SEARCH:   Advanced

Blood, 15 April 2007, Vol. 109, No. 8, pp. 3316-3324. Prepublished online as a Blood First Edition Paper on December 14, 2006; DOI 10.1182/blood-2006-07-038059. This Article Abstract Full Text (PDF) Supplemental Figures All Versions of this Article: blood-2006-07-038059v1 109/8/3316    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kishimoto, H. Articles by Suzuki, A. Search for Related Content PubMed PubMed Citation Articles by Kishimoto, H. Articles by Suzuki, A. Related Collections Signal Transduction Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  IMMUNOBIOLOGY The Pten/PI3K pathway governs the homeostasis of V14iNKT cells Hiroyuki Kishimoto1, Toshiaki Ohteki2, Nobuyuki Yajima1, Koichi Kawahara1, Miyuki Natsui1, Satoru Kawarasaki1, Koichi Hamada1, Yasuo Horie3, Yoshiaki Kubo4, Seiji Arase4, Masaru Taniguchi5, Bart Vanhaesebroeck6, Tak Wah Mak7, Toru Nakano8, Shigeo Koyasu9, Takehiko Sasaki10, and Akira Suzuki1 Departments of1 Molecular Biology 2 Immunology, and 3 Gastroenterology, Akita University School of Medicine, Japan; 4 Department of Dermatology, Institute of Health Bioscience, Graduate School of Medicine, The University of Tokushima, Japan; 5 RIKEN Research Center for Allergy and Immunology, Yokohama, Japan; 6 Ludwig Institute for Cancer Research, London, United Kingdom; 7 Campbell Family Institute for Breast Cancer Research and Departments of Immunology and Medical Biophysics, University of Toronto, ON, Canada; 8 Department of Pathology, Medical School and Graduate School of Frontier Biosciences, Osaka University, Suita, Japan; 9 Department of Microbiology and Immunology, Keio University School of Medicine, Tokyo, Japan; 10 Department of Microbiology, Akita University School of Medicine, Japan    Abstract Top Abstract Introduction Materials and methods Results Discussion Authorship References   The tumor suppressor PTEN is mutated in many human cancers. We previously used the Cre-loxP system to generate mice (LckCrePten mice) with a Pten mutation in T-lineage cells. Here we describe the phenotype of Pten-deficient V14iNKT cells. A failure in the development of V14iNKT cells occurs in the LckCrePten thymus between stage 2 (CD44highNK1.1–) and stage 3 (CD44highNK1.1+), resulting in decreased numbers of peripheral V14iNKT cells. In vitro, Pten-deficient V14iNKT cells show reduced proliferation and cytokine secretion in response to GalCer stimulation but enhanced inhibitory Ly49 receptor expression. Following interaction with dendritic cells (DCs) loaded with GalCer, Pten-deficient V14iNKT cells demonstrate activation of PI3K. Indeed, the effects of the Pten mutation require intact function of the PI3K subunits p110 and p110. In vivo, LckCrePten mice display reduced serum IFN after GalCer administration. Importantly, V14iNKT cell–mediated protection against the metastasis of melanoma cells to the lung was impaired in the absence of Pten. Thus, the Pten/PI3K pathway is indispensable for the homeostasis and antitumor surveillance function of V14iNKT cells.    Introduction Top Abstract Introduction Materials and methods Results Discussion Authorship References   V14iNKT cells, originally defined as NK1.1+ T cells,1–3 are a unique lymphoid lineage characterized by the expression of several receptors that distinguish them from T, B, and natural killer (NK) cells. These receptors include an invariant rearranged T-cell receptor (TCR) chain, NK1.1, the IL-2/IL-15Rß chain (CD122), and Ly49. The TCR chain expressed by V14iNKT cells is encoded by the V14 and J18 gene segments and is usually associated with a TCRß chain containing the Vß8 gene segment, a combination not expressed by conventional T cells.4 V14iNKT cells constitute more than half of the NK1.1+ T-cell population in the murine spleen and most NK1.1+ T cells in the thymus and liver.5 V14iNKT cells respond to glycolipid antigens such as the marine sponge–derived glycolipid -galactosylceramide (GalCer). GalCer is presented to NKT cells by CD1d, an antigen presentation molecule that structurally resembles major histocompatibility complex (MHC) class I. Because of their restricted specificity, V14iNKT cells can be readily identified by staining with CD1d tetramers or dimers loaded with GalCer.6,7 During their development in the thymus, V14iNKT precursors at the CD4+CD8+ (double positive [DP]) stage are positively selected by CD1d-mediated presentation of self-glycolipids (such as iGb3) by CD4+CD8+ thymocytes.8,9 V14iNKT precursors (CD44lowNK1.1–; stage 1) then develop sequentially into immature NKT cells (CD44highNK1.1–; stage 2) and finally into mature V14iNKT cells (CD44highNK1.1+; stage 3).10,11 V14iNKT cells apparently also undergo negative selection such that NKT cell maturation is severely reduced in situations in which activation signals for developing cells are enhanced to supraphysiological levels either in vitro or in vivo.12–16 In response to TCR ligation, mature peripheral V14iNKT cells promptly produce large amounts of Th1 cytokines (IFN, TNF) as well as Th2 cytokines (IL-4, IL-10, IL-13).17,18 V14iNKT cells therefore play important roles in a variety of innate and adaptive immune responses, including those mediating autoimmunity or combating microbial infections or tumor metastasis.5,19–21 Indeed, mice deficient in V14iNKT cells show impaired tumor rejection.21 This observation is consistent with the antitumor effect of GalCer administration on various tumor types, including melanomas and lung, colon, and hematopoietic cancers.22 Despite the importance of V14iNKT cells, little is known about their intracellular signaling pathways or how these pathways might be regulated. PTEN is a tumor suppressor gene23 that is mutated in many human sporadic cancers and in hereditary tumor susceptibility disorders such as Cowden disease.24 PTEN is a multifunctional phosphatase whose major substrate is phosphatidylinositol-3,4,5-triphosphate (PIP3),25 a lipid second messenger molecule. PIP3 is generated by the action of phosphoinositide-3-kinases (PI3Ks). Four distinct class I PI3K isotypes exist in mammals: PI3K, ß, , and .26 The function of the PIP3 generated by PI3K is to activate numerous downstream targets, including the serine-threonine kinase PKB/Akt that is involved in antiapoptosis, proliferation, and oncogenesis. By using its lipid phosphatase activity to dephosphorylate PIP3, PTEN negatively regulates the PI3K-PKB/Akt pathway and thereby exerts tumor suppression.27 Thus, murine T cells lacking Pten show enhanced proliferation and activation in vitro, and T cell–specific Pten-deficient mice develop T-cell lymphomas/leukemias and are prone to autoimmunity.28 Conversely, mice deficient for the PI3K subunit p110 show impaired T-cell development and activation,29 and T cells from animals with a kinase-inactivating mutation (D910A) of p110 have a defect in TCR-stimulated proliferation.30 Analyses of these and other genetically modified mice have begun to provide insights into the pathways and molecules mediating intracellular signaling in V14iNKT cells. However, the specific functions of PI3K and Pten in these cells are totally unknown. In this study, we demonstrate that mutation of Pten in murine V14iNKT cells results in developmental failure leading to increased numbers of immature V14iNKT cells and decreased numbers of mature V14iNKT cells of impaired functionality. Moreover, these defects depend on PI3K function. Our results indicate that the Pten/PI3K pathway is important for the homeostasis of V14iNKT cells and their antitumor surveillance function.