Life Sciences Research for Lifelong Health

Publications klaus-okkenhaug

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Obesity-Induced Metabolic Stress Leads to Biased Effector Memory CD4(+) T Cell Differentiation via PI3K p110δ-Akt-Mediated Signals.
Mauro C, Smith J, Cucchi D, Coe D, Fu H, Bonacina F, Baragetti A, Cermenati G, Caruso D, Mitro N, Catapano AL, Ammirati E, Longhi MP, Okkenhaug K, Norata GD, Marelli-Berg FM

Low-grade systemic inflammation associated to obesity leads to cardiovascular complications, caused partly by infiltration of adipose and vascular tissue by effector T cells. The signals leading to T cell differentiation and tissue infiltration during obesity are poorly understood. We tested whether saturated fatty acid-induced metabolic stress affects differentiation and trafficking patterns of CD4(+) T cells. Memory CD4(+) T cells primed in high-fat diet-fed donors preferentially migrated to non-lymphoid, inflammatory sites, independent of the metabolic status of the hosts. This was due to biased CD4(+) T cell differentiation into CD44(hi)-CCR7(lo)-CD62L(lo)-CXCR3(+)-LFA1(+) effector memory-like T cells upon priming in high-fat diet-fed animals. Similar phenotype was observed in obese subjects in a cohort of free-living people. This developmental bias was independent of any crosstalk between CD4(+) T cells and dendritic cells and was mediated via direct exposure of CD4(+) T cells to palmitate, leading to increased activation of a PI3K p110δ-Akt-dependent pathway upon priming.

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Cell metabolism, , 1932-7420, , 2017

PMID: 28190771

Targeting PI3K in Cancer: Impact on Tumor Cells, Their Protective Stroma, Angiogenesis, and Immunotherapy.
Okkenhaug K, Graupera M, Vanhaesebroeck B

The PI3K pathway is hyperactivated in most cancers, yet the capacity of PI3K inhibitors to induce tumor cell death is limited. The efficacy of PI3K inhibition can also derive from interference with the cancer cells' ability to respond to stromal signals, as illustrated by the approved PI3Kδ inhibitor idelalisib in B-cell malignancies. Inhibition of the leukocyte-enriched PI3Kδ or PI3Kγ may unleash antitumor T-cell responses by inhibiting regulatory T cells and immune-suppressive myeloid cells. Moreover, tumor angiogenesis may be targeted by PI3K inhibitors to enhance cancer therapy. Future work should therefore also explore the effects of PI3K inhibitors on the tumor stroma, in addition to their cancer cell-intrinsic impact.

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Cancer discovery, , 2159-8290, , 2016

PMID: 27655435

Ionic immune suppression within the tumour microenvironment limits T cell effector function.
Eil R, Vodnala SK, Clever D, Klebanoff CA, Sukumar M, Pan JH, Palmer DC, Gros A, Yamamoto TN, Patel SJ, Guittard GC, Yu Z, Carbonaro V, Okkenhaug K, Schrump DS, Linehan WM, Roychoudhuri R, Restifo NP

Tumours progress despite being infiltrated by tumour-specific effector T cells. Tumours contain areas of cellular necrosis, which are associated with poor survival in a variety of cancers. Here, we show that necrosis releases intracellular potassium ions into the extracellular fluid of mouse and human tumours, causing profound suppression of T cell effector function. Elevation of the extracellular potassium concentration ([K(+)]e) impairs T cell receptor (TCR)-driven Akt-mTOR phosphorylation and effector programmes. Potassium-mediated suppression of Akt-mTOR signalling and T cell function is dependent upon the activity of the serine/threonine phosphatase PP2A. Although the suppressive effect mediated by elevated [K(+)]e is independent of changes in plasma membrane potential (Vm), it requires an increase in intracellular potassium ([K(+)]i). Accordingly, augmenting potassium efflux in tumour-specific T cells by overexpressing the potassium channel Kv1.3 lowers [K(+)]i and improves effector functions in vitro and in vivo and enhances tumour clearance and survival in melanoma-bearing mice. These results uncover an ionic checkpoint that blocks T cell function in tumours and identify potential new strategies for cancer immunotherapy.

