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1.  Agonist-selected T cell development requires strong T-cell receptor signaling and store-operated calcium entry 
Immunity  2013;38(5):881-895.
Summary
T-cell receptor (TCR) signaling driven by interaction of the TCR with specific complexes of self-peptide and the major histocompatibility complex, determines T cell fate in thymic development. However, the signaling pathway through which TCR signal strength regulates distinct T cell lineages remains unknown. Here we have used mice lacking the endoplasmic reticulum Ca2+ sensors STIM1 and STIM2 to show that STIM-induced store-operated Ca2+ entry is not essential for thymic development of conventional TCRαβ+ T cells, but is specifically required for the development of agonist-selected T cells (regulatory T cells, invariant natural killer T cells and TCRαβ+ CD8αα+ intestinal intraepithelial lymphocytes). The severe impairment of agonist-selected T cell development is mainly due to a defect in interleukin-2 (IL-2) or IL-15 signaling. Thus, STIM1 and STIM2-mediated store-operated Ca2+ influx, leading to efficient activation of NFAT (nuclear factor of activated T-cells), is critical for the post-selection maturation of agonist-selected T cells.
doi:10.1016/j.immuni.2013.02.008
PMCID: PMC3669219  PMID: 23499491
2.  Generation of induced pluripotent stem cell-derived mice by reprogramming of a mature NKT cell 
International Immunology  2014;26(10):551-561.
The successful generation of iPSC-derived mouse strains to study NKT cells
NKT cells are characterized by their expression of an NKT-cell-specific invariant antigen-receptor α chain encoded by Vα14Jα18 gene segments. These NKT cells bridge the innate and acquired immune systems to mediate effective and augmented responses; however, the limited number of NKT cells in vivo hampers their analysis. Here, two lines of induced pluripotent stem cell-derived mice (NKT-iPSC-derived mice) were generated by reprogramming of mature NKT cells, where one harbors both rearranged Vα14Jα18 and Vβ7 genes and the other carries rearranged Vα14Jα18 on both alleles but germline Vβ loci. The analysis of NKT-iPSC-derived mice showed a significant increase in NKT cell numbers with relatively normal frequencies of functional subsets, but significantly enhanced in some cases, and acquired functional NKT cell maturation in peripheral lymphoid organs. NKT-iPSC-derived mice also showed normal development of other immune cells except for the absence of γδT cells and disturbed development of conventional CD4 αβT cells. These results suggest that the NKT-iPSC-derived mice are a better model for NKT cell development and function study rather than transgenic mouse models reported previously and also that the presence of a pre-rearranged Vα14Jα18 in the natural chromosomal context favors the developmental fate of NKT cells.
doi:10.1093/intimm/dxu057
PMCID: PMC4169672  PMID: 24854340
iPSC; NKT cell; TCR rearrangement
3.  NKT Cells as an Ideal Anti-Tumor Immunotherapeutic 
Human natural killer T (NKT) cells are characterized by their expression of an invariant T cell antigen receptor α chain variable region encoded by a Vα24Jα18 rearrangement. These NKT cells recognize α-galactosylceramide (α-GalCer) in conjunction with the MHC class I-like CD1d molecule and bridge the innate and acquired immune systems to mediate efficient and augmented immune responses. A prime example of one such function is adjuvant activity: NKT cells augment anti-tumor responses because they can rapidly produce large amounts of IFN-γ, which acts on NK cells to eliminate MHC negative tumors and also on CD8 cytotoxic T cells to kill MHC positive tumors. Thus, upon administration of α-GalCer-pulsed DCs, both MHC negative and positive tumor cells can be effectively eliminated, resulting in complete tumor eradication without tumor recurrence. Clinical trials have been completed in a cohort of 17 patients with advanced non-small cell lung cancers and 10 cases of head and neck tumors. Sixty percent of advanced lung cancer patients with high IFN-γ production had significantly prolonged median survival times of 29.3 months with only the primary treatment. In the case of head and neck tumors, 10 patients who completed the trial all had stable disease or partial responses 5 weeks after the combination therapy of α-GalCer-DCs and activated NKT cells. We now focus on two potential powerful treatment options for the future. One is to establish artificial adjuvant vector cells containing tumor mRNA and α-GalCer/CD1d. This stimulates host NKT cells followed by DC maturation and NK cell activation but also induces tumor-specific long-term memory CD8 killer T cell responses, suppressing tumor metastasis even 1 year after the initial single injection. The other approach is to establish induced pluripotent stem (iPS) cells that can generate unlimited numbers of NKT cells with adjuvant activity. Such iPS-derived NKT cells produce IFN-γ in vitro and in vivo upon stimulation with α-GalCer/DCs, and mediated adjuvant effects, suppressing tumor growth in vivo.
doi:10.3389/fimmu.2013.00409
PMCID: PMC3845015  PMID: 24348476
NKT cells; adjuvant effects; clinical trial; induced pluripotent stem cells; artificial adjuvant vector cells
4.  The transcription factor E4BP4 regulates the production of IL-10 and IL-13 in CD4+ T cells 
Nature immunology  2011;12(5):450-459.
