Identification of MHC class I sequences in Chinese-origin rhesus macaques

The rhesus macaque (Macaca mulatta) is an excellent model for human disease and vaccine research. Two populations exhibiting distinctive morphological and physiological characteristics, Indian- and Chinese-origin rhesus macaques, are commonly used in research. Genetic analysis has focused on the Indian macaque population, but the accessibility of these animals for research is limited. Due to their greater availability, Chinese rhesus macaques are now being used more frequently, particularly in vaccine and biodefense studies, although relatively little is known about their immunogenetics. In this study, we discovered major histocompatibility complex (MHC) class I cDNAs in 12 Chinese rhesus macaques and detected 41 distinct Mamu-A and Mamu-B sequences. Twenty-seven of these class I cDNAs were novel, while six and eight of these sequences were previously reported in Chinese and Indian rhesus macaques, respectively. We then performed microsatellite analysis on DNA from these 12 animals, as well as an additional 18 animals, and developed sequence specific primer PCR (PCR-SSP) assays for eight cDNAs found in multiple animals. We also examined our cohort for potential admixture of Chinese and Indian origin animals using a recently developed panel of single nucleotide polymorphisms (SNPs). The discovery of 27 novel MHC class I sequences in this analysis underscores the genetic diversity of Chinese rhesus macaques and contributes reagents that will be valuable for studying cellular immunology in this population.     

Diversity of MHC class I genes in Burmese-origin rhesus macaques

Rhesus macaques (Macaca mulatta) are widely used in developing a strategy for vaccination against human immunodeficiency virus by using simian immunodeficiency virus infection as a model system. Because the genome diversity of major histocompatibility complex (MHC) is well known to control the immune responsiveness to foreign antigens, MHC loci in Indian- and Chinese-origin macaques used in the experiments have been characterized, and it was revealed that the diversity of MHC in macaques was larger than the human MHC. To further characterize the diversity of Mamu-A and Mamu-B loci, we investigated a total of 73 different sequences of Mamu-A, 83 sequences of Mamu-B, and 15 sequences of Mamu-I cDNAs isolated from Burmese-origin macaques. It was found that there were one to five expressing genes in each locus. Among the Mamu-A, Mamu-B, and Mamu-I sequences, 44 (60.2%), 45 (54.2%), and 8 (53.3%), respectively, were novel, and most of the other known alleles were identical to those reported from Chinese- or Indian-origin macaques, demonstrating a genetic mixture between the geographically distinct populations of present day China and India. In addition, it was found that a Mamu haplotype contained at least two highly transcribed Mamu-A genes, because multiple Mamu-A1 cDNAs were obtained from one haplotype. These findings further revealed the diversity and complexity of MHC locus in the rhesus macaques.     

KIR polymorphisms modulate peptide-dependent binding to an MHC class I ligand with a Bw6 motif

Molecular interactions between killer immunoglobulin-like receptors (KIRs) and their MHC class I ligands play a central role in the regulation of natural killer (NK) cell responses to viral pathogens and tumors. Here we identify Mamu-A1*00201 (Mamu-A*02), a common MHC class I molecule in the rhesus macaque with a canonical Bw6 motif, as a ligand for Mamu-KIR3DL05. Mamu-A1*00201 tetramers folded with certain SIV peptides, but not others, directly stained primary NK cells and Jurkat cells expressing multiple allotypes of Mamu-KIR3DL05. Differences in binding avidity were associated with polymorphisms in the D0 and D1 domains of Mamu-KIR3DL05, whereas differences in peptide-selectivity mapped to the D1 domain. The reciprocal exchange of the third predicted MHC class I-contact loop of the D1 domain switched the specificity of two Mamu-KIR3DL05 allotypes for different Mamu-A1*00201-peptide complexes. Consistent with the function of an inhibitory KIR, incubation of lymphocytes from Mamu-KIR3DL05(+) macaques with target cells expressing Mamu-A1*00201 suppressed the degranulation of tetramer-positive NK cells. These observations reveal a previously unappreciated role for D1 polymorphisms in determining the selectivity of KIRs for MHC class I-bound peptides, and identify the first functional KIR-MHC class I interaction in the rhesus macaque. The modulation of KIR-MHC class I interactions by viral peptides has important implications to pathogenesis, since it suggests that the immunodeficiency viruses, and potentially other types of viruses and tumors, may acquire changes in epitopes that increase the affinity of certain MHC class I ligands for inhibitory KIRs to prevent the activation of specific NK cell subsets.     