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion Authorship References   Mice LckCrePtenflox/flox,28 Pten+/–,31 PI3K p110–/–,29 PI3K p110D910A/D910A,30 J18–/–,21 and AktPH-GFPtg/tg transgenic32 mice (all C57BL6/J background, H-2b) have been previously described. Because LckCrePtenflox/flox mice begin to develop T-cell lymphomas at age 3 months, all experiments were performed using lymphoma-free mice younger than 8 weeks of age. LckCrePten+/+ and Ptenflox/flox mice28 were indistinguishable in pilot experiments examining flow cytometry, cell proliferation, and cytokine production (data not shown), so that Ptenflox/flox mice were used as representative wild-type (WT) controls in this study. The Institutional Review Board of the Akita University School of Medicine approved all animal experiments. Reagents and melanoma cell line A synthetic form of GalCer (KRN7000) was provided by the Kirin Brewery (Gumma, Japan). Antibodies to TCRß-FITC (H57-597), NK1.1-FITC or -PE (PK136), CD24/HSA-biotin (30-F1), and PE-Cy5-streptavidin were purchased from eBioscience (San Diego, CA). Antibodies to TCRß-APC (H57-597), CD45R/B220-APC (RA3-6B2), Ly49C/I-biotin (5E6), Ly49A-biotin (A1), CD44-FITC (IM7), mouse IgG1–biotin (A85-1), and mouse CD1d dimmer–Ig fusion protein were purchased from BD PharMingen (San Diego, CA). The B16F10 mouse melanoma cell line was supplied by the Cell Resource Center for Biomedical Research, Tohoku University, Japan. Flow cytometry and cell sorting Mononuclear cells (MNCs) from the thymus, liver, and spleen were obtained as previously described.33 For routine staining of V14iNKT cells, MNCs were first preincubated with CD16/32 mAb to prevent nonspecific binding of antibodies to FcR. The cells were then incubated with CD1d dimmer–Ig (GalCer-CD1d)7 followed by anti–mouse IgG1–biotin, PE-Cy5-streptavidin, and anti-TCRß–APC. To analyze NKT cell development, cells were also stained with anti-NK1.1–PE and anti-CD44–FITC. To detect Ly49 expression, cells were stained with anti-NK1.1–FITC, anti-TCRß–APC, CD1d dimmer–Ig (GalCer-CD1d), anti–mouse IgG1–PE, anti-Ly49C/I–biotin (or anti-Ly49A–biotin), and streptavidin-PE-Cy5. All flow cytometry and sorting experiments were performed using MoFlo and Summit software (Dako Cytomation, Glostrup, Denmark). PCR and Western blotting For analyzing the deletion frequency of the Pten gene at the DNA level, genomic DNA samples from sorted TCRß+NK1.1+HSAlow thymocytes of 8-week-old Ptenflox/flox and LckCrePtenflox/flox mice were amplified by polymerase chain reaction (PCR) as described.34 Sense primer (5'-GTGAAAGTGCCCCAACATAAGG-3') and antisense primer (5'-CTCCCACCAATGAACAAACAGT-3') were used to detect the WT and floxed Pten alleles; sense primer (5'-GGCCTAGGACTCACTAGATAGC-3') and antisense primer (5'-CTCCCACCAATGAACAAACAGT-3') were used to detect the deleted Pten allele. Amplified fragments of 514 bp (Ptenflox allele) and 705 bp (Pten allele) were obtained. Total lysates (10 µg) of sorted TCRß+NK1.1+HSAlow thymocytes from 8-week-old Ptenflox/flox and LckCrePtenflox/flox mice were analyzed by Western blotting using antibodies directed against Pten (Cascade Bioscience, Winchester, MA) or actin (Santa Cruz Biotechnology, Santa Cruz, CA). In vitro and in vivo activation of NKT cells For in vitro activation of NKT cells, thymocytes were isolated from 8-week-old LckCrePtenflox.flox or Ptenflox.flox mice, and NK1.1+TCRß+HSAlow NKT cells were purified by cell sorting. GalCer-loaded dendritic cells (DCs) were prepared from spleens as described.35 Briefly, spleens from WT mice were digested using collagenase and erythrocytes, and MNCs were collected by step centrifugation using 28% bovine serum albumin and serumfree RPMI medium. Isolated MNCs were cultured for 2 hours, and adherent cells were further cultured overnight with or without 100 ng/mL GalCer. Nonadherent CD11c+ cells were plated at 2 x 104 per well in a 96-well plate and -irradiated (30 Gy). These cells were used as the GalCer-loaded DCs. Purified NKT cells (1 x 104 per well) were incubated with GalCer-loaded DCs in 96-well plates in RPMI 1640 medium containing 10% FCS. The GalCer-stimulated NKT cells were harvested on day 2, and their proliferation was assayed by pulsing for 12 hours with 0.5 µCi (0.0185 MBq) [3H]-thymidine (Amersham, Arlington Heights, IL) per well and measuring [3H]-thymidine incorporation by standard methods. Culture supernatants were assayed for the production of IFN and IL-4 by ELISA (Pierce Endogen, Rockford, IL). For in vivo activation of NKT cells, GalCer (2 µg) was intraperitoneally injected into 8-week-old Ptenflox/flox, Pten+/–, and LckCrePtenflox/flox mice. Serum IL-4 and IFN levels were examined by enzyme-linked immunosorbent assay (ELISA) at 2 hours and 12 hours after GalCer injection, respectively. NKT cell maturation in FTOC Fetal thymus organ culture (FTOC) was performed as described11 with some modifications. Fetal thymus lobes from embryonic day 14.5 J18–/– fetal mice were cultured for 3 days on Nucleopore membrane (Whatman, Middlesex, United Kingdom) in RPMI medium supplemented with 10% FCS, nonessential amino acids, pyruvate, and ß-mercaptoethanol (all from Invitrogen, Carlsbad, CA). Purified CD44highNK1.1–GalCer-CD1d+TCRß+ immature V14iNKT cells (1 x 105) isolated from the thymus of a 3-week-old LckCrePtenflox/flox or Ptenflox/flox mouse were microinjected into each thymic lobe of a J18–/– embryo and cultured for additional 9 days. The maturation of the injected NKT cells was evaluated by flow cytometry as described. Tumor immunity The B16F10 melanoma cell line was maintained in the same culture medium as that used for FTOC. J18–/– mice (8 weeks old) were intravenously inoculated with B16F10 cells (4 x 105). After 3 hours, these mice were intravenously injected with sorted GalCer-CD1d+TCRß+ thymocytes (1 x 106) from 8-week-old Ptenflox/flox, LckCrePtenflox/+, or LckCrePtenflox/flox mice. After 30 minutes, 2 µg GalCer was intraperitoneally injected into the mice. On days 4 and 8 after cell transfer, the mice received an additional 2 µg GalCer. The mice were killed on day 14, and the number of surface lung metastases was evaluated with aid of a dissecting microscope. The data were recorded as the mean number of metastases per lung ± SEM. Confocal video microscopy TCRß+NK1.1+HSAlow NKT cells (1 x 104) purified from thymocytes of 8-week-old AktPH-GFPtg/tg mice and DCs (1 x 105) either loaded or not with GalCer were coplated in fibronectin-coated glass-base dishes for 10 minutes. In some experiments, the PI3K inhibitor wortmannin (10 nM; Sigma, St Louis, MO) was added before the cells were mixed. Time-lapse studies to detect PIP3 identified by the AktPH-GFP bioprobe32 were performed at 37°C using a heating device (Harvard Apparatus, Holliston, MA) adapted to a DM IRE2 Leica (Wetzlar, Germany) microscope fitted with a confocal imaging system (Yokogawa, Tokyo, Japan) and equipped with a Leica HCX APO 100x/1.30 numerical aperture objective. Confocal fluorescent or differential interface contrast (Nomarski optics) images were acquired using an Orca-ER cooled charge-coupled-device (CCD) camera (Hamamatsu Photonics, Hamamatsu, Japan) driven by IPLab software version 3.0.4 (BD Bioscience). Statistical methods Statistical differences were determined using the Student t test.    