- [Show abstract] [Hide abstract] ABSTRACT: Peripheral T cell lymphomas (PTCLs) are a heterogeneous entity of neoplasms with poor prognosis, lack of effective therapies, and a largely unknown pathophysiology. Identifying the mechanism of lymphomagenesis and cell-of-origin from which PTCLs arise is crucial for the development of efficient treatment strategies. In addition to the well-described thymic lymphomas, we found that p53-deficient mice also developed mature PTCLs that did not originate from conventional T cells but from CD1d-restricted NKT cells. PTCLs showed phenotypic features of activated NKT cells, such as PD-1 up-regulation and loss of NK1.1 expression. Injections of heat-killed Streptococcus pneumonia , known to express glycolipid antigens activating NKT cells, increased the incidence of these PTCLs, whereas Escherichia coli injection did not. Gene expression profile analyses indicated a significant down-regulation of genes in the TCR signaling pathway in PTCL, a common feature of chronically activated T cells. Targeting TCR signaling pathway in lymphoma cells, either with cyclosporine A or anti-CD1d blocking antibody, prolonged mice survival. Importantly, we identified human CD1d-restricted lymphoma cells within Vδ1 TCR-expressing PTCL. These results define a new subtype of PTCL and pave the way for the development of blocking anti-CD1d antibody for therapeutic purposes in humans.
- [Show abstract] [Hide abstract] ABSTRACT: CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4(+), CD8α(+) and CD4(-)CD8α(-) double-negative (DN) subsets. CD4(+) iNKT cells expanded more readily than CD8α(+) and DN iNKT cells upon mitogen stimulation. CD8α(+) and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d(+) cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4(+) and CD8α(+) fractions, respectively. Only CD4(+) iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α(+), DN or CD4(+) iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.
- [Show abstract] [Hide abstract] ABSTRACT: Structural variants of α-galactosylceramide (αGC) that activate invariant natural killer T cells (iNKT cells) are being developed as potential immunomodulatory agents for a variety of applications. Identification of specific forms of these glycolipids that bias responses to favor production of proinflammatory vs anti-inflammatory cytokines is central to current efforts, but this goal has been hampered by the lack of in vitro screening assays that reliably predict the in vivo biological activity of these compounds. Here we describe a fluorescence-based assay to identify functionally distinct αGC analogues. Our assay is based on recent findings showing that presentation of glycolipid antigens by CD1d molecules localized to plasma membrane detergent-resistant microdomains (lipid rafts) is correlated with induction of interferon-γ secretion and Th1-biased cytokine responses. Using an assay that measures lipid raft residency of CD1d molecules loaded with αGC, we screened a library of ∼200 synthetic αGC analogues and identified 19 agonists with potential Th1-biasing activity. Analysis of a subset of these novel candidate Th1 type agonists in vivo in mice confirmed their ability to induce systemic cytokine responses consistent with a Th1 type bias. These results demonstrate the predictive value of this novel in vitro assay for assessing the in vivo functionality of glycolipid agonists and provide the basis for a relatively simple high-throughput assay for identification and functional classification of iNKT cell activating glycolipids.
