Hepatic CD1d expression in hepatitis C virus infection and recognition by resident proinflammatory CD1d-reactive T cells

A subset of CD161(+)CD56(+/-) NKT cells can recognize glycolipids presented by CD1d and positively or negatively regulate inflammatory responses, including those implicated in several models of hepatitis. CD1d is expressed at very low levels in the healthy liver, but there is a large fraction of CD161(+)CD56(+) NKT cells. There are high levels of nonclassical proinflammatory hepatic CD1d-reactive T cells in hepatitis C virus (HCV) infection. Hepatic inflammatory cells and biliary cells adjacent to portal tract fibrotic areas of HCV-infected donors specifically up-regulated CD1d. A hepatocyte cell line expressing minimal CD1d was efficiently recognized by hepatic CD1d-reactive T cells, suggesting a role for these cells in disease. Hepatic CD1d-reactive T cells from HCV-positive as well as negative donors produced large amounts of IFN-gamma with some IL-13, but only rarely detectable IL-4. We confirmed large numbers of hepatic CD161(+) T cells, lower levels of CD56(+) T cells, and small numbers of classic invariant NKT cells. However, hepatic CD1d-reactivity was not restricted to any of these populations. We suggest virally infected hepatic cells can process potent CD1d-presented liver Ag(s), for surveillance by resident Th1 hepatic CD1d-reactive T cells. This process may be beneficial in acute viral clearance, but in chronic infection could contribute to liver injury.     

CD1d-specific NK1.1+ T cells with a transgenic variant TCR

The majority of T lymphocytes carrying the NK cell marker NK1.1 (NKT cells) depend on the CD1d molecule for their development and are distinguished by their potent capacity to rapidly secrete cytokines upon activation. A substantial fraction of NKT cells express a restricted TCR repertiore using an invariant TCR Valpha14-Jalpha281 rearrangement and a limited set of TCR Vbeta segments, implying recognition of a limited set of CD1d-associated ligands. A second group of CD1d-reactive T cells use diverse TCR potentially recognizing a larger diversity of ligands presented on CD1d. In TCR-transgenic mice carrying rearranged TCR genes from a CD1d-reactive T cell with the diverse type receptor (using Valpha3. 2/Vbeta9 rearrangements), the majority of T cells expressing the transgenic TCR had the typical phenotype of NKT cells. They expressed NK1.1, CD122, intermediate TCR levels, and markers indicating previous activation and were CD4/CD8 double negative or CD4+. Upon activation in vitro, the cells secreted large amounts of IL-4 and IFN-gamma, a characteristic of NKT cells. In mice lacking CD1d, TCR-transgenic cells with the NKT phenotype were absent. This demonstrates that a CD1d-reactive TCR of the "non-Valpha 14" diverse type can, in a ligand-dependent way, direct development of NK1.1+ T cells expressing expected functional and cell-surface phenotype characteristics.     

A major fraction of human bone marrow lymphocytes are Th2-like CD1d-reactive T cells that can suppress mixed lymphocyte responses.

Murine bone marrow (BM) NK T cells can suppress graft-vs-host disease, transplant rejection, and MLRs. Human BM contains T cells with similar potential. Human BM was enriched for NK T cells, approximately 50% of which recognized the nonpolymorphic CD1d molecule. In contrast to the well-characterized blood-derived CD1d-reactive invariant NK T cells, the majority of human BM CD1d-reactive T cells used diverse TCR. Healthy donor invariant NK T cells rapidly produce large amounts of IL-4 and IFN-gamma and can influence Th1/Th2 decision-making. Healthy donor BM CD1d-reactive T cells were Th2-biased and suppressed MLR and, unlike the former, responded preferentially to CD1d(+) lymphoid cells. These results identify a novel population of human T cells which may contribute to B cell development and/or maintain Th2 bias against autoimmune T cell responses against new B cell Ag receptors. Distinct CD1d-reactive T cell populations have the potential to suppress graft-vs-host disease and stimulate antitumor responses.     

