NK-mediated elimination of mutant lymphocytes that have lost expression of MHC class I molecules

Mutant cells generated in vivo can be eliminated when mutated gene products are presented as altered MHC/peptide complexes and recognized by T cells. Diminished expression of MHC/peptide complexes enables mutant cells to escape recognition by T cells. In the present study, we tested the hypothesis that mutant lymphocytes lacking expression of MHC class I molecules are eliminated by autologous NK cells. In H-2b/k F1 mice, the frequency of H-2Kb-negative T cells was higher than that of H-2Kk-negative T cells. The frequency of H-2K-deficient T cells increased transiently after total body irradiation. During recovery from irradiation, H-2Kk-negative T cells disappeared more rapidly than H-2Kb-negative T cells. The disappearance of H-2K-deficient T cells was inhibited by administration of Ab against asialo-GM1. H-2Kk-negative T cells showed higher sensitivity to autologous NK cells in vitro than H-2Kb/k heterozygous or H-2Kb-negative T cells. Adding syngeneic NK cells to in vitro cultures prevented emergence of mutant cells lacking H-2Kk expression but had little effect on the emergence of mutant cells lacking H-2Kb expression. Results in the H-2b/k F1 strain correspond with the sensitivity of parental H-2-homozygous cells in models of marrow graft rejection. In H-2b/d F1 mice, there was no significant difference between the frequencies of H-2Kb-negative and H-2Kd-negative T cells, although the frequencies of mutant cells were different after radiation exposure among the strains examined. H-2b/d F1 mice also showed rapid disappearance of the mutant T cells after irradiation, and administration of Ab against asialo-GM1 inhibited the disappearance of H-2K-deficient T cells in H-2b/d F1 mice. Our results provide direct evidence that autologous NK cells eliminate mutant cell populations that have lost expression of self-MHC class I molecules.     

A novel expression vector for transfection of bovine MHC class I genes

A vector is described for the expression of genomic or cDNA copies of bovine major histocompatibility complex (MHC) class I genes in transfected mouse Ltk⁻ cells. Class I gene fragments are amplified by the polymerase chain reaction, using primers in conserved parts of exon 2 and the 3′-untranslated region of the gene. Amplified class I gene fragments can then be subcloned into the expression vector, pBoLA-21, which contains the necessary 5′-and 3′-sequences for correct expression. The vector was tested by subcloning and expressing genomic and cDNA clones.     

Analysis of MHC class I genes across horse MHC haplotypes

The genomic sequences of 15 horse major histocompatibility complex (MHC) class I genes and a collection of MHC class I homozygous horses of five different haplotypes were used to investigate the genomic structure and polymorphism of the equine MHC. A combination of conserved and locus-specific primers was used to amplify horse MHC class I genes with classical and nonclassical characteristics. Multiple clones from each haplotype identified three to five classical sequences per homozygous animal and two to three nonclassical sequences. Phylogenetic analysis was applied to these sequences, and groups were identified which appear to be allelic series, but some sequences were left ungrouped. Sequences determined from MHC class I heterozygous horses and previously described MHC class I sequences were then added, representing a total of ten horse MHC haplotypes. These results were consistent with those obtained from the MHC homozygous horses alone, and 30 classical sequences were assigned to four previously confirmed loci and three new provisional loci. The nonclassical genes had few alleles and the classical genes had higher levels of allelic polymorphism. Alleles for two classical loci with the expected pattern of polymorphism were found in the majority of haplotypes tested, but alleles at two other commonly detected loci had more variation outside of the hypervariable region than within. Our data indicate that the equine major histocompatibility complex is characterized by variation in the complement of class I genes expressed in different haplotypes in addition to the expected allelic polymorphism within loci.     

Marsupial MHC class II beta genes are not orthologous to the eutherian beta gene families

The major histocompatibility complex (MHC) class II DRB, DQB, DPB, and DOB gene clusters are shared by different eutherian orders. Such an orthologous relationship is not seen between the beta genes of birds and eutherians. A high degree of uncertainty surrounds the evolutionary relationship of marsupial class II beta sequences with eutherian beta gene families. In particular, it has been suggested that marsupials utilize the DRB gene cluster. A cDNA encoding an MHC class II beta molecule was isolated from a brushtail possum mesenteric lymph node cDNA library. This clone is most similar to Macropus rufogriseus DBB. Our analysis suggests that all known marsupial beta-chain genes, excluding DMB, fall into two separate clades, which are distinct from the eutherian DRB, DQB, DPB, or DOB gene clusters. We recommend that the DAB and DBB nomenclature be reinstated. DAB and DBB orthologs are not present in eutherians. It appears that the marsupial and eutherian lineages have retained different gene clusters following gene duplication events early in mammalian evolution.     

