C-type lectin Mincle-dependent and -independent activation of invariant NKT cells by exposure to Helicobacter pylori α-cholesteryl glucosides.

Helicobacter pylori extracts cholesterol from the host and converts it to its glycosides. We found that cholesteryl 6'-O-acyl α-glucoside (ChAcαG) produced by H. pylori is recognised by both invariant Vα14+ NKT (iNKT) cells and a C-type lectin receptor Mincle (Clec4e). However, it is unclear how these duplicated recognitions cooperate and contribute to host defence against H. pylori. Among T cell populations in the liver, iNKT cells predominantly expressed the T cell activation marker CD69 just after stimulation with ChAcαG. The production of IFN-γ and IL-4 was strictly dependent on both CD1d and Jα18 expressions, indicating the necessity of iNKT cell activation for the initiation of immune responses. Production of IFN-γ by iNKT cells was markedly reduced by the Mincle deficiency on antigen-presenting cells (APCs), while IL-4 production was not significantly influenced. IL-2 production by iNKT cell hybridomas was also diminished by the Mincle deficiency upon stimulation with APCs previously loaded with ChAcαG. Here, the immune responses of iNKT cell hybridomas stimulated with wild-type APCs were reduced by the addition of anti-IL-12 blocking antibody to the level stimulated with Mincle-deficient APCs. Collectively, these results suggest that iNKT cells can be activated with the cholesteryl glycosides via a Mincle-dependent, IL-12 signal-dependent pathway and a Mincle-independent, invariant TCR signal-dominant pathway. iNKT cells activated via the Mincle-dependent pathway produce IFN-γ-dominant cytokines; hence, they may contribute to enhancing proinflammatory responses against H. pylori infection.

Structure, metabolism and biological functions of steryl glycosides in mammals.

Steryl glycosides (SGs) are sterols glycosylated at their 3ß-hydroxy group. They are widely distributed in plants, algae, and fungi, but are relatively rare in bacteria and animals. Glycosylation of sterols, resulting in important components of the cell membrane SGs, alters their biophysical properties and confers resistance against stress by freezing or heat shock to cells. Besides, many biological functions in animals have been suggested from the observations of SG administration. Recently, cholesteryl glucosides synthesized via the transglycosidation by glucocerebrosidases (GBAs) were found in the central nervous system of animals. Identification of patients with congenital mutations in GBA genes or availability of respective animal models will enable investigation of the function of such endogenously synthesized cholesteryl glycosides by genetic approaches. In addition, mechanisms of the host immune responses against pathogenic bacterial SGs have partially been resolved. This review is focused on the biological functions of SGs in mammals taking into consideration their therapeutic applications in the future.

Invariant natural killer T cells stimulated with cholesteryl glycosides modulate immune responses in allergy and delayed-type hypersensitivity.

Invariant NKT cells were stimulated with cholesteryl O-acyl α-glycosides in the context of CD1d. The activated NKT cells have potential to sustain the homeostasis in the body exposed to excess in either Th1- or Th2-immunity.

Activation of invariant natural killer T cells stimulated with microbial α-mannosyl glycolipids.

Some synthetic and bacterial glycolipids presented by CD1d specifically activate invariant NKT (iNKT) cells bearing an invariant Vα14-Jα18 (mouse) or Vα24-Jα18 (human) TCR. The antigenic glycolipids identified to date consist of two hydrophobic chains and an α-glycoside in which the 2'-OH group is in the cis orientation toward the anomeric group, namely, either an α-galactoside or an α-glucoside. Several microbial α-mannosyl glycolipids, in which the 2'-OH group is in the trans orientation, were herein examined to establish whether they have potential to activate iNKT cells. We found that α-mannnosyl1-3 (6'-O-acyl α-mannosyl)-1-1 monoacylglycerol and cholesteryl 6'-O-acyl α-mannoside, found in Saccharopolyspora and Candida albicans, respectively, induced the activation of iNKT cells, dependent on CD1d. In contrast, α-mannosyldiacylglycerol found in Streptococcus suis or α-mannosylceramide demonstrated markedly less antigenicity for iNKT cells. The potentially antigenic α-mannosyl glycolipids contributed to the protection of mice against infection with S. pneumoniae in which iNKT cells have previously been found to participate. Furthermore, these glycolipids induced the production of proinflammatory cytokines by macrophages, thereby suggesting their recognition by specific pattern recognition receptors (PRRs). Collectively, these results suggest that these microbial α-mannosyl glycolipids are capable of being recognized by both the invariant TCR and PRRs and inducing immune responses.