DX5/CD49b-positive T cells are not synonymous with CD1d-dependent NKT cells

NKT cells are typically defined as CD1d-dependent T cells that carry an invariant TCR alpha-chain and produce high levels of cytokines. Traditionally, these cells were defined as NK1.1+ T cells, although only a few mouse strains express the NK1.1 molecule. A popular alternative marker for NKT cells has been DX5, an Ab that detects the CD49b integrin, expressed by most NK cells and a subset of T cells that resemble NKT cells. Interpretation of studies using DX5 as an NKT cell marker depends on how well DX5 defines NKT cells. Using a range of DX5 and other anti-CD49b Abs, we reveal major differences in reactivity depending on which Ab and which fluorochrome are used. The brightest, PE-conjugated reagents revealed that while most CD1d-dependent NKT cells expressed CD49b, they represented only a minority of CD49b+ T cells. Furthermore, CD49b+ T cell numbers were near normal in CD1d-/- mice that are completely deficient for NKT cells. CD1d tetramer- CD49b+ T cells differ from NKT cells by their activation and memory marker expression, tissue distribution, and CD4/CD8 coreceptor profile. Interestingly, both NKT cells and CD1d tetramer- CD49b+ T cells produce cytokines, but the latter are clearly biased toward Th1-type cytokines, in contrast to NKT cells that produce both Th1 and Th2 cytokines. Finally, we demonstrate that expression of CD49b by NKT cells does not dramatically alter with age, contrasting with earlier reports proposing DX5 as a maturation marker for NKT cells. In summary, our data demonstrate that DX5/CD49b is a poor marker for identifying CD1d-dependent NKT cells.     

NKT cells from normal and tumor-bearing human livers are phenotypically and functionally distinct from murine NKT cells

A major group of murine NK T (NKT) cells express an invariant Valpha14Jalpha18 TCR alpha-chain specific for glycolipid Ags presented by CD1d. Murine Valpha14Jalpha18(+) account for 30-50% of hepatic T cells and have potent antitumor activities. We have enumerated and characterized their human counterparts, Valpha24Vbeta11(+) NKT cells, freshly isolated from histologically normal and tumor-bearing livers. In contrast to mice, human NKT cells are found in small numbers in healthy liver (0.5% of CD3(+) cells) and blood (0.02%). In contrast to those in blood, most hepatic Valpha24(+) NKT cells express the Vbeta11 chain. They include CD4(+), CD8(+), and CD4(-)CD8(-) cells, and many express the NK cell markers CD56, CD161, and/or CD69. Importantly, human hepatic Valpha24(+) T cells are potent producers of IFN-gamma and TNF-alpha, but not IL-2 or IL-4, when stimulated pharmacologically or with the NKT cell ligand, alpha-galactosylceramide. Valpha24(+)Vbeta11(+) cell numbers are reduced in tumor-bearing compared with healthy liver (0.1 vs 0.5%; p < 0.04). However, hepatic cells from cancer patients and healthy donors release similar amounts of IFN-gamma in response to alpha-galactosylceramide. These data indicate that hepatic NKT cell repertoires are phenotypically and functionally distinct in humans and mice. Depletions of hepatic NKT cell subpopulations may underlie the susceptibility to metastatic liver disease.     

The effect of intracellular trafficking of CD1d on the formation of TCR repertoire of NKT cells

CD1 molecules belong to non-polymorphic MHC class I-like proteins and present lipid antigens to T cells. Five different CD1 genes (CD1a-e) have been identified and classified into two groups. Group 1 include CD1a-c and present pathogenic lipid antigens to αβ T cells reminiscence of peptide antigen presentation by MHC-I molecules. CD1d is the only member of Group 2 and presents foreign and self lipid antigens to a specialized subset of αβ T cells, NKT cells. NKT cells are involved in diverse immune responses through prompt and massive production of cytokines. CD1d-dependent NKT cells are categorized upon the usage of their T cell receptors. A major subtype of NKT cells (type I) is invariant NKT cells which utilize invariant Vα14-Jα18 TCR alpha chain in mouse. The remaining NKT cells (type II) utilize diverse TCR alpha chains. Engineered CD1d molecules with modified intracellular trafficking produce either type I or type II NKT cell-defects suggesting the lipid antigens for each subtypes of NKT cells are processed/generated in different intracellular compartments. Since the usage of TCR by a T cell is the result of antigen-driven selection, the intracellular metabolic pathways of lipid antigen are a key in forming the functional NKT cell repertoire. [BMB Reports 2014; 47(5): 241-248]     