Results Top Abstract Introduction Materials and methods Results Discussion Authorship References   Pten deficiency in NKT cells of LckCrePtenflox/flox mice We previously reported a conditional Pten knockout mouse in which the Pten gene was deleted by Lck, a T cell–specific promoter (LckCrePtenflox/flox mice).28 Genomic PCR examination of DNA from purified TCRß+NK1.1+HSAlow thymocytes from 8-week-old Ptenflox/flox and LckCrePtenflox/flox mice showed that highly efficient Cre-mediated recombination had occurred not only in conventional T cells but also in NKT cells (Figure 1A). The deletion of Pten was confirmed at the protein level by Western blotting (Figure 1B). View larger version (48K): [in this window] [in a new window]   Figure 1. Pten gene deletion in NKT cells in LckCrePtenflox/flox mice. (A) Genomic DNA from TCRß+NK1.1+HSAlow thymocytes of the indicated genotype was analyzed by PCR. In LckCrePtenflox/flox mice, most TCRß+NK1.1+HSAlow thymocytes contained the deleted Pten allele () rather than the WT Pten allele (flox). (B) Western blot analysis of Pten protein expression by the thymocytes in panel A. Actin loading control.   Impaired development of NKT cells in Pten-deficient mice To examine the role of Pten in NKT cell development, we analyzed the NK1.1+TCRß+ NKT population in the thymus, spleen, and liver of 7- to 8-week-old LckCrePtenflox/flox and Ptenflox/flox mice. LckCrePtenflox/flox mice showed reductions not only in the percentages of NK1.1+TCRß+ cells present in all 3 tissues (Figure 2A) but also in absolute numbers of these NKT cells in the thymus (4.5-fold decrease), spleen (2.1-fold), and liver (4.3-fold) (Figure 2B). View larger version (34K): [in this window] [in a new window]   Figure 2. Altered NKT cell populations in LckCrePtenflox/flox mice. (A-B) Decreased numbers of NK1.1+TCRß+ cells in LckCrePtenflox/flox mice. (A) MNCs were isolated from the thymus, spleen, and liver of mice of the indicated genotypes, and NK1.1+TCRß+ cells were analyzed by flow cytometry. Numbers within the panels indicate the relative percentages of NK1.1+TCRß+ cells in total lymphocyte populations. (B) Absolute numbers of the NK1.1+TCRß+ cells in panel A. (C-E) Altered maturation and numbers of V14iNKT cells. (C, left panel, and D) The percentage of V14iNKT cells in total MNCs is normal in the thymus of LckCrePtenflox/flox mice but decreased in spleen and liver. (C, right panel, and E) Stage 2 (CD44+NK1.1–) V14iNKT cells were increased, but stage 3 (CD44+NK1.1+) cells were decreased in the thymus of LckCrePtenflox/flox mice. The percentages of NKT subsets in GalCer-CD1d+TCRß+-gated thymocytes (C, right panel) and in total MNCs in thymus (E) are shown. For panels B, D, and E, results are expressed as the mean ± SEM for 3 mice per group. **P < .01.   We next investigated numbers of V14iNKT cells, a major subset of NKT cells, using the reagent CD1d dimer–Ig (GalCer-CD1d).7 The thymi of WT and LckCrePtenflox/flox mice contained equal numbers of GalCer-CD1d+TCRß+NKT cells (V14iNKT cells) (Figure 2C–D, left panels), indicating that Pten deficiency has no effect on total V14iNKT cell numbers. We then determined CD44/NK1.1 profiles10,11 to follow the development of V14iNKT cells in thymus. While about 80% of thymic V14iNKT cells in Ptenflox/flox mice were mature and had reached stage 3 (CD44highNK1.1+), only 9% of V14iNKT cells in LckCrePtenflox/floxmice were at stage 3 and most had arrested at stage 2 (immature CD44highNK1.1– cells) (Figure 2C, right panel, and E). No differences were observed between the WT and mutant thymus in the percentage of stage 1 NKT precursors (CD44lowNK1.1–) present. When proliferative capacity was examined, there was no difference between the WT and the mutant in the percentage of BrdU-positive cells in the stage 2 population (Figure S1A, available on the Blood website; see the Supplemental Materials link at the top of the online article). Thus, a block in differentiation is the most likely explanation for the observed increase in stage 2 cells in the mutant thymus. In the periphery, V14iNKT cells in LckCrePtenflox/flox mice were decreased in the spleen and liver to 51% and 48% of WT values, respectively (Figure 2C–D). These decreases had to be due to reductions in mature V14iNKT cells because no increases were observed in the percentage of NK1.1– V14iNKT cells in mutant spleen and liver (Figure S1B). V14iNKT cells are positively selected in the thymus via the interaction of their TCRs with CD1d expressed by DP cortical thymocytes and negatively selected by CD1d expressed by antigen-presenting cells such as DCs.8,12 Thus, normal CD1d expression on DP thymocytes and thymic/peripheral DCs is required for proper NKT cell development.8,9 Flow cytometric analysis revealed no differences in CD1d levels expressed by DP thymocytes or thymic or splenic DCs from Ptenflox/flox and LckCrePtenflox/flox mice (Figure S1C). These data indicate that the impaired development of Pten-deficient V14iNKT cells is not due to altered CD1d expression by antigen-presenting cells. The developmental failure of V14iNKT cells in LckCrePtenflox/flox mice is cell autonomous The Pten gene is deleted in both NKT cells and T cells in LckCrePtenflox/flox mice, and DP T cells are an important stromal component for normal NKT cell development in the thymus.8,9,12 To rule out the possibility that it was the Pten mutation in DP T cells that caused the decrease in NKT cells in LckCrePtenflox/flox mice, purified CD44highNK1.1–GalCer-CD1d+TCRß+ immature V14iNKT cells (stage 2) from Ptenflox/flox and LckCrePtenflox/flox mice were transferred into J18–/– fetal thymus (which lacks all V14iNKT cells) and cultured for 9 days using the FTOC method.11 As shown in Figure 3, more than 60% of immature Ptenflox/flox V14iNKT cells developed into mature V14iNKT cells (CD44highNK1.1+GalCer-CD1d+TCRß+; stage 3) in this environment. In contrast, almost all transferred immature LckCrePtenflox/flox V14iNKT cells remained at stage 2. This result clearly indicates that the reduced number of V14iNKT cells in LckCrePtenflox/flox mice was due to a cell-autonomous defect. View larger version (15K): [in this window] [in a new window]   Figure 3. Failure of Pten-deficient immature V14iNKT cells to mature in vitro. CD44highNK1.1– (stage 2, immature) V14iNKT cells were purified from the thymi of Ptenflox/flox or LckCrePtenflox/flox mice and injected into FTOCs derived from a J18–/– embryo. (Left) Flow cytometric analysis of cell populations injected. (Right) At 9 days after injection, V14iNKT cells in the FTOC were isolated and analyzed by flow cytometry to detect stage 3 (mature) cells. Mature V14iNKT cells were very rare in FTOCs receiving Pten-deficient stage 2 NKT cells. One result representative of 3 experiments is shown.   