Immunological functions of steryl glycosides.

Steryl glycosides, sterols glycosylated at the 3ß-hydroxy group, have been widely found in plants, algae, and fungi, but are rare in bacteria and animals. Glycosylation of sterols is known to modify properties of the cell membrane and confer resistance against stresses by freezing or heat-shock on cells. Furthermore, accumulating evidence obtained from recent research suggests important biological functions of steryl glycosides, including regulation of host defenses against pathogens, lipid metabolism, and developmental events. This review is focused on the immunological functions of steryl glycosides, such as modulation of host immune functions upon exposure to cholesteryl glycosides produced by pathogenic bacteria.

Modulation of immunoglobulin production by invariant Vα19-Jα33 TCR-bearing cells.

We have previously shown that invariant Vα19-Jα33 TCR(+) (Vα19i T) cells suppress the disease progress in some models for organ specific autoimmune diseases and type IV allergy that deteriorate along with decline to excess in Th1- or Th17- immunity. In this study, we examined the effects of over-generation of Vα19i T cells on the Th2-controlled immunoglobulin isotype production in the models for type I allergy. IgE production by invariant Vα19-Jα33 TCR transgenic (Tg) mice was suppressed compared with that by non-Tg controls following administration with goat anti-mouse IgD antiserum or OVA, while IgG2a production was not influenced by the introduction of the transgene into the recipients. IgE production by wild type mice was similarly reduced when they were subjected to adoptive transfer with invariant Vα19-Jα33 TCR Tg(+) but not Tg(-) cells prior to immunization. Furthermore, the suppression of IgE production by these recipients was enhanced when they were previously administered with a Vα19i T cell activator, one of the modified α-mannosyl ceramides. In summary, it is suggested that Vα19i T cells have potential to participate in the homeostasis of immunity and that they suppress disease progression resulting from not only Th1- but also Th2- immunity excess.

Regulation of immunological disorders by invariant Vα19-Jα33 TCR-bearing cells.

We have previously shown that over-expression of the invariant Vα19-Jα33 TCR α transgene (Tg) using a natural TCR α promoter in mice induces the development of NK1.1(+) T cells (Vα19 NKT cells) in lymphoid organs, including the liver and intestine. These cells produce different spectra of immunoregulatory cytokines such as IL-4, IL-10, IL-17, and IFN-γ depending on the duration and intensity of the invariant TCR stimulation. In this study, we examined the effects of over-expression of invariant Vα19-Jα33 TCR-bearing cells on disease progress in the models of immunological disorders. The introduction of invariant Vα19 TCR Tg into non-obese diabetic mice delayed the onset of the disease. In addition, delayed-type hypersensitivity (DTH) to sheep erythrocytes was suppressed in the Vα19 Tg mice. DTH was also suppressed in the wild type mice previously transferred with Vα19 Tg(+) but not non-Tg cells. Thus, invariant Vα19 TCR-bearing cells are suggested to participate in the homeostasis of immunity to suppress disease progression resulting from Th1-immunity excess.

Influenza infection in suckling mice expands an NKT cell subset that protects against airway hyperreactivity.

