NKT cells are a diverse group of cells that share. To Be or Not to Be NKT: Natural Killer T Cells in the Liver CONCISE REVIEW IN MECHANISMS OF DISEASE

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1 CONCISE REVIEW IN MECHANISMS OF DISEASE To Be or Not to Be NKT: Natural Killer T Cells in the Liver Mark A. Exley 1 and Margaret James Koziel 2 Much of the hepatology literature to date has focused on the adaptive, antigen-specific response mediated by classical T-cell populations in both the protection and pathogenesis of liver disease. However, the liver is selectively enriched for cells representative of innate immunity, including natural killer T (NKT) cells. In particular, certain CD1d-reactive T cells are present at much higher frequencies in the liver than in the peripheral blood. Although these cells have previously been defined mostly on the basis of phenotypic markers, recent emerging literature regarding NKT cell populations has revealed considerable functional complexity. This review summarizes the recent literature regarding NKT cells, which may have important roles in a variety of liver diseases. Although there is an abundance of literature on the phenotype, distribution, and function of these cells in mice, much less is known about them in human health or liver diseases. (HEPATOLOGY 2004;40: ) NKT cells are a diverse group of cells that share features of both classical T cells and natural killer (NK) cells, and have similarly diverse functions. One of the complexities in understanding NKT cells involves sorting through the various terminology that has been applied to them in the emerging literature. 1,2 Functionally, a major subset of these cells are defined by reactivity against CD1d, which is one of five Abbreviations: NKT, natural killer T; NK, natural killer; APC, antigen-presenting cell; GalCer, -galactosylceramide; IFN-, interferon gamma; IL, interleukin; DC, dendritic cell; TCR, T cell receptor; TNF-, tumor necrosis factor alpha; Th, T helper; Con-A, concanavalin A; PBC, primary biliary cirrhosis; HCV, hepatitis C virus. From the Divisions of 1 Hematology and Oncology and 2 Infectious Diseases, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA. Received June 23, 2004; accepted August 2, Financial support provided by NIH grants R01 DK068327, R01 AI (M.J.K.), and R01 DK (M.A.E.). M. A. E. is the recipient of an American Liver Foundation Seed award. Address reprint requests to: Mark A. Exley, Ph.D., Cancer Biology, New Research Building 1030L, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA mexley@bidmc.harvard.edu; fax: Copyright 2004 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience ( DOI /hep known glycolipid-binding nonpolymorphic major histocompatibility complex class 1 like glycoproteins: CD1a through e. 3 These glycoproteins appear to have evolved to facilitate recognition of nonprotein antigens specifically glycolipid antigens, including those present in the cell walls of mycobacterial species. CD1d is constitutively expressed on a number of hematopoietic-derived antigenpresenting cell (APC) types including B cells and myeloid cells and can be expressed by human T cells as well as healthy hepatocytes. Physiological ligands for CD1d remain poorly defined, although both glycolipids and hydrophobic peptides bind to CD1 family molecules. 4,5 Most antigen studies have used the synthetic glycolipid -galactosylceramide ( GalCer), which binds to CD1d and activates a specific subset of both murine and human NKT cells in vitro and in vivo. 5,6 CD1d is remarkably conserved across mammalian species, with mouse and human CD1d having greater than 90% sequence homology. 7 Consequently, murine CD1d-reactive T cells recognize human CD1d, and vice versa. 8 After activation, NKT cells are highly versatile and can produce large amounts of interferon gamma (IFN- ) and interleukin (IL)-4 9,10 as prototypic type 1 and type 2 cytokines, respectively, as well as display NK/lymphokine-activated killing like FasL-mediated or perforin-dependent CD1d-specific cytotoxicity thus potentially contributing to protective and pathogenic responses against multiple infectious agents and tumors. CD1d is expressed by dendritic cells (DCs), and Gal- Cer-loaded DCs mediate activation of invariant NKT cells Although peripheral NKT cells can recognize and respond to other CD1d cells, their in vivo activation may be initiated by interactions with DCs and possibly B cells. The interactions between invariant NKT cells and DCs share many features of interactions between conventional CD4 T cells and DCs. DCs pulsed with GalCer stimulate NKT cells through T cell receptor (TCR) ligation. 2,19,20 NKT cell activation and IFN- production are markedly enhanced by DC-produced IL-12, with an increase in the IL-12 receptor on activated NKT cells. 15,21,22 Conversely, invariant NKT cells also interact with Gal- Cer-loaded DCs to enhance CD4 and CD8 T-cell responses 16,17,23,24 and with B cells to provide help for antibody production. 25 However, Galcer is not a physiological antigen, and it is not yet clear whether physiological NKT DC interactions are mediated by specific CD1d-presented antigens or are antigen independent. The classical CD1d-reactive NKT cell population expresses a highly restricted TCR repertoire consisting of an invariant alpha chain rearrangement and restricted repertoire of V chains (invariant NKT cells). These TCRs are the murine V 14-J 18, which is preferentially paired 1033