The immunoregulatory cytokine interleukin 10 (IL-10) is expressed mainly by T helper type 2 (TH2) cells but also by TH1 cells during chronic infection. Here we observed plasticity in the expression of IL-10 and IL-13 after chronic TH1 stimulation; furthermore, the expression of Il10 and Il13 was regulated by the transcription factor E4BP4. Chronically stimulated E4BP4-deficient (Nfil3−/−; called ‘E4bp4−/−’ here) TH1 cells, regulatory T cells (Treg cells) and natural killer T cells (NKT cells) had attenuated expression of IL-10 and IL-13. Enforced expression of E4bp4 initiated the production of IL-10 and IL-13 by conventional TH1 cells. E4bp4−/− TH2 cells showed impairment of IL-10 production with no effect on IL-13. Our results indicate that E4BP4 has multiple functions in controlling the plasticity of IL-13 in TH1 cells and IL-10 in TH1 cells, TH2 cells, Treg cells and NKT cells.
doi:10.1038/ni.2020
PMCID: PMC3494493  PMID: 21460847
5.  Therapeutic Effects and Biomarkers in Sublingual Immunotherapy: A Review 
Journal of Allergy  2012;2012:381737.
Immunotherapy is considered to be the only curative treatment for allergic diseases such as pollinosis, perennial rhinitis, asthma, and food allergy. The sublingual route is widely applied for immunotherapy for allergy, instead of the conventional administration by subcutaneous route. A recent meta-analysis of sublingual immunotherapy (SLIT) has shown that this approach is safe, has positive clinical effects, and provides prolonged therapeutic effects after discontinuation of treatment. However, the mechanism of SLIT and associated biomarkers are not fully understood. Biomarkers that change after or during SLIT have been reported and may be useful for response monitoring or as prognostic indicators for SLIT. In this review, we focus on the safety, therapeutic effects, including prolonged effects after treatment, and new methods of SLIT. We also discuss response monitoring and prognostic biomarkers for SLIT. Finally, we discuss immunological mechanisms of SLIT with a focus on oral dendritic cells and facilitated antigen presentation.
doi:10.1155/2012/381737
PMCID: PMC3303629  PMID: 22500184
6.  Type II NKT Cells Stimulate Diet-Induced Obesity by Mediating Adipose Tissue Inflammation, Steatohepatitis and Insulin Resistance 
PLoS ONE  2012;7(2):e30568.
The progression of obesity is accompanied by a chronic inflammatory process that involves both innate and acquired immunity. Natural killer T (NKT) cells recognize lipid antigens and are also distributed in adipose tissue. To examine the involvement of NKT cells in the development of obesity, C57BL/6 mice (wild type; WT), and two NKT-cell-deficient strains, Jα18−/− mice that lack the type I subset and CD1d−/− mice that lack both the type I and II subsets, were fed a high fat diet (HFD). CD1d−/− mice gained the least body weight with the least weight in perigonadal and brown adipose tissue as well as in the liver, compared to WT or Jα18−/− mice fed an HFD. Histologically, CD1d−/− mice had significantly smaller adipocytes and developed significantly milder hepatosteatosis than WT or Jα18−/− mice. The number of NK1.1+TCRβ+ cells in adipose tissue increased when WT mice were fed an HFD and were mostly invariant Vα14Jα18-negative. CD11b+ macrophages (Mφ) were another major subset of cells in adipose tissue infiltrates, and they were divided into F4/80high and F4/80low cells. The F4/80low-Mφ subset in adipose tissue was increased in CD1d−/− mice, and this population likely played an anti-inflammatory role. Glucose intolerance and insulin resistance in CD1d−/− mice were not aggravated as in WT or Jα18−/− mice fed an HFD, likely due to a lower grade of inflammation and adiposity. Collectively, our findings provide evidence that type II NKT cells initiate inflammation in the liver and adipose tissue and exacerbate the course of obesity that leads to insulin resistance.
doi:10.1371/journal.pone.0030568
PMCID: PMC3284453  PMID: 22383967
7.  Development and Function of Invariant Natural Killer T Cells Producing TH2- and TH17-Cytokines 
PLoS Biology  2012;10(2):e1001255.