Infection with influenza A virus represents a major public health threat worldwide, particularly in patients with asthma. However, immunity induced by influenza A virus may have beneficial effects, particularly in young children, that might protect against the later development of asthma, as suggested by the hygiene hypothesis. Herein, we show that infection of suckling mice with influenza A virus protected the mice as adults against allergen-induced airway hyperreactivity (AHR), a cardinal feature of asthma. The protective effect was associated with the preferential expansion of CD4-CD8-, but not CD4+, NKT cells and required T-bet and TLR7. Adoptive transfer of this cell population into allergen-sensitized adult mice suppressed the development of allergen-induced AHR, an effect associated with expansion of the allergen-specific forkhead box p3+ (Foxp3+) Treg cell population. Influenza-induced protection was mimicked by treating suckling mice with a glycolipid derived from Helicobacter pylori (a bacterium associated with protection against asthma) that activated NKT cells in a CD1d-restricted fashion. These findings suggest what we believe to be a novel pathway that can regulate AHR, and a new therapeutic strategy (treatment with glycolipid activators of this NKT cell population) for asthma.

Altered production of immunoregulatory cytokines by invariant Valpha19 TCR-bearing cells dependent on the duration and intensity of TCR engagement.

Cells bearing invariant Valpha19-Jalpha33 TCR alpha chains are believed to participate in the regulation of inflammatory autoimmune diseases. In this study, the potential to produce immunoregulatory cytokines by these cells was characterized in order to find the mechanism underlying their immunoregulatory functions. Serum levels of IL-4, IL-10, transforming growth factor-beta, IFN-gamma and IL-17 increased in mice over-expressing an invariant Valpha19-Jalpha33 TCR alpha transgene (Valpha19 Tg) in response to anti-CD3 antibody injection. NK1.1(+) Valpha19 Tg(+), but not NK1.1(-) Valpha19 Tg(+) cells, promptly produced immunoregulatory IL-4, IFN-gamma and IL-17 upon invariant TCR engagement with immobilized anti-CD3 antibody in culture. The activation of Valpha19 Tg(+) cells then triggered the production of pro-inflammatory cytokines by bystander cells. Interestingly, the ratio of T(h)2 cytokines such as IL-4, IL-5 and IL-10, but not pro-inflammatory IL-17, to IFN-gamma was increased when the intensity of the stimulation to invariant TCR was attenuated. Collectively, these findings suggest that invariant Valpha19 TCR(+) cells have the potential to participate in the regulation of inflammatory autoimmunity by producing T(h)2-biased cytokines in certain circumstances.

Localization of NK1.1(+) invariant Valpha19 TCR(+) cells in the liver with potential to promptly respond to TCR stimulation.

Previously, we found that more than a half of the NK1.1(+) T cell lines prepared from CD1(-/-) livers expressed invariant Valpha19-Jalpha33 TCR alpha chains. Over-expression of the invariant Valpha19-Jalpha33 TCR alpha transgene (Tg) with a natural TCR alpha promoter and an enhancer in mice induced the development of NK1.1(+) T cells (Valpha19 NKT cells) in the lymphoid organs, especially in the liver. Preferential usage of the Valpha19 Tg by NKT cells in the transgenic mouse livers was indirectly indicated by the observation that few NK1.1(+) TCRalphabeta(+) cells of the Valpha19 Tg livers were stained with a cocktail of anti-TCR Valpha antibodies in the FACS analysis. Upon invariant TCR engagement in vivo following injection of mice with anti-CD3 antibody, NKT cells of the Tg mouse livers as well as spleens promptly produced immunoregulatory cytokines such as IL-4 and IFN-gamma and altered surface receptor expression. Collectively, localization of Valpha19 NKT cells in the liver is suggested that are ready to immediately response against antigen stimulation.

Non-reducing end alpha-mannosylated glycolipids as potent activators for invariant Valpha19 TCR-bearing natural killer T cells.