2 1034 EXLEY AND KOZIEL HEPATOLOGY, November 2004 Fig. 1. Model for protective role of CD1d-reactive NKT cells in acute hepatitis and pathogenic role during chronic hepatotropic infection. CD1d-reactive T cells may contribute to effector cell activation during acute infection because they can stimulate NK cells and antiviral cytotoxic T lymphocytes. However, where acute responses fail to clear infection, chronic stimulation of the large hepatic CD1d-reactive T-cell population may cause liver damage directly via CD1d-specific cytotoxicity as well as indirectly through continued inflammatory cell recruitment and/or stimulation. Finally, in advanced disease, CD1d-reactive T-cell IL-4 and IL-13 could contribute to profibrotic activity resulting in stellate cell stimulation, although the specific cytokines elaborated by NKT cells that might directly activate stellate cells have not been identified. NKT, natural killer T; Th, T helper; IFN-, interferon gamma; IL, interleukin; MIP1-, macrophage inflammatory protein 1- ; APC, antigen-presenting cell; ESLD, end stage liver disease. with polyclonal V 8.2, or the highly homologous human V 24-J 18, which is commonly associated with V 11. 9,26 28 Invariant TCR CD1d-reactive T cells (invariant NKT cells) represent up to 50% of rodent intrahepatic lymphocytes, 29 which is also consistent with expression of the restricting element CD1d within the mammalian liver. 3,7,30,31 Most rodent invariant NKT cells of appropriate strains express the CD161 (NKR-P1) NK1.1 allele, 9 as do the human orthologues, 12 in addition to a variety of cell surface markers classically associated with NK cells; these include CD56, CD69, CD94, and CD161, and, in some cases (at least in mice), killer inhibitory receptors of the C-type lectin NK locus Ly49- type. 9,10,12,26,32 Classical invariant NKT cells are typically either CD4 or are CD4 /CD8 (double negative), although in the human there is an appreciable CD8 subset. Both mouse and human invariant NKT cells can be highly selectively identified by use of CD1d tetramers loaded with -GalCer and human invariant NKT by use of an anti-invariant TCR monoclonal antibody. 36 Diversity of NKT Cells Recent data indicate that there is considerably more complexity within NKT cell and CD1d-reactive T-cell populations than was previously appreciated (Table 1). NKT is a phenotypic definition for a wide variety of cells, whereas CD1d-restricted is a functional definition. Many more human NKT cells are major histocompatibility complex restricted and not CD1d restricted. Conversely, not all CD1d-restricted NKT cells express NK markers. As a result, the literature can be confusing because of multiple nomenclatures and the specific subpopulation of cells under study. In rats and mice, which lack CD1 genes other than CD1d, the majority of NKT cells are invariant CD1d-restricted T cells, and most invariant T cells are NKT cells. Even within the classical invariant NKT subset, there may be substantial functional diversity. For example, CD4 but CD1d-tetramer binding cells can produce type 1 cytokines IFN- and tumor necrosis factor alpha (TNF- ), and express NKG2D, a marker associated with cytolysis of microbially infected and neoplastic cells. 37 In contrast, CD4 /tetramer invariant NKT cells produce both type 1 and type 2 cytokines. 34,35 There has been an increasing appreciation of subpopulations of CD1d-reactive T cells with diverse TCR that do not recognize GalCer and thus do not react with GalCer-loaded CD1d tetramers. 29,38 41 Other phenotypic NKT cells are recently activated classical class 1-restricted CD8 cells or can express TCR, most of which are not CD1d-restricted and are CD4/CD8-double negative. 3 Furthermore, some lineages of mice do not express the NK1.1 allele, yet a large number of hepatic T cells