Impaired activation of Pten-deficient NKT cells in vitro and in vivo We next investigated the functional status of Pten-deficient V14iNKT cells by determining their proliferation and cytokine production. When total cells from WT thymus were stained with CD1d dimer–Ig and anti-TCRß antibody, the V14iNKT fraction was found to be slightly activated prior to any exogenous stimulation. We therefore purified NK1.1+TCRß+ cells from Ptenflox/flox and LckCrePtenflox/flox thymi and activated these cells using DCs that had been loaded in vitro with GalCer. In response to this activation, NK1.1+TCRß+ mature NKT cells from Ptenflox/flox thymus showed high levels of proliferation (Figure 4A) as well as vigorous production of IL-4 (Figure 4B) and IFN (Figure 4C). In contrast, NK1.1+TCRß+ mature NKT cells from LckCrePtenflox/flox thymus showed severely impaired proliferation and cytokine production (Figure 4A–C). These results suggest that Pten is required not only for the normal development of NKT cells but also for their functions. View larger version (15K): [in this window] [in a new window]   Figure 4. Impaired activation of Pten-mutant NKT cells in vitro and in vivo. (A) Impaired proliferation. NK1.1+TCRß+ cells were purified from the thymi of Ptenflox/flox or LckCrePtenflox/flox mice and cocultured with DCs loaded with vehicle (DC) or GalCer (DC-GC). After 48 hours, cells were pulsed with [3H]-thymidine for 12 hours and thymidine uptake was measured. (B-C) Impaired cytokine production. IL-4 and IFN in the supernatants of the cultures in panel A were measured by ELISA. (D-E) Impaired response of V14iNKT cells to GalCer administration in vivo. GalCer was intraperitoneally injected into Ptenflox/flox, Pten+/–, or LckCrePtenflox/flox mice, and serum concentrations of IL-4 (D) and IFN (E) were evaluated at 2 hours and 12 hours after GalCer injection, respectively. For all panels, data are expressed as the mean value ± SEM for 3 mice per group. **P < .01.   To examine cytokine production by V14iNKT cells in response to GalCer in vivo, we intraperitoneally injected GalCer into Ptenflox/flox, Pten+/–, and LckCrePtenflox/flox mice. Administration of GalCer to WT mice led to a rapid increase in serum IL-4 and IFN levels within 2 hours and 12 hours, respectively (Figure 4D–E). It is known that V14iNKT cells are solely responsible for the production of these cytokines at these time points.22 Accordingly, in both Pten+/– and LckCrePtenflox/flox mice that received intraperitoneal injection of GalCer, serum levels of IFN were severely decreased. GalCer-treated LckCrePtenflox/flox mice also showed a significant decrease in serum IL-4 whereas Pten+/– mice showed only a slight (insignificant) reduction. These data indicate that, as well as reducing their numbers, mutation of Pten functionally impairs mature V14iNKT cells both in vitro and in vivo. Pten-deficient V14iNKT cells show defective antitumor immunity Because V14iNKT cells were abnormal in number and function in LckCrePtenflox/flox mice, we investigated whether these animals had a defect in V14iNKT-mediated antitumor immunity in vivo. First, we purified GalCer-CD1d+TCRß+ (V14iNKT) cells from Ptenflox/flox mice and adoptively transferred them into GalCer-treated J18–/– (V14iNKT cell–deficient) recipients that had been inoculated with B16F10 melanoma cells (Figure 5A). The presence of WT V14iNKT cells markedly inhibited the metastasis of B16F10 cells to the lungs (about 60% reduction; Figure 5B–C), consistent with previous observations.36 In contrast, only a minor inhibition of B16F10 metastasis was achieved using V14iNKT cells from GalCer-treated Pten+/– mice (16% reduction; Figure 5C) or GalCer-treated LckCrePtenflox/flox mice (8% reduction; Figure 5B). These findings show that mutation of Pten in V14iNKT cells impairs antitumor immunity. View larger version (36K): [in this window] [in a new window]   Figure 5. Defective antitumor surveillance by Pten-deficient V14iNKT cells. (A) Scheme describing the experimental protocol used to evaluate antitumor surveillance by NKT cells in vivo. V14iNKT cells were purified from mice of the indicated genotypes and adoptively transferred into J18–/– mice that had been inoculated with B16F10 melanoma cells. The mice were then injected with GalCer at 0, 4, and 8 days after NKT cell transfer. After 14 days, numbers of focal lung metastases were counted. (B-C, left panels) Increased lung metastasis. Quadrant scheme in the upper corners shows the labeling of the cell images. "None" indicates controls in which no cells were transferred. Representative images of lung metastases 2 weeks after the transfer of Ptenflox/flox or LckCrePtenflox/flox V14iNKT cells (B) or after the transfer of Pten+/+ or Pten+/– V14iNKT cells (C) are shown. (B-C, right panels) Relative numbers of the lung metastases identified in the left panels. Results shown are mean ± SEM of 18 mice that received no cells and 6 mice each that received V14iNKT cells. **P < .01.   PI3K signaling is responsible for the phenotypes of Pten-deficient V14iNKT cells To determine whether PI3K was activated in V14iNKT cells that interacted with GalCer-loaded DCs, we analyzed levels of the PI3K product PIP3. Purified V14iNKT cells expressing the fusion bioprobe AktPH-GFP32 were cultured with GalCer-loaded DCs in fibronectin-coated glass-base dishes. As shown in Figure 6A, the AktPH-GFP bioprobe was recruited to the plasma membrane of WT V14iNKT cells that interacted with GalCer-loaded DCs (solid arrow) but remained in the cytoplasm of WT V14iNKT cells that did not make contact with the DCs (dotted arrows). In addition, the bioprobe was not recruited to the plasma membrane of WT V14iNKT cells that interacted with GalCer-loaded DCs in the presence of wortmannin (a pan-PI3K inhibitor) or in V14iNKT cells that contacted DCs lacking GalCer (Figure 6B). These findings suggest that PI3K is activated in V14iNKT cells when these cells are stimulated by interaction with GalCer-loaded DCs. View larger version (103K): [in this window] [in a new window]   Figure 6. PI3K is activated in V14iNKT cells that interact with GalCer-loaded DCs. Localization of PIP3 to the NKT cell membrane. TCRß+NK1.1+HSAlow NKT cells were purified from thymocytes of 8-week-old AktPH-GFPtg/tg mice (WT for Pten) expressing the PIP3-binding AktPH-GFP bioprobe I (left). These V14iNKT cells were incubated with GalCer-loaded DCs, and PIP3 localization was determined by confocal fluorescence (left) and phase contrast (right) microscopy. (A) V14iNKT cells that made contact with GalCer-loaded DCs showed membrane localization of PIP3 (solid arrow), while PIP3 remained in the cytoplasm of V14iNKT cells that did not make contact with DCs (dotted arrows). (B) AktPH-GFP bioprobe localization to the plasma membrane of V14iNKT cells was inhibited by wortmannin or by contact with DCs lacking GalCer.   