Venkataraman Sriram

Cell wall glycosphingolipids of Sphingomonas paucimobilis are CD1d-specific ligands for NKT cells

The current consensus on characterization of NKT cells is based on their reactivity to the synthetic glycolipid, α‐galactosylceramide (α‐GalCer) in a CD1d‐dependent manner. Because of the limited availability of α‐GalCer, there is a constant search for CD1d‐presented ligands that activate NKT cells. The α‐anomericity of the carbohydrate is considered to be an important requisite for the CD1d‐specific activation of NKT cells. The gram‐negative, lipopolysaccharide‐free bacterium Sphingomonas paucimobilis is known to contain glycosphingolipids (GSL) with α‐anomeric sugars attached to the lipid chain. Here, we report that GSL extracted from this bacterium are able to stimulate NKT cells in a CD1d‐specific manner. In addition, soluble CD1d–Ig dimers loaded with this lipid extract specifically bind to NKT cells (but not conventional T cells). Further studies on the S. paucimobilis GSL could potentially lead to other natural sources of CD1d‐specific ligands useful for NKT cell analyses and aimed at identifying novel therapies for a variety of disease states.

Impaired Assembly yet Normal Trafficking of MHC Class I Molecules in Tapasin Mutant Mice

Loading of peptides onto major histocompatibility complex class I molecules involves a multifactorial complex that includes tapasin (TPN), a membrane protein that tethers empty class I glycoproteins to the transporter associated with antigen processing. To evaluate the in vivo role of TPN, we have generated Tpn mutant mice. In these animals, most class I molecules exit the endoplasmic reticulum (ER) in the absence of stably bound peptides. Consequently, mutant animals have defects in class I cell surface expression, antigen presentation, CD8+ T cell development, and immune responses. These findings reveal a critical role of TPN for ER retention of empty class I molecules. Tpn mutant animals should prove useful for studies on alternative antigen-processing pathways that involve post-ER peptide loading.

Defective presentation of the CD1d1-restricted natural Va14Ja18 NKT lymphocyte antigen caused by β-d-glucosylceramide synthase deficiency

Va14Ja18 natural T (NKT) cells play an immunoregulatory role, which is controlled by a self glycolipid(s) presented by CD1d. Although the synthetic antigen α-D-galactosylceramide (α-D-GalCer) stimulates all Va14Ja18 NKT cells, α-anomeric D-glycosylceramides are currently unknown in mammals. We have used β-D-GalCer-deficient mice and β-D-glucosylceramide (β-D-GlcCer)-deficient cells to define the chemical nature of a natural NKT cell antigen. β-D-GalCer-deficient mice exhibit normal NKT cell development and function, and cells from these animals potently stimulate NKT hybridomas. In striking contrast, the same hybridomas fail to react to CD1d1 expressed by a β-D-GlcCer-deficient cell line. Importantly, human β-D-GlcCer synthase cDNA transfer, and hence the biosynthesis of β-D-GlcCer, restores the recognition of mutant cells expressing CD1d1 by the Va14Ja18 NKT hybridomas. Additionally, suppression of β-D-GlcCer synthesis inhibits antigen presentation to Va14Ja18 NKT cells. The possibility that β-D-GlcCer itself is the natural NKT cell antigen was excluded because it was unable to activate NKT hybridomas in a cell-free antigen-presentation assay. These findings suggest that β-D-GlcCer may play an important role in generating and/or loading a natural Va14Ja18 NKT antigen.

Recycling CD1d1 Molecules Present Endogenous Antigens Processed in an Endocytic Compartment to NKT Cells

Mouse CD1d1 molecules present endogenous glycolipids to NKT cells. Although glycolipid presentation requires CD1d1 transport through the endocytic pathway, the processing requirements for such endogenous Ag presentation by CD1d1 molecules are undefined. We examined CD1d1 Ag presentation to NKT cells by disrupting endocytic trafficking and function in cells expressing normal and mutated CD1d1 expressed by recombinant vaccinia viruses. Consistent with previous studies, we found that preventing CD1d1 localization to endosomes by altering its cytoplasmic targeting sequences abrogated recognition by Vα14Jα281+ NKT cells without affecting recognition by Vα14− NKT cells. Increasing the pH of acidic compartments by incubating cells with chloroquine or bafilomycin A1 blocked CD1d1 recognition by Vα14+ (but not Vα14−) NKT cells without reducing levels of cell surface CD1d1. Similar results were obtained with primaquine, which interferes with the recycling of cell surface glycoproteins. These results suggest that the loading of a subset of glycolipid ligands onto CD1d1 molecules entails the delivery of cell surface CD1d1 molecules and an acidic environment in the endocytic pathway.

