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1.  Hepatitis C Virus-Specific T cell-Derived Transforming Growth Factor beta is Associated with Slow Hepatic Fibrogenesis 
Hepatology (Baltimore, Md.)  2012;56(6):2094-2105.
Background/Aims
Hepatitis C virus (HCV)-specific immune effector responses can cause liver damage in chronic infection. Hepatic stellate cells (HSC) are main effectors of liver fibrosis. We previously identified TGFβ, produced by HCV-specific CD8+ T cells, as key regulatory cytokine modulating HCV-specific effector T cells. Here we studied TGFβ as well as other factors produced by HCV-specific intrahepatic lymphocytes (IHL) and peripheral blood cells in hepatic inflammation and fibrogenesis.
Methods
Cross-sectional study of 2 well-defined groups of HCV-infected subjects with slow (≤0.1 Metavir units/year, n=13) or rapid (n=6) liver fibrosis progression. HCV-specific T cell responses were studied using IFNγ-ELISpot ±mAbs blocking regulatory cytokines, along with multiplex, ELISA and multi-parameter FACS. Effects of IHL stimulated with HCV-core peptides on HSC expression of pro-fibrotic and fibrolytic genes were determined.
Results
Blocking regulatory cytokines significantly raised detection of HCV-specific effector (IFNγ) responses only in slow fibrosis progressors, both in the periphery (p=0.003) and liver (p=0.01). Regulatory cytokine blockade revealed HCV-specific IFNγ responses strongly correlated with HCV-specific TGFβ, measured before blockade (R=0.84, p=0.0003), with only trend to correlation with HCV-specific IL-10. HCV-specific TGFβ was produced by CD8 and CD4 T cells. HCV-specific TGFβ, not IL-10, inversely correlated with liver inflammation (R=-0.63, p=0.008) and, unexpectedly, fibrosis (R=-0.46, p=0.05). In addition, supernatants from HCV-stimulated IHL of slow progressors specifically increased fibrolytic gene expression in HSC and treatment with anti-TGFβ mAb abrogated such expression.
Conclusion
Although TGFβ is considered a major profibrogenic cytokine, local production of TGFβ by HCV-specific T cells appeared to have a protective role in HCV-infected liver, together with other T-cell derived factors, ameliorating HCV liver disease progression.
doi:10.1002/hep.25951
PMCID: PMC3508175  PMID: 22806830
Treg; IL-17; MMP-1; liver fibrosis
2.  Circulating Microparticles as Disease-Specific Biomarkers of Severity of Inflammation in Patients with Hepatitis C or Nonalcoholic Steatohepatitis 
Gastroenterology  2012;143(2):448-458.
Background & Aims
Microparticles released into the bloodstream upon activation or apoptosis of CD4+ and CD8+ T cells correlate with inflammation, determined by histologic analysis, in patients with chronic hepatitis C (CHC). Patients with nonalcoholic fatter liver (NAFL) or nonalcoholic steatohepatitis (NASH) can be differentiated from those with CHC based on activation of distinct sets of immune cells in the liver.
Methods
We compared profiles of circulating microparticles from patients with NAFL and NASH (n=67) to those with CHC (n=42), compared with healthy individuals (controls) using flow cytometry; the profiles were correlated with inflammation grade and fibrosis stage, based on histologic analyses. We assessed the ability of the profiles determine the severity of inflammation and fibrosis, based on serologic and histologic analyses.
Results
Patients with CHC had increased levels of microparticles from CD4+ and CD8+ T cells; the levels correlated with disease severity, based on histologic analysis and levels of alanine aminotransferase (ALT). Patients with NAFL or NASH had significant increases in numbers of microparticles from invariant natural killer T (iNKT) cells and macrophages/monocytes (CD14+), which mediate pathogenesis of NASH. Microparticles from CD14+ and iNKT cells correlated with levels of ALT and severity of NASH (based on histology). Levels of microparticles could differentiate between patients with NAFL or NASH and those with CHC, or either group of patients and controls (area under the receiver operating characteristic curves ranging from 0.56 to 0.99).
Conclusions
Quantification of immune cell microparticles from serum samples can be used to assess the extent and characteristics of hepatic inflammation in patients with chronic liver disease.
doi:10.1053/j.gastro.2012.04.031
PMCID: PMC3404266  PMID: 22537612
Biomarker; CD1c; CD4+; CD8+; CD14+; CD15+; CD41+; CD16+; ectosome; fibrosis; HCV; inflammation; iNKT; NKT; liver; macrophage; microparticle; monocyte; NASH; plasma; serum; T cell; non-invasive assay; lymphocyte; serum assay; biomarker assay
3.  Islet allograft tolerance in the absence of invariant natural killer T cells 
Clinical immunology (Orlando, Fla.)  2011;141(3):268-272.