Four distinct subsets of invariant natural killer T (NKT) cells are shown to differentiate in the thymus, then migrate to peripheral tissues where they retain their phenotypic and functional characteristics.
There is heterogeneity in invariant natural killer T (iNKT) cells based on the expression of CD4 and the IL-17 receptor B (IL-17RB), a receptor for IL-25 which is a key factor in TH2 immunity. However, the development pathway and precise function of these iNKT cell subtypes remain unknown. IL-17RB+ iNKT cells are present in the thymic CD44+/− NK1.1− population and develop normally even in the absence of IL-15, which is required for maturation and homeostasis of IL-17RB− iNKT cells producing IFN-γ. These results suggest that iNKT cells contain at least two subtypes, IL-17RB+ and IL-17RB− subsets. The IL-17RB+ iNKT subtypes can be further divided into two subtypes on the basis of CD4 expression both in the thymus and in the periphery. CD4+ IL-17RB+ iNKT cells produce TH2 (IL-13), TH9 (IL-9 and IL-10), and TH17 (IL-17A and IL-22) cytokines in response to IL-25 in an E4BP4-dependent fashion, whereas CD4− IL-17RB+ iNKT cells are a retinoic acid receptor-related orphan receptor (ROR)γt+ subset producing TH17 cytokines upon stimulation with IL-23 in an E4BP4-independent fashion. These IL-17RB+ iNKT cell subtypes are abundantly present in the lung in the steady state and mediate the pathogenesis in virus-induced airway hyperreactivity (AHR). In this study we demonstrated that the IL-17RB+ iNKT cell subsets develop distinct from classical iNKT cell developmental stages in the thymus and play important roles in the pathogenesis of airway diseases.
Author Summary
T cells are a diverse group of immune cells involved in cell-mediated acquired immunity. One subset of T cells is the innate-like invariant natural killer T (iNKT) cells that recognize glycolipid ligands on target cells instead of peptides. We know that functionally distinct subtypes of iNKT cells are involved in specific pathologies, yet their development, phenotypes, and functions are not well understood. Here, we determine the relationship between various mouse iNKT cell subsets, identify reliable molecular markers for these subsets, and show that these contribute to their functional differences. We identify four iNKT cell subsets that we show arise via different developmental pathways and exhibit different cytokine profiles. Importantly, we show that these subsets can be isolated from the thymus (the organ of all T cells), as well as from peripheral tissues such as spleen, liver, lung, and lymph nodes. Contrary to the general understanding that iNKT cells mature after their exit from the thymus and their migration into peripheral tissues, we conclude that distinct phenotypic and functional iNKT cell subsets can be distinguished in the thymus by virtue of the presence or absence of the cytokine receptor IL-17RB and another cell surface molecule called CD4, and these subsets then migrate to peripheral tissues where they retain their phenotypic and functional characteristics. Regarding functional significance, we show that those iNKT cell subsets that lead to airway hyper-responsiveness to respiratory viruses are different to those that lead to allergen-induced airway hyperreactivity, which will enable researchers to focus on specific subsets as potential targets for therapeutic intervention.
doi:10.1371/journal.pbio.1001255
PMCID: PMC3274505  PMID: 22346732
8.  160 Candidate for Response Monitoring or Prognostic Biomarkers in Two-Year Sublingual Immunotherapy for Japanese Cedar Pollinosis 
Background
The aims of this study were to clarify the therapeutic effects and potential prognostic and response monitoring biomarkers of sublingual immunotherapy (SLIT) in a randomized, double-blind, placebo-controlled (DBPC) clinical trial for Japanese cedar (Cryptomeria japonica) pollinosis.
Methods
This trial was carried out for 2 pollinosis seasons in 2007 and 2008 in double-blind status. Carry-over therapeutic effects were analyzed in 2009 in single-blind manner. We recruited 130 participants diagnosed as Japanese cedar pollinosis based on clinical history and the presence of IgE specific to Japanese cedar pollen of at least class 2. Cytokine production from peripheral blood mononuclear cells and increase of iTregs were determined before and after 2008 pollen season. Clinical symptoms were estimated using a pollinosis-symptom diary and Quality-of-Life (QOL) questionnaire in 2007, 2008, and 2009 peak pollen seasons.
