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The molecules encoded by major histocompatibility complex (MHC) genes play an essential role in the adaptive immune response among vertebrates. We investigated the molecular evolution of MHC class I genes in the sable Martes zibellina. We isolated 26 MHC class I sequences, including 12 putatively functional sequences and 14 pseudogene sequences, from 24 individuals from two geographic areas of northeast China. The number of putatively functional sequences found in a single individual ranged from one to five, which might be at least 1–3 loci. We found that both balancing selection and recombination contribute to evolution of MHC class I genes in M. zibellina. In addition, we identified a candidate nonclassical MHC class I lineage in Carnivora, which may have preceded the divergence (about 52–57 Mya) of Caniformia and Feliformia. This may contribute to further understanding of the origin and evolution of nonclassical MHC class I genes. Our study provides important immune information of MHC for M. zibellina, as well as other carnivores.

Behaviors that increase an individual's exposure to pathogens are expected to have important effects on immunoactivity. Because sexual reproduction typically requires close contact among conspecifics, mating systems provide an ideal opportunity to study the immunogenetic correlates of behaviors with high versus low risks of pathogen exposure. Despite logical links between polygynandrous mating behavior, increased pathogen exposure, and greater immunoactivity, these relationships have seldom been examined in nonhuman vertebrates. To explore interactions among these variables in a different lineage of mammals, we used RNAseq to study the gene expression profiles of liver tissue—a highly immunoactive organ—from sympatric populations of the monogamous California mouse (Peromyscus californicus) and two polygynandrous congeners (P. maniculatus and P. boylii). Differential expression and co‐expression analyses revealed distinct patterns of gene activity among species, with much of this variation associated with differences in mating system. This tendency was particularly pronounced for MHC genes, with multiple MHC Class I genes being upregulated in the two polygynandrous species, as expected if exposure to sexually transmitted pathogens varies with mating system. Our results underscore the role of mating behavior in influencing patterns of gene expression and highlight the use of emerging transcriptomic tools in behavioral studies of free‐living animals.

The genomic structure of the Major Histocompatibility Complex (MHC) region and variation in selected MHC class I related genes in Old World camels, Camelus bactrianus and Camelus dromedaries were studied. The overall genomic organization of the camel MHC region follows a general pattern observed in other mammalian species and individual MHC loci appear to be well conserved. Selected MHC class I genes B-67 and BL3-7 exhibited unexpectedly low variability, even when compared to other camel MHC class I related genes MR1 and MICA. Interspecific SNP and allele sharing are relatively common, and frequencies of heterozygotes are usually low. Such a low variation in a genomic region generally considered as one of the most polymorphic in vertebrate genomes is unusual. Evolutionary relationships between MHC class I related genes and their counterparts from other species seem to be rather complex. Often, they do not follow the general evolutionary history of the species concerned. Close evolutionary relationships of individual MHC class I loci between camels, humans and dogs were observed. Based on the results of this study and on our data on MHC class II genes, the extent and the pattern of polymorphism of the MHC region of Old World camelids differed from most mammalian groups studied so far. Camels thus seem to be an important model for our understanding of the role of genetic diversity in immune functions, especially in the context of unique features of their immunoglobulin and T-cell receptor genes.

Pathogens are one of the main forces driving the evolution and maintenance of the highly polymorphic genes of the vertebrate major histocompatibility complex (MHC). Although MHC proteins are crucial in pathogen recognition, it is still poorly understood how pathogen-mediated selection promotes and maintains MHC diversity, and especially so in host species with highly duplicated MHC genes. Sedge warblers (Acrocephalus schoenobaenus) have highly duplicated MHC genes, and using data from high-throughput MHC genotyping, we were able to investigate to what extent avian malaria parasites explain temporal MHC class I supertype fluctuations in a long-term study population. We investigated infection status and infection intensities of two different strains of Haemoproteus, that is avian malaria parasites that are known to have significant fitness consequences in sedge warblers. We found that prevalence of avian malaria in carriers of specific MHC class I supertypes was a significant predictor of their frequency changes between years. This finding suggests that avian malaria infections partly drive the temporal fluctuations of the MHC class I supertypes. Furthermore, we found that individuals with a large number of different supertypes had higher resistance to avian malaria, but there was no evidence for an optimal MHC class I diversity. Thus, the two studied malaria parasite strains appear to select for a high MHC class I supertype diversity. Such selection may explain the maintenance of the extremely high number of MHC class I gene copies in sedge warblers and possibly also in other passerines where avian malaria is a common disease.

Major histocompatibility complex (MHC) genes in mammals include highly polymorphic class I and class II genes that are critical for donor-recipient matching for transplantation. Dogs have served as an effective, directly translatable model for stem/progenitor cell transplantation. Previous analyses of MHC class I genes in dogs point to a single highly polymorphic gene, dog leukocyte antigen (DLA)-88, as an important factor in the success or failure of hematopoietic stem cell transplants. Fifty-nine DLA-88 alleles have been identified and reported so far. Here, we extend this list by presenting 13 novel DLA-88 alleles found in domestic dogs.

Characterization of highly duplicated genes, such as genes of the major histocompatibility complex (MHC), where multiple loci often co-amplify, has until recently been hindered by insufficient read depths per amplicon. Here, we used ultra-deep Illumina sequencing to resolve genotypes at exon 3 of MHC class I genes in the sedge warbler (Acrocephalus schoenobaenus). We sequenced 24 individuals in two replicates and used this data, as well as a simulated data set, to test the effect of amplicon coverage (range: 500-20 000 reads per amplicon) on the repeatability of genotyping using four different genotyping approaches. A third replicate employed unique barcoding to assess the extent of tag jumping, that is swapping of individual tag identifiers, which may confound genotyping. The reliability of MHC genotyping increased with coverage and approached or exceeded 90% within-method repeatability of allele calling at coverages of >5000 reads per amplicon. We found generally high agreement between genotyping methods, especially at high coverages. High reliability of the tested genotyping approaches was further supported by our analysis of the simulated data set, although the genotyping approach relying primarily on replication of variants in independent amplicons proved sensitive to repeatable errors. According to the most repeatable genotyping method, the number of co-amplifying variants per individual ranged from 19 to 42. Tag jumping was detectable, but at such low frequencies that it did not affect the reliability of genotyping. We thus demonstrate that gene families with many co-amplifying genes can be reliably genotyped using HTS, provided that there is sufficient per amplicon coverage.

Major histocompatibility complex (MHC) class I and class II molecules play essential roles in the development and activation of the human adaptive immune system. An NLR protein, CIITA (MHC class II transactivator) has been recognized as a master regulator of MHC class II gene expression, albeit knowledge about the regulatory mechanism of MHC class I gene expression had been limited. Recently identified MHC class I transactivator (CITA), or NLRC5, also belongs to the NLR protein family and constitutes a critical regulator for the transcriptional activation of MHC class I genes. In addition to MHC class I genes, CITA/NLRC5 induces the expression of β2 -microglobulin, TAP1 and LMP2, essential components of the MHC class I antigen presentation pathway. Therefore, CITA/NLRC5 and CIITA are transcriptional regulators that orchestrate the concerted expression of critical components in the MHC class I and class II pathways, respectively. © 2016 BioFactors, 42(4):349-357, 2016.