Dataset: Figure S5[Show abstract] [Hide abstract] ABSTRACT: Comparison of cytokine responses of human iNKT cell clones between CD1d-transfected HeLa cells and Dendritic Cells, with a limited range of analog concentrations. CD1d-transfected HeLa cells (black bars) and monocyte-derived DCs (white bars) were incubated overnight in parallel with increasing concentrations of KRN7000, C20:2 (tested from left to right at 0.1, 1, 10 and 100 nM,) and higher concentrations of other analogs (10, 100 nM, 1 and 10 µM), then used as APC for iNKT cell stimulation. After 24 h of co-culture, supernatants were harvested, and cytokine levels were measured by ELISA. The sensitivity of detection was 27.4 pg/mL for IL-4 and 82.3 pg/mL for IL-13 and IFNγ. The data shown were generated with a CD4+ iNKT cell clone (HDD3) and a double negative (DN, HDE3). (0.12 MB TIF)
Dataset: Figure S7[Show abstract] [Hide abstract] ABSTRACT: Cytokine responses of human iNKT cell clones to a dose range of aGalCer analogs and costimulation by IL-12/IL-18. CD1d-transfected HeLa cells were incubated overnight with the indicated concentrations of KRN7000 and αGalCer analogs (concentration selected to provide suboptimal stimulation) and then used as APC for iNKT cell stimulation in absence (open bars) or presence of IL-12 and IL-18 (10 ng/mL and 50 ng/mL respectively, black bars). After 24 h of co-culture, supernatants were harvested, and cytokine levels were measured by ELISA. Average cytokine levels and standard deviations are indicated. The sensitivity of detection was 27.4 pg/mL for IL-4 and 82.3 pg/mL for IL-13, GM-CSF and IFNγ. The data shown were generated with a CD4+ iNKT cell clone (HDD3), and similar data (not shown) were obtained with a double negative clone (DN, HDE3). (0.11 MB TIF)
Dataset: Figure S2[Show abstract] [Hide abstract] ABSTRACT: Cytokine responses of a DN iNKT cell clone to a dose range of αGalCer analogs. The data shown were generated with the double negative (DN) HDE3 clone in the same manner as presented in Figure 2 for the CD4+ iNKT cell clone (HDD3). (0.12 MB TIF)
Dataset: Figure S1[Show abstract] [Hide abstract] ABSTRACT: Cytokine responses of human iNKT cell clones to CD1d-transfected HeLa cells. CD1d-transfected HeLa cells were used as APC for iNKT cell stimulation in the absence of added glycolipids. After 24h of co-culture, supernatants were harvested, and cytokine levels were measured by ELISA. The sensitivity of detection was 82.3 pg/mL for GM-CSF (white bars), IL-13 (black bars) and IFNγ (hatched bars). The data are shown for a variety of clones indicated at the bottom of the graph. No detectable reactivity was observed with mock-transfected HeLa cells (not shown). (0.32 MB TIF)
- [Show abstract] [Hide abstract] ABSTRACT: CD1d-restricted natural killer T cells with invariant T cell receptor α chains (iNKT cells) are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of α-galactosylceramide (αGalCer), the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-γ (IFNγ), and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-γ secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans.
Dataset: Figure S3[Show abstract] [Hide abstract] ABSTRACT: Cytokine responses of a CD8α+ iNKT cell clone to a dose range of αGalCer analogs. The data shown were generated with the CD8α+ clone HDA7, in the same manner as presented in Figure 2 for the CD4+ iNKT cell clone (HDD3). (0.12 MB TIF)
Dataset: Figure S6[Show abstract] [Hide abstract] ABSTRACT: Dose-dependent cytokine polarization of human iNKT cells with DCs. Variations in cytokine ratios depending on the analog concentration tested, IL-13/IFNγ ratios and IL-4/IFNγ ratios are presented on the bottom and top panel, respectively (calculated as explained in Materials and Methods). Ratios could be calculated only when IL-4 and/or IFNγ were detectable and the # symbol indicates ratios which could not be calculated. Concentrations of antigens are indicated as in Supplementary Figure S5, with low concentrations tested for KRN7000, C20:2 (0.1, 1, 10 and 100 nM) and high concentrations of other analogs (10, 100 nM, 1 and 10 µM). Data presented in this figure were interpreted from results shown in Supplementary Figure S5, and IL-13/IFNγ & IL-4/IFNγ ratios calculated for HDD3 (left panel) and HDE3 (right panel) clones with DCs are presented. (0.13 MB TIF)