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.     

Evidence that a mechanism for efficient flavivirus budding upregulates MHC class I

An appealing hypothesis for the biological role of flavivirus-induced, interferon-independent, upregulation of MHC class I on the surface of infected cells is that of viral immune evasion from NK cell recognition. Here we show that a mechanism for efficient flavivirus morphogenesis interferes with the MHC class I pathway, using a flavivirus budding mutant and recombinant expression of wild-type and mutant forms of the flavivirus structural proteins. We propose that the phenomenon of flavivirus-mediated MHC class I upregulation is a by-product of a unique assembly strategy evolved by flaviviruses and therefore did not evolve primarily as an immune escape mechanism for virus growth in the vertebrate host.     

Partial purification and characterization of a novel human factor that augments the expression of class I MHC antigens on tumour cells

A cytokine which augments the expression of major histocompatibility complex (MHC) I antigens on K562 and gastric carcinoma tumour (HR) cells, has been isolated from the culture supernatant of Concanavalin-A (Con-A) activated human peripheral blood mononuclear cells. The factor, termed MHC augmenting factor (MHC- AF) has been partially purified by Sephadex G- 100 column chromatography, preparative isoelectric focusing and HPLC with ion- exchange as well as sizing columns. MHC-AF activity is associated with a 35 kDa molecule which has pI of 6.0. Interferon (IFN)-α, \, tumour necrosis factor (TNF), Interleukin (IL)-2, IL-4, IL-5 and IL-7 had no significant effect in MHC- AF bioassay, but IFN-γ had significant MHC-AF activity. Antibodies to IFN-α, IFN-\ and TNF-α did not block the activity of MHC-AF, but anti-IFN-y antibodies could partially neutralize the activity. However, unlike IFN-γ, MHC-AF activity was resistant to pH 2.0 treatment. Purified MHC-AF preparations did not have any activity in WISH cell/encephalo myocarditis virus (EMC) IFN bioassays. In addition, anti-IFN-y affinity column did not retain MHC-AF activity. These results indicate that a MHC-AF distinct from IFN-γ, is produced by activated human mononuclear cells.     

Improved prediction of MHC class I and class II epitopes using a novel Gibbs sampling approach

MOTIVATION: Prediction of which peptides will bind a specific major histocompatibility complex (MHC) constitutes an important step in identifying potential T-cell epitopes suitable as vaccine candidates. MHC class II binding peptides have a broad length distribution complicating such predictions. Thus, identifying the correct alignment is a crucial part of identifying the core of an MHC class II binding motif. In this context, we wish to describe a novel Gibbs motif sampler method ideally suited for recognizing such weak sequence motifs. The method is based on the Gibbs sampling method, and it incorporates novel features optimized for the task of recognizing the binding motif of MHC classes I and II. The method locates the binding motif in a set of sequences and characterizes the motif in terms of a weight-matrix. Subsequently, the weight-matrix can be applied to identifying effectively potential MHC binding peptides and to guiding the process of rational vaccine design. RESULTS: We apply the motif sampler method to the complex problem of MHC class II binding. The input to the method is amino acid peptide sequences extracted from the public databases of SYFPEITHI and MHCPEP and known to bind to the MHC class II complex HLA-DR4(B1*0401). Prior identification of information-rich (anchor) positions in the binding motif is shown to improve the predictive performance of the Gibbs sampler. Similarly, a consensus solution obtained from an ensemble average over suboptimal solutions is shown to outperform the use of a single optimal solution. In a large-scale benchmark calculation, the performance is quantified using relative operating characteristics curve (ROC) plots and we make a detailed comparison of the performance with that of both the TEPITOPE method and a weight-matrix derived using the conventional alignment algorithm of ClustalW. The calculation demonstrates that the predictive performance of the Gibbs sampler is higher than that of ClustalW and in most cases also higher than that of the TEPITOPE method.