Classical major histocompatibility complex (MHC) class I allelic polymorphism is essential for competent antigen presentation. To improve the genotyping efforts in the golden pheasant, it is necessary to differentiate more accurately between classical and nonclassical class I molecules. In our study, all MHC class I genes were isolated from one golden pheasant based on two overlapping PCR amplifications. In total, six full-length class I nucleotide sequences (A-F) were identified, and four were novel. Two (A and C) belonged to the IA1 gene, two (B and D) were alleles derived from the IA2 gene through transgene amplification, and two (E and F) comprised a third novel locus, IA3 that was excluded from the core region of the golden pheasant MHC-B. IA1 and IA2 exhibited the broad expression profiles characteristic of classical loci, while IA3 showed no expression in multiple tissues and was therefore defined as a nonclassical gene. Phylogenetic analysis indicated that the three IA genes in the golden pheasant share a much closer evolutionary relationship than the corresponding sequences in other galliform species. This observation was consistent with high sequence similarity among them, which likely arises from the homogenizing effect of recombination. Our careful distinction between the classical and nonclassical MHC class I genes in the golden pheasant lays the foundation for developing locus-specific genotyping and establishing a good molecular marker system of classical MHC I loci.

The genetic diversity of the major histocompatibility complex (MHC) class I molecules of pigs has not been well characterized. Therefore, the influence of MHC genetic diversity on the immune-related traits of pigs, including disease resistance and other MHC-dependent traits, is not well understood. Here, we attempted to develop an efficient method for systemic analysis of the polymorphisms in the epitope-binding region of swine leukocyte antigens (SLA) class I genes. We performed a comparative analysis of the last 92 bp of the 5' untranslated region (UTR) to the beginning of exon 4 of six SLA classical class I-related genes, SLA-1, -2, -3, -4, -5, and -9, from 36 different sequences. Based on this information, we developed a genomic polymerase chain reaction (PCR) and direct sequencing-based comprehensive typing method for SLA-2. We successfully typed SLA-2 from 400 pigs and 8 cell lines, consisting of 9 different pig breeds, and identified 49 SLA-2 alleles, including 31 previously reported alleles and 18 new alleles. We observed differences in the composition of SLA-2 alleles among different breeds. Our method can be used to study other SLA class I loci and to deepen our knowledge of MHC class I genes in pigs.

The major histocompatibility complex is a diverse gene family that plays a crucial role in the adaptive immune system. In humans, the MHC class I genes consist of the classical loci of HLA-A, -B, and -C, and the nonclassical loci HLA-E, -F, and -G. In Platyrrhini species, few MHC class I genes have been described so far and were classified as MHC-E, MHC-F, and MHC-G, with MHC-G possibly representing a classical MHC class I locus while there were arguments about the existence of the MHC-B locus in Platyrrhini. In this study, MHC class I genes were identified in eight common marmosets (Callithrix jacchus) and two brown-headed spider monkeys (Ateles fusciceps). For common marmosets, 401 cDNA sequences were sequenced and 18 alleles were detected, including 14 Caja-G alleles and 4 Caja-B alleles. Five to eleven Caja-G alleles and one to three Caja-B alleles were detected in each animal. For brown-headed spider monkeys, 102 cDNA sequences were analyzed, and 9 new alleles were identified, including 5 Atfu-G and 4 Atfu-B alleles. Two or three Atfu-G and two Atfu-B alleles were obtained for each of animal. In phylogenetic analyses, the MHC-G and -B alleles from the two species and other Platyrrhini species show locus-specific clusters with bootstrap values of 86% and 50%. The results of pairwise sequence comparisons and an excess of non-synonymous nucleotide substitutions in the PBR region are consistent with the suggestion that Caja-G and Atfu-G may be classical MHC class I loci in the Platyrrhini species… But it appears that MHC-B locus of the two Platyrrhini species shares features with both classical and nonclasical MHC class I loci. Our results are an important addition to the limited MHC immunogenetic information available for the Platyrrhini species. Am. J. Primatol. 77:527–534, 2015. © 2015 Wiley Periodicals, Inc.

The NLR protein, NLRC5 is an important regulator of MHC class I gene expression, however, the role of NLRC5 in other innate immune responses is less well defined. In the present study, we report that NLRC5 binds RIG-I and that this interaction is critical for robust antiviral responses against influenza virus. Overexpression of NLRC5 in the human lung epithelial cell line, A549, and normal human bronchial epithelial cells resulted in impaired replication of influenza virus A/Puerto Rico/8/34 virus (PR8) and enhanced IFN-β expression. Influenza virus leads to induction of IFN-β that drives RIG-I and NLRC5 expression in host cells. Our results suggest that NLRC5 extends and stabilizes influenza virus induced RIG-I expression and delays expression of the viral inhibitor protein NS1. We show that NS1 binds to NLRC5 to suppress its function. Interaction domain mapping revealed that NLRC5 interacts with RIG-I via its N-terminal death domain and that NLRC5 enhanced antiviral activity in an leucine-rich repeat domain independent manner. Taken together, our findings identify a novel role for NLRC5 in RIG-I-mediated antiviral host responses against influenza virus infection, distinguished from the role of NLRC5 in MHC class I gene regulation.

The major histocompatibility complex (MHC) plays a central role in the adaptive immune system and provides a good model with which to understand the evolutionary processes underlying functional genes. Trans-species polymorphism and orthology are both commonly found in MHC genes; however, mammalian MHC class I genes tend to cluster by species. Concerted evolution has the potential to homogenize different loci, whereas birth-and-death evolution can lead to the loss of orthologs; both processes result in monophyletic groups within species. Studies investigating the evolution of MHC class I genes have been biased toward a few particular taxa and model species. We present the first study of MHC class I genes in a species from the superfamily Musteloidea. The European badger (Meles meles) exhibits moderate variation in MHC class I sequences when compared to other carnivores. We identified seven putatively functional sequences and nine pseudogenes from genomic (gDNA) and complementary (cDNA) DNA, signifying at least two functional class I loci. We found evidence for separate evolutionary histories of the alpha 1 and alpha 2/alpha 3 domains. In the alpha 1 domain, several sequences from different species were more closely related to each other than to sequences from the same species, resembling orthology or trans-species polymorphism. Balancing selection and probable recombination maintain genetic diversity in the alpha 1 domain, evidenced by the detection of positive selection and a recombination event. By comparison, two recombination breakpoints indicate that the alpha 2/alpha 3 domains have most likely undergone concerted evolution, where recombination has homogenized the alpha 2/alpha 3 domains between genes, leading to species-specific clusters of sequences. Our findings highlight the importance of analyzing MHC domains separately.