The PI3K signaling pathway leads to PKB/Akt activation, an event that is negatively regulated by Pten. Accordingly, Pten deficiency usually leads to enhanced PI3K signaling and cellular hyperactivation and hyperproliferation.26,28 Curiously, Pten deficiency in V14iNKT cells led to impaired cell numbers and functions. We therefore examined the role of PI3K in generating the phenotypes of Pten-deficient NKT cells. The PI3K subunit p110 is highly expressed in hematopoietic cells and is the major catalytic subunit of class IA PI3Ks in T-lineage cells.30 The PI3K subunit p110, which is a unique catalytic subunit responsible for class IB PI3K activation downstream of G protein–coupled receptors (GPCRs), is also highly expressed in lymphocytes and important for the normal development of T-lineage cells.29 To determine whether PI3K signaling was responsible for the defects of Pten-deficient V14iNKT cells, we examined double-mutant mice lacking Pten and PI3K p11029 (LckCrePtenflox/flox/p110–/–) or Pten and PI3K p11030 (LckCrePtenflox/flox/p110D910A/D910A). The mutation of p110 partially rescued V14iNKT cell numbers in LckCrePtenflox/flox mice (Figure 7A, upper panel) and completely rescued the proliferation defect of Pten-deficient V14iNKT cells (Figure 7B). A slightly weaker but still significant effect was seen upon mutation of p110. Moreover, numbers of peripheral Pten-deficient V14iNKT cells were restored to near-normal levels in both strains of double-mutant mice (data not shown). These results indicate that excessive PI3K signaling contributes to the phenotypes of Pten-deficient V14iNKT cells. View larger version (34K): [in this window] [in a new window]   Figure 7. The phenotypes observed in Pten-deficient NKT cells are PI3K dependent. (A, top) Decreased maturation. Thymic V14iNKT cells from mice of the indicated genotypes were analyzed by flow cytometry for percentages of stage 3 cells. (A, bottom) Increased expression of Ly49C/I receptors. The GalCer-CD1d+TCRß+NK1.1+ cells in the top panel were analyzed by flow cytometry to determine relative expression levels of Ly49C/I. (B) Impaired proliferation. The NK1.1+TCRß+ cells from panel A were stimulated by incubation with GalCer-loaded DCs, and [3H]-thymidine incorporation was measured. Data shown are the mean ± SEM for 3 mice per group. **P < .01. For both panels, the defects of Pten-deficient V14iNKT cells were partially rescued by mutation of a PI3K subunit (p110 or p110).   Enhanced expression of inhibitory Ly49 receptors by Pten-deficient V14iNKT cells Interaction between MHC class I and inhibitory Ly49 receptors (such as Ly49A and LY49C/I) can negatively regulate the responses of T cells to alloantigens,37 suggesting that signaling via the TCR can be regulated by a Ly49-dependent mechanism. With respect to NKT cells, NKT functions are impaired upon inhibitory Ly49–MHC class I interaction,38 and mice transgenic for Ly49A show impaired NKT cell maturation and selection.39 Thus, Ly49-mediated regulation is important for the development and functions of NKT cells, and this regulation may involve modulation of the strength of TCR signaling. These considerations led us to examine the expression of inhibitory Ly49 receptors by our Pten-deficient V14iNKT cells. Specifically, we determined the expression of Ly49C/I (which recognizes MHC class I, H-2Kb, H-2Dd) and Ly49A (which recognizes MHC class I, H-2Dd) by thymic V14iNKT cells of LckCrePtenflox/flox mice and the double mutants. As expected, the proportion of GalCer-CD1d+TCRß+NK1.1+ thymocytes showing strong expression of Ly49C/I (Figure 7A, bottom) and Ly49A (Figure S2) was increased in LckCrePtenflox/flox mice compared with Ptenflox/flox mice. At least for Ly49C/I, this increased expression was mitigated by the presence of the p110D910A/D910 or p110–/– mutation. There were no differences between LckCrePtenflox/flox mice and Ptenflox/flox mice in the H2-Kb levels expressed by thymic or splenic DCs (Figure S1C). Thus, PI3K signaling is responsible for the altered inhibitory Ly49 receptor repertoire of Pten-deficient thymic V14iNKT cells. Taken together, our results suggest that the reduced number and functions of Pten-deficient V14iNKT cells may be linked to their increased expression of inhibitory Ly49 receptors.    Discussion Top Abstract Introduction Materials and methods Results Discussion Authorship References   Many studies in mouse models have demonstrated that V14iNKT cells are activated in a broad range of disorders, including autoimmune diseases, microbial infections, graft rejection, and cancer progression/metastasis.5,19–21 However, little is known about intracellular signal transduction in NKT cells, particularly the role of the Pten/PI3K signaling pathway that is crucial in so many other cell types. Our results are the first to demonstrate that mutation of Pten in murine V14iNKT cells impairs the development and functions of these cells and that these defects depend on PI3K activity. An in vivo consequence of the defective V14iNKT compartment is enhanced tumor metastasis. The study of genetically modified mice has begun to yield important insights into the mechanisms controlling the maturation of V14iNKT cells. To date, investigations of diverse gene-deficient mice have revealed 3 general categories of V14iNKT cell abnormalities.40 In the first category (group 1), the mutant mice show a total absence or a severe reduction in numbers of both immature (stage 2) and mature (stage 3) V14iNKT cells. In the second category (group 2), the mutant animals exhibit a block in V14iNKT cell development between stage 2 and stage 3 such that stage 2 cells are increased while stage 3 cells are decreased. In the third category (group 3), numbers of stage 3 V14iNKT cells are decreased but there is no increase in stage 2 cell numbers. Knockout mice lacking CD1d, Fyn, SAP, IL-7, IKK2, NIK, or RelB all have the group 1 phenotype, indicating that these molecules are essential for the initial development of V14iNKT cells from DP precursors.40,41 Mice lacking T-bet42 or Itk43 are members of group 2. These molecules do not affect the earliest stages of NKT differentiation from DP precursors but are critical for