A novel invariant Valpha19-Jalpha33 T cell receptor alpha chain, first found in mammalian blood cells, was primarily expressed by natural killer T cell repertoire (Valpha19 NKT cell). Attempts have been made to find specific antigens for Valpha19 NKT cells. A series of alpha- and beta-glycosyl ceramides were synthesized and tested whether they had potential to stimulate the cells isolated from invariant Valpha19-Jalpha33 TCR transgenic mice (where the development of Valpha19 NKT cells is facilitated). Comprehensive examinations revealed substantial antigenic activity in alpha-ManCer that was presented by MR1, one of the MHC class Ib molecules. Next, naturally occurring and synthetic alpha-mannosyl glycolipids were further analyzed to determine structural requirements for natural ligands for Valpha19 NKT cells. As a result, alpha-mannosyl phosphatidyl inositols (PI) such as (alpha-Man)(2)-PI and alpha-Man-alpha-GlcNH(2)-PI (a partial structure of mycobacterial lipoarabinomannan and GPI-anchors) as well as alpha-ManCer derivatives were found to activate Valpha19 NKT cells in vivo and in vitro. Thus, Valpha19 NKT cells are possibly responsive to certain alpha-mannosyl glycolipids and may have roles in the innate and adaptative immune systems to protect against various antigens expressing alpha-mannosyl glycolipids and to regulate the adaptive immune system responding to the intracellular ligands.

Modulation of Valpha19 NKT cell immune responses by alpha-mannosyl ceramide derivatives consisting of a series of modified sphingosines.

We have demonstrated that analogues of alpha-mannosyl ceramide (alpha-ManCer) consisting of a series of immunosuppressive 2-aminoalcohol derivatives in place of sphingosine promote a greater immune response from mouse invariant Valpha19-Jalpha26 (AV19-AJ33) TCR-bearing NKT (Valpha19 NKT) cells than alpha-ManCer itself. To further characterize the immune responses of Valpha19 NKT cells to the alpha-ManCer analogues, cytokine production by the cells was examined in detail. We found that certain alpha-ManCer derivatives individually induced either Th1- or Th2-dominant cytokine production in culture. The Th1- or Th2-biased immune responses of Valpha19 NKT cells were dependent on MHC class I-like MR1, since they were induced by coculture with the MR1 transfectants previously loaded with the glycolipids and were inhibited in the presence of anti-MR1 antiserum. Presumably, the recognition of the alpha-mannosyl residue of the alpha-ManCer analogues by the invariant TCR is individually modulated, depending on the altered interaction with the groove of the antigen-presenting MR1. Priming of the Valpha19 invariant TCR-transgenic mice in vivo with these glycolipid derivatives resulted in the induction of the Th1- or Th2-biased immune responses. Thus, these alpha-ManCer derivatives are likely to be useful in immunotherapy for either Th1 or Th2 excess autoimmune diseases, modulating the function of Valpha19 NKT cells.

Glycolipids with nonreducing end alpha-mannosyl residues that have the potential to activate invariant Valpha19 NKT cells.

We have previously demonstrated that alpha-mannosyl ceramide and its derivatives promote immune responses of NK1.1(+) invariant Valpha19-Jalpha33 T cell receptor (TCR) alpha(+) T cells (Valpha19 NKT cells). In this study, attempts were made to determine the structural requirements for natural ligands for Valpha19 NKT cells. Naturally occurring and synthetic glycolipids were analyzed for their ability to stimulate the cells prepared from invariant Valpha19-Jalpha33 TCR transgenic mice, in which development of Valpha19 NKT cells is facilitated. As a result, alpha-mannosyl phosphatidylinositols such as 2,6-di-alpha-mannosyl phosphatidylinositol and alpha-mannosyl-4alpha-glucosaminyl-6-phosphatidylinositol (alpha-Man-GlcNH(2)-PtdIns) as well as alpha-mannosyl ceramide derivatives were found to activate the cells from the transgenic mouse liver, gut lamina propria and spleen in vivo and in vitro. Thus, glycolipids with nonreducing end alpha-mannosyl residues are suggested to be potent antigens for Valpha19 NKT cells. Next, a series of invariant Valpha19-Jalpha33 TCR(+) hybridomas, each with variations in the sequence of the Valpha-Jalpha junction and the TCR beta chain, were tested for responsiveness toward the alpha-mannosyl glycolipids. A loose correlation between the primary structure of the TCR and the reactive glycolipids was observed. For instance, hybridomas expressing TCRs consisting of an alpha chain with a variation in the Valpha19-Jalpha33 junction and a Vbeta6(+)beta chain showed affinity towards alpha-mannosyl ceramide and alpha-Man-GlcNH(2)-PtdIns, whereas those expressing TCRs with an invariant Valpha19-Jalpha33 alpha chain and a Vbeta8(+)beta chain responded to 2,6-di-alpha-mannosyl phosphatidylinositol. Thus, it is suggested that Valpha19 NKT cells with microheterogeneity in the TCR structure have been generated for defense against various antigens expressing alpha-mannosyl glycolipids.

