Life Sciences Research for Lifelong Health

Publications klaus-okkenhaug

Title / Authors / Details Open Access Download

Cross talk between phosphatidylinositol 3-kinase and cyclic AMP (cAMP)-protein kinase a signaling pathways at the level of a protein kinase B/beta-arrestin/cAMP phosphodiesterase 4 complex.
E Bjørgo, SA Solheim, H Abrahamsen, GS Baillie, KM Brown, T Berge, K Okkenhaug, MD Houslay, K Taskén

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 beta-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 beta-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/beta-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.

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Molecular and cellular biology, 30, 7, 1660-72, 2010

PMID: 20086095
DOI: 10.1128/MCB.00696-09

Open Access

PI3K p110delta regulates T-cell cytokine production during primary and secondary immune responses in mice and humans.
DR Soond, E Bjørgo, K Moltu, VQ Dale, DT Patton, KM Torgersen, F Galleway, B Twomey, J Clark, JS Gaston, K Taskén, P Bunyard, K Okkenhaug

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.

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Blood, 115, 11, 2203-13, 2010

PMID: 20081091
DOI: 10.1182/blood-2009-07-232330

Open Access

MAPK, phosphatidylinositol 3-kinase, and mammalian target of rapamycin pathways converge at the level of ribosomal protein S6 phosphorylation to control metabolic signaling in CD8 T cells.
RJ Salmond, J Emery, K Okkenhaug, R Zamoyska

Ribosomal protein S6 (rpS6) is a key component of the translational machinery in eukaryotic cells and is essential for ribosome biogenesis. rpS6 is phosphorylated on evolutionarily conserved serine residues, and data indicate that rpS6 phosphorylation might regulate cell growth and protein synthesis. Studies in cell lines have shown an important role for the serine kinase mammalian target of rapamycin (mTOR) in rpS6 phosphorylation, further linking rpS6 to control of cellular metabolism. rpS6 is essential in T cells because its deletion in mouse double-positive thymocyte cells results in a complete block in T cell development; however, the signaling pathway leading to rpS6 phosphorylation downstream of TCR stimulation has yet to be fully characterized. We show that maximal TCR-induced rpS6 phosphorylation in CD8 T cells requires both Lck and Fyn activity and downstream activation of PI3K, mTOR, and MEK/ERK MAPK pathways. We demonstrate that there is cross-talk between the PI3K and MAPK pathways as well as PI3K-independent mTOR activity, which result in differential phosphorylation of specific rpS6 serine residues. These results place rpS6 phosphorylation as a point of convergence for multiple crucial signaling pathways downstream of TCR triggering.

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Journal of immunology (Baltimore, Md. : 1950), 183, 11, 7388-97, 2009

PMID: 19917692
DOI: 10.4049/jimmunol.0902294

Open Access

Ribosomal protein S6 kinase 1 signaling regulates mammalian life span.
C Selman, JM Tullet, D Wieser, E Irvine, SJ Lingard, AI Choudhury, M Claret, H Al-Qassab, D Carmignac, F Ramadani, A Woods, IC Robinson, E Schuster, RL Batterham, SC Kozma, G Thomas, D Carling, K Okkenhaug, JM Thornton, L Partridge, D Gems, DJ Withers

Caloric restriction (CR) protects against aging and disease, but the mechanisms by which this affects mammalian life span are unclear. We show in mice that deletion of ribosomal S6 protein kinase 1 (S6K1), a component of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway, led to increased life span and resistance to age-related pathologies, such as bone, immune, and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian life-span and suggest that therapeutic manipulation of S6K1 and AMPK might mimic CR and could provide broad protection against diseases of aging.

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Science (New York, N.Y.), 326, 5949, 140-4, 2009

PMID: 19797661
DOI: 10.1126/science.1177221

Open Access

The p110delta isoform of phosphatidylinositol 3-kinase controls susceptibility to Leishmania major by regulating expansion and tissue homing of regulatory T cells.
D Liu, T Zhang, AJ Marshall, K Okkenhaug, B Vanhaesebroeck, JE Uzonna

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 p110delta isoform of PI3K (referred to as p110delta(D910A)) 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 p110delta(D910A) mice. Paradoxically, p110delta(D910A) 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-gamma and TNF) production. Interestingly, the ability of macrophages and dendritic cells from p110delta(D910A) mice to produce NO and destroy Leishmania parasites was similar to those of wild-type mice. We show that the enhanced resistance of p110delta(D910A) mice was due to impaired expansion and effector functions of regulatory T cells (Tregs). Adoptive transfer studies demonstrated that p110delta(D910A) 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.