Results
The final sample size included 88 subjects for on-treat analysis (SLIT; N = 51, placebo; N = 37) for the DBPC study, and a total of 63 patients completed a pollinosis-symptom diary for the single-blind follow-up study (SLIT; N = 36, placebo; N = 27). The symptom-medication score (SMS) in the SLIT group did not differ from that in the placebo group in the 2007 peak pollen season. However, the average SMS in 2008 and 2009 peak pollen seasons were significantly ameliorated in the SLIT group compared with the placebo group (4.2 vs. 5.3, P = 0.02 in 2008; 3.5 vs. 4.5, P = 0.03 in 2009). The ratio of Japanese cedar pollen-specific IgE to total IgE before treatment correlated with the SMS in the SLIT group in 2008. The patients with increased Cry j 1-specific iTregs in the SLIT group showed a significant reduction of QOL and QOL-symptom scores compared with those in the placebo group.
Conclusions
SLIT can ameliorate the clinical symptoms after 2-year administration with standardized extract of Japanese cedar pollen and the amelioration was observed for at least one pollen season after the treatment. The ratio of specific IgE to total IgE can be used as a prognostic biomarker and the increase of Cry j 1-specific iTregs may serve as a biomarker to monitor the clinical response to SLIT.
doi:10.1097/01.WOX.0000411917.58202.00
PMCID: PMC3513143
9.  THE ROLE OF NATURAL KILLER T (NKT) CELLS IN COSTIMULATION BLOCKADE-BASED MIXED CHIMERISM 
Distinct lymphocyte populations have been identified that either promote or impede the establishment of chimerism and tolerance through allogeneic bone marrow transplantation (BMT). NKT cells have pleiotropic regulatory properties capable of either augmenting or downmodulating various immune responses. We investigated here whether NKT cells affect outcome in mixed chimerism models employing fully mismatched non-myeloablative BMT with costimulation blockade (CB). The absence of NKT cells had no detectable effect on chimerism or skin graft tolerance after conditioning with 3Gy total body irradiation (TBI), and a limited positive effect with 1Gy TBI. Stimulation of NKT cells with alpha-galactosylceramide (alpha-gal) at the time of BMT prevented chimerism and tolerance. Activation of recipient (as opposed to donor) NKT cells was necessary and sufficient for the alpha-gal effect. The detrimental effect of NKT activation was also observed in the absence of T cells after conditioning with in vivo T cell depletion (TCD). NKT cells triggered rejection of BM via NK cells as chimerism and tolerance were not abrogated when NKT cells were stimulated in the absence of both NK cells and T cells. Thus, activation of NKT cells at the time of BMT overcomes the effects of CB, inhibiting the establishment of chimerism and tolerance.
doi:10.1111/j.1432-2277.2010.01120.x
PMCID: PMC2992941  PMID: 20536788
mixed chimerism; tolerance; costimulation blockade; NKT cells
10.  Murine induced pluripotent stem cells can be derived from and differentiate into natural killer T cells 
The Journal of Clinical Investigation  2010;120(7):2610-2618.
NKT cells demonstrate antitumor activity when activated to produce Th1 cytokines by DCs loaded with α-galactosylceramide, the prototypic NKT cell–activating glycolipid antigen. However, most patients do not have sufficient numbers of NKT cells to induce an effective immune response in this context, indicating a need for a source of NKT cells that could be used to supplement the endogenous cell population. Induced pluripotent stem cells (iPSCs) hold tremendous potential for cell-replacement therapy, but whether it is possible to generate functionally competent NKT cells from iPSCs has not been rigorously assessed. In this study, we successfully derived iPSCs both from embryonic fibroblasts from mice harboring functional NKT cell–specific rearranged T cell receptor loci in the germline and from splenic NKT cells from WT adult mice. These iPSCs could be differentiated into NKT cells in vitro and secreted large amounts of the Th1 cytokine IFN-γ. Importantly, iPSC-derived NKT cells recapitulated the known adjuvant effects of natural NKT cells and suppressed tumor growth in vivo. These studies demonstrate the feasibility of expanding functionally competent NKT cells via an iPSC phase, an approach that may be adapted for NKT cell–targeted therapy in humans.
doi:10.1172/JCI42027
PMCID: PMC2898602  PMID: 20516640
11.  High-mobility group box 1 is involved in the initial events of early loss of transplanted islets in mice 
Islet transplantation for the treatment of type 1 diabetes mellitus is limited in its clinical application mainly due to early loss of the transplanted islets, resulting in low transplantation efficiency. NKT cell–dependent IFN-γ production by Gr-1+CD11b+ cells is essential for this loss, but the upstream events in the process remain undetermined. Here, we have demonstrated that high-mobility group box 1 (HMGB1) plays a crucial role in the initial events of early loss of transplanted islets in a mouse model of diabetes. Pancreatic islets contained abundant HMGB1, which was released into the circulation soon after islet transplantation into the liver. Treatment with an HMGB1-specific antibody prevented the early islet graft loss and inhibited IFN-γ production by NKT cells and Gr-1+CD11b+ cells. Moreover, mice lacking either of the known HMGB1 receptors TLR2 or receptor for advanced glycation end products (RAGE), but not the known HMGB1 receptor TLR4, failed to exhibit early islet graft loss. Mechanistically, HMGB1 stimulated hepatic mononuclear cells (MNCs) in vivo and in vitro; in particular, it upregulated CD40 expression and enhanced IL-12 production by DCs, leading to NKT cell activation and subsequent NKT cell–dependent augmented IFN-γ production by Gr-1+CD11b+ cells. Thus, treatment with either IL-12– or CD40L-specific antibody prevented the early islet graft loss. These findings indicate that the HMGB1-mediated pathway eliciting early islet loss is a potential target for intervention to improve the efficiency of islet transplantation.