3 HEPATOLOGY, Vol. 40, No. 5, 2004 EXLEY AND KOZIEL 1035 Table 1. CD1d-Restricted and/or NKT Cell Populations: Population Known Locations NK Markers TCR Restriction Element Ligand* Role(s) Refs. Rodent Invariant NKT LN, liver,... V 14J 18 CD1d -Galcer Th1/2 (5, 9, 20, 22, 29, 32, 33, 93) Variant spleen, BM / Diverse CD1d not -Galcer Th1/2 (29, 38 41, 93) CD1d-restricted V 4 T LN, heart,...? V 4 CD1d not -Galcer Th1/2 (87) NKT cells wide Diverse MHC or CD1 diverse Cytolysis (1, 2, 4, 29, 32, 93) and Human Invariant NKT Blood, liver V 24J 18 CD1d -Galcer Th1/2 (10, 19, 34, 35, 43 48, 58) Variant CD1d Liver, BM / Diverse CD1d not -Galcer Th1/2 (54 56) V 3 T Liver / V 3 Unknown not -Galcer Th1/2 (1, 49) NKT cells Many Diverse and MHC or CD1 diverse Cytolysis (10, 19, 34, 35, 43 48) NOTE. Subsets of NKT in mouse and man. Primary locations cited: NK marker expression: indicates majority express NK markers. / indicates variable, some negative. indicates defined by presence of NK markers and TCR/CD3. Abbreviation: LN, lymph node. *Surrogate antigen, active natural ligands have yet to be reported. Original nomenclature: V 14J 18 V 14J 281; V 24J 18 V 24J Q. reactive to CD1d are found within this NK1.1-negative population. 33,42 In the mouse, classical invariant NKT cells appear to preferentially accumulate in the liver and thymus, 29,42 whereas CD1d-reactive T cells expressing more diverse TCRs are found in the spleen and bone marrow. 29,41,42 Therefore, relying solely on a phenotypic marker does not reflect the total functional activity of NKT. Unlike the rodent, there appear to be fewer invariant NKT cells within the human liver, and the phenotypic markers that best define hepatic NKT cells remain unclear. In humans, blood contamination may also complicate study. Most studies on NKT cells in humans have focused on the V 24 population, CD3 /CD56 cells, or CD3 /CD161 cells 44 from diseased tissue, and data regarding the distribution of V 24 cells in healthy human liver is limited. 47,49 We have recently identified and characterized human noninvariant CD1d-reactive T cells in the bone marrow, 54 the liver, 55,56 and the gut. 57 In contrast to human blood, bone marrow, or gut CD1d-reactive T cells, which express high amounts of type 2 cytokines like the homologous population in murine bone marrow, 10,25,41,54,57 human hepatic CD1d-reactive T cells are strongly type 1 biased. 55,56 CD1d reactivity is found in the noninvariant, CD56-negative, and even CD161-negative populations. 56 These functional data are consistent with data showing that CD3 / CD56 cells (often used as a marker of NKT cells) in normal human liver are type 1 biased 47,51,57 and with a recent report that hepatic invariant NKT cells behave like the major heterogeneous hepatic CD1d-reactive T cells. 58 Given that different subpopulations of NKT cells in mice have different functions and distribution, understanding the full spectrum of NKT cells in humans, whether defined phenotypically or by CD1d reactivity, 43 45,47 50,52 60 clearly requires further study. Roles of CD1d-Reactive NKT Cells Depending on location and stimulus, CD1d-reactive T cells have the novel property of modulating their spectrum of cytokine production toward either proinflammatory T helper (Th) 1 or anti-inflammatory Th2 cells. 41,45,49,54 56,58,61 By responding to infectious or other challenges prior to conventional adaptive T cells, CD1dreactive T cells could have the potential to influence the course of local and systemic bias of immune responses. Activated CD1d-reactive T cells can stimulate NK cells 59 and cytotoxic T lymphocytes, 23,24,62 actions dependent in part on the ability of the CD1d-reactive T cells to produce IFN-. In addition to the possible role of CD1d-reactive T-cell produced cytokines as facilitators of immune responses, IFN- and other cytokines may play a role in the clearance of pathogens, especially viral pathogens. CD1dreactive T cells may thus bridge the innate and adaptive arms of the immune response. Role in Control of Immune Responses Early interest in murine invariant CD1d-reactive T cells was sparked by their ability to rapidly produce large amounts of IL-4 and IFN- after stimulation, 9,10,32 suggesting a role in the development of Th2-

4 1036 EXLEY AND KOZIEL HEPATOLOGY, November 2004 type immune responses. Quantitative and qualitative defects in invariant NKT cells have been found in type 1 diabetes and a range of other human and model rodent autoimmune diseases. 63 CD1d-reactive T cells are also critical for immune tolerance to antigens at immune privileged sites and in neonatal tolerance. 64,65 Finally, invariant CD1d-reactive T cells promote tolerance in murine models of transplantation, such as cardiac allografts, 66 although the specific role of these cells in promoting tolerance of liver allografts is unknown. Therefore, under appropriate conditions, CD1d-reactive T cells can have anti-inflammatory and/or immunomodulatory properties. Role in Liver Disease In contrast to the anti-inflammatory effects observed in murine models of diabetes, CD1d-reactive T cells are also implicated in models of liver injury. Depletion of NK1.1 T cells ameliorates concanavalin A (Con-A) induced liver disease, 67 and CD1d-deficient mice are highly resistant to Con-A induced hepatitis. 