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Journal of immunology (Baltimore, Md. : 1950), 183, 3, 1921-33, 2009

PMID: 19596993
DOI: 10.4049/jimmunol.0901099

Open Access

CCL21 mediates CD4+ T-cell costimulation via a DOCK2/Rac-dependent pathway.
K Gollmer, F Asperti-Boursin, Y Tanaka, K Okkenhaug, B Vanhaesebroeck, JR Peterson, Y Fukui, E Donnadieu, JV Stein

CD4(+) T cells use the chemokine receptor CCR7 to home to and migrate within lymphoid tissue, where T-cell activation takes place. Using primary T-cell receptor (TCR)-transgenic (tg) CD4(+) T cells, we explored the effect of CCR7 ligands, in particular CCL21, on T-cell activation. We found that the presence of CCL21 during early time points strongly increased in vitro T-cell proliferation after TCR stimulation, correlating with increased expression of early activation markers. CCL21 costimulation resulted in increased Ras- and Rac-GTP formation and enhanced phosphorylation of Akt, MEK, and ERK but not p38 or JNK. Kinase-dead PI3Kdelta(D910A/D910A) or PI3Kgamma-deficient TCR-tg CD4(+) T cells showed similar responsiveness to CCL21 costimulation as control CD4(+) T cells. Conversely, deficiency in the Rac guanine exchange factor DOCK2 significantly impaired CCL21-mediated costimulation in TCR-tg CD4(+) T cells, concomitant with impaired Rac- but not Ras-GTP formation. Using lymph node slices for live monitoring of T-cell behavior and activation, we found that G protein-coupled receptor signaling was required for early CD69 expression but not for Ca(2+) signaling. Our data suggest that the presence of CCL21 during early TCR signaling lowers the activation threshold through Ras- and Rac-dependent pathways leading to increased ERK phosphorylation.

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Blood, 114, 3, 580-8, 2009

PMID: 19451552
DOI: 10.1182/blood-2009-01-200923

Open Access

p110gamma and p110delta isoforms of phosphoinositide 3-kinase differentially regulate natural killer cell migration in health and disease.
A Saudemont, F Garçon, H Yadi, M Roche-Molina, N Kim, A Segonds-Pichon, A Martín-Fontecha, K Okkenhaug, F Colucci

The mechanisms that regulate NK cell trafficking are unclear. Phosphoinositide-3 kinases (PI3K) control cell motility and the p110gamma and p110delta isoforms are mostly expressed in leukocytes, where they transduce signals downstream of G protein coupled receptors (GPCR) or tyrosine kinase receptors, respectively. Here, we set out to determine the relative contribution of p110gamma and p110delta to NK cell migration in mice. Using a combination of single-cell imaging analysis of transgenic cells reporting on PI3K activity in real time and small molecule inhibitors of p110gamma and p110delta, we show here that the tyrosine-kinase coupled p110delta is linked to GPCR signaling and, depending on the GPCR, may even be preferentially activated over p110gamma. Using gene-targeted mice, we showed that both isoforms were essential for NK cell chemotaxis to CXCL12 and to CCL3 and, in vivo, for normal NK cell migration during pregnancy and to the inflamed peritoneum. By contrast, only p110delta was indispensable for chemotaxis to S1P and CXCL10 and for NK cell distribution throughout lymphoid and nonlymphoid tissues and for extravasation to tumors. These results implicate p110delta downstream of GPCRs in NK cells and highlight its nonredundant role as a key regulator of NK cell trafficking in health and disease.