doi:10.1172/JCI41360
PMCID: PMC2827961  PMID: 20124731
12.  A novel subset of mouse NKT cells bearing the IL-17 receptor B responds to IL-25 and contributes to airway hyperreactivity 
The Journal of Experimental Medicine  2008;205(12):2727-2733.
Airway hypersensitive reaction (AHR) is an animal model for asthma, which is caused or enhanced by environmental factors such as allergen exposure. However, the precise mechanisms that drive AHR remain unclear. We identified a novel subset of natural killer T (NKT) cells that expresses the interleukin 17 receptor B (IL-17RB) for IL-25 (also known as IL-17E) and is essential for the induction of AHR. IL-17RB is preferentially expressed on a fraction of CD4+ NKT cells but not on other splenic leukocyte populations tested. IL-17RB+ CD4+ NKT cells produce predominantly IL-13 and Th2 chemokines upon stimulation with IL-25 in vitro. IL-17RB+ NKT cells were detected in the lung, and depletion of IL-17RB+ NKT cells by IL-17RB–specific monoclonal antibodies or NKT cell–deficient Jα18−/− mice failed to develop IL-25–dependent AHR. Cell transfer of IL-17RB+ but not IL-17RB− NKT cells into Jα18−/− mice also successfully reconstituted AHR induction. These results strongly suggest that IL-17RB+ CD4+ NKT cells play a crucial role in the pathogenesis of asthma.
doi:10.1084/jem.20080698
PMCID: PMC2585837  PMID: 19015310
13.  Contrasting roles for Vα14+ natural killer T cells in a viral model for multiple sclerosis 
Journal of neurovirology  2008;15(1):90-98.
Most natural killer (NK) T cells express an invariant Vα14 T-cell receptor. To explore the contribution of NKT cells in an animal model for multiple sclerosis, Theiler’s murine encephalomyelitis virus (TMEV) infection, TMEV-infected mice were treated with Vα14 antibody. Treatment during the early stage of infection delayed the onset of demyelinating disease with higher interleukin-4 production, whereas administration during the late stage or weekly resulted in more severe demyelination with enhanced virus persistence. The effect of in vivo depletion of NKT cells differed depending on the stage of infection, suggesting contrasting roles for NKT cells over the disease course.
doi:10.1080/13550280802400684
PMCID: PMC2671644  PMID: 19115130
CD1d; CNS autoimmune demyelinating diseases; cytokines; iNKT lymphocytes; Picornaviridae infections; Theiler’s virus
14.  Identification of CD4−CD8− Double-Negative Natural Killer T Cell Precursors in the Thymus 
PLoS ONE  2008;3(11):e3688.
Background
It is well known that CD1d-restricted Vα14 invariant natural killer T (NKT) cells are derived from cells in the CD4+CD8+ double-positive (DP) population in the thymus. However, the developmental progression of NKT cells in the earlier stages remains unclear, and the possible existence of NKT cell presursors in the earlier stages than DP stage remains to be tested.
Principal Findings
Here, we demonstrate that NKT cell precursors that express invariant Vα14-Jα18 transcripts but devoid of surface expression of the invariant Vα14 receptor are present in the late CD4−CD8− double-negative (DN)4 stage and have the potential to generate mature NKT cells in both in vivo and in vitro experimental conditions. Moreover, the DN4 population in CD1d knock-out (CD1dKO) mice was similar to those with an NKT cell potential in wild-type (WT) C57BL/6 (B6) mice, but failed to develop into NKT cells in vitro. However, these precursors could develop into NKT cells when co-cultured with normal thymocytes or in an in vivo experimental setting, indicating that functional NKT cell precursors are present in CD1dKO mice.