Invariant V(alpha)19i T cells regulate autoimmune inflammation.

T cells expressing an invariant V(alpha)19-J(alpha)33 T cell receptor alpha-chain (V(alpha)19i TCR) are restricted by the nonpolymorphic major histocompatibility complex class Ib molecule MR1. Whether V(alpha)19i T cells are involved in autoimmunity is not understood. Here we demonstrate that T cells expressing the V(alpha)19i TCR transgene inhibited the induction and progression of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis. Similarly, EAE was exacerbated in MR1-deficient mice, which lack V(alpha)19i T cells. EAE suppression was accompanied by reduced production of inflammatory mediators and increased secretion of interleukin 10. Interleukin 10 production occurred at least in part through interactions between B cells and V(alpha)19i T cells mediated by the ICOS costimulatory molecule. These results suggest an immunoregulatory function for V(alpha)19i T cells.

Induction of promotive rather than suppressive immune responses from a novel NKT cell repertoire Valpha19 NKT cell with alpha-mannosyl ceramide analogues consisting of the immunosuppressant ISP-I as the sphingosine unit.

A 2-substituted 2-aminopropane-1,3-diol or 2-aminoethanol is the minimum structure required for the immunosuppressive activity of ISP-I, an antibiotic isolated from the culture broth of Isaria sinclairil. A series of alpha-mannosyl ceramide (alpha-ManCer) analogues was derived from 2-substituted 2-aminopropane-1,3-diols or 2-aminoethanols in place of sphingosine. The newly synthesized glycosides were evaluated for their effects on immune responses. In contrast to the immunosuppressive activity of the precursors, the alpha-ManCer analogues induced immunopromotive responses from invariant Valpha19-Jalpha26 transgenic mouse lymphocytes more effectively than the original alpha-ManCer. Collectively, it is strongly suggested that the 2-substituted 2-aminopropane-1,3-diols and 2-aminoethanols mimic sphingosine in the alpha-ManCer analogues so that they potentially acquire specific antigenicity toward Valpha19 NKT cell, a novel NKT cell subset.

Synthetic alpha-mannosyl ceramide as a potent stimulant for an NKT cell repertoire bearing the invariant Valpha19-Jalpha26 TCR alpha chain.

A NKT cell repertoire is characterized by the expression of the Valpha19-Jalpha26 invariant TCR alpha chain (Valpha19 NKT cell). This repertoire, as well as a well-established Valpha14-Jalpha281 invariant TCR alpha(+) NKT cell subset (Valpha14 NKT cell), has been suggested to have important roles in the regulation of the immune system and, thus, is a major therapeutic target. Here, we attempted to find specific antigens for Valpha19 NKT cells. Valpha19 as well as Valpha14 NKT cells exhibited reactivity to alpha-galactosyl ceramide (alpha-GalCer). Thus, a series of monoglycosyl ceramides with an axially oriented glycosidic linkage between the sugar and ceramide moiety were synthesized and their antigenicity to Valpha19 NKT cells was determined by measuring their immune responses in culture with glycolipids. Comprehensive examinations revealed substantial antigenic activity for Valpha19 NKT cells by alpha-mannosyl ceramide.

Generation of Valpha14 NKT cells in vitro from hematopoietic precursors residing in bone marrow and peripheral blood.

