Complementary DNA sequences encoding the multimammate rat MHC class II DQ α and β chains and cross-species sequence comparison in rodents

Sequences of the complete open reading frame (ORF) for rodents major histocompatibility complex (MHC) class II genes are rare. Multimammate rat ( Mastomys natalensis ) complementary DNA (cDNA) encoding the α and β chains of MHC class II DQ gene was cloned from a rapid amplifications of cDNA Emds (RACE) cDNA library. The ORFs consist of 801 and 771 bp encoding 266 and 256 amino acid residues for DQB and DQA, respectively. The genomic structure of Mana-DQ genes is globally analogous to that described for other rodents except for the insertion of a serine residue in the signal peptide of Mana-DQB , which is unique among known rodents.     

Enhancement of class I HLA antigen expression by cytomegalovirus: role in amplification of virus infection

We have investigated the effect of cytomegalovirus (CMV) infection on the expression of class I HLA antigens on fibroblasts in vitro. Scanning and integrating microdensitometry was used to quantitate the level of cytoplasmic class I antigen expression, and an antibody binding assay was used to quantitate cell surface expression of class I HLA molecules. CMV infection resulted in a significant increase in the level of cytoplasmic and cell surface class I HLA antigen expression of fibroblast monolayers. The maximal effect was seen at 72 hours postinfection and was observed with both the laboratory strain of CMV, strain AD169, and with CMV purified directly from clinical specimens. Part of the increased HLA expression was mediated by interferon released from infected cells; however, an additional direct effect of the virus itself has not been ruled out. Interferon-induced increased expression of class I HLA antigens was accompanied by increased binding of CMV to the cells, consistent with our recent demonstration that class I HLA molecules can function as a cellular receptor for CMV.     

Induction of MHC class I molecule cell surface expression and epigenetic activation of antigen-processing machinery components in a murine model for human papilloma virus 16-associated tumours

Epigenetic events play an important role in tumour progression and also contribute to escape of the tumour from immune surveillance. In this study, we investigated the up-regulation of major histocompatibility complex (MHC) class I surface expression on tumour cells by epigenetic mechanisms using a murine tumour cell line expressing human E6 and E7 human papilloma virus 16 (HPV16) oncogenes and deficient in MHC class I expression, as a result of impaired antigen-presenting machinery (APM). Treatment of the cells with the histone deacetylase inhibitor Trichostatin A, either alone or in combination with the DNA demethylating agent 5-azacytidine, induced surface re-expression of MHC class I molecules. Consequently, the treated cells became susceptible to lysis by specific cytotoxic T lymphocytes. Further analysis revealed that epigenetic induction of MHC class I surface expression was associated with the up-regulation of APM genes [transporter associated with antigen processing 1 (TAP-1), TAP-2, low-molecular-mass protein 2 (LMP-2) and LMP-7]. The results demonstrate that expression of the genes involved in APM are modulated by epigenetic mechanisms and suggest that agents modifying DNA methylation and/or histone acetylation have the potential to change the effectiveness of antitumour immune responses and therapeutically may have an impact on immunological output.     

Lipopeptides: a novel antigen repertoire presented by major histocompatibility complex class I molecules

Post‐translationally modified peptides, such as those containing either phosphorylated or O‐glycosylated serine/threonine residues, may be presented to cytotoxic T lymphocytes (CTLs) by MHC class I molecules. Most of these modified peptides are captured in the MHC class I groove in a similar manner to that for unmodified peptides. N‐Myristoylated 5‐mer lipopeptides have recently been identified as a novel chemical class of MHC class I‐presented antigens. The rhesus classical MHC class I allele, Mamu‐B*098, was found to be capable of binding N‐myristoylated lipopeptides and presenting them to CTLs. A high‐resolution X‐ray crystallographic analysis of the Mamu‐B*098:lipopeptide complex revealed that the myristic group as well as conserved C‐terminal serine residue of the lipopeptide ligand functioned as anchors, whereas the short stretch of three amino acid residues located in the middle of the lipopeptides was only exposed externally with the potential to interact directly with specific T‐cell receptors. Therefore, the modes of lipopeptide–ligand interactions with MHC class I and with T‐cell receptors are novel and fundamentally distinct from that for MHC class I‐presented peptides. Another lipopeptide‐presenting MHC class I allele has now been identified, leading us to the prediction that MHC class I molecules may be separated on a functional basis into two groups: one presenting long peptides and the other presenting short lipopeptides. Since the N‐myristoylation of viral proteins is often linked to pathogenesis, CTLs capable of sensing N‐myristoylation may serve to control pathogenic viruses, raising the possibility for the development of a new type of lipopeptide vaccine.     