The invariant NKT cells are involved in both immunity and immune tolerance. However, their roles in transplant models remain controversial. We studied the role of NKT cells in the allograft response using two different strains of NKT deficient mice (CD1d−/− and Jα18−/− mice), and found that CD1d−/− and Jα18−/− mice rejected islet allografts with a similar kinetics as wild type B6 mice. Treatment of CD1d−/− and Jα18−/− mice with donor specific transfusion and anti-CD154 induced donor specific tolerance, which was identical to similarly treated wt B6 mice. The islet allograft tolerance requires Foxp3+ Tregs. In the periphery, Foxp3+ Tregs in CD1d−/−, Jα18−/−, and wt B6 mice were comparable both phenotypically and functionally. In addition, CD1d−/− and Jα18−/− CD4+ T cells (non-Tregs) could be readily converted to Foxp3+ Tregs by TGF-β in vitro. Our data suggest that islet allograft tolerance can be successfully established without invariant NKT cells.
doi:10.1016/j.clim.2011.09.003
PMCID: PMC3221878  PMID: 21996456
NK cells; Islets; Transplantation; Tolerance; Tregs
4.  Loss of Effector and Anti-Inflammatory Natural Killer T Lymphocyte Function in Pathogenic Simian Immunodeficiency Virus Infection 
PLoS Pathogens  2012;8(9):e1002928.
Chronic immune activation is a key determinant of AIDS progression in HIV-infected humans and simian immunodeficiency virus (SIV)-infected macaques but is singularly absent in SIV-infected natural hosts. To investigate whether natural killer T (NKT) lymphocytes contribute to the differential modulation of immune activation in AIDS-susceptible and AIDS-resistant hosts, we compared NKT function in macaques and sooty mangabeys in the absence and presence of SIV infection. Cynomolgus macaques had significantly higher frequencies of circulating invariant NKT lymphocytes compared to both rhesus macaques and AIDS-resistant sooty mangabeys. Despite this difference, mangabey NKT lymphocytes were functionally distinct from both macaque species in their ability to secrete significantly more IFN-γ, IL-13, and IL-17 in response to CD1d/α-galactosylceramide stimulation. While NKT number and function remained intact in SIV-infected mangabeys, there was a profound reduction in NKT activation-induced, but not mitogen-induced, secretion of IFN-γ, IL-2, IL-10, and TGF-β in SIV-infected macaques. SIV-infected macaques also showed a selective decline in CD4+ NKT lymphocytes which correlated significantly with an increase in circulating activated memory CD4+ T lymphocytes. Macaques with lower pre-infection NKT frequencies showed a significantly greater CD4+ T lymphocyte decline post SIV infection. The disparate effect of SIV infection on NKT function in mangabeys and macaques could be a manifestation of their differential susceptibility to AIDS. Alternately, these data also raise the possibility that loss of anti-inflammatory NKT function promotes chronic immune activation in pathogenic SIV infection, while intact NKT function helps to protect natural hosts from developing immunodeficiency and aberrant immune activation.
Author Summary
Several African nonhuman primate species such as sooty mangabeys are naturally infected with SIV and maintain high levels of viral replication without developing AIDS. SIV-infected natural hosts do not show evidence of increased chronic immune activation, a feature that distinguishes them from AIDS-susceptible SIV-infected Asian macaques. In this study we compared natural killer T (NKT) lymphocytes, a unique subset of innate T lymphocytes with anti-inflammatory properties, in AIDS-resistant and AIDS-susceptible hosts. Sooty mangabey NKT cells retained normal functionality following SIV infection and were more potent than macaque NKT cells in their ability to produce interferon-γ and secrete anti-inflammatory cytokines. In contrast, NKT cells of SIV-infected macaques were markedly hypo-functional with regards to secretion of anti-inflammatory and effector cytokines and showed an association between loss of CD4+ NKT cells and increased immune activation. These findings suggest that dysfunctional NKT cells may promote increased immune activation in AIDS-susceptible hosts while intact effector and anti-inflammatory NKT cells could help to prevent immunodeficiency and increased immune activation in natural hosts.
doi:10.1371/journal.ppat.1002928
PMCID: PMC3447755  PMID: 23028326
5.  Developing Understanding of the Roles of CD1d-restricted T cell Subsets in Cancer: Reversing Tumor-induced Defects 
Clinical immunology (Orlando, Fla.)  2011;140(2):184-195.
Summary
Invariant natural killer T-cells (‘iNKT’) are the best-known CD1d-restricted T-cells, with recently-defined roles in controlling adaptive immunity. CD1d-restricted T-cells can rapidly produce large amounts of Th1 and/or Th2//Treg/Th17-type cytokines, thereby regulating immunity. iNKT can stimulate potent anti-tumor immune responses via production of Th1 cytokines, direct cytotoxicity, and activation of effectors. However, Th2//Treg-type iNKT can inhibit anti-tumor activity. Furthermore, iNKT are decreased and/or reversibly functionally impaired in many advanced cancers. In some cases, CD1d-restricted T-cell cancer defects can be traced to CD1d+ tumor interactions, since hematopoietic, prostate, and some other tumors can express CD1d. Ligand and IL-12 can reverse iNKT defects and therapeutic opportunities exist in correcting such defects alone and in combination. Early stage clinical trials have shown potential for reconstitution of iNKT IFN-gamma responses and evidence of activity in a subset of patients, with rational new approaches to capitalize on this progress ongoing, as will be discussed here.
doi:10.1016/j.clim.2011.04.017
PMCID: PMC3143311  PMID: 21646050
cytokines; tumor immunity; CD1; CD1d-reactive T cells; iNKT; NKT
6.  Essential role for the prolyl isomerase Pin1 in Toll-like receptor signaling and type I interferon-mediated immunity 
Nature Immunology  2011;12(8):733-741.