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Proceedings of the National Academy of Sciences of the United States of America, 106, 14, 5795-800, 2009

PMID: 19297623
DOI: 10.1073/pnas.0808594106

Open Access

Cutting edge: the Foxp3 target miR-155 contributes to the development of regulatory T cells.
S Kohlhaas, OA Garden, C Scudamore, M Turner, K Okkenhaug, E Vigorito

Foxp3 is a transcription factor that is essential for the normal development of regulatory T cells (Tregs). In the absence of microRNAs (miRNAs), Foxp3(+) Tregs develop but fail to maintain immune homeostasis, leading to a scurfy-like disease. Global analysis of the network of genes regulated by Foxp3 has identified the miRNA miR-155, which is highly expressed in Tregs, as a direct target of Foxp3. In this study we report that miR-155-deficient mice have reduced numbers of Tregs, both in the thymus and periphery, due to impaired development. However, we found no evidence for defective suppressor activity of miR-155-deficient Tregs, either in vitro or in vivo. Our results indicate that miR-155 contributes to Treg development, but that additional miRNAs control Treg function.

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Journal of immunology (Baltimore, Md. : 1950), 182, 5, 2578-82, 2009

PMID: 19234151
DOI: 10.4049/jimmunol.0803162

Open Access

Genetic or pharmaceutical blockade of p110delta phosphoinositide 3-kinase enhances IgE production.
TT Zhang, K Okkenhaug, BF Nashed, KD Puri, ZA Knight, KM Shokat, B Vanhaesebroeck, AJ Marshall

Recent studies indicate that pharmaceutical blockade of phosphoinositide 3-kinase (PI3K) signaling enzymes might be effective in reducing allergic airway inflammation. Signals generated by the p110delta PI3K isoform play critical roles in signaling through antigen and cytokine receptors and were shown to be required for induction of type 2, but not type 1, cytokine responses.

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The Journal of allergy and clinical immunology, 122, 4, 811-819.e2, 2008

PMID: 19014771
DOI: 10.1016/j.jaci.2008.08.008

Proliferative signals mediated by CD28 superagonists require the exchange factor Vav1 but not phosphoinositide 3-kinase in primary peripheral T cells.
T Gogishvili, F Elias, JL Emery, K McPherson, K Okkenhaug, T Hünig, KM Dennehy

Almost all responses of naive T cells require co-stimulation, i.e. engagement of the clonotypic TCR with relevant antigen/MHC and the co-stimulatory molecule CD28. How CD28 contributes to T-cell proliferation remains poorly understood, with widely conflicting reports existing which may reflect different methods of co-ligating receptors. Some CD28 mAb, however, can stimulate T-cell proliferation without the need for TCR co-ligation, and thus provide unique tools to dissect proliferative signals mediated through CD28 alone. Using primary peripheral T cells from CD28-transgenic mice, we show that both the YMNM and Lck-binding motifs, but not the Itk-binding motif, in CD28 are required for proliferation. Given that the YMNM motif recruits both phosphoinositide 3-kinase (PI3K) and the exchange factor Vav1, we investigated the role of these two molecules in CD28-mediated proliferation. In p110delta(D910A/D910A) transgenic T cells, which are defective in PI3K activation following CD28 ligation, proliferation was comparable to that in wild-type cells. By contrast, T-cell proliferation was abolished in Vav1(-/-) cells. Although we did not address the role of Grb2 in CD28 signalling, these results indicate that CD28 can mediate Lck- and Vav1-dependent proliferative signals independently of PI3K.

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European journal of immunology, 38, 9, 2528-33, 2008

PMID: 18792405
DOI: 10.1002/eji.200838223

The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110gamma.
J Guillermet-Guibert, K Bjorklof, A Salpekar, C Gonella, F Ramadani, A Bilancio, S Meek, AJ Smith, K Okkenhaug, B Vanhaesebroeck

The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.

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Proceedings of the National Academy of Sciences of the United States of America, 105, 24, 8292-7, 2008

PMID: 18544649
DOI: 10.1073/pnas.0707761105

Open Access

Phosphatidylinositol-3-OH kinase and nutrient-sensing mTOR pathways control T lymphocyte trafficking.
LV Sinclair, D Finlay, C Feijoo, GH Cornish, A Gray, A Ager, K Okkenhaug, TJ Hagenbeek, H Spits, DA Cantrell

Phosphatidylinositol-3-OH kinase (PI(3)K) and the nutrient sensor mTOR are evolutionarily conserved regulators of cell metabolism. Here we show that PI(3)K and mTOR determined the repertoire of adhesion and chemokine receptors expressed by T lymphocytes. The key lymph node-homing receptors CD62L (L-selectin) and CCR7 were highly expressed on naive T lymphocytes but were downregulated after immune activation. CD62L downregulation occurred through ectodomain proteolysis and suppression of gene transcription. The p110delta subunit of PI(3)K controlled CD62L proteolysis through mitogen-activated protein kinases, whereas control of CD62L transcription by p110delta was mediated by mTOR through regulation of the transcription factor KLF2. PI(3)K-mTOR nutrient-sensing pathways also determined expression of the chemokine receptor CCR7 and regulated lymphocyte trafficking in vivo. Hence, lymphocytes use PI(3)K and mTOR to match metabolism and trafficking.

