Mutation of a positively charged cytoplasmic motif within CD1d results in multiple defects in antigen presentation to NKT cells

CD1d is an MHC class I-like molecule that presents glycolipid Ags to types I and II NKT cells. The YxxI motif in the cytoplasmic tail of CD1d contributes to its intracellular localization to the endolysosomal compartment and is important for Ag presentation to type I NKT cells. In this study, we identified the (327-329)RRR motif in CD1d and showed that it is critical for the control of CD1d intracellular trafficking and Ag presentation. The replacement of the arginines in this motif with alanines resulted in the extensive accumulation of CD1d in lysosomes but did not affect the cell surface expression. The defect in its cellular localization was accompanied by defects in Ag presentation to both type I and type II NKT cells. These results demonstrated that the (327-329)RRR motif of CD1d is required for proper cellular distribution of CD1d and optimal Ag presentation to both type I and type II NKT cells.     

Antigen presentation in Syrian hamster cells: substrate selectivity of TAP controlled by polymorphic residues in TAP1 and differential requirements for loading of H2 class I molecules

Expression of mouse major histocompatibility complex (MHC) class I molecules in different cell lines derived from Syrian hamsters has revealed antigen presentation deficiencies of some H2 allelic products in two cell lines (BHK and NIL-2) which were overcome by transient expression of the rat transporter associated with antigen processing (TAP; Lobigs et al. 1995). Here we show that in both cell lines the endogenous MHC class I cell surface expression was completely down-regulated. Lymphokine treatment induced endogenous and recombinant mouse MHC class I cell surface expression to levels similar to that in other Syrian hamster cell lines competent for antigen presentation through transduced H2 molecules. Accordingly, constitutive downregulation of expression of accessory molecules of the MHC class I pathway can reveal differences between H2 class I alleles in antigen presentation not encountered when the expression levels are augmented. In addition to the differential expression of MHC class I pathway genes, two cell lines representing competent (FF) and defective (BHK) antigen presentation phenotypes for mouse class I MHC restriction elements demonstrated substantial sequence polymorphism in Tap1 but not Tap2. Cytokine-treated FF or BHK cells and human TAP-deficient T2 cells transfected with FF or BHK TAP1 in combination with FF TAP2 differed in their preference for C-terminal peptide residues, as shown by an in vitro peptide transport assay. Thus, polymorphic residues in TAP1 can influence the substrate selectivity of the Syrian hamster peptide transporter.     

Distinct endosomal trafficking requirements for presentation of autoantigens and exogenous lipids by human CD1d molecules

CD1d molecules present both self Ags and microbial lipids to NKT cells. Previous studies have established that CD1d lysosomal trafficking is required for presentation of autoantigens to murine invariant NKT cells. We show in this study that this is not necessary for autoantigen presentation by human CD1d, but significantly affects the presentation of exogenous Ags. Wild-type and tail-deleted CD1d molecules stimulated similar autoreactive responses by human NKT clones, whereas presentation of exogenous lipids by tail-deleted CD1d was highly inefficient. Chloroquine treatment markedly inhibited exogenous Ag presentation by wild-type CD1d transfectants, but did not affect NKT autoreactive responses. Conversely, APC expression of HLA-DRalphabeta and the invariant chain (Ii) was associated with faster internalization of CD1d into the endocytic system and enhanced CD1d-mediated presentation of exogenous Ags, but did not appear to augment NKT autoreactivity. Knockdown of the Ii by small interfering RNA resulted in reduced CD1d surface expression and slower internalization in HLA-DR+ APCs, but not HLA-DR- APCs, demonstrating a direct effect of MHC/Ii complexes on CD1d trafficking. CD1d-mediated presentation of exogenous Ags was much more efficient in immature dendritic cells, which actively recycle MHC class II molecules through the endocytic system, than in mature dendritic cells that have stabilized MHC class II expression at the cell surface, suggesting a physiological role for MHC/Ii complexes in modulating CD1d function. These results indicate that autoantigens and exogenous lipids are acquired by human CD1d at distinct cellular locations, and that Ii trafficking selectively regulates CD1d-mediated presentation of extracellular Ags.     