Monte Carlo simulations of voltage-driven translocation of a signal sequence

CD1d-deficient (CD1d-/-) mouse lymphocytes were analyzed to classify the natural killer T (NKT) cells without reactivity to CD1d. The cells bearing a V(alpha)19.1-J(alpha)26 (AV19-AJ33) invariant TCR alpha chain, originally found in the peripheral blood lymphocytes, were demonstrated to be abundant in the NK1.1+ but not NK1.1- T cell population isolated from CD1d-/- mice. Moreover, more than half (11/21) of the hybrid cell lines established from CD1d-/- NKT cells expressed the V(alpha)19.1-J(alpha)26 invariant TCR alpha chain. The expression of the invariant V(alpha)19.1-J(alpha)26 mRNA was absent in beta2-microglobulin-deficient mice. Collectively, the present findings suggest the presence of a second NKT cell repertoire characterized by an invariant TCR alpha chain (V(alpha)19.1-J(alpha)26) that is selected by an MHC class I-like molecule other than CD1d.     

Structure-activity relationship studies of novel glycosphingolipids that stimulate natural killer T-cells

KRN7000, an anticancer drug candidate developed by Kirin Brewery Co. in 1995, is an α-galactosyl ceramide. It is a ligand making a complex with CD1d protein, and it stimulates invariant natural killer T (NKT) cells, which are one of the lineages of immunocytes. NKT cells activated by recognition of the CD1d/KRN7000 complex with its invariant T-cell receptor (TCR) can induce both protective and regulatory immune responses. To determine the recognition and activation mechanisms of NKT cells and to develop drug candidates more effective than KRN7000, a large number of analogs of KRN7000 have been synthesized. Some of them show potent bioactivities and have the potential of being utilized as therapeutic agents. In this review, structure-activity relationship studies of novel glycolipids which stimulate NKT cells efficiently are summarized.     

Activation of nonclassical CD1d-restricted NK T cells induces airway hyperreactivity in beta 2-microglobulin-deficient mice

Allergic asthma is characterized by Th2-driven eosinophilic airway inflammation and by a central feature called airway hyperreactivity (AHR), development of which requires the presence of classical type I invariant NK T (iNKT) cells. Allergen-induced AHR, however, develops in beta(2)-microglobulin (beta(2)m)(-/-) mice, which lack classical iNKT cells, suggesting that in some situations iNKT cells may be dispensable for the development of AHR. In contrast, our studies now suggest that a CD1d-restricted, NK1.1(+) noninvariant TCR NKT cell population is present in beta(2)m(-/-) mice and is responsible for the development of AHR but not for Th2 responses. Furthermore, treatment of beta(2)m(-/-) mice with anti-CD1d mAb or anti-NK1.1 mAb unexpectedly abolished allergen-induced AHR. The CD1-restricted NKT cells in these mice, which failed to respond to alpha-galactosylceramide and which therefore were not classical type I iNKT cells, appear to represent an NKT cell subset restricted by a beta(2)m-independent form of CD1d. These results indicate that, although classical type I iNKT cells are normally required for the development of AHR, under different circumstances other NKT cell subsets, including nonclassical NKT cells, may substitute for classical iNKT cells and induce AHR.     

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.     

CD1d-independent developmental acquisition of prompt IL-4 gene inducibility in thymus CD161(NK1)-CD44lowCD4+CD8- T cells is associated with complementarity determining region 3-diverse and biased Vbeta2/Vbeta7/Vbeta8/Valpha3.2 T cell receptor usage

Among Ag-inexperienced naive T cells, the CD1d-restricted NKT cell that uses invariant TCR-alpha-chain is the most widely studied cell capable of prompt IL-4 inducibility. We show in this study that thymus CD161-CD44lowCD4+CD8- T cells promptly produce IL-4 upon TCR stimulation, a response that displays biased Vbeta(2/7/8) and Valpha3.2 TCR usage. The association of Vbeta family bias and IL-4 inducibility in thymus CD161-CD44lowCD4+CD8- T cells is found for B6, B10, BALB/c, CBA, B10.A(4R), and ICR mouse strains. Despite reduced IL-4 inducibility, there is a similarly biased Vbeta(2/7/8) TCR usage by IL-4 inducibility+ spleen CD161-CD44lowCD4+CD8- T cells. Removal of alpha-galacotosylceramide/CD1d-binding cells from CD161-CD44lowCD4+CD8- thymocytes does not significantly affect their IL-4 inducibility. The development of thymus CD161-CD44lowCD4+CD8- T cells endowed with IL-4 inducibility and their associated use of Vbeta(2/7/8) are beta2-microglobulin-, CD1d-, and p59fyn-independent. Thymus CD161-CD44lowCD4+CD8- T cells produce low and no IFN-gamma inducibility in response to TCR stimulation and to IL-12 + IL-18, respectively, and they express diverse complementarity determining region 3 sequences for both TCR-alpha- and -beta-chains. Taken together, these results demonstrate the existence of a NKT cell distinct, TCR-repertoire diverse naive CD4+ T cell subset capable of prompt IL-4 inducibility. This subset has the potential to participate in immune response to a relatively large number of Ags. The more prevalent nature of this unique T cell subset in the thymus than the periphery implies roles it might play in intrathymic T cell development and may provide a framework upon which mechanisms of developmentally regulated IL-4 gene inducibility can be studied.     