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Nature immunology, 9, 5, 513-21, 2008

PMID: 18391955
DOI: 10.1038/ni.1603

Open Access

T cell receptor-induced phosphoinositide-3-kinase p110delta activity is required for T cell localization to antigenic tissue in mice.
SJ Jarmin, R David, L Ma, JG Chai, H Dewchand, A Takesono, AJ Ridley, K Okkenhaug, FM Marelli-Berg

The establishment of T cell-mediated inflammation requires the migration of primed T lymphocytes from the blood stream and their retention in antigenic sites. While naive T lymphocyte recirculation in the lymph and blood is constitutively regulated and occurs in the absence of inflammation, the recruitment of primed T cells to nonlymphoid tissue and their retention at the site are enhanced by various inflammatory signals, including TCR engagement by antigen-displaying endothelium and resident antigen-presenting cells. In this study, we investigated whether signals downstream of TCR ligation mediated by the phosphoinositide-3-kinase (PI3K) subunit p110delta contributed to the regulation of these events. T lymphocytes from mice expressing catalytically inactive p110delta displayed normal constitutive trafficking and migratory responses to nonspecific stimuli. However, these cells lost susceptibility to TCR-induced migration and failed to localize efficiently to antigenic tissue. Importantly, we showed that antigen-induced T cell trafficking and subsequent inflammation was abrogated by selective pharmacological inhibition of PI3K p110delta activity. These observations suggest that pharmacological targeting of p110delta activity is a viable strategy for the therapy of T cell-mediated pathology.

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The Journal of clinical investigation, 118, 3, 1154-64, 2008

PMID: 18259608
DOI: 10.1172/JCI33267

Open Access

CD28 provides T-cell costimulation and enhances PI3K activity at the immune synapse independently of its capacity to interact with the p85/p110 heterodimer.
F Garçon, DT Patton, JL Emery, E Hirsch, R Rottapel, T Sasaki, K Okkenhaug

Activation of PI3K is among the earliest signaling events observed in T cells after conjugate formation with antigen-presenting cells (APCs). The relevant PI3K catalytic isoform and relative contribution of the TcR and CD28 to PI3K activity at the immune synapse have not been determined unequivocally. Using a quantitative imaging-based assay, we show that the PI3K activity at the T cell-APC contact area is dependent on the p110delta, but not the p110gamma, isoform of PI3K. CD28 enhanced PIP3 production at the T-cell synapse independently of its YMNM PI3K-recruitment motif that instead was required for efficient PKC recruitment. CD28 could partially compensate for the lack of p110delta activity during T-cell activation, which indicates that CD28 and p110delta act in parallel and complementary pathways to activate T cells. Consistent with this, CD28 and p110delta double-deficient mice were severely immune compromised. We therefore suggest that combined pharmaceutic targeting of p110delta activity and CD28 costimulation has potent therapeutic potential.

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Blood, 111, 3, 1464-71, 2008

PMID: 18006698
DOI: 10.1182/blood-2007-08-108050

Open Access

Evidence for lifespan extension and delayed age-related biomarkers in insulin receptor substrate 1 null mice.
C Selman, S Lingard, AI Choudhury, RL Batterham, M Claret, M Clements, F Ramadani, K Okkenhaug, E Schuster, E Blanc, MD Piper, H Al-Qassab, JR Speakman, D Carmignac, IC Robinson, JM Thornton, D Gems, L Partridge, DJ Withers