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Nature, 537, 1476-4687, 539-543, 2016

PMID: 27626381

PI3Kδ and primary immunodeficiencies.
Lucas CL, Chandra A, Nejentsev S, Condliffe AM, Okkenhaug K

Primary immunodeficiencies are inherited disorders of the immune system, often caused by the mutation of genes required for lymphocyte development and activation. Recently, several studies have identified gain-of-function mutations in the phosphoinositide 3-kinase (PI3K) genes PIK3CD (which encodes p110δ) and PIK3R1 (which encodes p85α) that cause a combined immunodeficiency syndrome, referred to as activated PI3Kδ syndrome (APDS; also known as p110δ-activating mutation causing senescent T cells, lymphadenopathy and immunodeficiency (PASLI)). Paradoxically, both loss-of-function and gain-of-function mutations that affect these genes lead to immunosuppression, albeit via different mechanisms. Here, we review the roles of PI3Kδ in adaptive immunity, describe the clinical manifestations and mechanisms of disease in APDS and highlight new insights into PI3Kδ gleaned from these patients, as well as implications of these findings for clinical therapy.

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Nature reviews. Immunology, , 1474-1741, , 2016

PMID: 27616589

Clinical spectrum and features of activated phosphoinositide 3-kinase δ syndrome: A large patient cohort study.
Coulter TI, Chandra A, Bacon CM, Babar J, Curtis J, Screaton N, Goodlad JR, Farmer G, Steele CL, Leahy TR, Doffinger R, Baxendale H, Bernatoniene J, Edgar JD, Longhurst HJ, Ehl S, Speckmann C, Grimbacher B, Sediva A, Milota T, Faust SN, Williams AP, Hayman G, Kucuk ZY, Hague R, French P, Brooker R, Forsyth P, Herriot R, Cancrini C, Palma P, Ariganello P, Conlon N, Feighery C, Gavin PJ, Jones A, Imai K, Ibrahim MA, Markelj G, Abinun M, Rieux-Laucat F, Latour S, Pellier I, Fischer A, Touzot F, Casanova JL, Durandy A, Burns SO, Savic S, Kumararatne DS, Moshous D, Kracker S, Vanhaesebroeck B, Okkenhaug K, Picard C, Nejentsev S, Condliffe AM, Cant AJ

Activated phosphoinositide 3-kinase δ syndrome (APDS) is a recently described combined immunodeficiency resulting from gain-of-function mutations in PIK3CD, the gene encoding the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ).

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The Journal of allergy and clinical immunology, , 1097-6825, , 2016

PMID: 27555459

Open Access

BACH2 regulates CD8(+) T cell differentiation by controlling access of AP-1 factors to enhancers.
Roychoudhuri R, Clever D, Li P, Wakabayashi Y, Quinn KM, Klebanoff CA, Ji Y, Sukumar M, Eil RL, Yu Z, Spolski R, Palmer DC, Pan JH, Patel SJ, Macallan DC, Fabozzi G, Shih HY, Kanno Y, Muto A, Zhu J, Gattinoni L, O'Shea JJ, Okkenhaug K, Igarashi K, Leonard WJ, Restifo NP

T cell antigen receptor (TCR) signaling drives distinct responses depending on the differentiation state and context of CD8(+) T cells. We hypothesized that access of signal-dependent transcription factors (TFs) to enhancers is dynamically regulated to shape transcriptional responses to TCR signaling. We found that the TF BACH2 restrains terminal differentiation to enable generation of long-lived memory cells and protective immunity after viral infection. BACH2 was recruited to enhancers, where it limited expression of TCR-driven genes by attenuating the availability of activator protein-1 (AP-1) sites to Jun family signal-dependent TFs. In naive cells, this prevented TCR-driven induction of genes associated with terminal differentiation. Upon effector differentiation, reduced expression of BACH2 and its phosphorylation enabled unrestrained induction of TCR-driven effector programs.