Inhibition of MHC class I is a virulence factor in herpes simplex virus infection of mice

Herpes simplex virus (HSV) has a number of genes devoted to immune evasion. One such gene, ICP47, binds to the transporter associated with antigen presentation (TAP) 1/2 thereby preventing transport of viral peptides into the endoplasmic reticulum, loading of peptides onto nascent major histocompatibility complex (MHC) class I molecules, and presentation of peptides to CD8 T cells. However, ICP47 binds poorly to murine TAP1/2 and so inhibits antigen presentation by MHC class I in mice much less efficiently than in humans, limiting the utility of murine models to address the importance of MHC class I inhibition in HSV immunopathogenesis. To address this limitation, we generated recombinant HSVs that efficiently inhibit antigen presentation by murine MHC class I. These recombinant viruses prevented cytotoxic T lymphocyte killing of infected cells in vitro, replicated to higher titers in the central nervous system, and induced paralysis more frequently than control HSV. This increase in virulence was due to inhibition of antigen presentation to CD8 T cells, since these differences were not evident in MHC class I-deficient mice or in mice in which CD8 T cells were depleted. Inhibition of MHC class I by the recombinant viruses did not impair the induction of the HSV-specific CD8 T-cell response, indicating that cross-presentation is the principal mechanism by which HSV-specific CD8 T cells are induced. This inhibition in turn facilitates greater viral entry, replication, and/or survival in the central nervous system, leading to an increased incidence of paralysis.     

TAP1-deficiency does not alter atherosclerosis development in Apoe-/- mice

Antigen presenting cells (APC) have the ability to present both extra-cellular and intra-cellular antigens via MHC class I molecules to CD8(+) T cells. The cross presentation of extra-cellular antigens is reduced in mice with deficient Antigen Peptide Transporter 1 (TAP1)-dependent MHC class I antigen presentation, and these mice are characterized by a diminished CD8(+) T cell population. We have recently reported an increased activation of CD8(+) T cells in hypercholesterolemic Apoe(-/-) mice. Therefore, this study included TAP1-deficient Apoe(-/-) mice (Apoe(-/-)Tap1(-/-)) to test the atherogenicity of CD8(+) T cells and TAP1-dependent cross presentation in a hypercholesterolemic environment. As expected the CD8(+) T cell numbers were low in Apoe(-/-)Tap1(-/-) mice in comparison to Apoe(-/-) mice, constituting ~1% of the lymphocyte population. In spite of this there were no differences in the extent of atherosclerosis as assessed by en face Oil Red O staining of the aorta and cross-sections of the aortic root between Apoe(-/-)Tap1(-/-) and Apoe(-/-) mice. Moreover, no differences were detected in lesion infiltration of macrophages or CD3(+) T cells in Apoe(-/-)Tap1(-/-) compared to Apoe(-/-) mice. The CD3(+)CD4(+) T cell fraction was increased in Apoe(-/-)Tap1(-/-) mice, suggesting a compensation for the decreased CD8(+) T cell population. Interestingly, the fraction of CD8(+) effector memory T cells was increased but this appeared to have little impact on the atherosclerosis development.In conclusion, Apoe(-/-)Tap1(-/-) mice develop atherosclerosis equal to Apoe(-/-) mice, indicating a minor role for CD8(+) T cells and TAP1-dependent antigen presentation in the disease process.     