Natural killer T (NKT) cells and their role in antitumor immunity

Natural killer T (NKT) cells have become a major focus for those who study the innate immune response to tumors and infectious diseases, as well as autoimmunity. These novel T lymphocytes produce both Th1 and Th2 cytokines, recognize phospholipid and glycolipid antigens presented by CD1 molecules in a similar manner as peptides are recognized by cytotoxic T lymphocytes (CTL), and kill tumor cell targets by a perforin-dependent mechanism like NK cells and CTL. These ascribed functions thus demonstrate that NKT cells are a unique cytotoxic effector cell subpopulation with a kaleidoscope of activities. Because they can mediate antitumor effects in vivo with or without the collaboration of NK cells, the study of NKT cells in antitumor immunity may lead to novel treatments based on the ability to manipulate the generation and/or activity of these multifunctional lymphocytes.

Selective Loss of Natural Killer T Cells by Apoptosis following Infection with Lymphocytic Choriomeningitis Virus

ABSTRACT Natural killer T (NKT) cells, a unique subpopulation of T cells, coexpress markers also present on NK cells and recognize the major histocompatibility complex class I-like CD1d1 molecule. We studied the effect of an acute virus infection on NKT cells. Mice were infected with the nonhepatotropic Armstrong strain of lymphocytic choriomeningitis virus (LCMV), and at various times postinfection, mononuclear cells from the liver, peritoneum, and spleen were isolated. It was found that within 2 to 3 days, there was a selective loss of NKT cells from the liver with an apparent rapid recovery within 8 to 14 days. There was no increase in peritoneal or splenic NKT cells, indicating that NKT cells did not traffic to these tissues. This loss of NKT cells was independent of gamma interferon (IFN-γ) and interleukin 12 (IL-12) production, but did occur in mice treated with poly(I-C), a classical inducer of IFN-α/β. The reduction in NKT cells was CD28 and fas/fasL independent and occurred via apoptosis. It was not observed in LCMV-infected DNA fragmentation factor 45-deficient mice, and an increase in active caspase 3-specific staining was found in liver NKT cells from LCMV-infected and poly(I-C)-treated mice compared to uninfected wild-type mice. Interestingly, it was also found that liver NKT cells from LCMV-infected mice were themselves infected. These results suggest that the loss of NKT cells following an acute LCMV infection could be due to the induction of IFN-α/β resulting in NKT-cell apoptosis and is important for the hosts immune response to LCMV.

Inhibition of glycolipid shedding rescues recognition of a CD1+ T cell lymphoma by natural killer T (NKT) cells.

Neoplastic transformation of cells is accompanied by an aberration of cell surface glycolipid composition. These tumor-associated, altered glycosphingolipids are often shed into the tumor cell microenvironment and mediate immunosuppressive activity. The nature and form of glycolipids shed by a variety of tumor cell lines and the mechanism(s) of shedding have been well characterized. The murine T cell lymphoma line, L5178Y-R, is known to shed a tumor-associated glycolipid, gangliotriaosylceramide, into the culture medium. We analyzed the effect of glycolipids from L5178Y-R on antigen presentation by murine CD1d1 molecules. CD1d1 molecules present glycolipid antigens to a specialized class of T cells called natural killer T (NKT) cells that mainly express a T cell receptor α chain (Vα14Jα281) associated with Vβ chains of limited diversity. In the current report, we found that L5178Y-R cells express CD1 on their cell surface yet are unable to stimulate CD1d1-specific NKT cells. We hypothesized that the glycolipid(s) shed by L5178Y-R inhibited antigen presentation by CD1d1. Pretreatment of CD1d1+ cells with conditioned medium from L5178Y-R inhibited CD1-specific stimulation of canonical (Vα14+) but not noncanonical (Vα5+) NKT cells. Exogenous addition of lipids extracted from L5178Y-R cells as well as purified gangliotriaosylceramide mimicked this effect. Inhibition of glycolipid shedding in L5178Y-R cells with d-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol resulted in the rescue of CD1d1 recognition by canonical (but not noncanonical) NKT cells. These results suggest that one means by which certain tumor cells can evade the hosts innate antitumor immune response is by shedding glycolipids that inhibit CD1-mediated antigen presentation to NKT cells.