The role of BiP in endoplasmic reticulum-associated degradation of major histocompatibility complex class I heavy chain induced by cytomegalovirus proteins

Human cytomegalovirus (HCMV1) US11 and US2 proteins cause rapid degradation of major histocompatibility complex (MHC) molecules, apparently by ligating cellular endoplasmic reticulum (ER)-associated degradation machinery. Here, we show that US11 and US2 bind the ER chaperone BiP. Four related HCMV proteins, US3, US7, US9, and US10, which do not promote degradation of MHC proteins, did not bind BiP. Silencing BiP reduced US11- and US2-mediated degradation of MHC class I heavy chain (HC) without altering the synthesis or translocation of HC into the ER or the stability of HC in the absence of US11 or US2. Induction of the unfolded protein response (UPR) did not affect US11-mediated HC degradation and could not explain the stabilization of HC when BiP was silenced. Unlike in yeast, BiP did not act by maintaining substrates in a retrotranslocation-competent form. Our studies go beyond previous observations in mammalian cells correlating BiP release with degradation, demonstrating that BiP is functionally required for US2- and US11-mediated HC degradation. Further, US2 and US11 bound BiP even when HC was absent and degradation of US2 depended on HC. These data were consistent with a model in which US2 and US11 bridge HC onto BiP promoting interactions with other ER-associated degradation proteins.     

Positive selection is limited by available peptide-dependent MHC conformations

Recent data suggest that the diversity of self peptides presented in the thymus during development contributes to positive selection of a diverse T cell repertoire. We sought to determine whether a previously defined hole in the immunological repertoire could be explained by the absence of an appropriate selecting self peptide. The repertoire defect in question is the inability of bm8 mice to make an H-2K-restricted response to OVA. Like other OVA-specific, H-2K-restricted receptors, OT-I-transgenic T cells are not positively selected in bm8 mice. Using criteria we had previously established for identifying positive selection ligands, we found peptides that could restore positive selection of OT-I thymocytes in bm8 mice. Thus, the T cell repertoire can be limited by a requirement for specific self peptides during development. Data with MHC-specific Abs suggested that peptides might be able to force MHC residues to adopt different conformations in Kb vs Kbm8. This shows that peptides can potentially contribute to ligand diversity both directly (via variability in the solvent-exposed side chains) and indirectly (through their effect on the MHC conformation). Our data support a model where self peptide diversity allows selection of T cells specific for a broad range of MHC conformations.     

Conserved MHC class I peptide binding motif between humans and rhesus macaques

Since the onset of the HIV pandemic, the use of nonhuman primate models of infection has increasingly become important. An excellent model to study HIV infection and immunological responses, in particular cell-mediated immune responses, is SIV infection of rhesus macaques. CTL epitopes have been mapped using SIV-infected rhesus macaques, but, to date, a peptide binding motif has been described for only one rhesus class I MHC molecule, Mamu-A*01. Herein, we have established peptide-live cell binding assays for four rhesus MHC class I molecules: Mamu-A*11, -B*03, -B*04, and -B*17. Using such assays, peptide binding motifs have been established for all four of these rhesus MHC class I molecules. With respect to the nature and spacing of crucial anchor positions, the motifs defined for Mamu-B*04 and -B*17 present unique features not previously observed for other primate species. The motifs identified for Mamu-A*11 and -B*03 are very similar to the peptide binding motifs previously described for human HLA-B*44 and -B*27, respectively. Accordingly, naturally processed peptides derived from HLA-B*44 and HLA-B*27 specifically bind Mamu-A*11 and Mamu-B*03, respectively, indicating that conserved MHC class I binding capabilities exist between rhesus macaques and humans. The definition of four rhesus MHC class I-specific motifs expands our ability to accurately detect and quantitate immune responses to MHC class I-restricted epitopes in rhesus macaques and to rationally design peptide epitope-based model vaccine constructs destined for use in nonhuman primates.     