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Nature immunology, , 1529-2916, , 2016

PMID: 27158840

Inhibition of Phosphoinositide 3-Kinase p110delta Does Not Affect T Cell Driven Development of Type 1 Diabetes Despite Significant Effects on Cytokine Production.
Barbera Betancourt A, Emery JL, Recino A, Wong FS, Okkenhaug K, Wallberg M

Type 1 diabetes is caused by the destruction of insulin producing beta cells by the immune system. The p110δ isoform of PI3K is expressed primarily in cells of haematopoietic origin and the catalytic activity of p110δ is important for the activation of these cells. Targeting of this pathway offers an opportunity to reduce immune cell activity without unwanted side effects. We have explored the effects of a specific p110δ isoform inhibitor, IC87114, on diabetogenic T cells both in vitro and in vivo, and find that although pharmacological inhibition of p110δ has a considerable impact on the production of pro-inflammatory cytokines, it does not delay the onset of diabetes after adoptive transfer of diabetogenic cells. Further, we demonstrate that combination treatment with CTLA4-Ig does not improve the efficacy of treatment, but instead attenuates the protective effects seen with CTLA4-Ig treatment alone. Our results suggest that decreased IL-10 production by Foxp3+ CD4+ T cells in the presence of IC87114 negates individual anti-inflammatory effects of IC8114 and CTLA4-Ig.

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PloS one, 11, 1932-6203, e0146516, 2016

PMID: 26783747

Open Access

PI3Kδ promotes CD4(+) T cell interactions with antigen presenting cells by increasing LFA-1 binding to ICAM-1.
Garçon F, Okkenhaug K

Activation of T lymphocytes by peptide/MHC on antigen presenting cells (APCs) involves dynamic contacts between the two cells, during which T cells undergo dramatic morphological changes. These interactions are facilitated by integrins. Activation of the T cells increases the binding of the integrin LFA-1 expressed by T cells to ICAM-1 and ICAM-2 expressed by APCs. The signalling pathways that control integrin affinities are incompletely defined. The phosphoinositide 3-kinases (PI3Ks) generate second-messenger signalling molecules that control cell growth, proliferation, differentiation and trafficking. Here we show that in T cells, PI3Kδ attenuates the activation of Rac1, but sustains the activation of Rap1. Consequently, PI3Kδ increases LFA-1-dependent adhesion to form stable conjugates with antigen presenting cells. Increased Rap1 activity and LFA-1 adhesion were only in part mediated by the downstream kinase Akt, suggesting the involvement of additional PIP3-binding proteins. These results establish a link between PI3K activity, cytoskeletal changes and integrin binding and help explain the impaired T cell-dependent immune responses in PI3Kδ-deficient mice.Immunology and Cell Biology accepted article preview online, 07 January 2016. doi:10.1038/icb.2016.1.

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Immunology and cell biology, , 1440-1711, , 2016

PMID: 26740009

Open Access

The transcription factor BACH2 promotes tumor immunosuppression.
Roychoudhuri R, Eil RL, Clever D, Klebanoff CA, Sukumar M, Grant FM, Yu Z, Mehta G, Liu H, Jin P, Ji Y, Palmer DC, Pan JH, Chichura A, Crompton JG, Patel SJ, Stroncek D, Wang E, Marincola FM, Okkenhaug K, Gattinoni L, Restifo NP

The immune system has a powerful ability to recognize and kill cancer cells, but its function is often suppressed within tumors, preventing clearance of disease. Functionally diverse innate and adaptive cellular lineages either drive or constrain immune reactions within tumors. The transcription factor (TF) BACH2 regulates the differentiation of multiple innate and adaptive cellular lineages, but its role in controlling tumor immunity has not been elucidated. Here, we demonstrate that BACH2 is required to establish immunosuppression within tumors. Tumor growth was markedly impaired in Bach2-deficient mice and coincided with intratumoral activation of both innate and adaptive immunity. However, augmented tumor clearance in the absence of Bach2 was dependent upon the adaptive immune system. Analysis of tumor-infiltrating lymphocytes from Bach2-deficient mice revealed high frequencies of rapidly proliferating effector CD4+ and CD8+ T cells that expressed the inflammatory cytokine IFN-γ. Effector T cell activation coincided with a reduction in the frequency of intratumoral Foxp3+ Tregs. Mechanistically, BACH2 promoted tumor immunosuppression through Treg-mediated inhibition of intratumoral CD8+ T cells and IFN-γ. These findings demonstrate that BACH2 is a key component of the molecular program of tumor immunosuppression and identify therapeutic targets for the reversal of immunosuppression in cancer.