the continued maturation of stage 2 V14iNKT cells. Mice deficient for IL-15 or transgenic for mIkB fall into group 3 because these molecules are important for the survival of mature NKT cells.40,44 While the phenotypes observed in the V14iNKT cells of T-bet– or Itk-deficient mice are strikingly similar to those we observed for V14iNKT cells of LckCrePtenflox/flox mice, there are some fundamental differences. First, although the phenotypes observed in T-bet–deficient mice can be explained by a failure to express the IL-2R/IL-15R common ß subunit (CD122),42 the proliferative response to IL-15 by mature NKT cells of LckCrePtenflox/flox mice was normal (Figure S3). Second, Itk-deficient V14iNKT cells show reduced expression of the inhibitory NK receptors Ly49C/I and intact IFN secretion,43 but Pten-deficient V14iNKT cells exhibit enhanced Ly49C/I and Ly49A expression and reduced IFN secretion. In addition, because Itk is activated by Pten deficiency,45 reduction of Itk signaling is unlikely to have occurred in our LckCrePtenflox/flox mice. For these reasons, we believe that the phenotypes observed in V14iNKT cells of LckCrePtenflox/flox mice cannot be explained by effects attributable to altered T-bet or Itk function. Finally, in all the previously reported mutant mice, the molecules deleted by the gene targeting are known to activate cells. No other mutant mouse to date features the targeting of an intracellular signaling molecule that negatively regulates cellular activation in V14iNKT cells. Ly49 is a multigene receptor family expressed mainly by NK cells and stage 3 mature V14iNKT cells.44 Ly49A, Ly49C/I, and Ly49G2 are inhibitory NK receptors, while Ly49D and Ly49H are activatory NK receptors. Both groups of receptors interact with proteins encoded by specific MHC class I alleles.46 It has been clearly shown that the interaction between Ly49A and H-2Dd and between Ly49C and H-2Kb or H-2Dd negatively regulates NK effector functions such as cytotoxicity and bone marrow graft rejection.47,48 Similarly, the appropriate inhibitory Ly49–MHC class I interactions can negatively regulate the maturation, proliferation, and functions of NKT cells.37,38,39 In our study, we found that most GalCer-CD1d+TCRß+NK1.1+ thymocytes in LckCrePtenflox/flox mice showed an enhancement of Ly49C/I expression that was PIP3 dependent. The continuous accumulation of PIP3 that resulted from the Pten deficiency in our mutants may have up-regulated the expression of inhibitory Ly49 receptors. Thus, the phenotypes observed in Pten-deficient V14iNKT cells can plausibly be explained by their enhanced expression of inhibitory Ly49 receptors. Several lines of evidence suggest that developing V14iNKT cells undergo negative selection if they encounter high-avidity antigen or abundant self-antigen. First, the addition of GalCer to early-stage FTOC prevents the development of V14iNKT cells in a dose-dependent manner.14,16 Second, overexpression of CD1d on DCs or thymocytes in transgenic mice results in a reduced number of V14iNKT cells that exhibit both altered Vß repertoire usage and reduced sensitivity to antigen.12,14 Third, in mice doubly transgenic for the activatory Ly49D receptor and its ligand H2-Dd, V14iNKT cells are absent from the thymus and periphery due to developmental arrest between stage 2 and stage 3. The addition of the inhibitory Ly49A transgene to Ly49D/H2-Dd transgenic mice resulted in a partial rescue of NKT cell development.15 A possible interpretation of these findings is that V14iNKT cells with self-recognizing Ly49 inhibitory receptors preferentially survive as a result of decreased negative selection mediated by activatory Ly49. It may be that, as well as TCR avidity, an appropriate balance of signals delivered by activatory and inhibitory Ly49 receptors is required to set the affinity threshold and regulate proper V14iNKT cell development. Pten-deficient V14iNKT cells exposed to endogenous glycolipid antigens may transmit supraphysiological levels of signaling which, in turn, may drive enhanced negative selection of V14iNKT cells. The deficit of V14iNKT cells in the peripheral organs and thymus may then be partially restored by repopulation of the mature V14iNKT compartment with cells showing increased expression of inhibitory Ly49 receptors, altered Vß usage, and reduced sensitivity to antigen.14 Pre-TCR signaling in T cells of PI3K p110–/–/p110–/– double-mutant mice is greatly decreased compared with signaling in T cells of PI3K–/– or –/– single-mutant animals.49 In addition, T-cell responses induced by TCR stimulation are much weaker in p110–/–50 and p110–/–29 mice than in WT controls. In our study, we showed that the phenotypes observed in Pten-deficient NKT cells could be partially rescued by mutation of p110. These results suggest that TCR signaling may directly activate both PI3K and PI3K in V14iNKT cells. Alternatively, inhibitory Ly49 receptors may directly affect both PI3K and PI3K, which may eventually result in modulated TCR signaling. In support of the latter possibility, there is evidence that SHIP1 (another PIP3 phosphatase) as well as the p85 subunit of PI3K can both be recruited to Ly49A or Ly49C.51 The molecular mechanisms underlying the impaired function of V14iNKT cells in patients with autoimmune disease or cancer remain to be determined. Our work suggests that the continuous activation of the PI3K pathway in murine LckCrePtenflox/flox V14iNKT cells (due to decreased Pten function) may alter the negative selection, development, function, and tolerance of these cells. In humans, a parallel scenario could lead to the onset of these diseases. It is well known that GalCer has antitumor effects against cancer cells of various origins and that this protection depends on an initial burst of IFN production by NKT cells and subsequent ongoing IFN production by NK cells.36 IFN itself is not directly toxic to tumor cells, but the IFN produced by NKT cells is critical for the recruitment and proliferation of NK cells (and other leukocytes) that are cytolytic to tumor cells. In addition, IFN has potent antiangiogenic activity52 and induces other leukocytes to produce IFN-inducible factors such as IFN-inducible protein 10 (IP-10) and monokine induced by IFN.53 These cytokines may indirectly inhibit tumor neovascularization and thus induce tumor hypoxia. We have demonstrated that Pten-deficient V14iNKT cells fail to produce IFN both in vitro and in vivo, most likely accounting for the impaired antitumor surveillance observed in LckCrePtenflox/flox mice. Our study is the first to show that engagement of the TCRs of murine V14iNKT cells by GalCer-loaded DCs leads to the activation of PI3K in the NKT cells. We have also demonstrated that the loss of Pten or activation of PI3K signaling in V14iNKT cells leads to a failure in their development. The few mature V14iNKT cells that survive are tolerant to GalCer, resulting in defective antitumor surveillance and accelerated metastasis. Mice heterozygous for the Pten mutation (Pten+/–) showed a significant reduction in serum IFN production after administration of GalCer in vivo. In addition, adoptive transfer of Pten+/– V14iNKT cells into GalCer-treated, B16F10 melanoma cell–inoculated J18–/– recipients impaired the inhibition of B16F10 melanoma metastasis to the lungs. We speculate that an identical mechanism may be operating in humans with Cowden disease, a hereditary syndrome of cancer susceptibility caused by heterozygous mutations of PTEN: Namely, an individual who inherits a mutated PTEN allele is not only at risk for additional tumorigenic mutations due to loss of heterozygosity (LOH) of the PTEN gene but may also experience accelerated growth of any incipient tumors due to impaired NKT-mediated antitumor surveillance.    