Binding of H-2Kb-specific peptides to TAP and major histocompatibility complex class I in microsomes from wild-type, TAP1, and beta2-microglobulin mutant mice

Major histocompatibility complex (MHC) class I molecules are trimolecular complexes consisting of a heavy chain (HC), beta2-microglobulin (beta2m), and a short peptide. Assembly of MHC class I molecules is thought to take place early during biosynthesis. Deficiency in either beta2m or the transporter associated with antigen processing (TAP) results in accumulation of class I molecules in the endoplasmic reticulum (ER). In this study, we have assessed peptide binding to TAP and MHC class I in purified microsomes derived from wild-type, TAP1(-/-), beta2m-/-, and TAP1/beta2m-/- mice using a cross-linkable H-2Kb-binding peptide. This enabled us to study the influence of an intact TAP complex and beta2m on peptide binding to MHC class I and to analyze the stepwise interaction of peptide with TAP and MHC class I molecules. Peptide bound both immature and mature (terminally glycosylated) class I molecules in intact as well as permeabilized microsomes from wild-type mice. Efficient peptide binding to immature class I molecules was also detected in permeabilized microsomes from TAP1(-/-) mice. In contrast, no peptide binding to beta2m-free HC was detected in permeabilized microsomes from beta2m-/- and TAP1/beta2m-/- mice. However, the addition of exogenous beta2m allowed peptide binding to class I in permeabilized beta2m-/- and TAP1/beta2m-/- microsomes. These results demonstrate that a preformed class I HC middle dotbeta2m heterodimer is necessary for efficient peptide binding under physiological conditions. The observed peptide binding to class I in permeabilized TAP1(-/-) microsomes further suggests that TAP1 is not required for peptide binding to class I in the ER. Finally, kinetic studies allowed the demonstration of a stepwise binding of peptide to TAP, subsequent translocation across the ER membrane, a step that required ATP hydrolysis, and binding of peptide to preformed class I HC.beta2m heterodimers.     

Regulation of MHC class I membrane expression by beta 2-microglobulin

MHC-I binding peptides and beta 2 microglobulin (beta 2-m) can upregulate the MHC-I heavy chain expression on certain peptide transporter mutant cells. We have further studied this with normal cells and non-mutant cell lines. No MHC-I upregulation was seen with normal, resting or activated T cells. On mouse cell lines P815 and B16, both peptides and human beta 2-m gave an additive upregulation response. With the human small cell lung carcinoma H82, an optimal HLA.A2 binding peptide (GILGFVFTL) gave an upregulation response, whereas beta 2-m alone or in combination with this peptide had no effect. However, beta 2-m potentiated the response of H82 cells to a slightly longer peptide. Using mutant RMA-S cells, it was found that both Brefeldin A (BFA) and chloroquine, but not leupeptin, inhibited MHC-I upregulation response to both peptide and beta 2-m. In contrast to chloroquine, BFA also gave a reduction of background membrane MHC-I expression, presumably due to a block in Golgi transport. Human beta 2-m, which binds to RMA-S cells, and which is known to internalize into endosomes, did not reappear on the cell surface. When Db on RMA-S cells was upregulated by human beta 2-m, the sensitivity of these cells to Db restricted CTL cells increased. Even if beta 2-m did not upregulate the overall MHC-I expression on normal cells, it may still quantitatively increase the expression of optimally presented peptides and endosomal recycling many be important in this process.     

TAP1-independent loading of class I molecules by exogenous viral proteins

Presentation of peptides derived from endogenous proteins on class I molecules needs functional TAP peptide transporters. To reveal whether class I-associated presentation of exogenous proteins also required the presence of TAP transporters, we assessed in vitro the ability of spleen cells and macrophages from TAP1-deficient mice (TAP1-/-) to present peptides derived from exogenous recombinant viral proteins on their class I molecules. We found that recombinant glyco- and nucleoprotein from lymphocytic choriomeningitis virus and nucleoprotein of vesicular stomatitis virus were presented as efficiently by TAP1-/- cells as by control cells. Peptide regurgitation was not involved. Since particulate, non-replicating antigens can efficiently prime anti-viral cytotoxic T cells in vivo, this new, TAP-independent pathway of class I-associated antigen presentation may be applicable for vaccine strategies.     