Toll-like receptors (TLRs) shape innate and adaptive immunity to microorganisms. The enzyme IRAK1 transduces signals from TLRs, but its activation and regulation mechanisms remain unknown. We show that TLR7 and TLR9 activated the isomerase Pin1, which then bound to IRAK1, resulting in IRAK1 activation and facilitating its release from the receptor complex to activate the transcription factor IRF7 and induce type I interferons. Consequently, Pin1-null cells and mice failed to mount TLR-mediated, interferon-dependent innate and adaptive immune responses. Given the critical role of aberrant IRAK1 activation and type I interferons in various immune diseases, controlling IRAK1 activation via Pin1 inhibition may represent a useful therapeutic approach.
doi:10.1038/ni.2069
PMCID: PMC3298750  PMID: 21743479
7.  Heterogeneity in Phenotype and Function of CD8+ and CD4/CD8 Double-Negative Natural Killer T Cell Subsets in Sooty Mangabeys 
Journal of medical primatology  2010;39(4):224-234.
Background
We have recently reported the presence of CD8+ and CD4/8 double negative (DN) Natural Killer T (NKT) lymphocytes in sooty mangabeys. To investigate differences in the two NKT cell subsets, we compared the phenotype and function of sooty mangabey CD8+ and DN NKT cells.
Methods
Flow sorted NKT lymphocytes from one SIV-negative sooty mangabey were subjected to limiting dilution cloning. Invariant NKT clones were characterized by flow cytometry and cytokine-ELISA.
Results
The majority of NKT clones displayed an effector memory phenotype and expressed CXCR3 and NKG2D. While CD8+ NKT subsets expressed significantly higher levels of granzyme B and perforin and produced more IFN-γ, the DN NKT subsets secreted significantly more IL-4, IL-13, and IL-10.
Conclusions
The Th1 and Th2 cytokine bias of CD8+ and DN NKT cells respectively indicates the presence of functionally heterogeneous populations of NKT cells in sooty mangabeys.
doi:10.1111/j.1600-0684.2010.00431.x
PMCID: PMC2904642  PMID: 20618588
NKT cell subsets; α-GalCer; 6B11 mAb; sooty mangabey
8.  The A2aR adenosine receptor controls cytokine production in iNKT cells 
European journal of immunology  2010;40(3):682-687.
The purine nucleoside adenosine is an important anti-inflammatory molecule, inhibiting a variety of immune cells by adenosine receptor-mediated mechanisms. Invariant natural killer T (iNKT) cells recognize glycolipids presented on CD1d molecules and produce vigorous amounts of cytokines upon activation, hence regulating immune reactions. The mechanisms polarizing their cytokine pattern are elusive. Previous studies demonstrated that adenosine can suppress IFN-γ production by iNKT cells.
We describe the expression of all four known adenosine receptors A1R, A2aR, A2bR, and A3R, on mouse iNKT cells. We show that IL-4 production in primary mouse iNKT cells and a human iNKT line is efficiently inhibited by A2aR blockade with an inverse relation to IL-4. These data are supported by A2aR-deficient mice, which exhibit largely decreased levels of IL-4, IL-10 and TGF-β concomitantly with an increase of IFN-γ upon α-GalCer administration in vivo. While A2aR inhibits other lymphocyte populations, A2aR is required for the secretion of IL-4 and IL-10 by iNKT cells. These data suggest adenosine:A2aR-mediated mechanisms can control the cytokine secretion pattern of iNKT cells.
doi:10.1002/eji.200939897
PMCID: PMC2967447  PMID: 20039304
NKT cells; Cellular activation; Immune regulation
9.  DIRECT CD1d-MEDIATED STIMULATION OF APC IL-12 PRODUCTION AND PROTECTIVE IMMUNE RESPONSE TO VIRUS INFECTION In Vivo 
CD1d-restricted ‘NKT’ rapidly stimulate innate and adaptive immunity through production of Th1 and/or Th2 cytokines and induction of CD1d+ antigen-presenting cell (APC) maturation. However, therapeutic exploitation of NKT has been hampered by their paucity and defects in human disease. NKT:APC interactions can be modeled by direct stimulation of human APC through CD1d in vitro. We have now found that direct ligation with multiple CD1d mAbs also stimulated bioactive IL-12 release from CD1d+ but not CD1d KO murine splenocytes in vitro. Moreover, all CD1d mAbs tested also induced IL-12 as well as both IFN-γ and IFN-α in vivo from CD1d+ but not CD1d-deficient recipients. Unlike IFN-γ, CD1d-induced IFN-α was at least partially dependent on invariant NKT. Optimal resistance to infection with picornavirus encephalomyocarditis virus (EMCV) is known to require CD1d-dependent APC IL-12-induced IFN-γ as well as IFN-α. CD1d ligation in vivo enhanced systemic IL-12, IFN-γ, and IFN-α, and was protective against infection by EMCV, suggesting an alternative interpretation for previous results involving CD1d ‘blocking’ in other systems. Such protective responses, including elevations in Th1 cytokines, were also seen with CD1d FAb’2s in vivo, while an IgM mAb (with presumably minimal tissue penetration) was comparably effective at protection in vivo as well as cytokine induction both in vivo and in vitro. Although presumably acting immediately ‘downstream’, CD1d mAbs were protective later during infection than iNKT agonist α-galactosylceramide. These data indicate that NKT can be bypassed with CD1d-mediated induction of robust Th1 immunity, which may have therapeutic potential both directly and as adjuvant.