Pere David's deer (Elaphurus davidianus) is a highly inbred species that arose from 11 founders but now comprises a population of about 3,000 individuals, making it interesting to investigate the adaptive variation of this species from the major histocompatibility complex (MHC) perspective. In this study, we isolated Elda-MHC class I loci using magnetic bead-based cDNA hybridization, and examined the molecular variations of these loci using single-strand conformation polymorphism (SSCP) and sequence analysis. We obtained seven MHC class I genes, which we designated F1, F12, G2, I7, AF, I8, and C1. Our analyses of stop codons, phylogenetic trees, amino acid conservation, and G+C content revealed that F1, F12, G2, and I7 were classical genes, AF was a nonclassical gene, and I8 and C1 were pseudogenes. Our subsequent molecular examinations showed that the diversity pattern in the Pere David's deer was unusual. Most mammals have more polymorphic classical class I loci vs. the nonclassical and neutral genes. In contrast, the Pere David's deer was found to be monomorphic at classical genes F1, F12, G2, and I7, dimorphic at the nonclassical AF gene, dimorphic at pseudogene I8, and tetramorphic at pseudogene C1. The adverse polymorphism patterns of Elda-I genes might provide evidence for selection too faster deplete MHC variation than drift in the bottlenecked populations, while the postbottleneck tetramorphism of the C1 pseudogene appears to be evidence of strong historical balancing selection. J. Exp. Zool. (Mol. Dev. Evol.) 318:294–307, 2012. © 2012 Wiley Periodicals, Inc.

The major histocompatibility complex (MHC) genes code for proteins that play a critical role in the immune system response. The MHC genes are among the most polymorphic genes in vertebrates, presumably due to balancing selection. The two MHC classes appear to differ in the rate of evolution, but the reasons for this variation are not well understood. Here, we investigate the level of polymorphism and the evolution of sequences that code for the peptide-binding regions of MHC class I and class II DRB genes in the Alpine marmot (Marmota marmota). We found evidence for four expressed MHC class I loci and two expressed MHC class II loci. MHC genes in marmots were characterized by low polymorphism, as one to eight alleles per putative locus were detected in 38 individuals from three French Alps populations. The generally limited degree of polymorphism, which was more pronounced in class I genes, is likely due to bottleneck the populations undergone. Additionally, gene duplication within each class might have compensated for the loss of polymorphism at particular loci. The two gene classes showed different patterns of evolution. The most polymorphic of the putative loci, Mama-DRB1, showed clear evidence of historical positive selection for amino acid replacements. However, no signal of positive selection was evident in the MHC class I genes. These contrasting patterns of sequence evolution may reflect differences in selection pressures acting on class I and class II genes.

Genes of the highly dynamic major histocompatibility complex (MHC) are directly linked to individual fitness and are of high interest in evolutionary ecology and conservation genetics. Gene duplication and positive selection usually lead to high levels of polymorphism in the MHC region, making genotyping of MHC a challenging task. Here, we compare the performance of two methods for MHC class I genotyping in a passerine with highly duplicated MHC class I genes: capillary electrophoresis-single-strand conformation polymorphism (CE-SSCP) analysis and 454 GS FLX Titanium pyrosequencing. According to our findings, the number of MHC variants (called alleles for simplicity) detected by CE-SSCP is significantly lower than detected by 454. To resolve discrepancies between the two methods, we cloned and Sanger sequenced a MHC class I amplicon for an individual with high number of alleles. We found a perfect congruence between cloning/Sanger sequencing results and 454. Thus, in case of multi-locus amplification, CE-SSCP considerably underestimates individual MHC diversity. However, numbers of alleles detected by both methods are significantly correlated, although the correlation is weak (r = 0.32). Thus, in systems with highly duplicated MHC, 454 provides more reliable information on individual diversity than CE-SSCP.

Summary The major histocompatibility complex (MHC) is one of the most diverse regions of the mammalian genome. Diversity in MHC genes is integral to their function in the immune system, and while pathogens play a key role in shaping this diversity, the contribution of other selective forces remains unclear. The controlled breeding of cattle offers an excellent model for the identification and exploration of these forces. We characterized the MHC class I genes present in a sample of Canadian Holstein A.I. bulls and compared the results with those obtained in an earlier study. No evidence for a reduction in MHC diversity over 20 years was observed, but the relative frequency of some haplotypes had changed: the formerly rare A12 (w12B) haplotype had become the most common, together with A15, while A19, which dominated the earlier sample, had significantly reduced in frequency. Only 7% of bulls in the current study were MHC homozygous compared with the 14% expected under Hardy–Weinberg. To identify the selective forces at work, a gene substitution model was used to calculate the effects of MHC on selection traits using estimated breeding values for each bull. Significant associations between MHC and production, disease and fertility traits were identified, suggesting that MHC diversity is not merely shaped by disease in this controlled breeding system. The decrease in a common haplotype, the reduced number of homozygous bulls and the associations with disease and production traits together indicate that MHC diversity in dairy cattle is maintained by heterozygote advantage.

Natural killer (NK) cells are circulating lymphocytes that function in innate immunity and placental reproduction. Regulating both development and function of NK cells is an array of variable and conserved receptors that interact with major histocompatibility complex (MHC) class I molecules. Families of lectin-like and immunoglobulin-like receptors are determined by genes in the natural killer complex (NKC) and leukocyte receptor complex (LRC), respectively. As a consequence of the strong, varying pressures on the immune and reproductive systems, NK cell receptors and their MHC class I ligands evolve rapidly, are highly diverse and exhibit dramatic species-specific differences. The variable, polymorphic family of killer cell immunoglobulin-like receptors (KIR) that regulate human NK cell development and function arose recently, from a single-copy gene during the evolution of simian primates. Our studies of KIR and MHC class I genes in representative species show how these two unlinked but functionally intertwined genetic complexes have co-evolved. In humans, combinations of KIR and HLA class I factors are associated with infectious diseases, including HIV/AIDS, autoimmunity, reproductive success and the outcome of therapeutic transplantation. The extraordinary, and unanticipated, divergence of human NK cell receptors and MHC class I ligands from their mouse counterparts can in part explain the difficulties experienced in finding informative mouse models for human diseases. Non-human primate models have far greater potential, but to realize their promise will first require more complete definition of the genetics and function of KIR and MHC variation in non-human primate species, at a level comparable to that achieved for the human species.

Here we present the first isolation of major histocompatibility complex (MHC) class I genes from two ancient fish, paddlefish (Polyodon spathula) and Chinese sturgeon (Acipenser sinensis). Seventeen sequences obtained showed high polymorphism and positive natural selection with d(N)/d(S) > 1. Evolutionary relationships revealed that sequences from paddlefish and Chinese sturgeon distinguished from other vertebrate class I and had an intermingling of alleles, which indicates that Acipenseriformes have a common ancestral gene of class I and a trans-species polymorphism across Acipenseriformes. We also found clear evidence of recombination among class I genes of paddlefish and Chinese sturgeon. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The role and intensity of positive selection maintaining the polymorphism of major histocompatibility complex (MHC) class I genes in the three-spined stickleback Gasterosteus aculeatus was investigated. The highly polymorphic set of MHC class I genes found was organized in a single linkage group. Between 5 and 14 sequence variants per individual were identified by single-stranded conformation polymorphism (SSCP) analysis. Segregation analysis studied in 10 three-spined stickleback families followed the expected pattern of Mendelian inheritance. The gamete fusion in three-spined stickleback thus seems to be random with respect to the MHC class I genes. The DNA sequence analyses showed that the expressed MHC class I loci are under strong selection pressure, possibly mediated by parasites. Codons that were revealed to be under positive selection are potentially important in antigen binding. MHC class I sequences did not form significant supported clusters within a phylogenetic tree. Analogous to MHC class II genes, it was not possible to assign the class I sequences to a specific locus, suggesting that the class I genes may have been generated by recent gene duplication.