Conclusions
Together, these results demonstrate that thymic DN4 fraction contains NKT cell precursors. Our findings provide new insights into the early development of NKT cells prior to surface expression of the invariant Vα14 antigen receptor and suggest the possible alternative developmental pathway of NKT cells.
doi:10.1371/journal.pone.0003688
PMCID: PMC2577011  PMID: 18997862
15.  Cross-presentation of glycolipid from tumor cells loaded with α-galactosylceramide leads to potent and long-lived T cell–mediated immunity via dendritic cells 
The Journal of Experimental Medicine  2007;204(11):2641-2653.
We report a mechanism to induce combined and long-lived CD4+ and CD8+ T cell immunity to several mouse tumors. Surprisingly, the initial source of antigen is a single low dose of tumor cells loaded with α-galactosylceramide (α-GalCer) glycolipid (tumor/Gal) but lacking co-stimulatory molecules. After tumor/Gal injection intravenously (i.v.), innate NKT and NK cells reject the tumor cells, some of which are taken up by dendritic cells (DCs). The DCs in turn cross-present glycolipid on CD1d molecules to NKT cells and undergo maturation. For B16 melanoma cells loaded with α-GalCer (B16/Gal), interferon γ–producing CD8+ T cells develop toward several melanoma peptides, again after a single low i.v. dose of B16/Gal. In all four poorly immunogenic tumors tested, a single dose of tumor/Gal i.v. allows mice to become resistant to tumors given subcutaneously. Resistance requires CD4+ and CD8+ cells, as well as DCs, and persists for 6–12 mo. Therefore, several immunogenic features of DCs are engaged by the CD1d-mediated cross-presentation of glycolipid-loaded tumor cells, leading to particularly strong and long-lived adaptive immunity.
doi:10.1084/jem.20070458
PMCID: PMC2118481  PMID: 17923500
16.  OX40 ligand expressed by DCs costimulates NKT and CD4+ Th cell antitumor immunity in mice  
The Journal of Clinical Investigation  2007;117(11):3330-3338.
The exceptional immunostimulatory capacity of DCs makes them potential targets for investigation of cancer immunotherapeutics. We show here in mice that TNF-α–stimulated DC maturation was accompanied by increased expression of OX40 ligand (OX40L), the lack of which resulted in an inability of mature DCs to generate cellular antitumor immunity. Furthermore, intratumoral administration of DCs modified to express OX40L suppressed tumor growth through the generation of tumor-specific cytolytic T cell responses, which were mediated by CD4+ T cells and NKT cells. In the tumors treated with OX40L-expressing DCs, the NKT cell population significantly increased and exhibited a substantial level of IFN-γ production essential for antitumor immunity. Additional studies evaluating NKT cell activation status, in terms of IFN-γ production and CD69 expression, indicated that NKT cell activation by DCs presenting α-galactosylceramide in the context of CD1d was potentiated by OX40 expression on NKT cells. These results show a critical role for OX40L on DCs, via binding to OX40 on NKT cells and CD4+ T cells, in the induction of antitumor immunity in tumor-bearing mice.
doi:10.1172/JCI32693
PMCID: PMC2045612  PMID: 17975668
17.  IL-21–induced Bɛ cell apoptosis mediated by natural killer T cells suppresses IgE responses 
The Journal of Experimental Medicine  2006;203(13):2929-2937.
Epidemiological studies have suggested that the recent increase in the incidence and severity of immunoglobulin (Ig)E-mediated allergic disorders is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination; however, the underlying mechanisms remain uncertain. Here, we demonstrate that natural killer T (NKT) cells in mice and humans play a crucial role in the BCG-induced suppression of IgE responses. BCG-activated murine Vα14 NKT cells, but not conventional CD4 T cells, selectively express high levels of interleukin (IL)-21, which preferentially induces apoptosis in Bɛ cells. Signaling from the IL-21 receptor increases the formation of a complex between Bcl-2 and the proapoptotic molecule Bcl-2–modifying factor, resulting in Bɛ cell apoptosis. Similarly, BCG vaccination induces IL-21 expression by human peripheral blood mononuclear cells (PBMCs) in a partially NKT cell–dependent fashion. BCG-activated PBMCs significantly reduce IgE production by human B cells. These findings provide new insight into the therapeutic effect of BCG in allergic diseases.
doi:10.1084/jem.20062206
PMCID: PMC2118181  PMID: 17178921
18.  Functionally distinct NKT cell subsets and subtypes 
The Journal of Experimental Medicine  2005;202(12):1623-1626.