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The Journal of clinical investigation, , 1558-8238, , 2016

PMID: 26731475

Open Access

Oncogenic PI3Kα promotes multipotency in breast epithelial cells.
Okkenhaug K, Roychoudhuri R

The phosphoinositide 3-kinase (PI3K) signaling pathway is among the most frequently altered in cancer. Now, two studies show that a mutated oncogenic PI3Kα, commonly found in breast cancer, leads to dedifferentiation or destabilization of luminal and basal epithelial lineages, which in turn leads to increased cancer cell heterogeneity.

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Science signaling, 8, 1937-9145, pe3, 2015

PMID: 26535006

Open Access

PI3K Signaling in Normal B Cells and Chronic Lymphocytic Leukemia (CLL).
Okkenhaug K, Burger JA

B cells provide immunity to extracellular pathogens by secreting a diverse repertoire of antibodies with high affinity and specificity for exposed antigens. The B cell receptor (BCR ) is a transmembrane antibody, which facilitates the clonal selection of B cells producing secreted antibodies of the same specificity. The diverse antibody repertoire is generated by V(D)J recombination of heavy and light chain genes, whereas affinity maturation is mediated by activation-induced cytidine deaminase (AID)-mediated mutagenesis. These processes, which are essential for the generation of adaptive humoral immunity, also render B cells susceptible to chromosomal rearrangements and point mutations that in some cases lead to cancer. In this chapter, we will review the central role of PI3K s in mediating signals from the B cell receptor that not only facilitate the development of functional B cell repertoire, but also support the growth and survival of neoplastic B cells, focusing on chronic lymphocytic leukemia (CLL ) B cells. Perhaps because of the central role played by PI3K in BCR signaling, B cell leukemia and lymphomas are the first diseases for which a PI3K inhibitor has been approved for clinical use.

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Current topics in microbiology and immunology, 393, 0070-217X, 123-42, 2016

PMID: 26350103

Open Access

Editorial: Lipid Signaling in T Cell Development and Function.
Sauer K, Okkenhaug K

Frontiers in immunology, 6, 1664-3224, 410, 2015

PMID: 26322043

Open Access

PI3Kδ Regulates the Magnitude of CD8+ T Cell Responses after Challenge with Listeria monocytogenes.
Pearce VQ, Bouabe H, MacQueen AR, Carbonaro V, Okkenhaug K

PI3Ks regulate diverse immune cell functions by transmitting intracellular signals from Ag, costimulatory receptors, and cytokine receptors to control cell division, differentiation, survival, and migration. In this study, we report the effect of inhibiting the p110δ subunit of PI3Kδ on CD8(+) T cell responses to infection with the intracellular bacteria Listeria monocytogenes. A strong dependency on PI3Kδ for IFN-γ production by CD8(+) T cells in vitro was not recapitulated after Listeria infection in vivo. Inactivation of PI3Kδ resulted in enhanced bacterial elimination by the innate immune system. However, the magnitudes of the primary and secondary CD8 +: T cell responses were reduced. Moreover, PI3Kδ activity was required for CD8(+) T cells to provide help to other responding CD8(+) cells. These findings identify PI3Kδ as a key regulator of CD8(+) T cell responses that integrates extrinsic cues, including those from other responding cells, to determine the collective behavior of CD8(+) T cell populations responding to infection.

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Journal of immunology (Baltimore, Md. : 1950), , 1550-6606, , 2015

PMID: 26311905

Open Access

Cowden's syndrome with immunodeficiency.
Browning MJ, Chandra A, Carbonaro V, Okkenhaug K, Barwell J

Cowden's syndrome is a rare, autosomal dominant disease caused by mutations in the phosphoinositide 3-kinase and phosphatase and tensin homolog (PTEN) gene. It is associated with hamartomatous polyposis of the gastrointestinal tract, mucocutaneous lesions and increased risk of developing certain types of cancer. In addition to increased risk of tumour development, mutations in PTEN have also been associated with autoimmunity in both mice and humans. Until now, however, an association between Cowden's syndrome and immune deficiency has been reported in a single patient only.