Induction and functional characterization of beta2-microglobulin (beta2-mu)-free HLA class I heavy chains expressed by beta2-mu-deficient human FO-1 melanoma cells

The frequent loss of beta2-microglobulin (beta2-mu) in malignant cells has stimulated interest in the functional characteristics of beta2-mu-free HLA class I heavy chains, since this information contributes to assess the impact of beta2-mu abnormalities on the interaction of malignant cells with immune cells. Therefore, the present study has investigated the ability of beta2-mu-free HLA class I heavy chains to modulate NK cell-mediated lysis of melanoma cells and to present melanoma-associated antigen (MAA)-derived peptides to HLA class I-restricted, MAA-specific cytotoxic T lymphocytes (CTL). Beta2-mu-free HLA class I heavy chains were induced on beta2m null FO-1 cells by sequential incubation with IFN-alpha for 48 h at 37 degrees C and for 24 h at 26 degrees C. Transfection of cells with a wild-type H-2Ld gene (FO-1Ld) enhanced the induction of beta2-mu-free HLA class I heavy chains under such experimental conditions. Beta2-mu-free HLA class I heavy chains expressed on the cell membrane did not protect the B2m null FO-1 cells from NK cell-mediated lysis. Furthermore, FO-1 cells which express beta2-mu-free HLA-A2 heavy chains following transfection with a wild-type HLA-A2 gene were not lysed by HLA-A2-restricted, MAA-specific CTL lines and clones. These results indicate that association with beta2-mu is required for interaction of HLA class I molecules with NK inhibitory receptors and for peptide presentation to CTL.     

Functional comparison of bovine, murine, and human beta2-microglobulin: interactions with murine MHC I molecules

Fetal calf serum is a well known source of bovine beta2-microglobulin (beta2m) which can exchange with endogenous beta2m from, as well as promote peptide binding to, class I major histocompatibility (MHC I) molecules on cells cultured in vitro. Recombinant bovine beta2m was expressed and purified for direct functional comparison to human and murine beta2m for interactions with murine MHC I molecules H-2Kb, Db, Kd, Ld, and Dd. Bovine and human beta2m were equivalent in stabilizing MHC I heavy chains and facilitating peptide loading, suggesting similar affinities for murine MHC I heavy chains. The activity of murine beta2m was significantly weaker, consistent with previous work that demonstrated the lower affinity of murine human beta2m for murine heavy chains compared to human beta2m. Analysis of bovine beta2m in fetal calf serum revealed ten-fold higher concentrations than in adult bovine serum, levels shown to significantly affect MHC I stability and peptide loading. The ramifications for the study of MHC I molecules from cells in culture and the evolutionary implications of the higher affinity interactions of human and bovine beta2m are discussed.     

Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins

ER-to-Golgi transport in yeast may be reproduced in vitro with washed membranes, purified proteins (COPII, Uso1p and LMA1) and energy. COPII coated vesicles that have budded from the ER are freely diffusible but then dock to Golgi membranes upon the addition of Uso1p. LMA1 and Sec18p are required for vesicle fusion after Uso1p function. Here, we report that the docking reaction is sensitive to excess levels of Sec19p (GDI), a treatment that removes the GTPase, Ypt1p. Once docked, however, vesicle fusion is no longer sensitive to GDI. In vitro binding experiments demonstrate that the amount of Uso1p associated with membranes is reduced when incubated with GDI and correlates with the level of membrane-bound Ypt1p, suggesting that this GTPase regulates Uso1p binding to membranes. To determine the influence of SNARE proteins on the vesicle docking step, thermosensitive mutations in Sed5p, Bet1p, Bos1p and Sly1p that prevent ER-to-Golgi transport in vitro at restrictive temperatures were employed. These mutations do not interfere with Uso1p-mediated docking, but block membrane fusion. We propose that an initial vesicle docking event of ER-derived vesicles, termed tethering, depends on Uso1p and Ypt1p but is independent of SNARE proteins.     

Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b)

Natural killer (NK) cells preferentially lyse targets that express reduced levels of major histocompatibility complex (MHC) class I proteins. To date, the only known mouse NK receptors for MHC class I belong to the Ly49 family of C-type lectin homodimers. Here, we report the cloning of mouse NKG2A, and demonstrate it forms an additional and distinct class I receptor, a CD94/NKG2A heterodimer. Using soluble tetramers of the nonclassical class I molecule Qa-1(b), we provide direct evidence that CD94/NKG2A recognizes Qa-1(b). We further demonstrate that NK recognition of Qa-1(b) results in the inhibition of target cell lysis. Inhibition appears to depend on the presence of Qdm, a Qa-1(b)-binding peptide derived from the signal sequences of some classical class I molecules. Mouse NKG2A maps adjacent to CD94 in the heart of the NK complex on mouse chromosome six, one of a small cluster of NKG2-like genes. Our findings suggest that mouse NK cells, like their human counterparts, use multiple mechanisms to survey class I expression on target cells.     

Dissociation of beta 2-microglobulin from HLA class I heavy chains correlates with acquisition of epitopes in the cytoplasmic tail

A rabbit antiserum "ABR2" was raised against a peptide with sequence identity to 10 amino acids of the cytoplasmic tail of HLA class I heavy chains. Western blotting and immunoprecipitation analyses demonstrate that ABR2 reacts with HLA class I heavy chains. The antiserum reacts poorly with beta 2-microglobulin (beta 2-m)-associated heavy chains and reacts strongly with free heavy chains. ABR2 reacts with immature heavy chains from the endoplasmic reticulum that have yet to bind beta 2-m and mature heavy chains that have dissociated from beta 2-m at the plasma membrane. Comparison with HC10, a mAb that recognizes an epitope defined by polymorphism at residue 62 of the alpha 1 helix of free HLA class I heavy chains, shows that ABR2 reacts with overlapping populations of free heavy chains (for those allotypes that react with both Abs), but it also identifies populations that bind to one Ab and not the other. ABR2 induces dissociation of beta 2-m from HLA-B38 molecules expressed by the human B cell line "TEM," a phenomenon not detected with other allotypes or with the same allotype in a different cell line. This study shows that association of beta 2-m with the extracellular domains of HLA class I heavy chains can cause a change in the cytoplasmic tail that prevents binding of Abs present in the ABR2 antiserum. Similar findings have been made for mouse H-2 class I molecules, which suggests that this is a general property of class I MHC molecules.     

Dynamic change of beta 2-microglobulin in hemodialysis

A candidin, which is a suspension of killed yeast cells, is commonly used for intradermal tests of delayed hypersensitivity, to evaluate the immunological cellular competence of the patient, when the test is applied along with other similar tests. When working with a cellular antigen, the histopathology of positive skin tests reveals a cellular infiltrate which not only presents a characteristic hypersensitivity reaction but also a neutrophilic abscess in the central part. This research presents the results of a comparison between the yeast cell suspension and the polysaccharide antigens, both obtained from the same strains of Candida albicans. The results obtained by skin tests in one hundred individuals were 61.0% with the polysaccharide antigen and 69.0% with the yeast cell suspension antigen. Concordant results concerning the two antigens were observed in 82.0% of the individuals. The discussion section presents an assumption to explain the differences of positivity obtained with the two antigens. We conclude that the polysaccharide antigen can be utilized in the intradermal test of delayed hypersensitivity to Candida albicans.     

Expression of class II, but not class I, major histocompatibility complex molecules is required for granuloma formation in infection with Schistosoma mansoni

Previous studies have suggested that granulomatous inflammation in schistosomiasis is mediated by CD4+ T helper lymphocytes sensitized to parasite egg antigens. However, CD8+ T cells have also frequently been associated with the immune response to schistosome eggs. To examine more precisely the role of CD4+ and CD8+ T cells in the pathology of the schistosomal infection, we used mice with targeted mutations in major histocompatibility complex (MHC) class II or class I molecules. These mutations lead, respectively, to the virtual absence of CD4+ and CD8+ T cells. The results clearly show that schistosome-infected MHC class II mutant mice failed to form granulomas around parasite eggs. In contrast, infected MHC class I mutant mice displayed characteristic granulomatous lesions that were comparable to those in wild-type control mice. Moreover, lymphoid cells from MHC class II mutant mice were unable to react to egg antigens with either proliferative or cytokine [interferon-gamma, interleukin (IL)-4, IL-10] responses; nor were they able to present egg antigens to specifically sensitized CD4+ T helper cells from infected syngeneic control mice. By comparison, cells from MHC class I mutant mice exercised all these functions in a manner comparable with those from wild-type controls. These observations clearly demonstrate that schistosomal egg granulomas are mediated by MHC class II-restricted CD4+ T helper cells. They also suggest that CD8+ T cells do not become sensitized to egg antigens and play little role, if any, in the pathogenesis of schistosomiasis.     