Natural killer T (NKT) cells are a population of autoreactive cells that mediate both protective and regulatory immune functions. NKT cells comprise several subsets of cells, but it has been unclear whether these different NKT cell subsets possess distinct functions in vivo. New studies now demonstrate that subsets of NKT cells are indeed functionally distinct and that the specific functions of these cells may be dictated in part by organ-specific mechanisms.
doi:10.1084/jem.20051600
PMCID: PMC2212962  PMID: 16365145
19.  Vα14 NK T cell–triggered IFN-γ production by Gr-1+CD11b+ cells mediates early graft loss of syngeneic transplanted islets 
Pancreatic islet transplantation is a highly promising approach for the treatment of insulin-dependent diabetes mellitus. However, the procedure remains experimental for several reasons, including its low efficiency caused by the early graft loss of transplanted islets. We demonstrate that Gr-1+CD11b+ cells generated by transplantation and their IFN-γ production triggered by Vα14 NKT cells are an essential component and a major cause of early graft loss of pancreatic islet transplants. Gr-1+CD11b+ cells from Vα14 NKT cell–deficient (Jα281−/−) mice failed to produce IFN-γ, resulting in efficient islet graft acceptance. Early graft loss was successfully prevented through the repeated administration of α-galactosylceramide, a specific ligand for Vα14 NKT cells, resulting in dramatically reduced IFN-γ production by Gr-1+CD11b+ cells, as well as Vα14 NKT cells. Our study elucidates, for the first time, the crucial role of Gr-1+CD11b+ cells and the IFN-γ they produce in islet graft rejection and suggests a novel approach to improving transplantation efficiency through the modulation of Vα14 NKT cell function.
doi:10.1084/jem.20050448
PMCID: PMC2213168  PMID: 16186183
20.  Invariant NKT Cells Are Essential for the Regulation of Hepatic CXCL10 Gene Expression during Leishmania donovani Infection  
Infection and Immunity  2005;73(11):7541-7547.
Gamma interferon (IFN-γ)-regulated chemokines of the CXC family have been implicated as key regulators of a variety of T-cell-dependent inflammatory processes. However, the cellular source(s) of IFN-γ that regulates their early expression has rarely been defined. Here, we have directly addressed this question in mice after Leishmania donovani infection. Comparison of CXCL10 mRNA accumulation in normal and IFN-γ-deficient mice confirmed an absolute requirement for IFN-γ for sustained (24 h) expression of CXCL10 mRNA accumulation in this model. In normal mice, IFN-γ was produced by both CD3int NK1.1+ NKT cells and CD3− NK1.1+ NK cells, as detected by intracellular flow cytometry. Strikingly, B6.Jα281−/− mice lacking NKT cells that express the invariant Vα14Jα18 T-cell-receptor α chain, although retaining a significant population of IFN-γ-producing NK cells and NKT cells, were unable to sustain CXCL10 mRNA accumulation. These data indicate that invariant NKT cells are indispensable for the regulation of hepatic CXCL10 gene expression during L. donovani infection.
doi:10.1128/IAI.73.11.7541-7547.2005
PMCID: PMC1273891  PMID: 16239557
21.  Regulation of T helper type 2 cell differentiation by murine Schnurri-2 
Schnurri (Shn) is a large zinc finger protein implicated in cell growth, signal transduction, and lymphocyte development. Vertebrates possess at least three Shn orthologues (Shn-1, Shn-2, and Shn-3), which appear to act within the bone morphogenetic protein, transforming growth factor β, and activin signaling pathways. However, the physiological functions of the Shn proteins remain largely unknown. In Shn-2–deficient mice, mature peripheral T cells exhibited normal anti–T cell receptor–induced proliferation, although there was dramatic enhancement in the differentiation into T helper type (Th)2 cells and a marginal effect on Th1 cell differentiation. Shn-2–deficient developing Th2 cells showed constitutive activation of nuclear factor κB (NF-κB) and enhanced GATA3 induction. Shn-2 was able to compete with p50 NF-κB for binding to a consensus NF-κB motif and inhibit NF-κB–driven promoter activity. Thus, Shn-2 plays a crucial role in the control of Th2 cell differentiation by regulating NF-κB function.
doi:10.1084/jem.20040733
PMCID: PMC2213028  PMID: 15699073
22.  Invariant Vα14 Chain NKT Cells Promote Plasmodium berghei Circumsporozoite Protein-Specific Gamma Interferon- and Tumor Necrosis Factor Alpha-Producing CD8+ T Cells in the Liver after Poxvirus Vaccination of Mice  
Infection and Immunity  2005;73(2):849-858.