doi:10.4049/jimmunol.0800924
PMCID: PMC2967442  PMID: 19949077
antibodies; cell activation; cytokines; NKT cells; viral infection
10.  CD1d-Based Combination Therapy Eradicates Established Tumors in Mice1 
The use of Abs that induce tumor cell death together with immunostimulatory reagents to activate innate and adaptive immune cells has emerged as a potent approach for the treatment of cancer. We have previously demonstrated that the use of three mAbs (anti-DR5, anti-CD40, anti-CD137) termed TriMab can induce rejection in a majority of mice with established experimental or carcinogen-induced tumors. However, given the potential toxicity of CD40 agonists in the clinic, we tested an alternative approach to directly activate/mature APCs using anti-CD1d mAbs. In this study, we used a combination of three mAbs (anti-DR5, anti-CD137, anti-CD1d) that we termed 1DMab and demonstrated that this approach suppressed and/or eradicated established experimental renal, breast, and colon carcinomas in mice. Tumor suppression induced by 1DMab therapy required CD8+ T cells, IFN-γ, and CD1d, while NK cells and IL-12 were partially required. Interestingly 1DMab therapy was more effective than TriMab in tumor models regulated by CD1d-restricted type II NKT cells, but less efficacious against tumors where T regulatory cells were critical. Anti-CD1d mAbs could also be relatively effective in combination with anti-CD137 and conventional chemotherapeutics. This is the first study to illustrate the antitumor activity of CD1d-reactive mAbs in combination and our results strongly suggest that rational combination chemoimmunotherapies based on tumor immunoregulation may improve the efficacy of treatment.
doi:10.4049/jimmunol.0900796
PMCID: PMC2967455  PMID: 19620318
11.  CD1d Activation and Blockade: A New Antitumor Strategy1 
CD1d is expressed on APCs and presents glycolipids to CD1d-restricted NKT cells. For the first time, we demonstrate the ability of anti-CD1d mAbs to inhibit the growth of different CD1d-negative experimental carcinomas in mice. Anti-CD1d mAbs systemically activated CD1d+ APC, as measured by production of IFN-γ and IL-12. Tumor growth inhibition was found to be completely dependent on IFN-γ and IL-12 and variably dependent on CD8+ T cells and NK cells, depending upon the tumor model examined. Anti-CD1d mAb induced greater CD8+ T cell-dependent tumor suppression where regulatory CD1d-restricted type II NKT cells have been implicated, and were less effective in a NK cell-dependent manner against tumors where T regulatory cells were immunosuppressive. The ability of anti-CD1d mAbs to coincidently activate CD1d+ APCs to release IL-12 and inhibit CD1d-restricted type II NKT cells makes CD1d an exciting new target for immunotherapy of cancer based on tumor immunoregulation.
doi:10.4049/jimmunol.0802964
PMCID: PMC2967458  PMID: 19265113
12.  Generation of Antitumor Invariant NKT Cell Lines in Multiple Myeloma and Promotion of Their Functions via Lenalidomide: A Strategy for Immunotherapy 
Purpose
CD1d-restricted invariant NKT (iNKT) cells are important immunoregulatory cells in antitumor immune responses. However, the quantitative and qualitative defects of iNKT cells in advanced multiple myeloma (MM) hampered their antitumor effects. Therefore, the development of functional iNKT cells may provide a novel strategy for the immunotherapy in MM treatment.
Experimental Design
We activated and expanded iNKT cells from MM patients with α-galactosylceramide(α-GalCer)-pulsed-dendritic cells (DCs), characterized their antitumor effects by the cytokine production profile and cytotoxicity against MM cells, and explored the effects of immunomodulatory drug lenalidomide on these iNKT cells. We also investigated the expression of CD1d by primary MM cells and its function to activate iNKT cells.
Results
We established highly purified functional iNKT cell lines from newly diagnosed and advanced MM patients. These CD1d-restricted iNKT cell lines produced high level of antitumor Th1 cytokine in response to α-GalCer-pulsed-primary MM cells, CD1d-transfected MM1S cell line or DCs. Moreover, MM iNKT cell lines displayed strong cytotoxicity against α-GalCer-pulsed-primary MM cells. Importantly, lenalidomide further augmented the Th1-polarization by iNKT cell lines via the increased Th1 cytokine production and the reduced Th2 cytokine production. We also demonstrated that CD1d was expressed in primary MM cells at mRNA and protein levels from the majority of MM patients, but not in normal plasma cells and MM cell lines, and CD1d+ primary MM cells presented antigens to activate iNKT cell lines.
Conclusions
Taken together, our results provide the pre-clinical evidence for the iNKT cells-mediated immunotherapy and a rationale for their use in combination with lenalidomide in MM treatment.