Natural infections with influenza viruses have been reported in a variety of animal species including humans, pigs, horses, sea mammals, mustelids and birds. Occasionally, devastating pandemics occur in domestic chickens (broiler and layers) and in humans. From November 2003 to March 2004 in many countries in Asia, there were outbreaks of H5N1 avian influenza virus, causing death of infected patients, and devastating the poultry industry. Some groups of Thai indigenous chickens survived and were therefore classified as resistant. These traits were related to immunogenetics, in particular, the major histocompatibility complex (MHC) class I and class II molecules. The chicken MHC class I were investigated as candidate genes for avian influenza virus disease resistance. Seven hundred and thirty Thai indigenous chickens from smallholder farms in the rural area of avian influenza virus disease outbreaks in the central part of Thailand were used in the present study. They were separated into two groups, 340 surviving chickens and 390 dead chickens (resistant and susceptible). Genomic DNA were precipitated from blood samples and feathers. The DNA were used to amplify the MHC class I gene. Data were analyzed using χ 2 analysis to test significant differences of influences of MHC class I haplotypes on avian influenza virus disease traits. The results represented nine MHC class I haplotypes: A1, B12, B13, B15, B19, B21, B2, B6, and BA12, and included 10 of their heterozygotes. The homozygous B21 from these collected samples had a 100% survival rate and they were the major survival group. In addition, the heterozygous B21 also had a high survival rate because of co-dominant expression of these genes. In contrast, the homozygous B13 had a 100% mortality rate and they were the major mortality group. These results confirmed that MHC class I haplotypes influence avian influenza virus disease-resistant traits in Thai indigenous chicken. The MHC genes can be used as genetic markers to improve disease-resistant traits in chicken.

We have investigated the potential effects of H-2 and T-cell receptor (TCR) V beta family genes on induction of T-cell immunity and susceptibility to virally induced demyelinating disease by using BALB.S (H-2K(s)A(s)D(s)) and BALB.S 3 R (H-2K(s)A(s)D(d)/L(d)) mice. These parameters were compared with those of highly susceptible SJL/J (H-2K(s)A(s)D(s)) mice that contain only one-half of TCR V beta family genes compared with the above-mentioned strains. Our results demonstrate that BALB.S but not BALB.S 3 R mice are susceptible similar to SJL/J mice. Although the level of CD4(+) T-cell infiltration to the CNS was elevated in susceptible mice, virus-specific immune responses restricted with H-2(s) were similar in these mice. No preferential use of V beta families associated with differences in the major histocompatibility complex (MHC) components was apparent. However, the pattern and sequence of CDR 3 distribution shows T-cell clonal accumulation in the CNS associated with the H-2 components. Further anti-CD8 antibody treatment of resistant BALB.S 3 R mice abrogated resistance to demyelinating disease, indicating that CD8(+) T cells restricted with H-2D(d)/L(d) are most likely to exert resistance in BALB.S 3 R mice. These studies indicated that TCR V beta and MHC class II genes are the secondary to a particular MHC class I gene expression in susceptibility to virally induced demyelinating disease.

8 pages; International audience; The terminal investment hypothesis predicts that individuals should invest more in their present reproduction if they are less likely to survive to future reproductive events. Infections, which reduce viability, may be used by individuals as a cue of a diminishing residual reproductive value and could therefore theoretically trigger an intensification of breeding effort. We tested this hypothesis in a natural population of house sparrows (Passer domesticus). We manipulated the immune system of breeding females by injecting them with a vaccine against the Paramyxo virus, the agent of Newcastle disease. Females were captured and treated immediately after completion of their first clutch either with the vaccine (NDV) or with phosphate buffered saline (PBS). The entire clutch was subsequently removed. We also screened Mhc class I genes of females to assess possible genotype-by-immune treatment interactions on reproductive investment. Our results indicate that vaccinated females were more likely to lay replacement clutches and that the difference in number of eggs between first and replacement clutches was greater for NDV females than for controls. In addition, chick size, both in terms of tarsus length and body mass, was affected by immune activation but in interaction with nestling age and female body mass, respectively. Mhc genotype-by-immune treatment interactions were never significant; however, allelic diversity was positively correlated with nestling survival. These results show that immune system activation is potentially used as a cue of reduced survival prospect and appears to induce a costly terminal investment behavior, and Mhc diversity might be under selection in a natural population of house sparrows.

Summary Improved typing of horse classical MHC class I is required to more accurately define these molecules and to extend the number identified further than current serological assays. Defining classical MHC class I alleleic polymorphism is important in evaluating cytotoxic T lymphocyte (CTL) responses in horses. In this study, horse classical MHC class I genes were analyzed based on reverse transcription (RT)-PCR amplification of sequences encoding the polymorphic peptide binding region and the more conserved alpha 3, transmembrane and cytoplasmic regions followed by cloning and sequencing. Primer sets included a horse classical MHC class I-specific reverse primer and a forward primer conserved in all known horse MHC class I genes. Sequencing at least 25 clones containing MHC class I sequences from each of 13 horses identified 25 novel sequences and three others which had been described. Of these, nine alleles were identified from different horses or different RT-PCR and 19 putative alleles were identified in multiple clones from the same RT-PCR. The primer pairs did not amplify putative non-classical MHC class I genes as only classical MHC class I and related pseudogenes were found in 462 clones. This method also identified classical MHC class I alleles shared between horses by descent, and defined differences in alleles between horses varying in equine leukocyte antigen (ELA)-A haplotype as determined by serology. However, horses sharing ELA-A haplotypes defined by serotyping did not always share cDNA sequences, suggesting subhaplotypic variations within serologically defined ELA-A haplotypes. The 13 horses in this study had two to five classical MHC class I sequences, indicating that multiple loci code for these genes. Sequencing clones from RT-PCR with classical MHC class I-specific primers should be useful for selection of haplotype matched and mismatched horses for CTL studies, and provides sequence information needed to develop easier and more discriminating typing procedures.

A cattle BAC library derived from an MHC homozygous animal was screened for MHC class I genes. This revealed at least nine class I-related genes in a contig spanning approximately 400 kb, and several additional genes on other clones. The three classical class I genes expressed on this haplotype (A14) were shown to be distributed over a region at most 212 kb apart.