A cytomegalovirus glycoprotein re-routes MHC class I complexes to lysosomes for degradation

Mouse cytomegalovirus (MCMV) early gene expression interferes with the major histocompatibility complex class I (MHC class I) pathway of antigen presentation. Here we identify a 48 kDa type I transmembrane glycoprotein encoded by the MCMV early gene m06, which tightly binds to properly folded beta2-microglobulin (beta2m)-associated MHC class I molecules in the endoplasmic reticulum (ER). This association is mediated by the lumenal/transmembrane part of the protein. gp48-MHC class I complexes are transported out of the ER, pass the Golgi, but instead of being expressed on the cell surface, they are redirected to the endocytic route and rapidly degraded in a Lamp-1(+) compartment. As a result, m06-expressing cells are impaired in presenting antigenic peptides to CD8(+) T cells. The cytoplasmic tail of gp48 contains two di-leucine motifs. Mutation of the membrane-proximal di-leucine motif of gp48 restored surface expression of MHC class I, while mutation of the distal one had no effect. The results establish a novel viral mechanism for downregulation of MHC class I molecules by directly binding surface-destined MHC complexes and exploiting the cellular di-leucine sorting machinery for lysosomal degradation.     

Human cytomegalovirus immediate early glycoprotein US3 retains MHC class I molecules by transient association

Human cytomegalovirus (HCMV) interferes with major histocompatibility complex (MHC) class I antigen presentation by a sequential multistep process to escape T cell surveillance. During the immediate early phase of infection, the glycoprotein US3 prevents intracellular transport of MHC class I molecules. Interestingly, US3 displays a significantly shorter half-life than US3-retained MHC class I molecules. Here we show that US3 associates only transiently with MHC class I molecules, exits the ER, and is inefficiently retrieved from the Golgi. US3 was degraded in a post-Golgi compartment, most likely lysosomes, because: i) Brefeldin A treatment prolonged the half-life of US3; and ii) US3 co-localized with the lysosomal marker protein LAMP in chloroquine-treated cells. In contrast, MHC class I molecules remained stable in the ER. Upon inhibition of protein synthesis MHC class I molecules were released suggesting that a continuous supply of newly synthesized US3 molecules is required for inhibition of transport. Thus, US3 does not seem to retain MHC class I molecules by a retrieval mechanism. Instead, our observations are consistent with US3 preventing MHC class I trafficking by blocking forward transport.     

The cytoplasmic domain of rhesus cytomegalovirus Rh178 interrupts translation of major histocompatibility class I leader peptide-containing proteins prior to translocation

Cytomegalovirus (CMV) efficiently evades many host immune defenses and encodes a number of proteins that prevent antigen presentation by major histocompatibility complex class I (MHC-I) molecules in order to evade recognition and killing of infected cells by cytotoxic CD8(+) T cells. We recently showed that rhesus CMV-specific Rh178 intercepts MHC-I protein translation before interference of MHC-I maturation by homologues of the human CMV US6 family. Here, we demonstrate that Rh178 localizes to the membrane of the endoplasmic reticulum, displaying a short luminal and large cytosolic domain, and that the membrane-proximal cytosolic portion is essential for inhibition of MHC-I expression. We further observed that Rh178 does not require synthesis of full-length MHC-I heavy chains but is capable of inhibiting the translation of short, unstable amino-terminal fragments of MHC-I. Moreover, the transfer of amino-terminal fragments containing the MHC-I signal peptide renders recipient proteins susceptible to targeting by Rh178. The cytosolic orientation of Rh178 and its ability to target protein fragments carrying the MHC-I signal peptide are consistent with Rh178 intercepting partially translated MHC-I heavy chains after signal recognition particle-dependent transfer to the endoplasmic reticulum membrane. However, interference with MHC-I translation by Rh178 seems to occur prior to SEC61-dependent protein translocation, since inhibition of MHC-I translocation by eeyarestatin 1 resulted in a full-length degradation intermediate that can be stabilized by proteasome inhibitors. These data are consistent with Rh178 blocking protein translation of MHC-I heavy chains at a step prior to the start of translocation, thereby downregulating MHC-I at a very early stage of translation.     