Dataset: Figure S4[Show abstract] [Hide abstract] ABSTRACT: Human iNKT cell clone reactivity to OCH and C-Glycoside. (A) CD1d-transfected HeLa cells were incubated overnight with increasing concentrations of KRN7000 (ranging from 0.05 to 100 nM) and OCH or C-Glycoside (ranging from 39 nM to 10 µM) and then used as APC for iNKT cell stimulation. After 24 h of co-culture, supernatants were harvested, and cytokine levels were measured by ELISA. The sensitivity of detection was 27.4 pg/mL for IL-4 and 82.3 pg/mL for IL-13 and IFNγ. The data shown were generated with a CD4+ iNKT cell clone (HDD3), as well as a double negative (DN, HDE3). Dose-response curves are presented for IFNg, IL-4 and IL-13 (top to bottom panels respectively). Mean values measured from triplicate wells are indicated, and standard deviations are shown with brackets. Symbols for each glycolipid are indicated in the legend. (B) The same iNKT cell clones were stained with titrated amounts of analog-loaded hCD1d tetramers (1, 2.5, 5, 10, 20 and 40 nM final concentration), and the measured MFI is plotted for OCH-loaded tetramers (open inverted triangles) and C-Glycoside-loaded tetramers (black diamonds). (0.13 MB TIF)
- [Show abstract] [Hide abstract] ABSTRACT: Alpha-glucosyl ceramides 4 and 5 have been synthesised and evaluated for their ability to stimulate the activation and expansion of human iNKT cells. The key challenge in the synthesis of both target molecules was the stereoselective synthesis of the alpha-glycosidic linkage. Of the methods examined, glycosylation using per-TMS-protected glucosyl iodide 16 was completely alpha-selective and provided gram quantities of amine 11, from which alpha-glucosyl ceramides 4 and 5 were obtained by N-acylation. alpha-GlcCer 4, containing a C24 saturated acyl chain, stimulated a marked proliferation and expansion of human circulating iNKT cells in short-term cultures. alpha-GlcCer 5, which contains a C20 11,14-cis-diene acyl chain (C20:2), induced extremely similar levels of iNKT cell activation and expansion.
- [Show abstract] [Hide abstract] ABSTRACT: Several L-fucoglycolipids are associated with diseases such as cancer, cystic fibrosis and rheumatoid arthritis. Activation of iNKT cells is known to lead to the production of cytokines that can help alleviate or exacerbate these conditions. alpha-Galactosyl ceramide (alpha-GalCer) is a known agonist of iNKT cells and it is believed that its fucosyl counterpart might have similar immunogenic properties. We herein report the synthesis of alpha-L-fucosyl ceramide derivatives and describe their biological evaluation. The key challenge in the synthesis of the target molecules involved the stereoselective synthesis of the alpha-glycosidic linkage. Of the methods examined, the per-TMS-protected glycosyl iodide donor was completely alpha-selective, and could be scaled up to provide gram quantities of the azide precursor 11, from which a range of N-acylated alpha-L-fucosyl ceramides were readily obtained and evaluated for ex vivo expansion of human iNKT cells.
- [Show abstract] [Hide abstract] ABSTRACT: KRN7000 is an important ligand identified for CD1d protein of APC, and KRN7000/CD1d complex can stimulate NKT cells to release a broad range of bioactive cytokines. In an effort to understand the structure-activity relationships, we have carried out syntheses of 26 new KRN7000 analogues incorporating aromatic residues in either or both side chains. Structural variations of the phytosphingosine moiety also include varying stereochemistry at C3 and C4, and 4-deoxy and 3,4-dideoxy versions. Their biological activities are described.
- [Show abstract] [Hide abstract] ABSTRACT: The attenuated strain of Mycobacterium bovis known as bacille Calmette-Guérin (BCG) has been widely used as a vaccine for prevention of disease by Mycobacterium tuberculosis, but with relatively little evidence of success. Recent studies suggest that the failure of BCG may be due to its retention of immune evasion mechanisms that delay or prevent the priming of robust protective cell-mediated immunity. In this study, we describe an approach to enhance the immunogenicity of BCG by incorporating glycolipid activators of CD1d-restricted NKT cells, a conserved T cell subset with the potential to augment many types of immune responses. A method was developed for stably incorporating two forms of the NKT cell activator alpha-galactosylceramide into live BCG organisms, and the impact of this on stimulation of T cell responses and protective antimycobacterial immunity was evaluated. We found that live BCG containing relatively small amounts of incorporated alpha-galactosylceramide retained the ability to robustly activate NKT cells. Compared with immunization with unmodified BCG, the glycolipid-modified BCG stimulated increased maturation of dendritic cells and markedly augmented the priming of Ag-specific CD8(+) T cells responses. These effects were correlated with improved protective effects of vaccination in mice challenged with virulent M. tuberculosis. These results support the view that mycobacteria possess mechanisms to avoid stimulation of CD8(+) T cell responses and that such responses contribute significantly to protective immunity against these pathogens. Our findings raise the possibility of a simple modification of BCG that could yield a more effective vaccine for control of tuberculosis.