Summary: Humans express three highly polymorphic ‘classical’ (HLA-A,B and C) and three conserved ‘non-classical’ (HLA-E, F and G) MHC class I genes. Their comparison with the MHC class I genes of apes and monkeys reveals the differential extent to which MHC class I genes have been preserved during primate evolution. African apes have orthologues of all six human genes, and although allelic lineages of the A and C loci are shared, these species share none of the human alleles. In Asian apes, several MHC class I genes show significant differences from the human genes, a trend which continues with the Old World monkeys, and even more so in the New World monkeys, where E and F are the only human gene orthologues. The C locus is confined to humans and apes. Multiple A-related and B-related loci have been identified in apes and Old World monkeys showing that duplication of these loci has been a common event during primate evolution. Certain of the daughter loci exhibit low polymorphism, suggesting they have adopted a non-classical function. The differing rates at which MHC class I genes have evolved during primate evolution likely reflects their differing functions in the immune response.

MIC genes map to the major histocompatibility complex (MHC) and are distantly related to MHC class I genes. Recently, MICA/MICB-like genes have been described in nonhuman primates. In Macaca mulatta, three MICA/B-like genes could be identified: Mamu-MIC1, Mamu-MIC2, and Mamu-MIC3. We show here the isolation and characterization of rhesus macaque cosmid clones which carry the Mamu-MIC2 and Mamu-MIC3 genes. Neither the MIC2- and MIC3-coding sequences nor respective flanking sequences can be aligned unambiguously to either the human HLA-MICA or -MICB subregions, although MIC2 was found at a similar distance to the BAT1 gene as known for MICB in human. Thus, the characteristics allowing for a classification of primate MIC genes as being of the MICA or MICB types appear to have evolved after the separation of humans and rhesus monkeys from a common ancestor. Furthermore, also Mamu-MICD-containing cosmids could be isolated. In contrast to Mamu-MIC2 and Mamu-MIC3, the Mamu-MICD gene and its flanking sequences are highly conserved and orthologous to the human MICD subregion.

The polymorphic exon 2-exon 3 region of bovine major histocompatibility complex (MHC) class I genes was amplified by polymerase chain reaction (PCR) from genomic DNA samples with characterized class I polymorphism. The primers for amplification were designed in conserved regions at the borders of exons 2 and 3, based on all available cDNA sequences. The primers should, therefore, amplify most expressed class I genes, but may also amplify non-expressed class I genes. The PCR amplified class I gene fragments of 700 bp were characterized on the basis of restriction fragment length polymorphism (RFLP). The PCR-RFLP analysis of class I genes showed that the bands in each digestion could be classified as non-polymorphic, as shared between several bovine lymphocyte antigen (BoLA)-A types, or as specific to a single BoLA-A type. The same primers were then used for amplification of class I gene fragments from eight Sahiwal animals, a breed which originated in the Indian subcontinent. These studies showed that BoLA class I PCR-RFLP could be used to study class I polymorphism in family groups.

The neonatal Fc receptor, FcRn, is expressed in human placental syncytiotrophoblast, capillary endothelium, intestinal epithelium, and other tissues. By analogy with its role in the mouse, human FcRn is expected to transport maternal IgG to the foetus, and protect circulating IgG from catabolism. The larger subunit of FcRn is homologous to the alpha chains of the major histocompatibility complex (MHC) class I proteins, but is encoded outside the MHC on chromosome 19. We report the isolation of clones encoding the alpha chain of human FcRn from chromosome 19-specific libraries. The sequence revealed a similar organization to classical and non-classical MHC, and MHC-related genes. Compared with classical MHC class I genes, the human FcRn alpha chain gene has expanded by acquiring many repetitive sequences in its introns, including multiple Alu elements in the fourth intron. Primer extension analysis showed that there are two transcription initiation sites in the upstream flanking sequence.

The full-length cDNAs of woodchuck major histocompatibility complex (MHC) class I (MhcMamo-I or Mamo-I) genes were cloned by using cellular mRNA isolated from the peripheral blood mononuclear cells and liver tissues of woodchucks. DNA sequence analysis of Mamo-I cDNAs revealed that the coding regions of Mamo-I genes were about 1,080 bp long, encoding 359 amino acid residues. The deduced amino acid sequences of Mamo-I showed structural features like leader, alpha1, alpha2, alpha3, transmembrane and cytoplasmic domains, similar to their homologues in human and other mammals. Analysis of five full-length clones from unrelated woodchucks indicated a polymorphism within the alpha1 and alpha2 domains of Mamo-I heavy chain and a high conservation within the alpha3 and the transmembrane/cytoplasmic domains. Amino acid residues of the alpha2 and alpha3 domains that are supposed to be involved in the binding of MHC class I to CD8 molecule, were largely conserved in Mamo-I genes. Phylogenetic comparison of MHC class I genes of woodchuck and other mammals indicated a close evolutionary relationship between woodchuck and squirrel MHC class I. We tentatively named this region the locus A of Mamo-I genes (Mamo-A). Sequence analysis of 101 clones of alpha1 and alpha2 regions derived from 14 woodchucks revealed that at least 14 different alleles within Mamo-A exist. Among these 14 alleles identified so far, Mamo-A*01 and Mamo-A*09 were of the highest frequency of about 21.5% and 14.5%, respectively. Our results indicate that Mamo-I genes are of a similar molecular structure to those of human and other mammals.

Epstein-Barr virus (EBV) is present in Reed-Sternberg (RS) cells of a substantial proportion of Hodgkin's lymphoma cases. Most EBV-positive cases are also MHC class I-positive, whereas the majority of EBV-negative cases lack detectable levels of MHC class I expression. Application of the SAGE technique has led to the identification of tags corresponding to MHC class I and beta(2)-microglobulin genes in the EBV- and MHC class I-negative L428 Hodgkin's cell line. Further expression studies indicated that single RS cells that do not express HLA class I also lack beta(2)-microglobulins but frequently contain mRNA coding for these proteins. Another tag was identified corresponding to CD1a, a thymocyte and Langerhans cell antigen structurally related to the MHC class I genes. CD1a expression studies revealed mRNA in all cell lines and in several of the single cells, whereas immunostaining showed a cytoplasmic signal in only 2 of the 4 cell lines and in none of the Hodgkin's lymphoma tissue samples. In conclusion, RS cells frequently lack MHC class I, beta(2)-microglobulin and CD1a protein expression but contain mRNA coding for these proteins in some of the RS cells, suggesting a common mechanism affecting the translation of these antigen presentation-associated molecules. Int. J. Cancer 86:548-552, 2000. (C) 2000 Wiley-Liss, Inc.

Here we describe a nucleotide insertion in intron 2 of two rhesus major histocompatibility complex (MHC) class I alleles, Mamu-A*05 and Mamu-A*07 . This resulted in an intron 2 that was nearly twice the length of any other intron 2 of primate MHC class I genes sequenced to date. This insertion was most similar (93% identity) to the beginning of intron 3 of HLA-A alleles. It was also similar to intron 3 of several human MHC class I pseudogenes and MHC class I pseduogenes from cotton top tamarin and cat. The finding of this insertion in two rhesus MHC class I genes is surprising given the uniformity in length and sequence of introns of functional HLA-A, -B and -C genes.