CLIP-region mediated interaction of Invariant chain with MHC class I molecules

An association between the MHC class II chaperone molecule Invariant chain (Ii) and MHC class I molecules is known to occur, but the basis of the interaction is undetermined. Evidence is presented here that the CLIP region of Ii is involved in this interaction. A peptide encoding residues 91-99 of CLIP (MRMATPLLM) stabilised multiple MHC class I alleles, with the methionine residue at position 99 having a crucial role in binding to H2-K(b), whereas methionine at position 91 also appeared important in binding to RT1-A(a). Ii can also be detected in the class I MHC peptide loading complex. These data provide an explanation for the association of Ii and MHC class I molecules.     

Rapid high-resolution MHC class I genotyping of Chinese rhesus macaques by capillary reference strand-mediated conformational analysis

Rhesus macaques (Macaca mulatta) provide well-established models for studying human disease pathogenesis and vaccine development. When challenged with infectious agents, macaques exhibit individual differences in susceptibility. An important determinant of these differences is the complement of major histocompatability complex (MHC) class I sequences expressed by each animal. Although previous studies have reported strong associations between MHC expression and disease outcome, a rapid, cost-effective method for high-resolution MHC genotyping in macaques is lacking. In this study, we adapted a modified heteroduplex assay, reference strand-mediated conformational analysis (RSCA) to an ABI 3130xl capillary electrophoresis genetic analyzer for macaque MHC class I genotyping. For validation, we investigated the concordance of RSCA genotyping for 14 MHC class I sequences in 12 Chinese rhesus macaques whose genotypes were established through complementary DNA cloning and sequencing of MHC class I sequences. We observed a concordance greater than 98% between RSCA and the cloning and sequencing data. Furthermore, RSCA confirmed the presence of MHC haplotype sharing between three macaques as predicted previously by microsatellite analysis. RSCA genotyping of an additional 25 Chinese rhesus macaques demonstrated that the frequency of these 14 MHC class I sequences ranged from 5% to 32%, with the Mamu-A1*2601 sequence being most common in this cohort. Capillary RSCA genotyping has the potential to enable researchers to rapidly evaluate MHC class I genotypes in rhesus macaques and associate specific MHC sequences with disease susceptibility.     

HIV envelope protein inhibits MHC class I presentation of a cytomegalovirus protective epitope

CTL recognize peptides that derive from viral protein Ags by proteolytic processing and are presented by MHC class I molecules. In this study we tested whether coexpression of viral Ags in the same cell leads to competition between them. To this end, two L(d)-restricted epitopes derived from HIV-1 envelope gp160 (ENV) and from CMV pp89 phosphoprotein were coexpressed. HIV ENV strain IIIB, but not MN variant, impaired recognition by specific CTL of CMV pp89 epitope 9pp89. Susceptibility to inhibition after ENV coexpression was inversely related to the amount of antigenic 9pp89 peptide processed from different antigenic constructs. In line with it, competition decreased the yield of naturally processed antigenic 9pp89 peptide bound to MHC class I molecules in coinfected cells. Also, point mutants of the presenting MHC class I molecule differed in their competition pattern. Collectively, the data imply that competition operates at the step of MHC-peptide complex assembly or stabilization. We conclude that, although not the rule, in certain combinations there is interference between different Ags expressed in the same cell and presented by the same MHC class I allele. These studies have implications for vaccine development and for understanding immunodominance.     

Signal transduction through class I MHC by a monoclonal antibody that detects multiple murine and human class I molecules.