doi:10.1158/1078-0432.CCR-07-5290
PMCID: PMC2967459  PMID: 18980990
iNKT cells; multiple myeloma; lenalidomide; immunotherapy
13.  Circulating Myeloid Dendritic Cells of Advanced Cancer Patients Result in Reduced Activation and a Biased Cytokine Profile in Invariant NKT Cells1 
CD1d-restricted invariant NKT (iNKT) cells play important regulatory roles in various immune responses, including antitumor immune responses. Previous studies have demonstrated quantitative and qualitative defects in iNKT cells of cancer patients, and these defects are clinically relevant as they are associated with poor prognosis. In this study we demonstrate that defects in the iNKT cell population can, at least in part, be attributed to defective interactions between iNKT cells and CD1d-expressing circulating myeloid dendritic cells (mDC), as mDC of patients with advanced melanoma and renal cell cancer reduced the activation and Th1 cytokine production of healthy donor-derived iNKT cells. Interestingly, this reduced activation of iNKT cells was restricted to patients with low circulating iNKT cell numbers and could be reversed by IL-12 and in part by the neutralization of TGF-β, but it was further reduced by the neutralization of IL-10 in vitro. Additional experiments revealed discordant roles for TGF-β and IL-10 on human iNKT cells, because TGF-β suppressed iNKT cell activation and proliferation and IFN-γ production while IL-10 was identified as a cytokine involved in stimulating the activation and expansion of iNKT cells that could subsequently suppress NK cell and T cell responses.
PMCID: PMC2967461  PMID: 18490728
14.  Natural Killer T Cell Dysfunction in CD39-Null Mice Protects Against Concanavalin A–Induced Hepatitis 
Hepatology (Baltimore, Md.)  2008;48(3):841-852.
Concanavalin A (Con A)–induced injury is an established natural killer T (NKT) cell–mediated model of inflammation that has been used in studies of immune liver disease. Extracellular nucleotides, such as adenosine triphosphate, are released by Con A–stimulated cells and bind to specific purinergic type 2 receptors to modulate immune activation responses. Levels of extracellular nucleotides are in turn closely regulated by ectonucleotidases, such as CD39/NTPDase1. Effects of extracellular nucleotides and CD39 on NKT cell activation and upon hepatic inflammation have been largely unexplored to date. Here, we show that NKT cells express both CD39 and CD73/ecto-5’-nucleotidase and can therefore generate adenosine from extracellular nucleotides, whereas natural killer cells do not express CD73. In vivo, mice null for CD39 are protected from Con A–induced liver injury and show substantively lower serum levels of interleukin-4 and interferon-γ when compared with matched wild-type mice. Numbers of hepatic NKT cells are significantly decreased in CD39 null mice after Con A administration. Hepatic NKT cells express most P2X and P2Y receptors; exceptions include P2X3 and P2Y11. Heightened levels of apoptosis of CD39 null NKT cells in vivo and in vitro appear to be driven by unimpeded activation of the P2X7 receptor.
Conclusion
CD39 and CD73 are novel phenotypic markers of NKT cells. Deletion of CD39 modulates nucleotide-mediated cytokine production by, and limits apoptosis of, hepatic NKT cells providing protection against Con A–induced hepatitis. This study illustrates a further role for purinergic signaling in NKT-mediated mechanisms that result in liver immune injury.
doi:10.1002/hep.22401
PMCID: PMC2929828  PMID: 18752325
15.  Primary deficiency of microsomal triglyceride transfer protein in human abetalipoproteinemia is associated with loss of CD1 function 
The Journal of Clinical Investigation  2010;120(8):2889-2899.
Abetalipoproteinemia (ABL) is a rare Mendelian disorder of lipid metabolism due to genetic deficiency in microsomal triglyceride transfer protein (MTP). It is associated with defects in MTP-mediated lipid transfer onto apolipoprotein B (APOB) and impaired secretion of APOB-containing lipoproteins. Recently, MTP was shown to regulate the CD1 family of lipid antigen-presenting molecules, but little is known about immune function in ABL patients. Here, we have shown that ABL is characterized by immune defects affecting presentation of self and microbial lipid antigens by group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 molecules. In dendritic cells isolated from ABL patients, MTP deficiency was associated with increased proteasomal degradation of group 1 CD1 molecules. Although CD1d escaped degradation, it was unable to load antigens and exhibited functional defects similar to those affecting the group 1 CD1 molecules. The reduction in CD1 function resulted in impaired activation of CD1-restricted T and invariant natural killer T (iNKT) cells and reduced numbers and phenotypic alterations of iNKT cells consistent with central and peripheral CD1 defects in vivo. These data highlight MTP as a unique regulator of human metabolic and immune pathways and reveal that ABL is not only a disorder of lipid metabolism but also an immune disease involving CD1.
doi:10.1172/JCI42703
PMCID: PMC2912200  PMID: 20592474
16.  Defective NKT Cell Activation by CD1d+ TRAMP Prostate Tumor Cells Is Corrected by Interleukin-12 with alpha-Galactosylceramide 
PLoS ONE  2010;5(6):e11311.
Numerical and functional defects of invariant natural killer T cells (iNKT) have been documented in human and mouse cancers, resulting in a defect in IFN production in several malignancies. iNKT cells recognize glycolipids presented on CD1d molecules by dendritic and related cells, leading to their activation and thereby regulating immune reactions. Activated iNKT cells cytokine secretion and cytotoxicity can inhibit existing and spontaneous tumor growth, progression, and metastasis. We have identified functional iNKT cell defects in the murine TRAMP prostate cancer model. We found that iNKT cells show the ability to migrate into TRAMP prostate tumors. This infiltration was mediated through CCL2: CCR5 chemokine: receptor interaction. Prostate tumor cells expressing CD1d partially activated iNKT cells, as appreciated by up-regulation of CD25, PD-1 and the IL-12R. However, despite inducing up-regulation of these activation markers and, hence, delivering positive signals, prostate tumor cells inhibited the IL-12-induced STAT4 phosphorylation in a cell-cell contact dependent but CD1d-independent manner. Consequently, tumor cells did not induce secretion of IFNγ by iNKT cells. Blocking the inhibitory Ly49 receptor on iNKT cells in the presence of α-GalCer restored their IFNγ production in vivo and in vitro. However, Ly49 blockade alone was not sufficient. Importantly, this defect could be also be reversed into vigorous secretion of IFNγ by the addition of both IL-12 and the exogenous CD1d ligand alpha-galactosylceramide, but not by IL-12 alone, both in vivo and in vitro. These data underscore the potential to optimize iNKT-based therapeutic approaches.