During the development of an HLA-A polymerase chain reaction using sequence-specific oligonucleotide probes (PCR-SSOP) method for the identification of HLA-A*24 and -A*30 alleles, group amplification resulted in the formation of an unusual PCR product in certain individuals. This fragment was approximately 900 bp smaller than the expected product and was also detected in some non-HLA-A*24- and -A*30-positive individuals acting as negative controls for the group specific amplification. Nucleotide sequence analysis of this product identified it as a unique class I gene sequence displaying homology to both primate and human class I A-locus genes. The entire gene was amplified using PCR and the complete DNA sequence information from exon 1 to exon 8, including introns, was determined. A recombination event was identified which results in the fusion of intron 2 with intron 3, causing a deletion of the intervening exon 3 sequence. In addition, there are two cytosine insertions in the poly-cytosine stretch at the start of exon 4 which cause a frameshift and premature termination. The exon 1 and 2 sequences most closely align with the gorilla allele A*0501, displaying only five mismatches. PCR analysis has established that the gene is associated with the following HLA-A types: HLA-A*3001, -A*3301, -A*3303, -A*6802, -A*2901, -A*0203, -A*0205 and -A*31012. Reverse transcription (RT)-PCR analysis of individuals containing this gene failed to detect any mRNA transcription, suggesting that this is a previously undescribed non-expressed class I pseudogene which we have provisionally named HLA-BEL. Its unique gene structure gives a possible insight into the evolutionary pathway that created HLA class I genes.

In swine, the major histocompatibility complex (Mhc) or swine leukocyte antigen (SLA) is located on chromosome 7 and divided by the centromere. Thus, the telomeric class I and more centromeric class III regions are located on the p arm and the class II region is located on the q arm. The SLA region spans about 2 Mb, in which more than 70 genes have so far been characterized. Despite its division by the centromere, the spatial relationships between the genes in the class II and class III regions, and between the well-conserved non-class I genes of the class I region, are similar to those found in the human HLA complex. On the other hand, no orthologous relationships have been found between the Mhc class I genes in man and swine. In swine, the 12 SLA class I sequences constitute two distinct clusters. One cluster comprises six classical class I-related sequences, while the other comprises five class I-distantly related sequences including two swine homologous genes of the HLA Mhc class I chain-related gene (MIC) sequence family. The number of functional SLA classical class I genes, as defined by serology, probably varies from one to four, depending on the haplotype. Some of the SLA class I-distantly related sequences are clearly transcribed. As regards the SLA class II genes, some of them clearly code for at least one functional SLA-DR and one SLA-DQ heterodimer product, but none code for any DP product. The amino acid alignment of the variable domains of 33 SLA classical class I chains, and 62 DR beta and 20 DQ beta chains confirmed the exceptionally polymorphic pattern of these polypeptides. Among the class II genes, the genes are either monomorphic, like the DRA gene, or oligomorphic, like the DQA genes. In contrast, the DRB and DQB genes display considerable polymorphism, which seems more marked in DRB than DQB genes.

Summary: Three MHC class I genes have been characterized in salmonids: A, B, and UA. Levels of polymorphism vary among the genes, but they all share one common feature: a lack of sequence diversity. Although individual species can carry over 3.0 alleles at a given locus (A), intraspecific diversity is generally less than 5% in Pacific salmon (genus Oncorhynchus). and less Chan 10% in Atlantic salmon (genus Salmo). These levels of diversity suggest that few ancient allelic lineages have persisted within species, and that most of the allelic radiation has occurred during or since speciation. Also apparent is the greater retention of allelic lineages in Atlantic salmon than Pacific salmon, which reflects historic differences of the two genera. Comparison of the salmonid class I sequences with those of other teleosts reveals two well supported groups: one containing the Cypriniformes and the salmonid UA, and the other containing the neoteleosts and the salmonid A and B, There is no homology between known Cypriniformes; and neoteleostean sequences. If this relationship is borne out, it offers strong support for the hypothesis that the higher teleosts diverged more recently from the Salmoniformes than the Cypriniformes, The salmonid MHC may provide a snapshot of the neoteleostean MHC prior to the extensive class I duplication that has taken place in at least some of the more advanced species.

The analysis of cattle MHC (BoLA) class I gene expression is an essential component of studies on immune responses and susceptibility to disease. International BoLA workshops have generated data and reagents that allow discrimination of class I molecules at the haplotype level, but progress has been limited by difficulties encountered in defining single alleles. Our aim in this study was to develop a DNA-based system for improved identification of expressed class I alleles, utilizing available cDNA sequences derived from cattle carrying a series of serologically defined class I specificities. This method has allowed more accurate typing of animals for expression of the class I genes present within a small number of haplotypes. The method has also reliably differentiated between allelic variants (identified by prior sequence analysis) and has split existing serological specificities. The data show that MHC class I genes in cattle are more polymorphic than demonstrated by serology and biochemical analysis.

In order to understand the molecular basis of the synergistic action of interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) on rat oligodendrocyte development, we studied some aspects of the signalling pathways involved in the regulation of the major histocompatibility complex (MHC) class I and the interferon regulatory factor 1 (IRF-1) gene expression. Two well-defined inducible enhancers of the MHC class I gene promoter, the MHC class I regulatory element (MHC-CRE) and the interferon consensus sequence (ICS), were analysed. Neither IFN-gamma nor TNF-alpha was capable of inducing MHC-CRE binding activity when administrated alone. Following the exposure of oligodendrocytes to IFN-gamma, TNF-R1 expression was transcriptionally induced by the binding of signal transducer and activator of transcription (STAT-1) homodimers to the IFN-gamma activated site (GAS) present in the gene promoter. The upregulation of TNF-R1 allowed TNF-alpha to induce the binding of nuclear factor-kappaB (NF-kappaB) to the MHC-CRE site. With respect to ICS element, IFN-gamma induced IRF-1 binding, that was further enhanced upon co-treatment with TNF-alpha. The existence of a synergism between IFN-gamma and TNF-alpha in stimulating IRF-1 expression at the transcriptional level was supported by IRF-1 promoter analysis: IFN-gamma directly induced the binding of STAT-1 homodimers to the GAS element, while NF-kappaB binding to the kappaB sequence was activated by TNF-alpha only after IFN-gamma treatment. This transcriptional regulation of IRF-1 gene by IFN-gamma and TNF-alpha was confirmed at the mRNA level. The synergism demonstrated in the present study highlights the importance of cytokine interactions in magnifying their biological effects during brain injury and inflammation.