Differential regulation of Th1 and Th2 functions of NKT cells by CD28 and CD40 costimulatory pathways

Valpha14 NKT cells produce large amounts of IFN-gamma and IL-4 upon recognition of their specific ligand alpha-galactosylceramide (alpha-GalCer) by their invariant TCR. We show here that NKT cells constitutively express CD28, and that blockade of CD28-CD80/CD86 interactions by anti-CD80 and anti-CD86 mAbs inhibits the alpha-GalCer-induced IFN-gamma and IL-4 production by splenic Valpha14 NKT cells. On the other, the blockade of CD40-CD154 interactions by anti-CD154 mAb inhibited alpha-GalCer-induced IFN-gamma production, but not IL-4 production. Consistent with these findings, CD28-deficient mice showed impaired IFN-gamma and IL-4 production in response to alpha-GalCer stimulation in vitro and in vivo, whereas production of IFN-gamma but not IL-4 was impaired in CD40-deficient mice. Moreover, alpha-GalCer-induced Th1-type responses, represented by enhanced cytotoxic activity of splenic or hepatic mononuclear cells and antimetastatic effect, were impaired in both CD28-deficient mice and CD40-deficient mice. In contrast, alpha-GalCer-induced Th2-type responses, represented by serum IgE and IgG1 elevation, were impaired in the absence of the CD28 costimulatory pathway but not in the absence of the CD40 costimulatory pathway. These results indicate that CD28-CD80/CD86 and CD40-CD154 costimulatory pathways differentially contribute to the regulation of Th1 and Th2 functions of Valpha14 NKT cells in vivo.     

Valpha24-JalphaQ-independent,CD1d-restricted recognition of alpha-galactosylceramide by human CD4(+) and CD8alphabeta(+) T lymphocytes

Human CD1d molecules present an unknown ligand, mimicked by the synthetic glycosphingolipid alpha-galactosylceramide (alphaGC), to a highly conserved NKT cell subset expressing an invariant TCR Valpha24-JalphaQ paired with Vbeta11 chain (Valpha24(+)Vbeta11(+) invariant NK T cell (NKT(inv))). The developmental pathway of Valpha24(+)Vbeta11(+)NKT(inv) is still unclear, but recent studies in mice were consistent with a TCR instructive, rather than a stochastic, model of differentiation. Using CD1d-alphaGC-tetramers, we demonstrate that in humans, TCR variable domains other than Valpha24 and Vbeta11 can mediate specific recognition of CD1d-alphaGC. In contrast to Valpha24(+)Vbeta11(+)NKT(inv) cells, Valpha24(-)/CD1d-alphaGC-specific T cells express either CD8alphabeta or CD4 molecules, but they are never CD4 CD8 double negative. We show that CD8alphabeta(+)Valpha24(-)/CD1d-alphaGC-specific T cells exhibit CD8-dependent specific cytotoxicity and have lower affinity TCRs than Valpha24(+)/CD1d-alphaGC-specific T cells. In conclusion, our results demonstrate that, contrary to the currently held view, recognition of CD1d-alphaGC complex in humans is not uniformly restricted to the Valpha24-JalphaQ/Vbeta11 NKT cell subset, but can be mediated by a diverse range of Valpha and Vbeta domains. The existence of a diverse repertoire of CD1d-alphaGC-specific T cells in humans strongly supports their Ag-driven selection.     

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.     