Regulation of NKT cells by Ly49: analysis of primary NKT cells and generation of NKT cell line

TCRalphabeta(+)NK1.1(+) (NKT) cells are known to express various NK cell-associated molecules including the Ly49 family of receptors for MHC class I, but its functional significance has been unclear. Here, we examined the expression of Ly49A, C/I and G2 on various NKT cell populations from normal and MHC class I-deficient C57BL/6 mice as well as their responsiveness to alpha-galactosylceramide (alpha-GalCer), a potent stimulator of CD1d-restricted NKT cells. The frequency and the level of Ly49 expression varied among NKT cells from different tissues, and were regulated by the expression of MHC class I and CD1d in the host. Stimulation of various NKT cells with alpha-GalCer suggested that Ly49 expression inversely correlates with the responsiveness of NKT cells to alpha-GalCer. Moreover, alpha-GalCer presented by normal dendritic cells stimulated purified Ly49(-), but not Ly49(+), splenic NKT cells, whereas MHC class I-deficient dendritic cells presented alpha-GalCer to both Ly49(+) and Ly49(-) NKT cells equally well. Therefore, MHC class I on APCs seems to inhibit activation of NKT cells expressing Ly49. To further characterize CD1d-restricted NKT cells, we generated an alpha-GalCer-responsive NKT cell line from thymocytes. The line could only be generated from Ly49(-)NK1.1(+)CD4(+) thymocytes but not from other NKT cell subsets, and it lost expression of NK1.1 and CD4 during culture. Together, these results indicate the functional significance of Ly49 expression on NKT cells.     

The transporter associated with antigen processing (TAP): structural integrity, expression, function, and its clinical relevance

BACKGROUND: The transporter associated with antigen processing (TAP), a member of the family of ABC transporters, plays a crucial role in the processing and presentation of the major histocompatibility complex (MHC) class I restricted antigens. TAP transports peptides from the cytosol into the endoplasmic reticulum, thereby selecting peptides matching in length and sequence to respective MHC class I molecules. Upon loading on MHC class I molecules, the trimeric MHC class I/beta2-microglobulin/ peptide complex is then transported to the cell surface and presented to CD8+ cytotoxic T cells. Abnormalities in MHC class I surface expression have been found in a number of different malignancies, including tumors of distinct histology, viral infections, and autoimmune diseases, and therefore represent an important mechanism of malignant or virus-infected cells to escape proper immune response. In many cases, this downregulation has been attributed to impaired TAP expression, which could be due to structural alterations or dysregulation. This review summarizes the physiology and pathophysiology of TAP, thereby focusing on its function in immune responses and its role in human diseases.     

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]     

CD1d expression by hepatocytes is a main restriction element for intrahepatic T-cell recognition

The liver has specific mechanisms to protect itself from infectious agents and to avoid autoimmunity, indicating an important role of the hepatic tissues in antigen presentation and tolerance induction. Since intrahepatic lymphocytes may contribute to the innate immunity and to the liver pathology, it is of interest to analyze the expression of antigen presenting molecules and of the related T cell recognition in liver, and how these change in relation to different diseases. We analyzed the expression of MHC class I, and of CD1-a, -b, -c, and -d proteins on liver tissues from patients with different hepatic diseases. Moreover, in the same patients we studied the intrahepatic and peripheral NKT cell recognition of alpha-galactosyl ceramide antigen in the context of CD1d. Unlike in other tissues, classical MHC class I molecules were poorly expressed in the hepatic compartment, suggesting that inflamed hepatocytes may trigger weak MHC-restricted T cell responses. Nevertheless, we observed a prevalent expression of HLA class I-like CD1d isoform on the hepatocyte surface, indicating that CD1d is the main restriction element in the liver. In patients with viral hepatitis, the intrahepatic CD1d expression parallels the recruitment of CD56+Valpha24Vbeta11+ NKT cells in the liver which recognize CD1d presenting glycolipids such as alpha-galactosyl ceramide, suggesting that the intrahepatic T cell immunity may focus on glycolipid antigens.     

Retention of empty MHC class I molecules by tapasin is essential to reconstitute antigen presentation in invertebrate cells.