Type I Streptococcus pneumoniae carbohydrate utilizes a nitric oxide and MHC II-dependent pathway for antigen presentation

Some pathogenic bacteria form thick capsules that both block immune responses through inhibition of complement deposition and phagocytosis and stimulate a weak response resulting from a lack of T-cell involvement. Contrary to this model, capsular polysaccharides from 23 serotypes of Streptococcus pneumoniae have been successfully used in a multivalent vaccine in the absence of a carrier protein. Furthermore, type I pneumococcal polysaccharide (Sp1) has been shown to activate T cells in vivo and in vitro via an uncharacterized mechanism. In the present report, we demonstrate that Sp1 utilizes the major histocompatibility complex (MHC) class II pathway in antigen-presenting cells (APCs) for processing and presentation. APCs internalize and process Sp1 through a nitric oxide-dependent mechanism and, once inside the cell, it associates with MHC II proteins in an H-2M-dependent manner that leads to in vivo T-cell activation. These results establish that Sp1 activates T cells which can lead to abscess formation in mice through an H-2M-dependent polysaccharide antigen presentation pathway in APCs, potentially contributing to pneumococcal polysaccharide vaccine efficacy through the recruitment of T-cell help.     

Peptide–MHC Complexes Assembled Following Multiple Pathways:: An Opportunity for the Design of Vaccines and Therapeutic Molecules

Antigen degradation and peptide loading to major histocompatibility complex class I and class II molecules are described with special emphasis on “noncanonical” pathways. Examples of specific peptide loading for measles proteins are provided. In addition, characterization of defined epitopes presented to T cells can lead to the design of products of special interest in medicine and, in particular, in development of vaccines.     

Major histocompatibility complex class I binding predictions as a tool in epitope discovery

Over the last decade, in silico models of the major histocompatibility complex (MHC) class I pathway have developed significantly. Before, peptide binding could only be reliably modelled for a few major human or mouse histocompatibility molecules; now, high‐accuracy predictions are available for any human leucocyte antigen (HLA) ‐A or ‐B molecule with known protein sequence. Furthermore, peptide binding to MHC molecules from several non‐human primates, mouse strains and other mammals can now be predicted. In this review, a number of different prediction methods are briefly explained, highlighting the most useful and historically important. Selected case stories, where these ‘reverse immunology’ systems have been used in actual epitope discovery, are briefly reviewed. We conclude that this new generation of epitope discovery systems has become a highly efficient tool for epitope discovery, and recommend that the less accurate prediction systems of the past be abandoned, as these are obsolete.     

The interface between tapasin and MHC class I: identification of amino acid residues in both proteins that influence their interaction

Prior to the binding of antigenic peptide, a complex of chaperone proteins associates with the Major Histocompatibility Complex (MHC) class I heavy chain/β₂m heterodimer. Although each dornain of the MHC class I heavy chain contains amino acid resid uses that influence chaperone binding, there are several pieces of evidence that point to an interaction between the MHC clas 1α2/α3 domains and tapasin. In egard to the site on tapasin involved in the tapasin/MHC interface, we have found that a particular region of tapasin (containing amino acid residues 334–342) is necessary for the binding of tapasin to the MHC class I heavy chain. Our results also indicate that amino acids in this region of tapasin also affect the proportion of MHC class I open forms expressed at the cell surface and MHC class I egress from the endoplasmic reticulurn. Based on these results and those obtained by other laboratories, a model for MHC class I/tapasin interaction is proposed.     