doi:10.1371/journal.pone.0011311
PMCID: PMC2892484  PMID: 20593019
17.  Selective activation, expansion, and monitoring of human iNKT cells with a monoclonal antibody specific for the TCR α-chain CDR3 loop 
European journal of immunology  2008;38(6):1756-1766.
Summary
A significant fraction of CD1d-restricted T cells express an invariant T cell receptor (TCR) α-chain. These highly conserved ‘iNKT’ populations are important regulators of a wide spectrum of immune responses. The ability to directly identify and manipulate iNKT is essential to understanding their function and to exploit their therapeutic potential. To that end, we sought monoclonal and polyclonal antibodies specific for iNKT by immunizing CD1d KO mice, which lack iNKT, with a cyclic peptide modeled after the TCR-α CDR3 loop. One monoclonal antibody (mAb; 6B11) was specific for cloned and primary human but not rodent iNKT and the human invariant TCR-α, as shown by transfection and reactivity with human invariant TCR-α transgenic T cells ex vivo and in situ. 6B11 was utilized to identify, purify, and expand iNKT from an otherwise minor component of human peripheral blood lymphocytes and to specifically identify human iNKT in tissue. Thus, we report a novel and general strategy for the generation of monoclonal antibodies specific for the CDR3 loop encoded by the TCR of interest. Specifically, an anti-Vα24Jα18 CDR3 loop clonotypic TCR mAb is available for the enumeration and therapeutic manipulation of human and non-human primate iNKT populations.
doi:10.1002/eji.200737389
PMCID: PMC2864538  PMID: 18493987
anti-TCR; CD161; clonotypic; cyclic peptide; IL-4; invariant; NKT
18.  Paucity of CD4+ Natural Killer T (NKT) Lymphocytes in Sooty Mangabeys Is Associated with Lack of NKT Cell Depletion after SIV Infection 
PLoS ONE  2010;5(3):e9787.
Lack of chronic immune activation in the presence of persistent viremia is a key feature that distinguishes nonpathogenic simian immunodeficiency virus (SIV) infection in natural hosts from pathogenic SIV and HIV infection. To elucidate novel mechanisms downmodulating immune activation in natural hosts of SIV infection, we investigated natural killer T (NKT) lymphocytes in sooty mangabeys. NKT lymphocytes are a potent immunoregulatory arm of the innate immune system that recognize glycolipid antigens presented on the nonpolymorphic MHC-class I-like CD1d molecules. In a cross-sectional analysis of 50 SIV-negative and 50 naturally SIV-infected sooty mangabeys, ligand α-galactosylceramide loaded CD1d tetramers co-staining with Vα24-positive invariant NKT lymphocytes were detected at frequencies ≥0.002% of circulating T lymphocytes in approximately half of the animals. In contrast to published reports in Asian macaques, sooty mangabey NKT lymphocytes consisted of CD8+ and CD4/CD8 double-negative T lymphocytes that were CXCR3-positive and CCR5-negative suggesting that they trafficked to sites of inflammation without being susceptible to SIV infection. Consistent with these findings, there was no difference in the frequency or phenotype of NKT lymphocytes between SIV-negative and SIV-infected sooty mangabeys. On stimulation with α-galactosylceramide loaded on human CD1d molecules, sooty mangabey NKT lymphocytes underwent degranulation and secreted IFN-γ, TNF-α, IL-2, IL-13, and IL-10, indicating the presence of both effector and immunoregulatory functional capabilities. The unique absence of CD4+ NKT lymphocytes in sooty mangabeys, combined with their IL-10 cytokine-secreting ability and preservation following SIV infection, raises the possibility that NKT lymphocytes might play a role in downmodulating immune activation in SIV-infected sooty mangabeys.
doi:10.1371/journal.pone.0009787
PMCID: PMC2844411  PMID: 20352088
19.  Peripheral Blood Progenitor Cell Product Contains Th1-Biased Non-Invariant CD1d-Reactive NKT Cells 
Experimental hematology  2008;36(4):464-472.
OBJECTIVE
Bone marrow (BM) Th1 populations can contribute to graft-versus-leukemia (GvL) responses. G/GM-CSF-mobilized peripheral blood progenitor cells (PBPC) have become widely accepted alternatives to BM transplantation (BMT). T cells co-expressing NK proteins (NKT) include a CD1d-reactive subset which influence immunity by rapidly producing large amounts of Th1 and/or Th2 cytokines dependent upon microenvironment and disease. There are two types of CD1d-reactive NKT. “iNKT” express a semi-invariant TCR-α. Other “non-invariant” CD1d-reactive NKT from BM and liver produce large amounts of IL-4 or IFN-γ respectively, and within the intestine can be biased in either direction. Recent data suggests that NKT might contribute to clinical benefits of PBPC.