The T/B cell interaction involved in induction of the mouse IgG2ab suppression is restricted by major histocompatibility complex class I, but not class II molecules

To determine the major histocompatibility complex (MHC) restriction of the T/ B cell interaction involved in a negative regulation of Ig production, we used mouse model of T cell-induced IgG2ab suppression in vivo. Normal or specifically triggered T splenocytes from mice of the Igha haplotype, when neonatally transferred into histocompatible Igha/b heterozygotes, are able to induce a specific and total suppression of the IgG2ab allotype. Nevertheless, only transfer of IgG2ab-primed Igha T splenocytes induces this suppression in Ighb/b homozygous congenic mice in which the whole IgG2a isotype production is inhibited. This suppression is chronically maintained by CD8+ T cells, but can be experimentally reversed. We have established that the suppression induction required a CD4+CD8+ T cell cooperation and operated via the recognition by the involved TCR of C gamma 2ab-derived peptides presented by the target B cells in an MHC haplotype-restricted manner. Here, by using Ighb mice genetically deficient for MHC class I (beta 2-microglobulin%, or beta 2m%) or class II (I-A beta%) molecules, we demonstrate functionally that the suppression induction implicates an MHC class I-, but not class II-restricted interaction. Indeed, the anti-IgG2ab T cells transferred into Ighb H-2b I-A beta% mice carry out the suppression process normally, while in Ighb H-2b beta 2m% recipients, their suppression induction capacity is significantly inhibited. Moreover, the C gamma 2ab 103-118 peptide, identified as the sole C gamma 2ab-derived peptide able to amplify the anti-IgG2ab T cell reactivity in Igha H-2b mice, is also able to stabilize the H-2Db, but not the H-2Kb class I molecules at the surface of RMA-S (TAP2-, H-2b) cells. These results indicate that, despite the CD4+/CD8+ T cell cooperation during the induction phase of suppression only MHC class I molecule expression is required at the surface of IgG2ab+ B cells for suppression establishment.     

IFN-gamma action on pancreatic beta cells causes class I MHC upregulation but not diabetes

We have generated transgenic nonobese diabetic (NOD) mice expressing dominant negative mutant IFN-gamma receptors on pancreatic beta cells to investigate whether the direct effects of IFN-gamma on beta cells contribute to autoimmune diabetes. We have also quantitated by flow cytometry the rise in class I MHC on beta cells of NOD mice with increasing age and degree of islet inflammatory infiltrate. Class I MHC expression increases gradually with age in wild-type NOD mice; however, no such increase is observed in the transgenic beta cells. The transgenic mice develop diabetes at a similar rate to that of wild-type animals. This study dissociates class I MHC upregulation from progression to diabetes, shows that the rise in class I MHC is due to local IFN-gamma action, and eliminates beta cells as the targets of IFN-gamma in autoimmune diabetes.     

Ultrastructural organization of hemodialysis-associated beta 2-microglobulin amyloid fibrils

Fibrils of hemodialysis-associated beta 2-microglobulin amyloid were examined by high resolution electron microscopy and immunohistochemical labeling. The amyloid containing tissues obtained through autopsy were prepared for thin section observations. In contrast to other forms of amyloid, the most conspicuous feature of these fibrils were their curved conformations. The fibril core showed ultrastructural and immunohistochemical features in common with the core of connective tissue microfibrils and of previously observed fibrils of experimental murine AA amyloidosis and familial amyloid polyneuropathy (FAP). The core was wrapped in a layer of 3 nm wide ribbon-like "double tracked" structures identified as chondroitin sulfate proteoglycan (CSPG) with immunogold labeling as well as from the results of previous in vitro experiments. Finally, the outer surface of the fibril was associated with a loose assembly of 1 nm wide filaments immunohistochemically identified as beta 2-microglobulin. This is similar to the manner in which AA protein and transthyretin filaments are associated with their respective fibrils. The results of this study provide an additional example for the concept that amyloid fibrils in general are microfibril-like structures externally associated with amyloid protein filaments. An unusual feature of the fibrils of hemodialysis-associated amyloid, however, is the presence of a peripheral layer composed of CSPG rather than of heparan sulfate proteoglycan (HSPG) as in the case of the other two amyloids above. These chondroitin sulfate chains in the outer CSPG layer may be less effective in providing rigidity to the fibril core, thus allowing for the curved conformations of beta 2-microglobulin amyloid fibrils.     