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Journal of medical genetics, 52, 1468-6244, 856-9, 2015

PMID: 26246517

Open Access

PI3K inhibitors in inflammation, autoimmunity and cancer.
Stark AK, Sriskantharajah S, Hessel EM, Okkenhaug K

The healthy immune system protects against infection and malignant transformation without causing significant damage to host tissues. Immune dysregulation results in diverse pathologies including autoimmune disease, chronic inflammatory disorders, allergies as well as immune deficiencies and cancer. Phosphoinositide 3-kinase (PI3K) signalling has been shown to be a key pathway in the regulation of the immune response and continues to be the focus of intense research. In recent years we have gained detailed understanding of PI3K signalling, and saw the development of potent and highly selective small molecule inhibitors, of which several are currently in clinical trials for the treatment of immune-related disorders and cancer. The role of PI3K signalling in the immune response has been the subject of detailed reviews; here we focus on relevant recent progress in pre-clinical and clinical development of PI3K inhibitors.

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Current opinion in pharmacology, 23, 1471-4973, 82-91, 2015

PMID: 26093105

Open Access

Immunomodulation of Selective Naive T Cell Functions by p110δ Inactivation Improves the Outcome of Mismatched Cell Transplantation.
Doisne JM, Hüber CM, Okkenhaug K, Colucci F

Allogeneic hematopoietic stem cell transplantation (HSCT) can treat certain hematologic malignancies due to the graft versus leukemia (GvL) effect but is complicated by graft versus host disease (GvHD). Expression of the p110δ catalytic subunit of the phosphoinositide 3-kinase pathway is restricted to leukocytes, where it regulates proliferation, migration, and cytokine production. Here, in a mouse model of fully mismatched hematopoietic cell transplantation (HCT), we show that genetic inactivation of p110δ in T cells leads to milder GvHD, whereas GvL is preserved. Inactivation of p110δ in human lymphocytes reduced T cell allorecognition. We demonstrate that both allostimulation and granzyme B expression were dependent on p110δ in naive T cells, which are the main mediators of GvHD, whereas memory T cells were unaffected. Strikingly, p110δ is not mandatory for either naive or memory T cells to mediate GvL. Therefore, immunomodulation of selective naive T cell functions by p110δ inactivation improves the outcome of allogeneic HSCT.

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Cell reports, , 2211-1247, , 2015

PMID: 25660021

Open Access

PI3K Signaling in B Cell and T Cell Biology.
Okkenhaug K, Turner M, Gold MR

Frontiers in immunology, 5, 1664-3224, 557, 2014

PMID: 25404931

Open Access

Inactivation of PI(3)K p110δ breaks regulatory T-cell-mediated immune tolerance to cancer.
K Ali, DR Soond, R Piñeiro, T Hagemann, W Pearce, EL Lim, H Bouabe, CL Scudamore, T Hancox, H Maecker, L Friedman, M Turner, K Okkenhaug, B Vanhaesebroeck

Inhibitors against the p110δ isoform of phosphoinositide-3-OH kinase (PI(3)K) have shown remarkable therapeutic efficacy in some human leukaemias. As p110δ is primarily expressed in leukocytes, drugs against p110δ have not been considered for the treatment of solid tumours. Here we report that p110δ inactivation in mice protects against a broad range of cancers, including non-haematological solid tumours. We demonstrate that p110δ inactivation in regulatory T cells unleashes CD8(+) cytotoxic T cells and induces tumour regression. Thus, p110δ inhibitors can break tumour-induced immune tolerance and should be considered for wider use in oncology.

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Nature, 510, , 407-411, 2014

PMID: 24919154
DOI: 10.1038/nature13444

Open Access

Haematological cancer: Idelalisib-targeting PI3Kδ in patients with B-cell malignancies.
JA Burger and K Okkenhaug

Idelalisib, the first PI3Kδ inhibitor in clinical use, has excellent activity in patients with chronic lymphocytic leukaemia and indolent B-cell lymphomas, heralding a new era of targeted therapy for these types of cancer. Idelalisib intercepts critical communications between B cells and the microenvironment, including B-cell receptor signalling and chemokine networks.