68 Similar findings were observed in a model of Salmonella-induced liver injury. 69 Activation of CD1dreactive T cells, whether through Con-A or GalCer, results in a rapid induction of apoptosis of liver NKT cells and concomitant liver damage, 68,70 which appears to be based on both IFN- and TNF- production by NKT cells. 71,72 In addition, this process is dependent upon Fas FasL interactions, because NKT cells from gld mice (mutation in FasL) cannot induce Con-A mediated damage. 68 Alternatively, the production of cytokines may activate Kupffer cells and conventional T cells, resulting in further production of TNF- and liver injury. 72 Another model of liver injury is that of partial hepatectomy, and NKT cells may also play a role in the liver injury observed in that model. Following partial hepatectomy, there is expansion of NKT cells, and NKT-deficient mice have decreased serum aminotransferase levels compared with wild-type mice. 73,74 GalCer-enhanced liver damage during the early phase of regeneration in this model appears to be due to production of TNF- (instead of IFN- ) by NKT cells and high TNF receptor expression on hepatocytes, although TNF- may also contribute to liver regeneration. 75 The complexity of understanding the functional role of NKT cells in different models of liver disease pathogenesis is illustrated by findings in the ob/ob mouse, a murine model of fatty liver in which there is leptin deficiency. A recent report demonstrated that ob/ob mice have a deficiency of hepatic NKT cells due to reduced IL-15 and increased IL-12 production by Kupffer cells, 76 and the inability to regulate IL-12 may explain the sensitivity of these animals to endotoxin. 77 However, ob/ob mice are resistant to Con-A mediated hepatitis, which appears to be related to reduced production of TNF-. 78,79 Whether or not a deficiency and functional role of NKT cells are also found in human patients who have nonalcoholic fatty liver disease and excess leptin remains to be determined. In the human, there is evidence that NKT cells may play a role in autoimmune conditions such as primary biliary cirrhosis (PBC). Both CD1d expression and invariant NKT numbers increase in PBC compared with livers of healthy subjects. 80,84 CD1d is expressed at very low levels in healthy human as well as rodent hepatocytes, 7,30,31 but increases markedly on epithelioid granuloma cells in PBC. 81 CD1d is expressed on cholangiocytes and epithelial cells of the small bile ducts in earlier stage PBC and is lost in the late stages. A subsequent study demonstrated that CD3 /CD57 cells are increased in the portal tracts, in particular around the interlobular bile ducts, in patients with PBC. 82 However, the functional characteristics of these cells and their role in PBC and other forms of liver injury such as nonalcoholic fatty liver disease, alcohol-induced liver injury, and autoimmune hepatitis have not been explored in detail. Role in Infections of the Liver CD1d-reactive T cells within the liver have been shown to play a protective role in the clearance of multiple intrahepatic and other pathogens. Mice that lack CD1d or invariant NKT are more susceptible to specific infections, such as picornaviruses, 83 herpes simplex virus, 84 cerebral (though not systemic) malaria, 85 or Pseudomonas aeruginosa, 86 whereas in other circumstances a lack of NKT cells appears to ameliorate disease, such as viral myocarditis 87 or respiratory syncitia virus infection. 88 Of most interest to hepatologists is the role these cells might play in viral and other forms of hepatitis. In transgenic mouse models of hepatitis B virus replication, invariant NKT cells play a role in IFN- / and IFN- dependent inhibition of hepatitis B virus. 89 Within 24 hours of GalCer treatment, there is an increase in hepatic cytokines preceding the influx of leukocytes into the liver, supporting the role of resident invariant NKT cell populations in controlling hepatitis B virus. However, an elegant series of adoptive transfer experiments by Baron et al. demonstrated the potential of the hepatic nonclassical subset in the inflammation of acute hepatitis B. 90 Similarly, NKT cell deficiency results in higher bacterial burdens following Listeria infection, whereas CD1 antibody reduces the associated liver disease, 91,92 again potentially