Authorship Top Abstract Introduction Materials and methods Results Discussion Authorship References   Conflict-of-interest disclosure: The authors declare no competing financial interests. H.K. and T.O. contributed equally to this work. Correspondence: Akira Suzuki, Hondo 1-1-1, Akita, 0108543 Japan; e-mail: suzuki{at}med.akita-u.ac.jp.    Acknowledgments   This work was supported by grants from the Ministry of Education, Science, Sports and Culture, Japan; the Kowa Life Science Foundation; the Daiwa Securities Health Foundation; the Suzuken Memorial Foundation; and the Mochida Memorial Foundation for Medical and Pharmaceutical Research. We thank Dr Tetsuo Noda (Tohoku University), Dr Kiminori Hasagawa, Dr Masato Sasaki, Dr Junko Sasaki, Dr Shunsuke Takasuga, Ms Miki Nishio, Mr Seiichi Kuwajima, and Mr Tomoharu Kanie (all of Akita University) for helpful discussions and technical support.    Footnotes   Submitted July 27, 2006; accepted December 7, 2006. Prepublished online as Blood First Edition Paper, December 14, 2006 DOI: 10.1182/blood-2006-07-038059 The online version of this article contains a data supplement. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 USC section 1734.    References Top Abstract Introduction Materials and methods Results Discussion Authorship References   Imai K, Kanno M, Kimoto H, Shigemoto K, Yamamoto S, Taniguchi M. Sequence and expression of transcripts of the T-cell antigen receptor alpha-chain gene in a functional, antigen-specific suppressor-T-cell hybridoma. Proc Natl Acad Sci U S A 1986; 83:8708–8712.[Abstract/Free Full Text] Budd RC, Miescher GC, Howe RC, Lees RK, Bron C, MacDonald HR. Developmentally regulated expression of T cell receptor beta chain variable domains in immature thymocytes. J Exp Med 1987; 166:577–582.[Abstract/Free Full Text] Fowlkes BJ, Kruisbeek AM, Ton-That H, et al. A novel population of T-cell receptor alpha beta-bearing thymocytes which predominantly expresses a single V beta gene family. Nature 1987; 329:251–254.[CrossRef][Medline] [Order article via Infotrieve] Taniguchi M, Koseki H, Tokuhisa T, et al. Essential requirement of an invariant V alpha 14 T cell antigen receptor expression in the development of natural killer T cells. Proc Natl Acad Sci U S A 1996; 93:11025–11028.[Abstract/Free Full Text] Kronenberg M and Gapin L. The unconventional lifestyle of NKT cells. Nat Rev Immunol 2002; 2:557–568.[Medline] [Order article via Infotrieve] Matsuda JL, Naidenko OV, Gapin L, et al. Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J Exp Med 2000; 192:741–754.[Abstract/Free Full Text] Schumann J, Voyle RB, Wei BY, MacDonald HR. Cutting edge: influence of the TCR V beta domain on the avidity of CD1d:alpha-galactosylceramide binding by invariant V alpha 14 NKT cells. J Immunol 2003; 170:5815–5819.[Abstract/Free Full Text] Bendelac A. Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes. J Exp Med 1995; 182:2091–2096.[Abstract/Free Full Text] Coles MC and Raulet DH. NK1.1+ T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4+CD8+ cells. J Immunol 2000; 164:2412–2418.[Abstract/Free Full Text] Benlagha K, Kyin T, Beavis A, Teyton L, Bendelac A. A thymic precursor to the NK T cell lineage. Science 2002; 296:553–555.[Abstract/Free Full Text] Pellicci DG, Hammond KJ, Uldrich AP, Baxter AG, Smyth MJ, Godfrey DI. A natural killer T (NKT) cell developmental pathway involving a thymus-dependent NK1.1(-)CD4(+) CD1d-dependent precursor stage. J Exp Med 2002; 195:835–844.[Abstract/Free Full Text] Schumann J, Pittoni P, Tonti E, Macdonald HR, Dellabona P, Casorati G. Targeted expression of human CD1d in transgenic mice reveals independent roles for thymocytes and thymic APCs in positive and negative selection of Valpha14i NKT cells. J Immunol 2005; 175:7303–7310.[Abstract/Free Full Text] Xu H, Chun T, Colmone A, Nguyen H, Wang CR. Expression of CD1d under the control of a MHC class Ia promoter skews the development of NKT cells, but not CD8+ T cells. J Immunol 2003; 171:4105–4112.[Abstract/Free Full Text] Chun T, Page MJ, Gapin L, et al. CD1d-expressing dendritic cells but not thymic epithelial cells can mediate negative selection of NKT cells. J Exp Med 2003; 197:907–918.[Abstract/Free Full Text] Voyle RB, Beermann F, Lees RK, et al. Ligand-dependent inhibition of CD1d-restricted NKT cell development in mice transgenic for the activating receptor Ly49D. J Exp Med 2003; 197:919–925.[Abstract/Free Full Text] Pellicci DG, Uldrich AP, Kyparissoudis K, et al. Intrathymic NKT cell development is blocked by the presence of alpha-galactosylceramide. Eur J Immunol 2003; 33:1816–1823.[CrossRef][Medline] [Order article via Infotrieve] Kawano T, Cui J, Koezuka Y, et al. CD1d-restricted and TCR-mediated activation of valpha14 NKT cells by glycosylceramides. Science 1997; 278:1626–1629.[Abstract/Free Full Text] Burdin N, Brossay L, Koezuka Y, et al. Selective ability of mouse CD1 to present glycolipids: alpha-galactosylceramide specifically stimulates V alpha 14+ NK T lymphocytes. J Immunol 1998; 161:3271–3281.[Abstract/Free Full Text] Taniguchi M, Harada M, Kojo S, Nakayama T, Wakao H. The regulatory role of Valpha14 NKT cells in innate and acquired immune response. Annu Rev Immunol 2003; 21:483–513.[CrossRef][Medline] [Order article via Infotrieve] Godfrey DI and Kronenberg M. Going both ways: immune regulation via CD1d-dependent NKT cells. J Clin Invest 2004; 114:1379–1388.[CrossRef][Medline] [Order article via Infotrieve] Cui J, Shin T, Kawano T, et al. Requirement for Valpha14 NKT cells in IL-12-mediated rejection of tumors. Science 1997; 278:1623–1626.[Abstract/Free Full Text] Elewaut D and Kronenberg M. Molecular biology of NK T cell specificity and development. Semin Immunol 2000; 12:561–568.[CrossRef][Medline] [Order article via Infotrieve] Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 1997; 275:1943–1947.[Abstract/Free Full Text] Liaw D, Marsh DJ, Li J, et al. Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome. Nat Genet 1997; 16:64–67.[CrossRef][Medline] [Order article via Infotrieve] Maehama T and Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 1998; 273:13375–13378.