METHODS
To address these issues, we phenotypically and functionally studied PBPC NKT.
RESULTS
Similarly to BM, NKT-like cells were common in allogeneic and autologous PBPC, there were relatively few classical iNKT, but high CD1d-reactivity concentrated in NKT fractions. Significantly, PBPC CD1d-reactive cells were relatively Th1-biased and their presence was associated with better prognosis. G-CSF treatment of BM to yield PBPC in vivo as well as in vitro Th2-polarizes conventional T cells and iNKT. However, G-CSF treatment of BM in vitro produced Th1-biased NKT, providing a mechanism for opposite polarization of NKT from BM versus PBPC.
CONCLUSIONS
These results suggest distinct Th1 CD1d-reactive NKT cells could stimulate anti-tumor responses from those previously described, which can suppress GvHD.
doi:10.1016/j.exphem.2007.12.010
PMCID: PMC2390922  PMID: 18261838
20.  Hepatitis C Virus (HCV)-Specific CD8+ Cells Produce Transforming Growth Factor β That Can Suppress HCV-Specific T-Cell Responses▿  
Journal of Virology  2007;81(11):5882-5892.
Hepatitis C virus (HCV)-specific T-cell responses are rarely detected in peripheral blood, especially in the presence of human immunodeficiency virus (HIV) coinfection. Based on recent evidence that T-regulatory cells may be increased in chronic HCV, we hypothesized that functional blockade of regulatory cells could raise HCV-specific responses and might be differentially regulated in the setting of HIV coinfection. Three groups of subjects were studied: HCV monoinfected, HCV-HIV coinfected, and healthy controls. Frequencies of peripheral T cells specific for peptides derived from HCV core, HIV type 1 p24, and recall antigens were analyzed by gamma interferon (IFN-γ) enzyme-linked immunospot assay. HCV-specific T-cell responses were very weak in groups with HCV and HCV-HIV infections. Addition of blocking antibodies against transforming growth factor β1 (TGF-β1), -2, and -3 and interleukin-10 specifically increased the HCV-specific T-cell responses in both infected groups; however, this increase was attenuated in the group with HCV-HIV coinfection compared to HCV infection alone. No increase in recall antigen- or HIV-specific responses was observed. Flow cytometric sorter analysis demonstrated that regulatory-associated cytokines were produced by HCV-specific CD3+CD8+CD25− cells. Enhancement of the IFN-γ effect was observed for both CD4 and CD8 T cells and was mediated primarily by TGF-β1, -2, and -3 neutralization. In conclusion, blockade of TGF-β secretion could enhance peripheral HCV-specific T-cell responses even in the presence of HIV coinfection.
doi:10.1128/JVI.02202-06
PMCID: PMC1900307  PMID: 17376924
21.  CD1d Mediates T-Cell-Dependent Resistance to Secondary Infection with Encephalomyocarditis Virus (EMCV) In Vitro and Immune Response to EMCV Infection In Vivo 
Journal of Virology  2006;80(14):7146-7158.
The innate and adaptive immune responses have evolved distinct strategies for controlling different viral pathogens. Encephalomyocarditis virus (EMCV) is a picornavirus that can cause paralysis, diabetes, and myocarditis within days of infection. The optimal innate immune response against EMCV in vivo requires CD1d. Interaction of antigen-presenting cell CD1d with distinct natural killer T-cell (“NKT”) populations can induce rapid gamma interferon (IFN-γ) production and NK-cell activation. The T-cell response of CD1d-deficient mice (lacking all NKT cells) against acute EMCV infection was further studied in vitro and in vivo. EMCV persisted at higher levels in CD1d-knockout (KO) splenocyte cultures infected in vitro. Furthermore, optimal resistance to repeat cycles of EMCV infection in vitro was also shown to depend on CD1d. However, this was not reflected in the relative levels of NK-cell activation but rather by the responses of both CD4+ and CD8+ T-cell populations. Repeated EMCV infection in vitro induced less IFN-γ and alpha interferon (IFN-α) from CD1d-deficient splenocytes than with the wild type. Furthermore, the level of EMCV replication in wild-type splenocytes was markedly and specifically increased by addition of blocking anti-CD1d antibody. Depletion experiments demonstrated that dendritic cells contributed less than the combination of NK and NKT cells to anti-EMCV responses and that none of these cell types was the main source of IFN-α. Finally, EMCV infection in vivo produced higher levels of viremia in CD1d-KO mice than in wild-type animals, coupled with significantly less lymphocyte activation and IFN-α production. These results point to the existence of a previously unrecognized mechanism of rapid CD1d-dependent stimulation of the antiviral adaptive cellular immune response.