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Nature Reviews Clinical Oncology, 11, , 184-186, 2014

PMID: 24642682
DOI: 10.1038/nrclinonc.2014.42

Open Access

IL-21 promotes CD4 T cell responses by phosphatidylinositol 3-kinase-dependent upregulation of CD86 on B cells.
Attridge K, Kenefeck R, Wardzinski L, Qureshi OS, Wang CJ, Manzotti C, Okkenhaug K, Walker LS

The cytokine IL-21 is a potent immune modulator with diverse mechanisms of action on multiple cell types. IL-21 is in clinical use to promote tumor rejection and is an emerging target for neutralization in the setting of autoimmunity. Despite its clinical potential, the biological actions of IL-21 are not yet fully understood and the full range of effects of this pleiotropic cytokine are still being uncovered. In this study, we identify a novel role for IL-21 as an inducer of the costimulatory ligand CD86 on B lymphocytes. CD86 provides critical signals through T cell-expressed CD28 that promote T cell activation in response to Ag engagement. Expression levels of CD86 are tightly regulated in vivo, being actively decreased by regulatory T cells and increased in response to pathogen-derived signals. In this study, we demonstrate that IL-21 can trigger potent and sustained CD86 upregulation through a STAT3 and PI3K-dependent mechanism. We show that elevated CD86 expression has functional consequences for the magnitude of CD4 T cell responses both in vitro and in vivo. These data pinpoint CD86 upregulation as an additional mechanism by which IL-21 can elicit immunomodulatory effects.

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Journal of immunology (Baltimore, Md. : 1950), 192, 1550-6606, 2195-201, 2014

PMID: 24470500

Open Access

Two Birds with One Stone: Dual p110δ and p110γ Inhibition.
K Okkenhaug

In this issue of Chemistry & Biology, Winkler and colleagues describe the discovery and preclinical development of IPI-145, a new inhibitor of the phosphoinositide 3-kinase (PI3K) isoforms p110δ and p110γ that have entered clinical trials.

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Chemistry & biology, 20, 11, 1309-10, 2013

PMID: 24267274
DOI: 10.1016/j.chembiol.2013.11.002

Open Access

Phosphoinositide 3-Kinase δ Gene Mutation Predisposes to Respiratory Infection and Airway Damage.
I Angulo, O Vadas, F Garçon, E Banham-Hall, V Plagnol, TR Leahy, H Baxendale, T Coulter, J Curtis, C Wu, K Blake-Palmer, O Perisic, D Smyth, M Maes, C Fiddler, J Juss, D Cilliers, G Markelj, A Chandra, G Farmer, A Kielkowska, J Clark, S Kracker, M Debré, C Picard, I Pellier, N Jabado, JA Morris, G Barcenas-Morales, A Fischer, L Stephens, P Hawkins, JC Barrett, M Abinun, M Clatworthy, A Durandy, R Doffinger, E Chilvers, AJ Cant, D Kumararatne, K Okkenhaug, RL Williams, A Condliffe, S Nejentsev

Genetic mutations cause primary immunodeficiencies (PIDs), which predispose to infections. Here we describe Activated PI3K-δ Syndrome (APDS), a PID associated with a dominant gain-of-function mutation in which lysine replaced glutamic acid at residue 1021 (E1021K) in the p110δ protein, the catalytic subunit of phosphoinositide 3-kinase δ (PI3Kδ), encoded by the PIK3CD gene. We found E1021K in 17 patients from seven unrelated families, but not among 3346 healthy subjects. APDS was characterized by recurrent respiratory infections, progressive airway damage, lymphopenia, increased circulating transitional B cells, increased immunoglobulin M and reduced immunoglobulin G2 levels in serum and impaired vaccine responses. The E1021K mutation enhanced membrane association and kinase activity of p110δ. Patient-derived lymphocytes had increased levels of phosphatidylinositol 3,4,5-trisphosphate and phosphorylated AKT protein and were prone to activation-induced cell death. Selective p110δ inhibitors IC87114 and GS-1101 reduced the activity of the mutant enzyme in vitro, which suggested a therapeutic approach for patients with APDS.