[Abstract/Free Full Text] Vanhaesebroeck B, Ali K, Bilancio A, Geering B, Foukas LC. Signalling by PI3K isoforms: insights from gene-targeted mice. Trends Biochem Sci 2005; 30:194–204.[CrossRef][Medline] [Order article via Infotrieve] Stambolic V, Suzuki A, de la Pompa JL, et al. Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN. Cell 1998; 95:29–39.[CrossRef][Medline] [Order article via Infotrieve] Suzuki A, Yamaguchi MT, Ohteki T, et al. T cell-specific loss of Pten leads to defects in central and peripheral tolerance. Immunity 2001; 14:523–534.[CrossRef][Medline] [Order article via Infotrieve] Sasaki T, Irie-Sasaki J, Jones RG, et al. Function of PI3Kgamma in thymocyte development, T cell activation, and neutrophil migration. Science 2000; 287:1040–1046.[Abstract/Free Full Text] Okkenhaug K, Bilancio A, Farjot G, et al. Impaired B and T cell antigen receptor signaling in p110delta PI 3-kinase mutant mice. Science 2002; 297:1031–1034.[Abstract/Free Full Text] Suzuki A, de la Pompa JL, Stambolic V, et al. High cancer susceptibility and embryonic lethality associated with mutation of the PTEN tumor suppressor gene in mice. Curr Biol 1998; 8:1169–1178.[CrossRef][Medline] [Order article via Infotrieve] Nishio M, Watanabe K, Sasaki J, et al. Control of cell polarity and motility by the PtdIns(3,4,5)P(3) phosphatase SHIP1. Nat Cell Biol 2007; 9:36–44.[CrossRef][Medline] [Order article via Infotrieve] Ohteki T and MacDonald HR. Major histocompatibility complex class I related molecules control the development of CD4+8- and CD4–8- subsets of natural killer 1.1+ T cell receptor-alpha/beta+ cells in the liver of mice. J Exp Med 1994; 180:699–704.[Abstract/Free Full Text] Hamada K, Sasaki T, Koni PA, et al. The PTEN/PI3K pathway governs normal vascular development and tumor angiogenesis. Genes Dev 2005; 19:2054–2065.[Abstract/Free Full Text] Ohteki T, Suzue K, Maki C, Ota T, Koyasu S. Critical role of IL-15-IL-15R for antigen-presenting cell functions in the innate immune response. Nat Immunol 2001; 2:1138–1143.[CrossRef][Medline] [Order article via Infotrieve] Smyth MJ, Crowe NY, Pellicci DG, et al. Sequential production of interferon-gamma by NK1.1(+) T cells and natural killer cells is essential for the antimetastatic effect of alpha-galactosylceramide. Blood 2002; 99:1259–1266.[Abstract/Free Full Text] Held W, Cado D, Raulet DH. Transgenic expression of the Ly49A natural killer cell receptor confers class I major histocompatibility complex (MHC)-specific inhibition and prevents bone marrow allograft rejection. J Exp Med 1996; 184:2037–2041.[Abstract/Free Full Text] Ortaldo JR, Winkler-Pickett R, Mason AT, Mason LH. The Ly-49 family: regulation of cytotoxicity and cytokine production in murine CD3+ cells. J Immunol 1998; 160:1158–1165.[Abstract/Free Full Text] Robson MacDonald H, Lees RK, Held W. Developmentally regulated extinction of Ly-49 receptor expression permits maturation and selection of NK1.1+ T cells. J Exp Med 1998; 187:2109–2114.[Abstract/Free Full Text] Matsuda JL and Gapin L. Developmental program of mouse Valpha14i NKT cells. Curr Opin Immunol 2005; 17:122–130.[CrossRef][Medline] [Order article via Infotrieve] Pasquier B, Yin L, Fondaneche MC, et al. Defective NKT cell development in mice and humans lacking the adapter SAP, the X-linked lymphoproliferative syndrome gene product. J Exp Med 2005; 201:695–701.[Abstract/Free Full Text] Townsend MJ, Weinmann AS, Matsuda JL, et al. T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells. Immunity 2004; 20:477–494.[CrossRef][Medline] [Order article via Infotrieve] Gadue P and Stein PL. NK T cell precursors exhibit differential cytokine regulation and require Itk for efficient maturation. J Immunol 2002; 169:2397–2406.[Abstract/Free Full Text] Stanic AK, Bezbradica JS, Park JJ, et al. NF-kappa B controls cell fate specification, survival, and molecular differentiation of immunoregulatory natural T lymphocytes. J Immunol 2004; 172:2265–2273.[Abstract/Free Full Text] Shan X, Czar MJ, Bunnell SC, et al. Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation. Mol Cell Biol 2000; 20:6945–6957.[Abstract/Free Full Text] Yokoyama WM and Seaman WE. The Ly-49 and NKR-P1 gene families encoding lectin-like receptors on natural killer cells: the NK gene complex. Annu Rev Immunol 1993; 11:613–635.[Medline] [Order article via Infotrieve] Karlhofer FM, Ribaudo RK, Yokoyama WM. MHC class I alloantigen specificity of Ly-49+ IL-2-activated natural killer cells. Nature 1992; 358:66–70.[CrossRef][Medline] [Order article via Infotrieve] Yu YY, George T, Dorfman JR, Roland J, Kumar V, Bennett M. The role of Ly49A and 5E6(Ly49C) molecules in hybrid resistance mediated by murine natural killer cells against normal T cell blasts. Immunity 1996; 4:67–76.[CrossRef][Medline] [Order article via Infotrieve] Webb LM, Vigorito E, Wymann MP, Hirsch E, Turner M. Cutting edge: T cell development requires the combined activities of the p110gamma and p110delta catalytic isoforms of phosphatidylinositol 3-kinase. J Immunol 2005; 175:2783–2787.[Abstract/Free Full Text] Okkenhaug K, Patton DT, Bilancio A, Garcon F, Rowan WC, Vanhaesebroeck B. The p110delta isoform of phosphoinositide 3-kinase controls clonal expansion and differentiation of Th cells. J Immunol 2006; 177:5122–5128.[Abstract/Free Full Text] Wang JW, Howson JM, Ghansah T, et al. Influence of SHIP on the NK repertoire and allogeneic bone marrow transplantation. Science 2002; 295:2094–2097.[Abstract/Free Full Text] Ruegg C, Yilmaz A, Bieler G, Bamat J, Chaubert P, Lejeune FJ. Evidence for the involvement of endothelial cell integrin alphaVbeta3 in the disruption of the tumor vasculature induced by TNF and IFN-gamma. Nat Med 1998; 4:408–414.[CrossRef][Medline] [Order article via Infotrieve] Farber JM. Mig and IP-10: CXC chemokines that target lymphocytes. J Leukoc Biol 1997; 61:246–257.[Abstract] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: H. Guo, A. Samarakoon, B. Vanhaesebroeck, and S. Malarkannan The p110{delta} of PI3K plays a critical role in NK cell terminal maturation and cytokine/chemokine generation J. Exp. Med., September 29, 2008; 205(10): 2419 - 2435. [Abstract] [Full Text] [PDF] T. A. Johnson, S. Tsutsui, and F. R. Jirik Antigen-Induced Pten Gene Deletion in T Cells Exacerbates Neuropathology in Experimental Autoimmune Encephalomyelitis Am. J. Pathol., April 1, 2008; 172(4): 980 - 992. [Abstract] [Full Text] [PDF] This Article Abstract Full Text (PDF) Supplemental Figures All Versions of this Article: blood-2006-07-038059v1 109/8/3316    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kishimoto, H. Articles by Suzuki, A. Search for Related Content PubMed PubMed Citation Articles by Kishimoto, H. Articles by Suzuki, A. Related Collections Signal Transduction Social Bookmarking                   What's this?

	Copyright © 2007 by American Society of Hematology         Online ISSN: 1528-0020