The MHC class I genes of the New World primate the cotton-top tamarin (Saguinus oedipus) are an exception to the high polymorphism and variability usually displayed by this multigene family. In the present work, the cloning and sequencing of a new pseudogene, tentatively named Saoe-Mhc-N4, in this primate species are reported. This new sequence has two characteristic deletions at exon 2, making it very unlikely that any putative protein from this sequence was an antigen-presenting molecule. Comparison of intron 1, intron 2, partial exon1, exon 2 and partial exon 3 showed little similarity with those of classical class I genes and pseudogenes in S. oedipus and in other primates. Phylogenetic analysis grouped this Saoe-Mhc-N4 sequence with other pseudogenes in S. oedipus. Thus, it seems that Saoe-Mhc-N4 is an inactivated gene or a pseudogene which has been originated by the common process of duplication and subsequent inactivation of MHC class I loci in this primate species. The major histocompatibility complex (MHC) class I genes encode cell surface glycoproteins that present peptides to cytotoxic T cells. On the basis of the level of expression and polymorphism, MHC class I mammalian genes may be grouped within three different categories: (1) classical class I loci, which are highly polymorphic with products expressed on the surface of all nucleated somatic cells, (2) non-classical class I loci, which are monomorphic or show very little polymorphism and whose products, of unknown function, have a more limited tissue distribution, and (3) class I pseudogenes which include complete genes whose expression is prevented by frameshift, premature stop codons or other defects and also gene fragments lacking one or more of the exons characteristic of expressed class I genes (Le Bouteiller, 1994). The MHC class I genes of the New World primate cotton-top tamarin (S. oedipus) show low polymorphism and the variation among alleles of their three described MHC class I loci is comparable to that seen for alleles at the same locus in human classical class I loci (Cadavid et al., 1996b). Additionally, the expressed MHC class I DNA sequences of cotton-top tamarins have been shown to be more closely related to the human non-classical HLA-G gene (Watkins et al., 1991), and to HLA-E (Alvarez et al., 1997), than to the human classical HLA-A, -B or -C genes. It has been proposed that the unusually limited polymorphism in the cotton top tamarins’ MHC originated because they may come from a small founder population and recently duplicated genes have been fixed and quickly inactivated thereafter. Only three possible functional Saoe class I genes and four pseudogenes have been described so far (Cadavid et al., 1996b). In the present work, a MHC class I sequencing study in S. oedipus was carried out to define possible new alleles or genes and investigate the nature and evolution of the MHC class I genes in these New World monkeys. A new sequence has been found, Saoe-Mhc-N4, which carries two deletions at exon 2 and several stop codons in exons 1 and 3; this suggests that Saoe-Mhc-N4 is a new pseudogene. Genomic DNA from EBV cell lines of three different unrelated S. oedipus individuals (B95, 3F1 and 74E7) was extracted using standard methods (Arnaiz-Villena et al., 1992). Partial exon 1, intron 1, exon 2, intron 2 and partial exon 3 from this new Saoe-Mhc nucleotide sequence gene were obtained by polymerase chain reaction (PCR) using SAOEX1 41–60 (5'-CGAGGGCCCTGGTCCTGAT-3') and SAOEX3197–215 (5'-GC C- C TCCAGGTAGGCTTC-3') primers; the first primer corresponds to exon 1 of classical class I genes at base positions 41–60 and the second to exon 3 at base positions 197–215 (Alvarez et al., 1997). PCR amplification fragments had a size of 825 bp. Random sequencing of the amplified products was carried out and the PCR products purified and inserted into the pMOS ‘blue vector’ (Amersham, UK) following the manufacturers’ protocols. Double-stranded DNA stretches were automatically sequenced in an Applied Biosystems (Foster City, CA) machine as previously described (Arnaiz-Villena et al., 1992; Castro et al., 1996; Alvarez et al., 1997). A phylogenetic tree based on exon 2 and partial exon 3 was obtained by using the neighbour-joining method (Saitou & Nei, 1987) and the computer program NJBOOT (kindly provided by Masatoshi Nei, Mol. Evol. Genetics, Pennsylvania, USA). The phylogenetic tree was based on a genetic distance matrix of nucleotide substitutions per site, obtained with the Kimura’s biparametric method. The bootstrap analysis for tree topology (Felsenstein, 1985) was used to evaluate the statistical significance of each internal branch of the tree. Twenty clones were obtained from each of the following S. oedipus individuals: B95, 74e7 and 3F1, all of which were subsequently sequenced. Three, five and four clones, respectively, were identical and corresponded to a new MHC-DNA sequence, tentatively named Saoe-Mhc-N4. Other sequences obtained were already described (Saoe-G*02, *04, *06, and *08). This sequence (Fig. 1) has two characteristic deletions at exon 2, the first between codon 14 and the first base in codon 23 (28 bp), and the second one between the second base of codon 66 and codon 71 (17 bp). A putative protein resulting from a hypothetical transcription and translation would not bear part of the β sheet (amino acids 14–22) at the floor of the MHC molecule groove and seven amino acids of the α helix (amino acids 66–71); also, the expected reading frame has three premature stop codons located at position – 2 in exon 1 and positions 101 and 123 in exon 3. However, Saoe-Mhc-N4 has an 81-codon open reading frame, starting at codon 5. The other two possible reading frames show also several premature stop codons. Thus, a theoretical Saoe-Mhc-N4 protein, if transcribed and translated, would not be an antigen-presenting molecule. A comparison of exon 1, exon 2 and partial exon 3 DNA sequences with those of classical class I genes and pseudogenes in S. oedipus (Fig. 1) and other primates (not shown) showed that this new sequence shows little similarity to any of them. Introns 1 and 2 of Saoe-Mhc-N4 were compared with those of classical and non-classical human class I genes to study intron sequence similarities (Geraghty et al., 1987; Cereb et al., 1996; Gomez-Casado et al., 1997), since it has been shown that MHC gene intron-specific motifs may be conserved for more than 33 million years (Castro et al., 1996). The nature of substitutions present in introns 1 and 2 of Saoe-Mhc-N4 are not conserved in any of the compared genes (Fig. 2), showing a specific deletion at intron 2 between positions 208 and 227 absent in all the MHC classical or non-classical class I genes analysed. The relationship of Saoe-Mhc-N4 exons 2 and 3 with other class I loci was further studied; 36 exon 2 and exon 3 classical and non-classical class I DNA sequences from different primates were included to construct a relatedness NJ dendrogram (Watkins et al., 1991; Otting & Bontrop, 1993; Watkins et al., 1993; Boyson et al., 1995; Cadavid et al., 1996a,b; Castro et al., 1996; Alvarez et al., 1997) (Fig. 3). The tree showed that the Saoe-Mhc-N4 sequence was grouped with Saoe-PS1 and So-N1, two pseudogenes already found in S. oedipus; however, this grouping has a low bootstrap value and may not be definitive. Moreover, the branch length leading to Saoe-Mhc-N4 is longer (approximately 2-fold) than that leading to Saoe-PS1 and So-N1; this suggests that the nucleotide substitution rate could be faster in Saoe-Mhc-N4 than in other pseudogenes found in S. oedipus, or that this pseudogene is older. In summary, Saoe-Mhc-N4 seems to be an inactivated gene or a pseudogene, which may be a result of the common process of duplication and subsequent inactivation of MHC class I loci in S. oedipus (Cadavid et al., 1996b) because: 1Saoe-Mhc-N4 is grouped with Saoe-PS1 and So-N1, two previously described pseudogenes (Fig. 1), although its mutation rate seems to be much higher (probably due to a longer period of inactivation); 2 it contains deletions that would disrupt the correct folding of a putative MHC molecule; 3 it has several premature stop codons in all possible reading frames; 4 the percentage of G + C at the third codon position was low (82.8%), further suggesting that Saoe-Mhc-N4 is an inactivated class I gene or a pseudogene (Hughes & Nei, 1989).