Regulation by Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 of alpha-galactosylceramide-induced antimetastatic activity and Th1 and Th2 responses of NKT cells

Interaction of alpha-galactosylceramide (alpha-GalCer) presented by CD1d on dendritic cells (DCs) with the invariant TCR of NKT cells activates NKT cells. We have now investigated the role of Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), a transmembrane protein abundantly expressed on DCs, in regulation of NKT cells with the use of mice that express a mutant form of SHPS-1. The suppression by alpha-GalCer of experimental lung metastasis was markedly attenuated in SHPS-1 mutant mice compared with that apparent in wild-type (WT) mice. The antimetastatic effect induced by adoptive transfer of alpha-GalCer-pulsed DCs from SHPS-1 mutant mice was also reduced compared with that apparent with WT DCs. Both the production of IFN-gamma and IL-4 as well as cell proliferation in response to alpha-GalCer in vitro were greatly attenuated in splenocytes or hepatic mononuclear cells from SHPS-1 mutant mice compared with the responses of WT cells. Moreover, CD4+ mononuclear cells incubated with alpha-GalCer and CD11c+ DCs from SHPS-1 mutant mice produced markedly smaller amounts of IFN-gamma and IL-4 than did those incubated with alpha-GalCer and CD11c+ DCs from WT mice. SHPS-1 on DCs thus appears to be essential for alpha-GalCer-induced antimetastatic activity and Th1 and Th2 responses of NKT cells. Moreover, our recent findings suggest that SHPS-1 on DCs is also essential for the priming of CD4+ T cells by DCs.     

Expression of CD1d molecules by human schwann cells and potential interactions with immunoregulatory invariant NK T cells

CD1d-restricted NKT cells expressing invariant TCR alpha-chains (iNKT cells) produce both proinflammatory and anti-inflammatory cytokines rapidly upon activation, and are believed to play an important role in both host defense and immunoregulation. To address the potential implications of iNKT cell responses for infectious or inflammatory diseases of the nervous system, we investigated the expression of CD1d in human peripheral nerve. We found that CD1d was expressed on the surface of Schwann cells in situ and on primary or immortalized Schwann cell lines in culture. Schwann cells activated iNKT cells in a CD1d-dependent manner in the presence of alpha-galactosylceramide. Surprisingly, the cytokine production of iNKT cells stimulated by alpha-galactosylceramide presented by CD1d+ Schwann cells showed a predominance of Th2-associated cytokines such as IL-5 and IL-13 with a marked deficiency of proinflammatory Th1 cytokines such as IFN-gamma or TNF-alpha. Our findings suggest a mechanism by which iNKT cells may restrain inflammatory responses in peripheral nerves, and raise the possibility that the expression of CD1d by Schwann cells could be relevant in the pathogenesis of infectious and inflammatory diseases of the peripheral nervous system.     

NKT cells in the rat: organ-specific distribution of NK T cells expressing distinct V alpha 14 chains

Rat invariant TCR alpha-chains and NKT cells were investigated to clarify whether CD1d-mediated recognition by NKT cells is conserved further in evolution. Rats had multiple-copies of TRAV14 genes, which can be categorized into two types according to the diversity accumulated in the CDR2 region. Rats retained invariant TCR alpha forms with the homogeneous junctional region similar to mouse invariant TRAV14-J281. The proportion of invariant TCR among V alpha 14+ clones was 12.9% in the thymus and increased in the periphery, 31% in the spleen and 95% in hepatic sinusoidal cells. The invariant TRAV14-J281 was expressed by liver sinusoidal and splenic NKT cells with CD8, CD44high, and TCR V beta 8. Type 1 invariant TCR alpha was expressed more frequently in hepatic lymphocytes, while type 2 invariant TCR alpha was expressed predominantly in the spleen. Both types of cells cytolyzed to and were stimulated to proliferate by CD1d-expressing cells in a CD1d-restricted manner. These results suggested that rat NKT cells bearing distinct V alpha 14 chains are distributed in a tissue-specific pattern. NKT cell populations in rats were more variable than those in mice, indicating that they play novel roles in nature. The implication of the molecular interaction between the structurally diverse invariant TCR alpha and CD1d/ligand complex in different organs is discussed.     

