Tonya J. Webb

Virus-induced inhibition of CD1d1-mediated antigen presentation: Reciprocal regulation by p38 and ERK

A critical component of the host’s innate immune response involves lipid Ag presentation by CD1d molecules to NK T cells. In this study we used murine CD1d1-transfected L (L-CD1) cells to study the effect of viruses on CD1d-mediated Ag presentation to NKT cells and found that an infection with vesicular stomatitis and vaccinia (but not lymphocytic choriomeningitis) virus inhibited murine CD1d1-mediated Ag presentation. This was under the reciprocal control of the MAPKs, p38 and ERK, and was due to changes in the intracellular trafficking of CD1d1. The reciprocal regulation of CD1d1-mediated Ag presentation by MAPK suggests that the targeting of these pathways is a novel means of immune evasion by viruses.

Inhibition of CD1d1-mediated antigen presentation by the vaccinia virus B1R and H5R molecules

Vaccinia virus (VV) has been most commonly used as the vaccine to protect individuals against the causative agent of smallpox (variola virus), but it also uses a number of strategies meant to evade or blunt the hosts antiviral immune response. Natural killer T (NKT) cells are a subset of immunoregulatory CD1d‐restricted T lymphocytes believed to bridge the innate and adaptive immune responses. It is shown here that the VV‐encoded molecules, B1R and H5R, play a role in the ability of VV to inhibit CD1d‐mediated antigen presentation to NKT cells. These are the first poxvirus‐encoded molecules identified that can play such a role in the evasion of an important component of the innate immune response.

Molecular identification of GD3 as a suppressor of the innate immune response in ovarian cancer

Tumors often display mechanisms to avoid or suppress immune recognition. One such mechanism is the shedding of gangliosides into the local tumor microenvironment, and a high concentration of circulating gangliosides is associated with poor prognosis. In this study, we identify ganglioside GD3, which was isolated from the polar lipid fraction of ovarian cancer-associated ascites, as an inhibitory factor that prevents innate immune activation of natural killer T (NKT) cells. Purified GD3 displayed a high affinity for both human and mouse CD1d, a molecule involved in the presentation of lipid antigens to T cells. Purified GD3, as well as substances within the ascites, bound to the CD1d antigenic-binding site and did not require additional processing for its inhibitory effect on NKT cells. Importantly, in vivo administration of GD3 inhibited α-galactosylceramide (α-GalCer)-induced NKT cell activation in a dose-dependent manner. These data therefore indicate that ovarian cancer tumors may use GD3 to inhibit the antitumor NKT cell response as an early mechanism of tumor immune evasion.

Dietary fatty acids modulate antigen presentation to hepatic NKT cells in nonalcoholic fatty liver disease

Dietary fatty acids are major contributors to the development and progression of insulin resistance and nonalcoholic fatty liver disease (NAFLD). Dietary fatty acids also alter hepatic NKT cells that are activated by antigens presented by CD1d. In the current study, we examine the mechanism of dietary fatty acid induced hepatic NKT cell deficiency and its causal relationship to insulin resistance and NAFLD. We discover that dietary saturated fatty acids (SFA) or monounsaturated fatty acids (MUFA), but not polyunsaturated fatty acids (PUFA), cause hepatic NKT cell depletion with increased apoptosis. Dietary SFA or MUFA also impair hepatocyte presentation of endogenous, but not exogenous, antigen to NKT cells, indicating alterations of the endogenous antigen processing or presenting pathway. In vitro treatment of normal hepatocytes with fatty acids also demonstrates impaired ability of CD1d to present endogenous antigen by dietary fatty acids. Furthermore, dietary SFA and MUFA activate the NFκB signaling pathway and lead to insulin resistance and hepatic steatosis. In conclusion, both dietary SFA and MUFA alter endogenous antigen presentation to hepatic NKT cells and contribute to NKT cell depletion, leading to further activation of inflammatory signaling, insulin resistance, and hepatic steatosis.

Differential innate immune cell activation and proinflammatory response in Anaplasma phagocytophilum infection.

