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1.  Physiological control of NKT cell-dependent hepatitis induction by extracellular adenosine 
European journal of immunology  2014;44(4):1119-1129.
Extracellular adenosine regulates inflammatory responses via A2A adenosine receptor (A2AR). A2AR-deficiency results in much exaggerated acute hepatitis, indicating non-redundancy of adenosine-A2AR pathway in inhibitory mechanisms of immune activation. To identify a critical target of immunoregulatory effect of extracellular adenosine, we focused on NKT cells, which play an indispensable role in hepatitis. A2AR agonist abolished NKT cell-dependent induction of acute hepatitis by Con A or α-galactosylceramide (α-GalCer), corresponding to down-regulation of activation markers and cytokines in NKT cells and of NK cell co-activation. These results show that A2AR signaling can down-regulate NKT cell activation and suppress NKT cell-triggered inflammatory responses. Next, we hypothesized that NKT cells might be under physiological control of the adenosine-A2AR pathway. Indeed, both Con A and α-GalCer induced more severe hepatitis in A2AR−/− mice than in wild-type controls. Transfer of A2AR−/− NKT cells into A2AR-expressing recipients resulted in exaggeration of Con A-induced liver damage, suggesting that NKT cell activation is controlled by endogenous adenosine via A2AR, and this physiological regulatory mechanism of NKT cells is critical in the control of tissue-damaging inflammation. The current study suggests the possibility to manipulate NKT cell activity in inflammatory disorders through intervention to the adenosine-A2AR pathway.
PMCID: PMC4482763  PMID: 24448964
NKT cell; adenosine; A2A adenosine receptor; hepatitis; immunoregulation
2.  Involvement of the iNKT Cell Pathway Is Associated With Early-Onset Eosinophilic Esophagitis and Response to Allergen Avoidance Therapy 
Recent experimental evidence suggests that environmental microbial factors early in life determine susceptibility to allergic diseases through inappropriate chemotaxis and local activation of CD1d-restricted, invariant chain natural killer T (iNKT) cells. In this study, we analyzed the involvement of these pathways in pediatric patients with eosinophilic esophagitis (EoE) before and after dietary allergen elimination.
mRNA expression levels of components of the C-X-C motif chemokine ligand 16 (CXCL16)–iNKT–CD1d axis were compared in esophageal biopsies from EoE patients vs. normal or inflammatory controls and before and after treatment.
CXCL16, iNKT cell–associated cell marker Vα24, and CD1d were significantly upregulated in esophageal biopsies from EoE patients and correlated with the expression of inflammatory mediators associated with allergy. Upregulation of each of these factors was significantly more pronounced in patients aged < 6 years at diagnosis, and this early-onset EoE subpopulation was characterized by a more prominent food allergic disease phenotype in a cohort-wide analysis. Successful, but not unsuccessful, treatment of early-onset EoE patients with dietary elimination of instigating allergens led to reduction in infiltrating iNKT cells and complete normalization of mRNA expression levels of CXCL16 and CD1d.
Our observations place iNKT cells at the center of allergic inflammation associated with EoE, which could have profound implications for our understanding, treatment and prevention of this and other human allergic diseases.
PMCID: PMC4132949  PMID: 24513807
3.  Human invariant NKT cell subsets differentially promote differentiation, antibody production and T cell stimulation by B cells in vitro1 
Invariant natural killer T (iNKT) cells can provide help for B cell activation and antibody production. Since B cells are also capable of cytokine production, antigen presentation and T cell activation, we hypothesized that iNKT cells will also influence these activities. Furthermore, subsets of iNKT cells based on CD4 and CD8 expression that have distinct functional activities may differentially affect B cell functions. We investigated the effects of co-culturing expanded human CD4+, CD8α+ and CD4−CD8α− double negative (DN) iNKT cells with autologous peripheral B cells in vitro. All iNKT cell subsets induced IgM, IgA and IgG release by B cells without needing the iNKT cell agonist ligand α-galactosylceramide (α-GC). Additionally, CD4+ iNKT cells induced expansions of cells with phenotypes of regulatory B cells. When co-cultured with α-GC-pulsed B cells, CD4+ and DN iNKT cells secreted Th1 and Th2 cytokines but at 10–1,000-fold lower levels than when cultured with dendritic cells. CD4+ iNKT cells reciprocally induced IL-4 and IL-10 production by B cells. DN iNKT cells expressed the cytotoxic degranulation marker CD107a upon exposure to B cells. Remarkably, while iNKT cell subsets could induce CD40 and CD86 expression by B cells, iNKT cell-matured B cells were unable to drive proliferation of autologous and alloreactive conventional T cells, as seen with B cells cultured in the absence of iNKT cells. Therefore, human CD4+, CD8α+ and DN iNKT cells can differentially promote and regulate the induction of antibody and T cell responses by B cells.