Resistance to malarial infection is achieved by the cooperation of NK1.1(+) and NK1.1(-) subsets of intermediate TCR cells which are constituents of innate immunity

We previously reported that the major expanding lymphocytes were intermediate TCR (TCR(int)) cells (mainly NK1.1(-)) during malarial infection in mice. Cell transfer experiments of TCR(int) cells indicated that these T cells mediated resistance to malaria. However, TCR(int) cells always contain NK1.1(+)TCR(int) cells (i.e., NKT cells) and controversial results (NKT cells were effective or not for resistance to malaria) have been reported by different investigators. In this study, we used CD1d((-/-)) mice, which almost completely lack NKT cells in the liver and other immune organs. Parasitemia was prolonged in the blood of CD1d((-/-)) mice and the expansion of lymphocytes in the liver of these mice was more prominent after an injection of Plasmodium yoelii-infected erythrocytes. However, these mice finally recovered from malaria. In contrast to B6 mice, CD4(-)8(-) NKT cells as well as NK1.1(-)CD3(int) cells expanded in CD1d((-/-)) mice after malarial infection, instead of CD4(+) (and CD8(+)) NKT cells. These newly generated CD4(-)8(-)NKT cells in CD1d((-/-)) mice did not use an invariant chain of Valpha14Jalpha281 for TCRalpha. Other evidence was that severe thymic atrophy and autoantibody production were accompanied by malarial infection, irrespective of the mice used. These results suggest that both NK1.1(-) and NK1.1(+) subsets of TCR(int) cells (i.e., constituents of innate immunity) are associated with resistance to malaria and that an autoimmune-like state is induced during malarial infection.     

CD1 expression and CD1-restricted T cell activity in normal and tumour-bearing human liver

CD1d-restricted natural killer T (NKT) cells expressing invariant Vα14Jα18 T cell receptor α-chains are abundant in murine liver and are implicated in the control of malignancy, infection and autoimmunity. Invariant NKT cells have potent anti-metastatic effects in mice and phase I clinical trials involving their homologues in humans are ongoing. However, invariant NKT cells are less abundant in human liver (∼0.5% of hepatic T cells) than in murine liver (up to 50%) and it is not known if other hepatic T cells are CD1-restricted. We have examined expression of CD1a, CD1b, CD1c and CD1d mRNA and protein in human liver and evaluated the reactivity of mononuclear cells (MNC) from histologically normal and tumour-bearing human liver specimens against these CD1 isoforms. Messenger RNA for all CD1 isotypes was detectable in all liver samples. CD1c and CD1d were expressed at the protein level by hepatic MNC. CD1d, only, was detectable at the cell surface, but CD1c and CD1d were found at an intracellular location in significant numbers of liver MNC. CD1b was not expressed by MNC from healthy livers but was detectable within MNC in all tumour samples tested. Hepatic T cells exhibited reactivity against C1R cells expressing transfected CD1c and CD1d, but neither CD1a nor CD1b. These cells secreted interferon-γ (IFN-γ) but not interleukin-4 (IL-4) upon stimulation. In contrast, similar numbers of peripheral T cells released 13- and 16-fold less IFN-γ in response to CD1c and CD1d, respectively. CD1c and CD1d expression and T cell reactivity were not altered in tumour-bearing liver specimens compared to histologically normal livers. These data suggest that, in addition to invariant CD1d-restricted NKT cells, autoreactive T cells that recognise CD1c and CD1d and release inflammatory cytokines are abundant in human liver.     

A subset of NKT cells that lacks the NK1.1 marker, expresses CD1d molecules, and autopresents the alpha-galactosylceramide antigen

In the present report, we characterize a novel T cell subset that shares with the NKT cell lineage both CD1d-restriction and high reactivity in vivo and in vitro to the alpha-galactosylceramide (alpha-GalCer) glycolipid. These cells preferentially use the canonical Valpha14-Jalpha281 TCR-alpha-chain and Vbeta8 TCR-beta segments, and are stimulated by alpha-GalCer in a CD1d-dependent fashion. However, in contrast to classical NKT cells, they lack the NK1.1 marker and express high surface levels of CD1d molecules. In addition, this NK1.1(-) CD1d(high) T subset, further referred to as CD1d(high) NKT cells, can be distinguished by its unique functional features. Although NK1.1(+) NKT cells require exogenous CD1d-presenting cells to make them responsive to alpha-GalCer, CD1d(high) NKT cells can engage their own surface CD1d in an autocrine and/or paracrine manner. Furthermore, in response to alpha-GalCer, CD1d(high) NKT cells produce high amounts of IL-4 and moderate amounts of IFN-gamma, a cytokine profile more consistent with a Th2-like phenotype rather than the Th0-like phenotype typical of NK1.1(+) NKT cells. Our work reveals a far greater level of complexity within the NKT cell population than previously recognized and provides the first evidence for T cells that can be activated upon TCR ligation by CD1d-restricted recognition of their ligand in the absence of conventional APCs.