ABSTRACT Human granulocytic anaplasmosis (HGA) is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. The critical role of gamma interferon (IFN-γ) for induction of severe inflammatory histopathology, even in the absence of a significant bacterial load, was previously demonstrated in a murine model of HGA. We hypothesized that NK, NKT, and possibly CD8+ cytotoxic T cells participate in the development of histopathologic lesions with A. phagocytophilum infection. Mice were mock infected or infected with low- or high-passage A. phagocytophilum and assayed for hepatic histopathology and splenocyte immunophenotype during the first 21 days after infection. Compared to high-passage A. phagocytophilum-infected mice, low-passage A. phagocytophilum-infected mice had more severe hepatic lesions and increased apoptosis. The hepatic histopathology severity in low-passage A. phagocytophilum-infected mice peaked on day 2 at the time of peak plasma IFN-γ levels and gradually decreased through day 21. Low-passage A. phagocytophilum-infected mice also showed significantly increased levels of lymphocyte NK1.1/FasL expression on days 4 to 7 corresponding to early, severe hepatic inflammation, whereas the levels of NKT cells were substantially lower on day 4, suggesting that there was NKT cell involvement. This result supports the concept that NK1.1+ cells, including NK and NKT cells, are major components in the early pathogenesis of A. phagocytophilum infection.

Raising the Roof: The Preferential Pharmacological Stimulation of Th1 and Th2 Responses Mediated by NKT Cells

Natural killer T (NKT) cells serve as a bridge between the innate and adaptive immune systems, and manipulating their effector functions can have therapeutic significances in the treatment of autoimmunity, transplant biology, infectious disease, and cancer. NKT cells are a subset of T cells that express cell‐surface markers characteristic of both natural killer cells and T cells. These unique immunologic cells have been demonstrated to serve as a link between the innate and adaptive immune systems through their potent cytokine production following the recognition of a range of lipid antigens, mediated through presentation of the major histocompatibility complex (MHC) class I like CD1d molecule, in addition to the NKT cells cytotoxic capabilities upon activation. Although a number of glycolipid antigens have been shown to complex with CD1d molecules, most notably the marine sponge derived glycolipid alpha‐galactosylceramide (α‐GalCer), there has been debate as to the identity of the endogenous activating lipid presented to the T‐cell receptor (TCR) via the CD1d molecule on antigen‐presenting cells (APCs). This review aims to survey the use of pharmacological agents and subsequent structure–activity relationships (SAR) that have given insight into the binding interaction of glycolipids with both the CD1d molecules as well as the TCR and the subsequent immunologic response of NKT cells. These studies not only elucidate basic binding interactions but also pave the way for future pharmacological modulation of NKT cell responses.

Ex vivo induction and expansion of natural killer T cells by CD1d1-Ig coated artificial antigen presenting cells

Natural killer T (NKT) cells play a pivotal role in maintaining immune homostasis. They recognize lipid antigen in the context of CD1d molecules and subsequently produce cytokines that activate cells of both the innate and adaptive immune responses. Many studies examining patients with autoimmune disease or cancer have shown that there is a reduction in both NKT cell number and function. Due to the complexities of manipulating NKT cells in vivo, ex vivo expanded effector NKT cells would be an excellent therapeutic modality. To date, immunotherapy utilizing the NKT/CD1d system has been dependent on the use of autologous DC in the presence or absence of a synthetic glycolipid, alpha-galactocylceramide. Here we report a novel technique that facilitates the growth and analysis of NKT cells through the use of CD1d-expressing aAPC. CD1d-based aAPC can effectively propagate both canonical (iNKT cells) and noncanonical (Valpha14(-)) NKT cells. Importantly, CD1d-Ig aAPC can expand NKT cells from cancer patients. Thus, CD1d-expressing aAPC will enhance our knowledge of NKT cell biology and could potentially be used as a novel tool in adoptive immunotherapeutic strategies.