5 HEPATOLOGY, Vol. 40, No. 5, 2004 EXLEY AND KOZIEL 1037 highlighting the importance of the different functional activities of different subsets. The role of CD1d-reactive T cells in hepatitis C virus (HCV) is currently unknown, because the available reports have focused primarily on analysis of phenotypic markers rather than functional analysis. Using V 24 as a surrogate marker for NKT cells, a relatively high frequency of these cells was found in the liver of patients with chronic HCV; furthermore, most of these cells appeared to have been recently activated, because they were undergoing apoptosis. 43,48,50 Conversely, a decreased frequency of CD3 /CD56 and V 24 cells in chronic HCV compared with healthy liver has been reported. 47 We have found that HCV human liver from patients with chronic infection has large numbers of diverse Th1 proinflammatory CD1d-reactive T cells, 55 which supports the hypothesis that these cells have the potential to cause hepatitis both directly through hepatic CD1d recognition and indirectly by stimulating other inflammatory immune cells. Focal CD1d staining was increased on cholangiocytes and inflammatory cells in chronic HCV infection, 56 as previously described in PBC. 80,81 Furthermore, nonclassical variant CD1d-reactive T cells were found in uninfected livers, although they were found more frequently in HCV-infected donors. Although CD1d-reactive T cells from mild cases had a strong Th1 bias, Th2 cytokines were also produced in advanced fibrosis and cirrhosis. Finally, the CD1d reactivity of CD161- negative populations was comparable to that of CD161 T cells, showing that CD1d-reactive T cells and NKT cells represent overlapping but not equivalent populations in humans 56 as well as mice. 2,29,93 Role in Tumor Surveillance In murine models, NKT cells appear to play an important role in tumor surveillance. Stimulation of invariant NKT cells with IL-12 increases their cytotoxic capability and protects against liver metastasis in animal models. 94 This conclusion was confirmed genetically with J 281 knockout mice, which cannot form the invariant V 14- J 18 TCR. These mice had fewer NKT cells and could not mediate IL-12 induced tumor rejection. 11 An increased incidence of mutagen-induced tumors was observed in invariant NKT / mice, and the spectrum of tumors is similar to that found in IL-12 / mice, implicating a physiological role for invariant NKT cells in IL- 12 mediated antitumor responses These results have been further supported by numerous studies showing that antitumor immunity can be augmented in normal mice by stimulating invariant NKT cells with GalCer. Indeed, GalCer was initially isolated from marine sponge in a screen for biological products with antitumor activity and was subsequently discovered to be a CD1d-presented antigen recognized specifically by invariant NKT cells. 5,6 NKT-like cells have been shown to mediate suppression of hepatocellular carcinoma following the adoptive transfer of tumor lines. 98,99 Production of IFN- and IL-12 by NKT cells and dendritic cells, respectively, appears to be responsible for the antitumor effect against either primary or metastatic disease in these models, 13,97,100,101 although activated invariant NKT cells can have NK/lymphokine-activated killing like and/or CD1d-specific cytoxic activity, 11,12,14,55 which could in principle contribute to antitumor responses. In addition to murine models of tumor development, data obtained from patients with various malignancies supports an antitumor function for invariant NKT cells. For example, NKT cells are reduced in advanced prostate cancer patients and have decreased proliferation and cytokine production compared with healthy controls. 36 Subsequent reports showed decreased proliferative invariant NKT cell responses from patients with a variety of neoplastic diseases 102 and showed that freshly isolated invariant NKT from patients with specifically progressive multiple myeloma had a marked decrease in IFN- production. 103 Livers from patients with metastatic disesase show a reduced frequency of V 24 NKT cells compared with healthy controls. 49 A phase 1 trial of GalCer-pulsed, monocyte-derived DCs in patients with advanced cancer demonstrated in vivo activation of invariant NKT cells, NK cells, and T cells, increased serum IFN- and IL-12, and several encouraging clinical responses. 104 Systemic treatment with GalCer or GalCer-loaded DCs do not cause increased serum aminotransferase levels or other evidence of liver damage in adult cancer patients, 104,105 presumably because of the small number of invariant NKT cells within the human liver. Conclusions CD1d-reactive T cells can have systemic anti-inflammatory or proinflammatory properties depending on location, disease, and stimulus. This may be based on the expression of different adhesion molecules and chemokine receptors on the surface of different subsets, which direct Th1 or Th2 cells to different locations. Other than the classical invariant NKT cells, the phenotypic markers of different subtypes of NKT cells have yet to be identified. A challenge for the liver disease research community will be defining the role of these cells in other inflammatory conditions of the liver, such as alcoholic hepatitis, autoimmune hepatitis, and steatohepatitis.

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