Myeloid marker expression on antiviral CD8+ T cells following an acute virus infection

CD11b, CD11c, and F4/80 are normally used to define dendritic cell and/or macrophage populations. In this study, the expression of all three markers was observed on CD8+ T cells following infection of mice with several distinct viruses. Using lymphocytic choriomeningitis virus as a model virus, it was found that relatively more CD11b+CD8+ and CD11c+CD8+ T cells were present in the periphery than in primary lymphoid organs; in contrast, the F4/80+CD8+ T cell population was more prevalent in the spleen. All three myeloid markers were detected on virus‐specific CTL. The expression of CD11b and CD11c on CD8+ T cells correlated with their level of CTL activity, whereas the F4/80+CD8+ T cell population increased after the peak of the CTL response but did not have higher CTL activity. These data suggest that there is a differential induction of CD11b, CD11c, and F4/80 on virus‐specific CD8+ T cells following an acute virus infection.

Generation of cellular immunity to lymphocytic choriomeningitis virus is independent of CD1d1 expression

CD1 molecules are cell surface glycoproteins, structurally similar to major histocompatibility complex (MHC) class I molecules. The murine CD1d1 molecule has been shown to be essential for the positive selection of a unique subpopulation of T cells [the natural killer (NK) T cells], as CD1d1‐deficient mice lack NK T cells. These cells have recently been suggested to play an important role in the induction of innate immunity (i.e. NK cells) and the regulation of immune homeostasis. As such, it was asked whether NK T cells were necessary for the generation of cellular immunity to an acute virus infection. In these studies, the Armstrong strain of lymphocytic choriomeningitis virus (LCMV), a classic inducer of NK cells, and its pathogenic variant clone 13 were used. When NK‐cell activity was assessed on day 3 post‐LCMV infection, surprisingly, it was found that CD1d1‐deficient mice could generate NK‐cell activity at wild‐type levels. Likewise, LCMV‐specific cytotoxic T‐lymphocyte (CTL) activity in CD1d1‐deficient mice was indistinguishable from that generated in wild‐type mice. Additionally, viral titres in the spleen (LCMV Armstrong) and blood (LCMV clone 13) of infected CD1d1‐deficient mice were at comparable levels to those found in wild‐type mice, as were virus infection‐induced increases in cell surface H‐2Kb in the spleen. Therefore, these results suggest that the LCMV‐induced generation of NK‐cell and virus‐specific CTL activity, as well as viral clearance, are independent of CD1d1 expression.

Inhibition of antitumor immunity by invariant natural killer T cells in a T-cell lymphoma model in vivo

We have investigated the role of the hosts CD1d‐dependent innate antitumor immune response in a murine T‐cell lymphoma model in vivo. We found that C57BL/6 wildtype (WT) mice inoculated with RMA/S cells transfected with murine CD1d1 died at the same rate as mice inoculated with vector‐transfected cells. In contrast, natural killer T (NKT) cell‐deficient CD1d or Jα18 knockout mice inoculated with CD1d‐transfected RMA/S cells survived significantly longer than mice inoculated with vector‐transfected RMA/S cells, implicating the involvement of invariant NKT (iNKT) cells in inhibiting antitumor activity in vivo. In contrast to the mutant mice, which produced more of the proinflammatory cytokines IFN‐γ and GM‐CSF, WT mice produced significantly elevated amounts of IL‐13. Antitumor activity in the knockout mice was not due to the development of CD1d‐specific cytotoxic T lymphocytes or circulating antibodies. However, iNKT cell numbers were elevated in tumor‐bearing mice. Thus, iNKT cells may be playing a negative role in the hosts antitumor immune response against T‐cell lymphomas in a CD1d‐dependent manner.