- [Show abstract] [Hide abstract] ABSTRACT: An alpha-galactosyl ceramide (alpha-GalCer) 2 was synthesized and evaluated for its ability to stimulate iNKT-cell proliferation and elicit T-helper cytokines, IL-4 and IFNgamma. Compound 2 combines the acyl chain of the potent, Th2 biasing alpha-GalCer 1 with a sphingoid base of the same length as that found in OCH, which also exhibits Th2 skewing, Such complementation may enhance cytokine bias, which is thought to be important for therapeutic applications of iNKT cell stimulation. Two related alpha-GalCers, 3 and 4, with saturated acyl chains were prepared for comparison.
- [Show abstract] [Hide abstract] ABSTRACT: CD1d-restricted natural killer T cells (NKT cells) possess a wide range of effector and regulatory activities that are related to their ability to secrete both T helper 1 (Th1) cell- and Th2 cell-type cytokines. We analyzed presentation of NKT cell activating alpha galactosylceramide (alphaGalCer) analogs that give predominantly Th2 cell-type cytokine responses to determine how ligand structure controls the outcome of NKT cell activation. Using a monoclonal antibody specific for alphaGalCer-CD1d complexes to visualize and quantitate glycolipid presentation, we found that Th2 cell-type cytokine-biasing ligands were characterized by rapid and direct loading of cell-surface CD1d proteins. Complexes formed by association of these Th2 cell-type cytokine-biasing alphaGalCer analogs with CD1d showed a distinctive exclusion from ganglioside-enriched, detergent-resistant plasma membrane microdomains of antigen-presenting cells. These findings help to explain how subtle alterations in glycolipid ligand structure can control the balance of proinflammatory and anti-inflammatory activities of NKT cells.
Dataset: Document S1. Five Figures
- [Show abstract] [Hide abstract] ABSTRACT: Invariant NKT cells (iNKT cells) recognize glycolipid Ags via an invariant TCR alpha-chain and play a central role in various immune responses. Although human CD4(+) and CD4(-) iNKT cell subsets both produce Th1 cytokines, the CD4(+) subset displays an enhanced ability to secrete Th2 cytokines and shows regulatory activity. We performed an ex vivo analysis of blood, liver, and tumor iNKT cells from patients with hepatocellular carcinoma and metastases from uveal melanoma or colon carcinoma. Frequencies of Valpha24/Vbeta11 iNKT cells were increased in tumors, especially in patients with hepatocellular carcinoma. The proportions of CD4(+), double negative, and CD8alpha(+) iNKT cell subsets in the blood of patients were similar to those of healthy donors. However, we consistently found that the proportion of CD4(+) iNKT cells increased gradually from blood to liver to tumor. Furthermore, CD4(+) iNKT cell clones generated from healthy donors were functionally distinct from their CD4(-) counterparts, exhibiting higher Th2 cytokine production and lower cytolytic activity. Thus, in the tumor microenvironment the iNKT cell repertoire is modified by the enrichment of CD4(+) iNKT cells, a subset able to generate Th2 cytokines that can inhibit the expansion of tumor Ag-specific CD8(+) T cells. Because CD4(+) iNKT cells appear inefficient in tumor defense and may even favor tumor growth and recurrence, novel iNKT-targeted therapies should restore CD4(-) iNKT cells at the tumor site and specifically induce Th1 cytokine production from all iNKT cell subsets.
- [Show abstract] [Hide abstract] ABSTRACT: KRN7000 is an important ligand identified for CD1d protein of APC, and KRN7000/CD1d complex can stimulate NKT cells to release Th1 and Th2 cytokines. In an effort to understand the structure-activity relationships, we have carried out the synthesis of a complete set of the eight KRN7000 stereoisomers, and their biological activities have been examined.