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Science (New York, N.Y.), , , , 2013

PMID: 24136356
DOI: 10.1126/science.1243292

Open Access

PI3K p110δ is expressed by gp38(-)CD31(+) and gp38(+)CD31(+) spleen stromal cells and regulates their CCL19, CCL21, and LTβR mRNA levels.
Zotes TM, Spada R, Mulens V, Pérez-Yagüe S, Sorzano CO, Okkenhaug K, Carrera AC, Barber DF

The role of p110δ PI3K in lymphoid cells has been studied extensively, showing its importance in immune cell differentiation, activation and development. Altered T cell localization in p110δ-deficient mouse spleen suggested a role for p110δ in non-hematopoietic stromal cells, which maintain hematopoietic cell segregation. We tested this hypothesis using p110δ(WT/WT) mouse bone marrow to reconstitute lethally irradiated p110δ(WT/WT) or p110δ(D910A/D910A) (which express catalytically inactive p110δ) recipients, and studied localization, number and percentage of hematopoietic cell subsets in spleen and lymph nodes, in homeostatic conditions and after antigen stimulation. These analyses showed diffuse T cell areas in p110δ(D910A/D910A) and in reconstituted p110δ(D910A/D910A) mice in homeostatic conditions. In these mice, spleen CD4(+) and CD8(+) T cell numbers did not increase in response to antigen, suggesting that a p110δ(D910A/D910A) stroma defect impedes correct T cell response. FACS analysis of spleen stromal cell populations showed a decrease in the percentage of gp38(-)CD31(+) cells in p110δ(D910A/D910A) mice. qRT-PCR studies detected p110δ mRNA expression in p110δ(WT/WT) spleen gp38(-)CD31(+) and gp38(+)CD31(+) subsets, which was reduced in p110δ(D910A/D910A) spleen. Lack of p110δ activity in these cell populations correlated with lower LTβR, CCL19 and CCL21 mRNA levels; these molecules participate in T cell localization to specific spleen areas. Our results could explain the lower T cell numbers and more diffuse T cell areas found in p110δ(D910A/D910A) mouse spleen, as well as the lower T cell expansion after antigen stimulation in p110δ(D910A/D910A) compared with p110δ(WT/WT) mice.

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PloS one, 8, 1932-6203, e72960, 2013

PMID: 24009720

Open Access

A protocol for construction of gene targeting vectors and generation of homologous recombinant embryonic stem cells.
H Bouabe, K Okkenhaug

The completion of human and mouse genome sequencing has confronted us with huge amount of data sequences that certainly need decades and many generations of scientists to be reasonably interpreted and assigned to physiological functions, and subsequently fruitfully translated into medical application. A means to assess the function of genes provides gene targeting in mouse embryonic stem cells (ESCs) that enables to introduce site-specific modifications in the mouse genome, and analyze their physiological consequences. Gene targeting enables almost any type of genetic modifications of interest, ranging from gene insertion (e.g., insertion of human-specific genes or reporter genes), gene disruption, point mutations, and short- and long-range deletions, inversions. Site-specific modification into the genome of ESCs can be reached by homologous recombination using targeting vectors. Here, we describe a protocol to generate targeting constructs and homologous recombinant ESCs.

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Methods in molecular biology (Clifton, N.J.), 1064, , 337-54, 2013

PMID: 23996269
DOI: 10.1007/978-1-62703-601-6_24

Open Access

Gene targeting in mice: a review.
H Bouabe, K Okkenhaug

The ability to introduce DNA sequences (e.g., genes) of interest into the germline genome has rendered the mouse a powerful and indispensable experimental model in fundamental and medical research. The DNA sequences can be integrated into the genome randomly or into a specific locus by homologous recombination, in order to: (1) delete or insert mutations into genes of interest to determine their function, (2) introduce human genes into the genome of mice to generate animal models enabling study of human-specific genes and diseases, e.g., mice susceptible to infections by human-specific pathogens of interest, (3) introduce individual genes or genomes of pathogens (such as viruses) in order to examine the contributions of such genes to the pathogenesis of the parent pathogens, (4) and last but not least introduce reporter genes that allow monitoring in vivo or ex vivo the expression of genes of interest. Furthermore, the use of recombination systems, such as Cre/loxP or FRT/FLP, enables conditional induction or suppression of gene expression of interest in a restricted period of mouse's lifetime, in a particular cell type, or in a specific tissue. In this review, we will give an updated summary of the gene targeting technology and discuss some important considerations in the design of gene-targeted mice.

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Methods in molecular biology (Clifton, N.J.), 1064, , 315-36, 2013

PMID: 23996268
DOI: 10.1007/978-1-62703-601-6_23

Open Access