doi:10.1128/JVI.02745-05
PMCID: PMC1489038  PMID: 16809320
22.  Microsomal triglyceride transfer protein lipidation and control of CD1d on antigen-presenting cells 
Microsomal triglyceride transfer protein (MTP), an endoplasmic reticulum (ER) chaperone that loads lipids onto apolipoprotein B, also regulates CD1d presentation of glycolipid antigens in the liver and intestine. We show MTP RNA and protein in antigen-presenting cells (APCs) by reverse transcription–polymerase chain reaction and by immunoblotting of mouse liver mononuclear cells and mouse and human B cell lines. Functional MTP, demonstrated by specific triglyceride transfer activity, is present in both mouse splenocytes and a CD1d-positive mouse NKT hybridoma. In a novel in vitro transfer assay, purified MTP directly transfers phospholipids, but not triglycerides, to recombinant CD1d. Chemical inhibition of MTP lipid transfer does not affect major histocompatibility complex class II presentation of ovalbumin, but considerably reduces CD1d-mediated presentation of α-galactosylceramide (α-galcer) and endogenous antigens in mouse splenic and bone marrow–derived dendritic cells (DCs), as well as in human APC lines and monocyte-derived DCs. Silencing MTP expression in the human monocyte line U937 affects CD1d function, as shown by diminished presentation of α-galcer. We propose that MTP acts upstream of the saposins and functions as an ER chaperone by loading endogenous lipids onto nascent CD1d. Furthermore, our studies suggest that a small molecule inhibitor could be used to modulate the activity of NKT cells.
doi:10.1084/jem.20050183
PMCID: PMC2212858  PMID: 16087713
23.  Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis 
Journal of Clinical Investigation  2004;113(10):1490-1497.
While Crohn disease (CD) has been clearly identified as a Th1 inflammation, the immunopathogenesis of its counterpart inflammatory bowel disease, ulcerative colitis (UC), remains enigmatic. Here we show that lamina propria T (LPT) cells from UC patients produce significantly greater amounts of IL-13 (and IL-5) than control cells and little IFN-γ, whereas comparable cells from CD patients produce large amounts of IFN-γ and small amounts of IL-13. We then show that stimulation of UC LPT cells bearing an NK marker (CD161) with anti-CD2/anti-CD28 or with B cells expressing transfected CD1d induces substantial IL-13 production. While this provided firm evidence that the IL-13–producing cell is an NK T (NKT) cell, it became clear that this cell does not express invariant NKT cell receptors characteristic of most NKT cells since there was no increase in cells binding α-galactosylceramide–loaded tetramers, and α-galactosylceramide did not induce IL-13 secretion. Finally, we show that both human NKT cell lines as well as UC CD161+ LPT cells are cytotoxic for HT-29 epithelial cells and that this cytotoxicity is augmented by IL-13. These studies show that UC is associated with an atypical Th2 response mediated by nonclassical NKT cells producing IL-13 and having cytotoxic potential for epithelial cells.
doi:10.1172/JCI200419836
PMCID: PMC406524  PMID: 15146247
24.  Multiple immuno-regulatory defects in type-1 diabetes 
Susceptibility to immune-mediated diabetes (IMD) in humans and NOD mice involves their inherently defective T cell immunoregulatory abilities. We have followed natural killer (NK) T cell numbers in patients with IMD, both by flow cytometry using mAbs to the characteristic junctions found in the T cell receptors of this cell subtype, and by semiquantitative RT-PCR for the corresponding transcripts. Both before and after clinical onset, the representation of these cells in patients’ PBMCs is reduced. We also report low numbers of resting CD4+ CD25+ T cells in IMD patients, a subset of T cells shown to have important immunoregulatory functions in abrogating autoimmunities in 3-day thymectomized experimental mice. Whereas a biased Th1 to Th2 cytokine profile has been suggested to underlie the pathogenesis of IMD in both species, we found defective production of IFN-γ in our patients after in vitro stimulation of their PBMCs by phorbol-myristate acetate and ionomycin and both IFN-γ and IL-4 deficiencies in Vα24+ NK T–enriched cells. These data suggest that multiple immunoregulatory T (Treg) cell defects underlie islet cell autoimmunity leading to IMD in humans and that these lesions may be part of a broad T cell defect.
doi:10.1172/JCI13605
PMCID: PMC150819  PMID: 11781358
25.  Cd1-Reactive Natural Killer T Cells Are Required for Development of Systemic Tolerance through an Immune-Privileged Site 
The Journal of Experimental Medicine  1999;190(9):1215-1226.
Systemic tolerance can be elicited by introducing antigen into an immune-privileged site, such as the eye, or directly into the blood. Both routes of immunization result in a selective deficiency of systemic delayed type hypersensitivity. Although the experimental animal model of anterior chamber–associated immune deviation (ACAID) occurs in most mouse strains, ACAID cannot be induced in several mutant mouse strains that are coincidentally deficient in natural killer T (NKT) cells. Therefore, this model for immune-privileged site–mediated tolerance provided us with an excellent format for studying the role of NKT cells in the development of tolerance. The following data show that CD1-reactive NKT cells are required for the development of systemic tolerance induced via the eye as follows: (a) CD1 knockout mice were unable to develop ACAID unless they were reconstituted with NKT cells together with CD1+ antigen-presenting cells; (b) specific antibody depletion of NKT cells in vivo abrogated the development of ACAID; and (c) anti-CD1 monoclonal antibody treatment of wild-type mice prevented ACAID development. Significantly, CD1-reactive NKT cells were not required for intravenously induced systemic tolerance, thereby establishing that different mechanisms mediate development of tolerance to antigens inoculated by these routes. A critical role for NKT cells in the development of systemic tolerance associated with an immune-privileged site suggests a mechanism involving NKT cells in self-tolerance and their defects in autoimmunity.
PMCID: PMC2195676  PMID: 10544194
innate immunity ; immune deviation ; anterior chamber–associated immune deviation ; autoimmunity ; i.v. tolerance

Results 1-25 (27)