Presentation of antigen-derived peptides by major histocompatibility complex (MHC) class I molecules is dependent on an endoplasmic reticulum (ER) resident glycoprotein, tapasin, which mediates their interaction with the transporter associated with antigen processing (TAP). Independently of TAP, tapasin was required for the presentation of peptides targeted to the ER by signal sequences in MHC class I-transfected insect cells. Tapasin increased MHC class I peptide loading by retaining empty but not peptide-containing MHC class I molecules in the ER. Upon co-expression of TAP, this retention/release function of tapasin was sufficient to reconstitute MHC class I antigen presentation in insect cells, thus defining the minimal non-housekeeping functions required for MHC class I antigen presentation.     

TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD4-8+ T cells.

The transporter associated with the antigen processing 1 (TAP1) gene encodes a subunit for a transporter, presumed to be involved in the delivery of peptides across the endoplasmic reticulum membrane to class I molecules. We have generated mice with a disrupted TAP1 gene using embryonic stem cell technology. TAP1-deficient mice are defective in the stable assembly and intracellular transport of class I molecules and consequently show severely reduced levels of surface class I molecules. These properties are strikingly similar to those described for the TAP2 mutant cell line RMA-S. Cells from the TAP1-deficient mice are unable to present cytosolic antigens to class I-restricted cytotoxic T cells. As predicted from the near absence of class I surface expression, TAP1-deficient mice lack CD4-8+ T cells.     

Pseudomonas aeruginosa Cif Protein Enhances the Ubiquitination and Proteasomal Degradation of the Transporter Associated with Antigen Processing (TAP) and Reduces Major Histocompatibility Complex (MHC) Class I Antigen Presentation

Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8⁺ T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.     

Glycosylphosphatidylinositol-anchored mucin-like glycoproteins from Trypanosoma cruzi bind to CD1d but do not elicit dominant innate or adaptive immune responses via the CD1d/NKT cell pathway

It has been proposed that self and protozoan-derived GPI anchors are natural ligands of CD1d. In this study, we investigated the ability of GPI anchors from Trypanosoma cruzi to bind to CD1d and mediate activation of NKT cells. We observed that GPI-anchored mucin-like glycoproteins (GPI mucins), glycoinositolphospholipids (GIPLs), and their phosphatidylinositol moieties bind to rCD1d and inhibit the stimulation of a NKT hybridoma by the alpha-galactosylceramide-CD1 complex. However, these GPI anchors and related structures were unable to activate NKT cells in vitro or in vivo. We found that high titers of Ab anti-GPI mucins, but not anti-GIPLs, were detected in sera from wild-type as well as in TAP1(-/-), CD1d(-/-), and MHC class II(-/-) mice after immunization. However, T-dependent anti-GPI mucin Ab isotypes, such as IgG1, IgG2a, IgG2b, and IgG3, were absent on MHC class II(-/-), but were conserved in CD1d(-/-) and TAP1(-/-) mice. Furthermore, we found that CD1d(-/-) mice presented a robust cytokine as well as anti-GPI mucins and anti-GIPL Ab responses, upon infection with T. cruzi parasites. These results indicate that, despite binding to CD1d, GPI mucins and related structures expressed by T. cruzi appear not to evoke dominant CD1d-restricted immune responses in vivo. In contrast, MHC class II is critical for the production of the major Ig G isotypes against GPI mucins from T. cruzi parasites.     