Recent evidence suggests that alterations in insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) can increase mammalian life span. For example, in several mouse mutants, impairment of the growth hormone (GH)/IGF1 axis increases life span and also insulin sensitivity. However, the intracellular signaling route to altered mammalian aging remains unclear. We therefore measured the life span of mice lacking either insulin receptor substrate (IRS) 1 or 2, the major intracellular effectors of the IIS receptors. Our provisional results indicate that female Irs1-/- mice are long-lived. Furthermore, they displayed resistance to a range of age-sensitive markers of aging including skin, bone, immune, and motor dysfunction. These improvements in health were seen despite mild, lifelong insulin resistance. Thus, enhanced insulin sensitivity is not a prerequisite for IIS mutant longevity. Irs1-/- female mice also displayed normal anterior pituitary function, distinguishing them from long-lived somatotrophic axis mutants. In contrast, Irs2-/- mice were short-lived, whereas Irs1+/- and Irs2+/- mice of both sexes showed normal life spans. Our results therefore suggest that IRS1 signaling is an evolutionarily conserved pathway regulating mammalian life span and may be a point of intervention for therapies with the potential to delay age-related processes.

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FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 22, 3, 807-18, 2008

PMID: 17928362
DOI: 10.1096/fj.07-9261com

Open Access

Inactivation of PI3Kgamma and PI3Kdelta distorts T-cell development and causes multiple organ inflammation.
H Ji, F Rintelen, C Waltzinger, D Bertschy Meier, A Bilancio, W Pearce, E Hirsch, MP Wymann, T Rückle, M Camps, B Vanhaesebroeck, K Okkenhaug, C Rommel

Mice lacking both the p110gamma and p110delta isoforms display severe impairment of thymocyte development. Here, we show that this phenotype is recapitulated in p110gamma-/-/p110delta(D910A/D910A) (p110gamma(KO)delta(D910A)) mice where the p110delta isoform has been inactivated by a point mutation. Moreover, we have examined the pathological consequences of the p110gammadelta deficiency, which include profound T-cell lymphopenia, T-cell and eosinophil infiltration of mucosal organs, elevated IgE levels, and a skewing toward Th2 immune responses. Using small-molecule selective inhibitors, we demonstrated that in mature T cells, p110delta, but not p110gamma, controls Th1 and Th2 cytokine secretion. Thus, the pathology in the p110gammadelta-deficient mice is likely to be secondary to a developmental block in the thymus that leads to lymphopenia-associated inflammatory responses.

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Blood, 110, 8, 2940-7, 2007

PMID: 17626838
DOI: 10.1182/blood-2007-04-086751

Open Access

A two-signal model for T cell trafficking.
FM Marelli-Berg, K Okkenhaug, V Mirenda

T-cell-receptor triggering and the delivery of co-stimulation are essential events leading to T cell expansion, differentiation and effector function. The influence that such signals exert on T cell migration during and following priming has been highlighted by recent reports. Moreover, induction of peripheral tolerance might act in part by affecting T cell migration. Here, we propose that the integration of co-stimulatory signals, which regulate the ability of primed T cells to access nonlymphoid tissue, and cognate recognition of the endothelium, which determines the selective recruitment of specific T cells, contribute to the anatomy of T cell-mediated immunity and tolerance. The implications for therapeutic strategies manipulating these signals are discussed.

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Trends in immunology, 28, 6, 267-73, 2007

PMID: 17481953
DOI: 10.1016/j.it.2007.04.008

Requirement of bic/microRNA-155 for normal immune function.
A Rodriguez, E Vigorito, S Clare, MV Warren, P Couttet, DR Soond, S van Dongen, RJ Grocock, PP Das, EA Miska, D Vetrie, K Okkenhaug, AJ Enright, G Dougan, M Turner, A Bradley

MicroRNAs are a class of small RNAs that are increasingly being recognized as important regulators of gene expression. Although hundreds of microRNAs are present in the mammalian genome, genetic studies addressing their physiological roles are at an early stage. We have shown that mice deficient for bic/microRNA-155 are immunodeficient and display increased lung airway remodeling. We demonstrate a requirement of bic/microRNA-155 for the function of B and T lymphocytes and dendritic cells. Transcriptome analysis of bic/microRNA-155-deficient CD4+ T cells identified a wide spectrum of microRNA-155-regulated genes, including cytokines, chemokines, and transcription factors. Our work suggests that bic/microRNA-155 plays a key role in the homeostasis and function of the immune system.