Binding domain regulation of MHC class II molecule assembly, trafficking, fate, and function

Major histocompatibility complex class II molecules are heterodimeric type I integral membrane glycoproteins whose primary function is the capture of fragments of antigen in the endocytic pathway, and the presentation of these peptides to CD4+ αβ TCR-bearing T cells. The biochemical features of the class II peptide binding domain optimize it for this function by allowing interaction with denatured proteins prior to extensive degradation in endosomes and lysosomes. These same properties pose problems for αβ heterodimer assembly, avoidance of non-productive interactions with self-proteins, intracellular transport and dimer stability. This review discusses how coevolution of α and β chain binding domain polymorphism and the extrinsic control of binding site function by invariant chain occupancy deal with these problems and permit the efficient functioning of the class II presentation system.     

Activation of human CD4+ T cells by targeting MHC class II epitopes to endosomal compartments using human CD1 tail sequences

Distinct CD4(+) T-cell epitopes within the same protein can be optimally processed and loaded into major histocompatibility complex (MHC) class II molecules in disparate endosomal compartments. The CD1 protein isoforms traffic to these same endosomal compartments as directed by unique cytoplasmic tail sequences, therefore we reasoned that antigen/CD1 chimeras containing the different CD1 cytoplasmic tail sequences could optimally target antigens to the MHC class II antigen presentation pathway. Evaluation of trafficking patterns revealed that all four human CD1-derived targeting sequences delivered antigen to the MHC class II antigen presentation pathway, to early/recycling, early/sorting and late endosomes/lysosomes. There was a preferential requirement for different CD1 targeting sequences for the optimal presentation of an MHC class II epitope in the following hierarchy: CD1b > CD1d = CD1c > > > CD1a or untargeted antigen. Therefore, the substitution of the CD1 ectodomain with heterologous proteins results in their traffic to distinct intracellular locations that intersect with MHC class II and this differential distribution leads to specific functional outcomes with respect to MHC class II antigen presentation. These findings may have implications in designing DNA vaccines, providing a greater variety of tools to generate T-cell responses against microbial pathogens or tumours.     

A simple method to eliminate the antigenicity of surface class I MHC molecules from the membrane of viable cells by acid treatment at pH 3

We describe here a simple, reproducible method which specifically eliminates the antigenecity of surface class I major histocompatibility complex (MHC) molecules by acid treatment at pH 3 from the membrane of viable cells. When fresh mononuclear cells (MNC) or established cultured cell lines were treated at 4°C for 2 min with citric acid buffer at pH 3 containing 1% bovine serum albumin, the antigenicity of class I MHC molecules, but not those of class II MHC and the other non-MHC antigens, was eliminated from the surface membrane without significant cell death. This method was effective for both human and murine cells with various origins. Monoclonal and polyclonal antibodies were used to identify the expression of surface antigens in conjunction with immunofluorescence tests. The eliminated antigenicity of human class I MHC antigens (i.e., HLA-A,B,C) on MNC regenerated when cells were incubated in the medium at 37°C for 10 h. The pretreatment of cells with emetine (10⁻⁴ M), a protein synthesis inhibitor, was found to be effective in inhibiting this regeneration. The acid treatment method might be useful for future studies on the functional characterization of surface class I MHC antigens.     

The structure and location of SIMP/STT3B account for its prominent imprint on the MHC I immunopeptidome

Proteins show drastic discrepancies in their contribution to the collection of self-peptides that shape the repertoire of CD8 T cells (MHC I self-immunopeptidome). To decipher why selected proteins are the foremost sources of MHC I-associated self-peptides, we chose to study SIMP/STT3B because this protein generates very high amounts of MHC I-associated peptides in mice and humans. We show that the endoplasmic reticulum (ER)-associated degradation pathway and MHC I processing intersect at SIMP/STT3B. Relevant key features of SIMP/STT3B are its lysine-rich region, its propensity to misfold and its location in the ER membrane in close proximity to the immunoproteasome. Moreover, we show that coupling to SIMP/STT3B can be used to foster MHC I presentation of a selected peptide, here the ovalbumin peptide SIINFEKL. These data yield novel insights into relations between the cell proteome and the MHC I immunopeptidome. They suggest that the contribution of a given protein to the MHC I immunopeptidome results from the interplay of at least three factors: the presence of degrons (degradation signals), the tendency of the protein to misfold and its subcellular localization. Furthermore, they indicate that substrates of the ER-associated degradation pathway may have a prominent imprint on the MHC I self-immunopeptidome.     