We previously reported the generation of Valpha14 invariant TCR+ (Valpha14i) NK1.1+ natural killer T (NKT) cells in the cytokine-activated suspension culture of murine fetal liver cells. In this study, we attempted to apply this finding to the induction of Valpha14i NKT cell differentiation in the culture of hematopoietic precursors residing in bone marrow or peripheral blood. Preferential generation of NKT cells was found in the culture of Thy-1(+)-depleted bone marrow cells in the presence of culture supernatant from Con A-stimulated spleen T cells and a combination of recombinant IL-3, IL-4, IL-7 and GM-CSF. NKT cell development from peripheral blood hematopoietic precursors was induced when they were cultured on stromal cell monolayers prepared from Thy-1(+)-depleted bone marrow or fetal liver cells, suggesting that certain environments derived from hematopoietic organs are required for the induction of NKT cells from precursors in vitro. A significant fraction of NKT cells generated in the culture were positive for staining with CD1-alpha-galactosylceramide tetramer, indicating that Valpha14i NKT cells were the major subset among the NKT cells. The present methods for obtaining NKT cells in the culture of bone marrow or peripheral blood cells are applicable to the treatment of patients suffering from diseases with numerical and functional disorders of NKT cells.

Accumulation of Valpha7.2-Jalpha33 invariant T cells in human autoimmune inflammatory lesions in the nervous system.

T cells expressing an invariant TCR alpha chain and NK cell markers are expected to exhibit unique functions. Whereas much attention has been paid to CD1d-restricted NKT cells, a second NKT cell population bearing an invariant AV19-AJ33 TCR has recently been identified in mice and humans. Selection and/or expansion of this population require B cells, and would involve a non-classical class I-related molecule MR1. Although their preferential distribution in the gut mucosa indicates their role in the host response at the site of pathogen entry, it remains unknown whether they play an alternative role at different sites or in immunological disorders. Using single-strand conformation polymorphism clonotype analysis, we investigated the presence of the human AV19-AJ33 T cells (V(alpha)7.2-J(alpha)33 T cells) in autopsy samples from multiple sclerosis (MS) patients as well as in nerve biopsy samples from chronic inflammatory demyelinating polyneuropathy (CIDP) patients. Here we report that the V(alpha)7.2-J(alpha)33 T cells are accumulated in some of the central nervous system lesions of MS and in the majority of the peripheral nerve samples from CIDP. We have previously revealed that CD1d-restricted, V(alpha)24-J(alpha)Q NKT cells are remarkably reduced in the peripheral blood from MS. However, V(alpha)7.2-J(alpha)33 T cells are not reduced in the peripheral blood from MS and could be detected in a large majority of the cerebrospinal fluid samples obtained during relapse of MS. The present results indicate that the V(alpha)7.2-J(alpha)33 T cells are involved in the autoimmune inflammatory lesions.

Antibody production in early life supported by maternal lymphocyte factors.

To examine the influence of maternal lymphocyte factors on the immune responses in offspring in early life, antibody production in neonates born to either normal or lymphocyte-deficient mothers was analyzed. Recombination activating gene (Rag)-2(+/-) mouse neonates born to Rag-2(+/+), Rag-2(+/-)or Rag-2(-/-)mothers were injected with goat anti-mouse IgD antiserum, and IgE and IgG(1) production was evaluated. The levels of IgE and IgG(1) were higher in the pups born to Rag-2(+/+)and Rag-2(+/-) dams than to lymphocyte-deficient Rag-2(-/-) dams. The enhanced antibody production in the former compared with the latter neonates was also found following immunization with ovalbumin or TNP-Ficoll. Thus, the presence of maternal lymphocyte factors was suggested in neonates that augmented antigen-specific antibody production in both T cell-dependent and -independent pathways. A reduction in antibody production was observed in normal neonates when they were foster-nursed by Rag-2(-/-) mothers. Thus, the maternal lymphocyte factors enhancing the immune responses in newborns were shown to be present in breast-milk.

Presence of a novel subset of NKT cells bearing an invariant V(alpha)19.1-J(alpha)26 TCR alpha chain.

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.