Understanding the protective mechanism in the liver induced by recombinant vaccines against the pre-erythrocytic stages of malaria is important for vaccine development. Most studies in mice have focused on splenic and peripheral blood T cells and identified gamma interferon (IFN-γ)-producing CD8+ T cells as correlates of protection, which can be induced by prime-boost vaccination with recombinant poxviruses. Invariant natural killer T (Vα14iNKT) cells can also protect against liver stage malaria, when activated, and are abundant in the liver. Since poxviruses have nonspecific immunomodulating effects, which are incompletely understood, we investigated whether recombinant poxviruses affect the protective properties of hepatic Vα14iNKT cells and thus vaccine efficacy. We show that intradermal vaccination with recombinant poxviruses activated Vα14iNKT cells and NK cells in the livers of BALB/c mice while inducing IFN-γ- and tumor necrosis factor alpha (TNF-α)-producing pre-erythrocytic stage antigen-specific CD8+ T cells. Greater numbers of hepatic Vα14iNKT cells secreted interleukin-4 than IFN-γ. Vaccinated Vα14iNKT-cell-deficient mice had lower, but still protective levels of hepatic and splenic IFN-γ+ and TNF-α+ CD8+ T cells and better protection rates later after challenge with Plasmodium berghei sporozoites. Therefore, vaccine-activated hepatic Vα14iNKT cells help in generating specific T cells but are not required for protection induced by recombinant poxviruses. Furthermore, double-positive INF-γ+/TNF-α+ CD8+ T cells were enriched in protected livers, suggesting that cells expressing both of these cytokines may be most relevant for protection.
doi:10.1128/IAI.73.2.849-858.2005
PMCID: PMC546932  PMID: 15664925
24.  Bone Marrow Allograft Rejection Mediated by a Novel Murine NK Receptor, NKG2I 
Natural killer (NK) cells mediate bone marrow allograft rejection. However, the molecular mechanisms underlying such a rejection remain elusive. In previous analyses, it has been shown that NK cells recognize allogeneic target cells through Ly-49s and CD94/NKG2 heterodimers. Here, we describe identification and characterization of a novel murine NK receptor, NKG2I, belonging to the NKG2 family. NKG2I, which was composed of 226 amino acids, showed ∼40% homology to the murine NKG2D and CD94 in the C-type lectin domain. Flow cytometric analysis with anti-NKG2I monoclonal antibody (mAb) revealed that expression of NKG2I was largely confined to NK and NKT cells, but was not seen in T cells. Furthermore, anti-NKG2I mAb inhibited NK cell–mediated cytotoxicity, whereas cross-linking of NKG2I enhanced interleukin 2– and interleukin 12–dependent interferon-γ production. Similarly, the injection of anti-NKG2I mAb before the allogeneic bone marrow transfer in vivo impinged on the function of NKG2I, resulting in the enhanced colony formation in the spleen. NKG2I is a novel activating receptor mediating recognition and rejection of allogeneic target cells.
doi:10.1084/jem.20030851
PMCID: PMC1887729  PMID: 14707119
C-type lectin family; NK cells; activating receptor; IFN-γ; NKT cells
25.  Gamma Interferon Production by Hepatic NK T Cells during Escherichia coli Infection Is Resistant to the Inhibitory Effects of Oxidative Stress  
Infection and Immunity  2003;71(5):2468-2477.
The reductive-oxidative status of tissues regulates the expression of many inflammatory genes that are induced during gram-negative bacterial infections. The cytokine gamma interferon (IFN-γ) is a potent stimulus for host inflammatory gene expression, and oxidative stress has been shown to inhibit its production in mice challenged with Escherichia coli bacteria. The objective of the present study was to characterize the cells that produced IFN-γ in a mouse bacterial peritonitis model and determine the effects of oxidative stress on their activation. The liver contained large numbers of IFN-γ-expressing lymphocytes following challenge with viable E. coli bacteria. The surface phenotypes of IFN-γ-expressing hepatic lymphocytes were those of natural killer (NK) cells (NK1.1+ CD3−), conventional T cells (NK1.1− CD3+), and NK T cells (NK1.1+ CD3+). Treating mice with diethyl maleate to deplete tissue thiols significantly impaired IFN-γ production by NK cells, conventional T cells, and CD1d-restricted NK T cells in response to E. coli challenge. However, IFN-γ expression by a subset of NK T cells, which did not bind α-galactosylceramide-CD1d tetramers, was resistant to the inhibitory effects of tissue oxidative stress. Stress-resistant IFN-γ-expressing cells were also predominantly CD8+ and bore γδ T-cell antigen receptors. The residual IFN-γ response by NK T cells may explain previous reports of hepatic gene expression following gram-negative bacterial challenge in thiol-depleted mice. The finding also demonstrates that innate immune cells differ significantly in their responses to altered tissue redox status.
doi:10.1128/IAI.71.5.2468-2477.2003
PMCID: PMC153215  PMID: 12704118

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