruitment of a PDE4/β-arrestin complex to rafts in a phosphatidylinositol 3-kinase (PI3K)-dependent manner, resulting in the downregulation of cAMP levels. Thus, the activities of both PKA and PDE4 seem to be important for regulation of TCR-induced signaling and T-cell function. The use of selective inhibitors has revealed that PDEs are important drug targets in several diseases with an inflammatory component where immune function is important such as asthma, chronic obstructive pulmonary disease (COPD), cardiovascular diseases, and neurological disorders. PDEs are also interesting drug targets in immunosuppression following transplantation and for modulation of immune responses.

Cellular localisation and nuclear export of the human bZIP transcription factor TCF11

TCF11 is a ubiquitous transcription factor of the CNC-bZIP family. The activity of this vital protein is strictly regulated and we have previously published that the two major translated protein forms show a clearly different transactivation ability in transient transfections. Only the full-length form is active in a variety of mammalian cells [J. Biol. Chem. 276 (2001) 17641]. Here we further investigate the complex regulation of TCF11, studying the cellular localisation of some of the different protein isoforms. The full-length form is located both in the cytoplasm and the nucleus, while the internally initiated shorter protein form is restricted to nuclear localisation. A nuclear export signal (NES) localised in the N-terminus of TCF11 is responsible for the active nuclear export of the protein. This export is highly sensitive to leptomycin B (LMB) and is largely blocked by mutating three of the leucine residues in the signal region. These results indicate that export occurs through the Crm1-mediated pathway. Due to alternative splicing within the tcf11 gene, different isoforms of the longer protein form are produced. Some of these isoforms, one identical to Nrf1, lack the NES and are thereby restricted to nuclear localisation.

Quantitative proteome analysis of detergent-resistant membranes identifies the differential regulation of protein kinase C isoforms in apoptotic T cells

Several lines of evidence suggest that detergent‐resistant membranes (DRMs) (also known as lipid rafts and glycosphingolipid‐enriched microdomains) may have a role in signaling pathways of apoptosis. Here, we developed a method that combines DRMs isolation and methanol/chloroform extraction with stable isotope labeling with amino acids in cell culture‐based quantitative proteome analysis of DRMs from control and cisplatin‐induced apoptotic Jurkat T cells. This approach enabled us to enrich proteins with a pivotal role in cell signaling of which several were found with increased or decreased amounts in DRMs upon induction of apoptosis. Specifically, we show that three isoforms of protein kinase C (PKC) are regulated differently upon apoptosis. Although PKCα which belongs to the group of conventional PKCs is highly up‐regulated in DRMs, the levels of two novel PKCs, PKCη and PKCθ, are significantly reduced. These alterations/differences in PKC regulation are verified by immunoblotting and confocal microscopy. In addition, a specific enrichment of PKCα in apoptotic blebs and buds is shown. Furthermore, we observe an increased expression of ecto‐PKCα as a result of exposure to cisplatin using flow cytometry. Our results demonstrate that in‐depth proteomic analysis of DRMs provides a tool to study differential localization and regulation of signaling molecules important in health and disease.