PMCID: PMC4201948  PMID: 23851681
4.  Ex vivo Analysis of Resident Hepatic Pro-inflammatory CD1d-reactive T cells & Hepatocyte Surface CD1d Expression in Hepatitis C 
Journal of viral hepatitis  2013;20(8):556-565.
Hepatic CD1d-restricted and natural killer T cell populations are heterogeneous. Classical ‘Type 1’ α-galactosylceramide-reactive CD1d-restricted T cells express ‘invariant’ TCRα (‘iNKT’). iNKT dominating rodent liver are implicated in inflammation, including in hepatitis models. Low levels of iNKT are detected in human liver, decreased in subjects with chronic hepatitis C (CHC). However, high levels of human hepatic CD161±CD56± non-invariant pro-inflammatory CD1d-restricted ‘Type 2’ T cells have been identified in vitro. Unlike rodents, healthy human hepatocytes only express trace and intracellular CD1d. Total hepatic CD1d appears to be increased in CHC and primary biliary cirrhosis.
Direct ex vivo analysis of human intra-hepatic lymphocytes (IHL), including matched ex vivo versus in vitro expanded IHL, demonstrated detectable non-invariant CD1d-reactivity in substantial proportions of HCV-positive livers and significant fractions of HCV-negative livers. However, α-galactosylceramide-reactive iNKT were detected only relatively rarely. Liver CD1d-restricted IHL produced IFNγ, variable levels of IL-10, and modest levels of Th2 cytokines IL-4 and IL-13 ex vivo. In a novel FACS assay, a major fraction (10–20%) of hepatic T cells rapidly produced IFNγ and up-regulated activation marker CD69 in response to CD1d. As previously only shown with murine iNKT, non-invariant human CD1d-specific responses were augmented by IL-12. Interestingly, CD1d was also found selectively expressed on the surface of hepatocytes in CHC, but not those CHC subjects with history of alcohol usage or resolved CHC. In contrast to hepatic iNKT, non-invariant IFNγ-producing Type 2 CD1d-reactive NKT cells are commonly detected in CHC, together with cognate ligand CD1d, implicating them in CHC liver damage.
PMCID: PMC4054599  PMID: 23808994
CD1; chronic; human; inflammation; NKT
5.  CD1d restriction and Th1/Th2/Th17 cytokine secretion by human Vδ3 T cells1 
Human γδ T cells expressing the Vδ3 TCR comprise a minor lymphocyte subset in blood but are enriched in liver and in patients with some chronic viral infections and leukemias. We analysed the frequencies, phenotypes, restriction elements and functions of fresh and expanded peripheral blood Vδ3 T cells. Vδ3 T cells accounted for ~0.2% of circulating T cells, included CD4+, CD8+ and CD4−CD8− subsets, and variably expressed CD56, CD161, HLA-DR and NKG2D, but not NKG2A nor NKG2C. Vδ3 T cells were sorted and expanded by mitogen stimulation in the presence of IL-2. Expanded Vδ3 T cells recognised CD1d, but not CD1a, CD1b nor CD1c. Upon activation, they killed CD1d+ target cells, released Th1, Th2 and Th17 cytokines and induced maturation of dendritic cells into APCs. Thus, Vδ3 T cells are glycolipid-reactive T cells with distinct antigen specificities but functional similarities to natural killer T cells.