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Science (New York, N.Y.), 316, 5824, 608-11, 2007

PMID: 17463290
DOI: 10.1126/science.1139253

Open Access

p110delta is required for innate immunity to transplantable lymphomas.
A Saudemont, K Okkenhaug, F Colucci

NK cell (natural killer cells) are lymphocytes of innate immunity that kill tumour cells and respond to infections, without prior stimulation. A balance of activating and inhibitory signals regulates NK cell cytotoxicity, but the molecular mechanisms are not fully understood. General inhibitors of PI3K (phosphoinositide 3-kinase) suppress cytotoxicity in human and mouse NK cells. However, which isoforms and how they regulate NK cell activation is unknown, and no data have been published on mice carrying PI3K mutations. p110delta expression is restricted to leucocytes, where it plays central roles in lymphocyte development and signalling. We have used mice carrying a catalytically inactive mutant form of p110delta in order to determine its role in NK cell biology. We show here that p110delta is not required to kill tumour cells, but unexpectedly p110delta mutant mice failed to fully reject transplanted lymphomas. Our results show for the first time a critical role for p110delta in NK cell biology in vivo.

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Biochemical Society transactions, 35, Pt 2, 183-5, 2007

PMID: 17371233
DOI: 10.1042/BST0350183

The PI3K p110delta controls T-cell development, differentiation and regulation.
DT Patton, F Garçon, K Okkenhaug

PI3Ks (phosphoinositide 3-kinases) regulate diverse cellular functions such as metabolism, growth, gene expression and migration. The p110delta isoform of PI3K is mainly expressed in cells of the immune system and contributes to cellular and humoral immunity. In the thymus, p110delta and p110gamma play complementary roles in regulating the transition through key developmental checkpoints. In addition, p110delta regulates the differentiation of peripheral Th (helper T-cells) towards the Th1 and Th2 lineages. Moreover, p110delta is critical for Treg (regulatory T-cell) function. Here, we review the role of PI3Ks in T-cell development and function.

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Biochemical Society transactions, 35, Pt 2, 167-71, 2007

PMID: 17371229
DOI: 10.1042/BST0350167

Requirement for phosphoinositide 3-kinase p110delta signaling in B cell antigen receptor-mediated antigen presentation.
MM Al-Alwan, K Okkenhaug, B Vanhaesebroeck, JS Hayflick, AJ Marshall

The BCR serves to both signal cellular activation and enhance uptake and presentation of Ags by B cells; however, the intracellular signaling mechanisms linking the BCR to Ag presentation functions have been controversial. PI3Ks are critical signaling enzymes controlling many cellular processes, with the p110delta isoform playing a critical role in BCR signaling. In this study, we used pharmacological and genetic approaches to evaluate the role of p110delta signaling in Ag presentation by primary B lymphocytes. It was found that activation of allogeneic T cells is significantly reduced when B cells are pretreated with global PI3K inhibitors, but was intact when p110delta signaling was specifically inactivated. In contrast, inactivation of p110delta significantly impaired the ability of B cells to activate T cells in a BCR-mediated Ag uptake and presentation model. Prestimulation of p110delta-inactivated B cells with anti-CD40 or LPS could not rescue their BCR-mediated Ag presentation ability to normal levels. p110delta signaling was required for efficient presentation of either anti-Ig or protein Ag via a lysozyme-specific BCR. p110delta-inactivated B cells were able to internalize Ag normally, and no defects in association of Ag with lysosome-associated membrane protein 1(+) late endosomes were observed; however, these cells were less effective in forming polarized conjugates with Ag-specific T cells. Our data demonstrate a role for p110delta signaling in B cell Ag presentation function, implicating 3-phosphoinositides and their targets in the latter stages of this process.

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Journal of immunology (Baltimore, Md. : 1950), 178, 4, 2328-35, 2007

PMID: 17277138

Open Access

Role of the phosphoinositide 3-kinase p110delta in generation of type 2 cytokine responses and allergic airway inflammation.
BF Nashed, T Zhang, M Al-Alwan, G Srinivasan, AJ Halayko, K Okkenhaug, B Vanhaesebroeck, KT Hayglass, AJ Marshall

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 p110delta 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 p110delta mutants, whereas type 1 cytokine responses (IFN-gamma and CXCL10) were robust. Elevated IFN-gamma production during the primary response to ovalbumin (OVA) was associated with reduced production of the regulatory cytokine IL-10. IFN-gamma 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 p110delta-inactivated mice. Adoptive transfer of OVA-primed splenocytes from normal but not p110delta-inactivated mice could induce airway eosinophilia in naive, 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.