Expression of CD1d under the control of a MHC class Ia promoter skews the development of NKT cells, but not CD8+ T cells

Although CD1d and MHC class Ia share similar overall structure, they have distinct levels and patterns of surface expression. While the expression of CD1d1 is known to be essential for the development of NKT cells, the contribution of CD1d1 to the development of CD8(+) T cells appears to be inconsequential. To investigate whether CD1d tissue distribution and expression levels confer differential capacity in selecting these two T cell subsets, we analyzed CD8 and NKT cell compartments in K(b)-CD1d-transgenic mice that lack endogenous MHC class Ia and CD1d, respectively. We found that MHC class Ia-like expression pattern and tissue distribution are not sufficient for CD1d to rescue the development of CD8(+) T cells, suggesting that unique structural features of CD1d preclude its active participation in selection of CD8(+) T cells. Conversely, cell type-specific CD1d surface density is important for the selection of NKT cells, as the NKT cell compartment was only partially rescued by the K(b)-CD1d transgene. We have previously demonstrated that increased CD1d expression on dendritic cells enhanced negative selection of NKT cells. In this study, we show that cell type-specific expression levels of CD1d establish a narrow window between positive and negative selection, suggesting that the distinct CD1d expression pattern may be selected evolutionarily to ensure optimal output of NKT cells.     

Activating immunity in the liver. I. Liver dendritic cells (but not hepatocytes) are potent activators of IFN-gamma release by liver NKT cells

A prominent subset of the hepatic innate immune system is alpha-galactosylceramide (alphaGalCer)-reactive, (CD4(+) and CD4(-)CD8(-)) CD1d-restricted NKT cells. We investigated in C57BL/6 (B6) mice which hepatic cell type stimulates hepatic NKT cell activation. Surface expression of CD1d but not CD40, CD80, or CD86 costimulator molecules was detected in hepatocytes. Pulsed in vitro or in vivo with alphaGalCer, hepatocytes triggered IL-4 release by liver NKT cells but required exogenous IL-12 to trigger IFN-gamma release by NKT cells. Liver dendritic cells (DC) isolated from nontreated mice showed low surface expression of MHC, CD1d, and CD40, CD80, or CD86 costimulator molecules that were strikingly up-regulated after alphaGalCer injection. Although liver CD11c(+) DC displayed lower CD1d surface expression than hepatocytes, they were potent stimulators of IFN-gamma and IL-4 release by liver NKT when pulsed with alphaGalCer in vitro or in vivo. Liver DC are thus potent stimulators of proinflammatory cytokine release by NKT cells, are activated themselves in the process of NKT cell activation, and express an activated phenotype after the NKT cell population is eliminated following alphaGalCer stimulation.     

In vivo priming of natural killer T cells by dendritic cells pulsed with hepatoma-derived acid-eluted substances

Many murine tumor cells express not only individual haplotype-matched class I MHC molecules, but also species-specific CD1d molecules. The former class I MHC molecules generally present internally synthesized tumor-derived peptide antigens to highly specific CD8⁺ cytotoxic T lymphocytes (CTLs) in acquired immunity. In contrast, the latter CD1d molecules may present tumor-associated glycolipid antigens to broadly crossreactive natural killer T (NKT) cells, which might correlate with controlling tumor metastasis. Here, we showed that murine hepatoma cell line Hepa1-6-derived acid-eluted substances might contain both D^b class I MHC-restricted antigens and CD1d-restriced substances, which could sensitize not only syngeneic bone marrow-derived DCs (BM-DCs), but also allogeneic BM-DCs expressing haplotype-mismatched class I MHC and species-specific CD1d molecules. To our surprise, intravenous (i.v.) immunization of C57BL/6 mice with the former syngeneic BM-DCs carrying acid-eluted materials primed both CD4^–CD8^– and CD8⁺ NKT cells in the spleen, whereas immunization with the latter allogeneic BM-DCs loaded the tumor-derived substances primed CD4^–CD8^–, but not CD8⁺ NKT cells. The findings shown in the present study will open a new area for cancer immunotherapy using allogeneic DCs and tumor-derived acid-eluted substances.Abbreviations CTLs cytotoxic T lymphocytes - NKT natural killer T - BM-DCs bone marrow-derived dendritic cells - CTM complete T-cell medium - FCS fetal calf serum - MMC mitomycin C - TCRs T cell receptors