The Interaction between Regulatory T Cells and NKT Cells in the Liver: A CD1d Bridge Links Innate and Adaptive Immunity

Background/Aims Regulatory T cells (Tregs) and natural killer T (NKT) cells are two distinct lymphocyte subsets that independently regulate hepatic adaptive and innate immunity, respectively. In the current study, we examine the interaction between Tregs and NKT cells to understand the mechanisms of cross immune regulation by these cells. Methods The frequency and function of Tregs were evaluated in wild type and NKT cell deficient (CD1dko) mice. In vitro lymphocyte proliferation and apoptosis assays were performed with NKT cells co-cultured with Tregs. The ability of Tregs to inhibit NKT cells in vivo was examined by adoptive transfer of Tregs in a model of NKT cell mediated hepatitis. Results CD1dko mice have a significant reduction in hepatic Tregs. Although, the Tregs from CD1dko mice remain functional and can suppress conventional T cells, their ability to suppress activation induced NKT cell proliferation and to promote NKT cell apoptosis is greatly diminished. These effects are CD1d dependent and require cell to cell contact. Adoptive transfer of Tregs inhibits NKT cell-mediated liver injury. Conclusions NKT cells promote Tregs, and Tregs inhibit NKT cells in a CD1d dependent manner requiring cell to cell contact. These cross-talk immune regulations provide a linkage between innate and adaptive immunity.

Ascites Specific Inhibition of CD1d-Mediated Activation of Natural Killer T Cells

Purpose: Natural killer T (NKT) cells recognize lipid antigen presented by CD1 molecules. NKT cells can both directly, through cytotoxicity, and indirectly, through activation of other effector cells, mediate antitumor immunity. It has been shown, however, that tumor-associated lipids are frequently shed into the tumor microenvironment, which can mediate immunosuppressive activity. Given that ovarian cancer–associated ascites has been reported to have increased levels of gangliosides, we examined the effect of tumor-associated and other ascites on CD1d-mediated antigen presentation to NKT cells. Experimental Design: To investigate the effects of ascites on NKT cell activation, we pretreated CD1d-expressing cells with the ascites and measured their ability to stimulate cytokine production in NKT cells. To determine whether antigen processing or editing was necessary, CD1d-immunoglobulin–based artificial antigen presenting cells (aAPC) were also incubated with ascites. In addition, to examine specificity, we analyzed whether ascites fluid could influence the activation of classic CD8+ T cells. Results: Pretreatment of CD1d-expressing cells with ascites from the majority of patients inhibited the ability of the cells to stimulate/activate NKT cells in a dose-dependent manner. Ascites treatment also partially blocked the ability of α-galactosylceramide–loaded CD1d-immunoglobulin–based aAPC to activate NKT cells. In addition, our data show that treatment with ascites does not inhibit HLA-A2–mediated activation of classic CD8+ T cells. Conclusions: Together, these data suggest that ovarian and other cancers may have developed immune evasion mechanisms specifically targeting the CD1/NKT cell system.

Probiotic antigens stimulate hepatic natural killer T cells

Increasing evidence suggests that gut flora play an important role in the pathogenesis of non‐alcoholic fatty liver disease (NAFLD). Our previous studies show that hepatic natural killer T (NKT) cells play a significant role in the pathogenesis of NAFLD. In this study, we explore the mechanism by which modification of gut flora leads to the alteration of hepatic NKT cells and improvement of steatosis. Mice were fed a high‐fat (HF) diet to induce NAFLD. Some of them also received different doses of mixed‐strain probiotics (VSL#3); single‐strain probiotic (Bifidobacterium infantis) or antibiotics. Animal weight, glucose tolerance, liver steatosis and hepatic NKT cells were assessed. Lipid extracts from probiotics were tested for their ability to activate NKT cells. Toll‐like receptor 4 (TLR4) knockout mice were also evaluated for their responses to HF diet. High‐dose VSL#3 was more effective than low‐dose VSL#3 and B. infantis for the improvement of hepatic NKT cell depletion and steatosis. The lipids extracted from VSL#3 stimulated NKT cells both in vivo and in vitro. In contrast, lipids from B. infantis decreased α‐GalCer‐mediated NKT cell activation in vitro, but were able to stimulate NKT cells. TLR4 knockout mice have a similar response to HF‐diet‐induced NKT cell depletion and obesity. These results suggest that alterations in the gut flora have profound effects on hepatic NKT cells and steatosis, which are both strain‐specific and dose‐dependent, but not through TLR4 signalling. Furthermore, these data suggest that probiotics may contain bacterial glycolipid antigens that directly modulate the effector functions of hepatic NKT cells.