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European journal of immunology, 37, 2, 416-24, 2007

PMID: 17236236
DOI: 10.1002/eji.200636401

Antigen receptor signalling: a distinctive role for the p110delta isoform of PI3K.
K Okkenhaug, K Ali, B Vanhaesebroeck

The activation of antigen receptors triggers two important signalling pathways originating from phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)]. The first is phospholipase Cgamma (PLCgamma)-mediated hydrolysis of PtdIns(4,5)P(2), resulting in the activation of Ras, protein kinase C and Ca(2+) flux. This culminates in profound alterations in gene expression and effector-cell responses, including secretory granule exocytosis and cytokine production. By contrast, phosphoinositide 3-kinases (PI3Ks) phosphorylate PtdIns(4,5)P(2) to yield phosphatidylinositol(3,4,5)-trisphosphate, activating signalling pathways that overlap with PLCgamma or are PI3K-specific. Pathways that are PI3K-specific include Akt-mediated inactivation of Foxo transcription factors and transcription-independent regulation of glucose uptake and metabolism. The p110delta isoform of PI3K is the main source of PI3K activity following antigen recognition by B cells, T cells and mast cells. Here, we review the roles of p110delta in regulating antigen-dependent responses in these cell types.

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Trends in immunology, 28, 2, 80-7, 2007

PMID: 17208518
DOI: 10.1016/j.it.2006.12.007

Open Access

Physiologic and aberrant regulation of memory T-cell trafficking by the costimulatory molecule CD28.
V Mirenda, SJ Jarmin, R David, J Dyson, D Scott, Y Gu, RI Lechler, K Okkenhaug, FM Marelli-Berg

Productive T-cell immunity requires both the activation and the migration of specific T cells to the antigenic tissue. The costimulatory molecule CD28 plays an essential role in the initiation of T-cell-mediated immunity. We investigated the possibility that CD28 may also regulate migration of primed T cells to target tissue. In vitro, CD28-mediated signals enhanced T-cell transendothelial migration, integrin clustering, and integrin-mediated migration. In vivo, T cells bearing a mutation in the CD28 cytoplasmic domain, which abrogates PI3K activation, displayed normal clonal expansion but defective localization to antigenic sites following antigenic rechallenge. Importantly, antibody-mediated CD28 stimulation led to unregulated memory T-cell migration to extra-lymphoid tissue, which occurred independently of T-cell receptor (TCR)-derived signals and homing-receptor expression. Finally, we provide evidence that CD28- and CTLA-4-mediated signals exert opposite effects on T-cell trafficking in vivo. These findings highlight a novel physiologic function of CD28 that has crucial implications for the therapeutic manipulation of this and other costimulatory molecules.

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Blood, 109, 7, 2968-77, 2007

PMID: 17119120
DOI: 10.1182/blood-2006-10-050724

Open Access

Cutting edge: the phosphoinositide 3-kinase p110 delta is critical for the function of CD4+CD25+Foxp3+ regulatory T cells.
Patton DT, Garden OA, Pearce WP, Clough LE, Monk CR, Leung E, Rowan WC, Sancho S, Walker LS, Vanhaesebroeck B, Okkenhaug K

CD4+CD25+Foxp3+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance by inhibiting the expansion and function of conventional T cells. Treg development and homeostasis are regulated by the Ag receptor, costimulatory receptors such as CD28 and CTLA-4, and cytokines such as IL-2, IL-10, and TGF-beta. Here we show that the proportions of Tregs in the spleen and lymph nodes of mice with inactive p110delta PI3K (p110deltaD910A/D910A) are reduced despite enhanced Treg selection in the thymus. p110deltaD910A/D910A CD4+CD25+Foxp3+ Tregs showed attenuated suppressor function in vitro and failed to secrete IL-10. In adoptive transfer experiments, p110deltaD910A/D910A T cells failed to protect against experimental colitis. The identification of p110delta as an intracellular signaling protein that regulates the activity of CD4+CD25+Foxp3+ Tregs may facilitate the further elucidation of the molecular mechanisms responsible for Treg-mediated suppression.

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Journal of immunology (Baltimore, Md. : 1950), 177, 0022-1767, 6598-602, 2006

PMID: 17082571

Open Access