Anti-MUC1 antibodies react directly with MUC1 peptides presented by class I H2 and HLA molecules

Codon usage bias, the preferential use of particular codons within each codon family, is characteristic of synonymous base composition in many species, including Drosophila, yeast, and many bacteria. Preferential usage of particular codons in these species is maintained by natural selection acting largely at the level of translation. In Drosophila, as in bacteria, the rate of synonymous substitution per site is negatively correlated with the degree of codon usage bias, indicating stronger selection on codon usage in genes with high codon bias than in genes with low codon bias. Surprisingly, in these organisms, as well as in mammals, the rate of synonymous substitution is also positively correlated with the rate of nonsynonymous substitution. To investigate this correlation, we carried out a phylogenetic analysis of substitutions in 22 genes between two species of Drosophila, Drosophila pseudoobscura and D. subobscura, in codons that differ by one replacement and one synonymous change. We provide evidence for a relative excess of double substitutions in the same species lineage that cannot be explained by the simultaneous mutation of two adjacent bases. The synonymous changes in these codons also cannot be explained by a shift to a more preferred codon following a replacement substitution. We, therefore, interpret the excess of double codon substitutions within a lineage as being the result of relaxed constraints on both kinds of substitutions in particular codons.     

Hepatitis C virus envelope glycoprotein E1 originates in the endoplasmic reticulum and requires cytoplasmic processing for presentation by class I MHC molecules

We investigated whether hepatitis C virus envelope glycoprotein E1 is transported from the endoplasmic reticulum (ER) to the cytoplasm of infected cells for class I MHC processing. Target cells expressing E1 were killed by CTL lines from a hepatitis C virus-infected chimpanzee, and synthetic peptides were used to define an epitope (amino acids 233-GNASRCWVA-241) presented by the Patr-B*1601 class I MHC molecule. An unusually high concentration (>100 nM) of this nonameric peptide was required for target cell lysis, but this could be reduced at least 1000-fold by replacing the asparagine at amino acid position 234 (Asn234) with aspartic acid (Asp), the anticipated anchor residue for NH2-terminal peptide binding to Patr-B*1601. Conspicuously, position 234 is part of an N-glycosylation motif (Asn-Xaa-Ser/Thr), suggesting that the Asn234 to Asp substitution might occur naturally within the cell due to deglycosylation/deamidation of this amino acid by the cytosolic enzyme peptide N-glycanase. In support of this model, we demonstrate that presentation of the epitope depended on 1) cotranslational synthesis of E1 in the ER, 2) glycosylation of the E1 molecule, and 3) a functional TAP transporter to shuttle peptide from the cytosolic to ER compartment. These results indicate for the first time that during infection of the host, viral envelope glycoproteins originating in the ER are processed in the cytoplasm for class I MHC presentation. That a posttranslational change in amino acid sequence from Asn to Asp alters the repertoire of peptides presented to CD8+ CTL has implications for the design of antiviral vaccines.     

Possible assortment of a1 and a2 region gene segments in human MHC class I molecules

Using pair-wise comparison of aligned nucleotide sequences of distinct and complete human MHC class I molecules, we have constructed triangular tables to study the similarities and differences of various a1 (exon 2) and a2 (exon 3) region sequences. There are two HLA-A (A*6901 and A*6601) and 13 HLA-B (B*4201, B*8101, B*4102, B*4801, B*4007, B*4001, B*4802, Dw53, B*4406, B*4402, B*3901, B*1514 and B*3702) sequences that have identical a1 sequences with other known MHC class I molecules, while their a2 sequences are the same as those of different ones. Of these 15, A*6901, B*4001 and B*4802 have previously been suggested as the results of recombination between A*6801 and A*0201, B*4101 and B*8101, and B*4801 and B*3501, respectively. However, many other sequences can also be used to generate them by recombination. Furthermore, their reciprocal products have never been identified. Thus, gene conversion has subsequently been suggested as an alternative. Another possible genetic mechanism for generating these nucleotide sequence similarities can be assortment, or that some gene segments can be duplicated or multiplicated to be used in different human MHC class I molecules. Interestingly, this genetic mechanism is probably absent for the generation of different mouse MHC class I molecules.