We have generated a hamster anti-mouse class I reactive mAb that is capable of activating T cells in the presence of the cofactor PMA, as assayed by both IFN-gamma production and cellular proliferation. This mAb detects an epitope present on the majority of murine class I molecules, with the known exceptions of H-2Kk and H-2Kq, and is therefore not beta 2-microglobulin-specific. It also recognizes multiple human class I molecules. The epitope recognized by this antibody maps to the class I alpha 1 domain. The activation properties of this mAb are not mediated exclusively through the glycosylphosphatidylinositol-linked Qa-2 molecule, as the antibody activates spleen cells from Qa-2 negative strains. Although class I molecules are not usually considered as activation Ag, these data demonstrate their potential for involvement in signal transduction.     

Peptide presentation by MHC class I molecules

The presentation of peptides by class I histocompatibility molecules plays a central role in the cellular immune response to virally infected or transformed cells. The main steps in this process include the degradation of both self and 'foreign' proteins to short peptides in the cytosol, translocation of peptides into the lumen of the endoplasmic reticulum, binding of a subset of peptides to assembling class I molecules and expression of class-I-peptide complexes at the cell surface for examination by cytotoxic T cells. A molecular understanding of most of these steps is emerging, revealing a remarkable coordination between the processes of peptide translocation, delivery and binding to class I molecules.     

MHC class I multimers

T lymphocytes play a key role in the immune response to both foreign and self peptide antigens, which they recognize in combination with MHC molecules. In the past it has been difficult to analyse objectively the specificity, frequency and intensity of T cell responses. The recent application of fluorescent-labelled MHC class I multimers, however, has provided a powerful experimental approach to the direct visualisation of antigen-specific T cells. As a result, our perspective of how T cells respond to both viruses and other antigens in vivo has been greatly enhanced.     

Induction of assembly of MHC class I heavy chains with beta 2microglobulin by interferon-gamma.

Assembly of histocompatibility class I heavy chains with beta 2microglobulin (beta 2m) is known to be necessary for cell surface expression. Studies on the H-2 class I deficient but interferon-gamma (IFN-gamma) inducible fibrosarcoma BC2 and the lung carcinoma CMT 64.5 showed that after transfection with allogeneic H-2 class I genes the class I proteins are expressed, but only intracellularly and not on the cell surface. In spite of the presence of beta 2m in the cells no association of the transfected class I chain with beta 2m was observed. However, stimulation with IFN-gamma induced assembly and subsequent surface expression. These findings show that the assembly of class I heavy chains with beta 2m is not a spontaneous event but appears to be regulated by cellular mechanisms the nature of which is still unknown.     

Human cytomegalovirus disrupts constitutive MHC class II expression

CD8(+) and CD4(+) T lymphocytes are important in controlling human CMV (HCMV) infection, but the virus has evolved protean mechanisms to inhibit MHC-based Ag presentation and escape T lymphocyte immunosurveillance. Herein, the interaction of HCMV with the MHC class II Ag presentation pathway was investigated in cells stably transfected with class II transactivator. Flow cytometry experiments demonstrate that HCMV infection decreases cell-surface MHC class II expression. HCMV down-regulates MHC class II surface expression without a significant effect on class II RNA or steady-state protein levels. SDS-stability and confocal microscopy experiments demonstrate normal levels of steady-state peptide-loaded class II molecules in infected cells and that class II molecules reach late endosomal and HLA-DM positive peptide-loading compartments. However, MHC class II positive vesicles are retained in an abnormal perinuclear distribution. Finally, experiments with a mutant HCMV strain demonstrate that this novel mechanism of decreased MHC class II expression is not mediated by one of the known HCMV immunomodulatory genes. These defects in MHC class II expression combined with previously identified CMV strategies for decreasing MHC class I expression enables infected cells to evade T lymphocyte immunosurveillance.     

Presentation of antigenic peptides by MHC class I molecules

The basis for the immune response against intracellular pathogens is the recognition by cytotoxic T lymphocytes of antigenic peptides derived from cytosolic proteins, which are presented on the cell surface by major histocompatibility complex (MHC) class I molecules. The understanding of MHC class I-restricted peptide presentation has recently improved dramatically with the elucidation of the structural basis for the specificity of peptide binding to MHC class I molecules and the identification of proteins encoded in the class II region of the MHC that are putatively involved in the production of peptides and their transport into the endoplasmic reticulum, where they assemble with class I molecules.