Association of MHC dimorphic Alu insertions with HLA class I and MIC genes in Japanese HLA-B48 haplotypes

A large proportion of Japanese with the HLA-B48 allele have a MICA gene deletion associated with a MICB null allele within the class I region of the Major Histocompatibility Complex (MHC). Here, we report for the first time a novel positive association between the presence of a polymorphic Alu insertion, AluyMICB, within the first intron of the MICB gene and the MICAdel/MICBnull/HLA-B48 haplotype for five of six well-characterized Japanese cell-lines. The AluyMICB insertion was found to be present at a frequency of 0.242 in 86 Japanese tissue donors and in four of the five individuals with the HLA-B48 allele. The AluyMICB insertion was also associated with at least three different MICB alleles, *0102, *0107N and *0105, and three different HLA-B alleles, B13, B48 and B57, respectively, in the seven Workshop cell-lines (the 4th Asia-Oceania Histocompatibility Workshop, and the 10th International Histocompatibility Workshop) and the six Japanese cell-lines that were selected for this study. Based on the analysis of associations between different polymorphic markers within the beta block, the MICB*0102 allele was inferred to be the ancestral form of the MICB*0105 and MICB*0107N alleles. The AluyMICB polymorphism can now be used to further investigate its relationship with other MICB alleles and consequently their origins. In addition, we have examined the absence and presence of three other polymorphic Alu markers distributed within the alpha block of the class I region of the HLA-B48/AluyMICB haplotype. We conclude that the extended HLA-B haplotypes are best defined by considering multiple genomic sites including the four polymorphic Alu insertions described in this study.     

Mapping and restriction of a dominant viral CD4+ T cell core epitope by both MHC class I and MHC class II

Virus-specific CD4(+) T cells contribute to effective virus control through a multiplicity of mechanisms including direct effector functions as well as "help" for B cell and CD8(+) T cell responses. Here, we have used the lymphocytic choriomeningitis virus (LCMV) system to assess the minimal constraints of a dominant antiviral CD4(+) T cell response. We report that the core epitope derived from the LCMV glycoprotein (GP) is 11 amino acids in length and provides optimal recognition by epitope-specific CD4(+) T cells. Surprisingly, this epitope is also recognized by LCMV-specific CD8(+) T cells and thus constitutes a unique viral determinant with dual MHC class I- and II-restriction.     

The regulation of exogenous and endogenous class I MHC genes in a human tumor cell line, K562

Previous studies have implied the existence of a trans-dominant intracellular repressor able to down-regulate the expression of the entire family of class I MHC genes in the genome of the K562 erythroleukemia cell line. This study demonstrates, however, that the transfection of human or murine class I genes into K562 cells leads to the cell surface expression of the transfected MHC gene product in all situations, even when several kilobases of 5' flanking sequence were included in the transfected genes. The endogenous cellular class I MHC genes remained repressed in the transfected cells. These findings suggest that repression of class I MHC gene expression in K562 may not be mediated predominantly by a trans-dominant repressor of MHC gene expression; rather, other more complex regulatory influences might exist.     

Differential contribution of TAP and tapasin to HLA class I antigen expression

Expression of class I human leucocyte antigens (HLA) on the surface of malignant cells is critical for their recognition and destruction by cytotoxic T lymphocytes. Surface expression requires assembly and folding of HLA class I molecules in the endoplasmic reticulum with the assistance of proteins such as Transporter associated with Antigen Processing (TAP) and tapasin. Interferon-gamma induces both TAP and tapasin so dissection of which protein contributes more to HLA class I expression has not been possible previously. In this study, we take advantage of a human melanoma cell line in which TAP can be induced, but tapasin cannot. Interferon-gamma increases TAP protein levels dramatically but HLA class I expression at the cell surface does not increase substantially, indicating that a large increase in peptide supply is not sufficient to increase HLA class I expression. On the other hand, transfection of either allelic form of tapasin (R240 or T240) enhances HLA-B*5001 and HLA-B*5701 antigen expression considerably with only a modest increase in TAP. Together, these data indicate that in the presence of minimal TAP activity, tapasin can promote substantial HLA class I expression at the cell surface.