PMCID: PMC3721026  PMID: 23740951
6.  Pulmonary Natural Killer T Cells Play an Essential Role in Mediating Hyperoxic Acute Lung Injury 
Critically ill patients are routinely exposed to high concentrations of supplemental oxygen for prolonged periods of time, which can be life-saving in the short term, but such exposure also causes severe lung injury and increases mortality. To address this therapeutic dilemma, we studied the mechanisms of the tissue-damaging effects of oxygen in mice. We show that pulmonary invariant natural killer T (iNKT) cells are unexpectedly crucial in the development of acute oxygen-induced lung injury. iNKT cells express high concentrations of the ectonucleotidase CD39, which regulates their state of activation. Both iNKT cell–deficient (Jα18−/−) and CD39-null mice tolerate hyperoxia, compared with wild-type control mice that exhibit severe lung injury. An adoptive transfer of wild-type iNKT cells into Jα18−/− mice results in hyperoxic lung injury, whereas the transfer of CD39-null iNKT cells does not. Pulmonary iNKT cell activation and proliferation are modulated by ATP-dependent purinergic signaling responses. Hyperoxic lung injury can be induced by selective P2X7-receptor blockade in CD39-null mice. Our data indicate that iNKT cells are involved in the pathogenesis of hyperoxic lung injury, and that tissue protection can be mediated through ATP-induced P2X7 receptor signaling, resulting in iNKT cell death. In conclusion, our data suggest that iNKT cells and purinergic signaling should be evaluated as potential novel therapeutic targets to prevent hyperoxic lung injury.
PMCID: PMC3707383  PMID: 23349052
lung injury; immunology; ATP; P2X7; oxygen
7.  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.
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.
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.
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.
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.
PMCID: PMC3508175  PMID: 22806830
Treg; IL-17; MMP-1; liver fibrosis
8.  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.
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.
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).
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.
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
9.  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.
PMCID: PMC3221878  PMID: 21996456
NK cells; Islets; Transplantation; Tolerance; Tregs
10.  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.
PMCID: PMC3447755  PMID: 23028326
11.  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.
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.
PMCID: PMC3143311  PMID: 21646050
cytokines; tumor immunity; CD1; CD1d-reactive T cells; iNKT; NKT
12.  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.
PMCID: PMC3298750  PMID: 21743479
13.  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.
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.
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.
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.
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.
PMCID: PMC2904642  PMID: 20618588
NKT cell subsets; α-GalCer; 6B11 mAb; sooty mangabey
14.  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.
PMCID: PMC2967447  PMID: 20039304
NKT cells; Cellular activation; Immune regulation
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.
PMCID: PMC2967442  PMID: 19949077
antibodies; cell activation; cytokines; NKT cells; viral infection
16.  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.
PMCID: PMC2967455  PMID: 19620318
17.  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.
PMCID: PMC2967458  PMID: 19265113
18.  Generation of Antitumor Invariant NKT Cell Lines in Multiple Myeloma and Promotion of Their Functions via Lenalidomide: A Strategy for Immunotherapy 
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.
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.
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.
PMCID: PMC2967459  PMID: 18980990
iNKT cells; multiple myeloma; lenalidomide; immunotherapy
19.  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
20.  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.
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.
PMCID: PMC2929828  PMID: 18752325
21.  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.
PMCID: PMC2912200  PMID: 20592474
22.  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.
PMCID: PMC2892484  PMID: 20593019
23.  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.
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.
PMCID: PMC2864538  PMID: 18493987
anti-TCR; CD161; clonotypic; cyclic peptide; IL-4; invariant; NKT
24.  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.
PMCID: PMC2844411  PMID: 20352088
25.  Peripheral Blood Progenitor Cell Product Contains Th1-Biased Non-Invariant CD1d-Reactive NKT Cells 
Experimental hematology  2008;36(4):464-472.
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.
To address these issues, we phenotypically and functionally studied PBPC NKT.
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.
These results suggest distinct Th1 CD1d-reactive NKT cells could stimulate anti-tumor responses from those previously described, which can suppress GvHD.
PMCID: PMC2390922  PMID: 18261838

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