The Tapasin molecule is a member of the immunoglobulin (Ig) superfamily required for the association of TAP transporters and MHC class I heterodimers in the endoplasmic reticulum. In this study, the Tapasin gene was precisely mapped in relation to the MHC. The gene was centromeric of the HLA-DP locus between the HSET and HKE1.5 genes and within 500 kbp of the TAP1 and TAP2 genes. A homologous mouse EST was mapped to a syntenic position on chromosome 17, centromeric of the H-2 K locus. Similarly, the rat Tapasin gene was shown to be in an equivalent location with respect to the RT1.A focus. The localization of Tapasin, TAP, LMP and class I genes within such a short distance of each other on the chromosome implies some regulatory or functional significance. We determined the Tapasin gene sequence for comparison of its structure to that of other Ig superfamily members, such as MHC class I genes. The IgC domain was encoded by a separate exon. However, the positions of the other introns were not characteristic of other Ig superfamily genes, indicating that Tapasin has a distinct phylogeny.

Summary: The missing self model predicts chat NK cells adapt somatically to the type as well as levels of MHC class I products expressed by their host, Transgenic and gene knock-out mice have provided conclusive evidence chat MHC class I genes control specificity and tolerance of NK cells. The article describes this control and discusses the POSSIBLE Mechanisms behind it. starting from a genetic model to study how natural resistance to tumors is influenced by MHC class I expression in the host as well as in the target cells. Data on host gene regulation of NK-cell functional specificity as well as Lγ49 receptor expression are reviewed, leading up to the central question: how does the system develop and maintain “useful” NK cells, while avoiding “harmful” and “useless” ones? The available data can be fitted with in each of two mutually non-exclusive models: cellular adaptation and clonal selection. Recent studies supporting cellular adaptation bring the focus on different possibilities within this general mechanism, such as energy, receptor calibration and, most importantly, whether the specificity of each NK cell is permanently fixed or subject to continuous regulation.

The classical major histocompatibility complex (MHC) class I genes are conserved in higher primates. Motifs common to human, chimpanzee and gorilla alleles indicate that class I alleles diverged from ancestral sequences that existed before separation of these species. Analysis of native human populations such as Australian Aborigines and Amerindians shows that HLA-B is characterized by rapid generation of new alleles. HLA-A and -C appear to be evolving more slowly. Comparison of alleles for orthologous class I genes in humans and other primates confirms that similar mechanisms contribute to the generation of new alleles in these species.

The major histocompatibility complex (MHC) is a multigene family that plays an essential role in the innate and adaptive immune system. The MHC has been intensely studied in a variety of vertebrate species and comparative analyses of the MHC in distantly related species have revealed changes in gene complexity and organisation during the evolution of this gene cluster. Marsupials occupy a unique phylogenetic position that bridges the gap between eutherians and nonmammal species. Characterisation of MHC genes and genomic regions in marsupials has greatly improved our knowledge on MHC evolution, including our understanding of changes in genomic organisation, the effect of translocation or loss of genes on gene function and the influence of natural selection on genetic variations at MHC loci. Key Concepts: The genomic organisation of MHC class I genes differs between nonmammalian and eutherian species. The organisation of the marsupial MHC resembles that of nonmammals. Separation of class I genes from class II and antigen-processing genes occurred after the divergence of marsupial and eutherian mammals. Classical class I genes are not linked to the MHC in the tammar wallaby. The nonclassical class I gene UK is conserved in marsupials and likely plays a significant functional role. Loss of a class II gene family in the Tasmanian devil may have an adverse impact on immunity in this species. Balancing selection acts to maintain high MHC diversity in the Kangaroo island tammar wallaby population. Selective sweeps may have contributed to the low MHC diversity in the Tasmanian devil. Keywords: comparative analysis; MHC; evolution; marsupial; grey short-tailed opossum; tammar wallaby; Tasmanian devil

Nineteen horse MHC class I specificities have been serologically identified previously at a single locus (ELA-A), and two other specificities appear to be coded at other loci. Biochemical studies indicate that there are at least two expressed loci. In order to establish the number of transcribed horse MHC class I genes, we made a cDNA library from a heterozygous animal (ELA-A3/A7), and screened for positive clones using a bovine class I probe. More than 200 class I clones were isolated in this way, and so far seven unique full length sequences have been identified. All of the sequences are predicted to code for surface expressed, functional molecules. The number of different sequences identified demonstrate that at least four genes are transcribed, although variations in transmembrane length (which is generally conserved in class I loci) suggest that five genes could be represented. Evolutionary analysis of these sequences (and two additional sequences known to represent different horse class I loci) reveals no firm relationships, such that the division between the different loci cannot be discerned. These results suggest an unusual evolutionary history for the horse MHC, the precise nature of which may be revealed only following further cross-species comparisons.

Enhancement of major histocompatibility complex (MHC) class I expression leads to protection from recognition by natural killer (NK) cells in several systems. MHC class I gene products can be expressed in different forms at the cell surface--for example as "empty" beta 2-microglobulin (beta 2m)-associated heterodimers or free heavy chains. To study the role of different class I heavy chain forms in NK target interactions, we have used lymphoblastoid target cell lines preincubated with beta 2m. This was found to shift the equilibrium between beta 2m-associated and non-associated--heavy chains in favor of the former. In parallel, there was a significant increase in NK sensitivity. The recognition of MHC class I-deficient cell lines was not affected by beta 2m, arguing against a general nonspecific effect of beta 2m on NK sensitivity. Our data indicate that protection against NK recognition correlates with target cell expression of free heavy chains (i.e. devoid of beta 2m) rather than with expression of complexes.

In neuroblastoma, N-myc suppresses the expression of major histocompatibility complex (MHC) Class I antigens by reducing the binding of a nuclear factor to the enhancer-A element in the MHC Class I gene promoter. We show here that the p50 subunit of NF-kappa B is part of this complex and that expression of p50 mRNA is suppressed by N-myc. Transfection of a p50 expression vector in neuroblastoma cells that express N-myc at a high level leads to restoration of factor binding to the MHC Class I gene enhancer, restores enhancer activity and leads to re-expression of MHC Class I antigens at the cell surface. These data indicate that the p50 subunit of NF-kappa B is involved in the regulation of MHC Class I antigen expression and that N-myc down-regulates MHC Class I gene expression primarily through suppression of p50 expression.

The major histocompatibility complex (MHC) of the mouse, which is called the H2 complex, is located on chromosome 17. It contains genes critical to the functioning of the immune system, the products of which are intimately involved in the initiation of immune responses. The proteins made by these genes display a significant amount of polymorphism, or sequence variability. Few genes possess this degree of polymorphism, but it is critical to the immune response and to the survival of a species. The chief characteristic of MHC molecules is their ability to bind small peptides. These bound peptides along with specific areas of the MHC molecule are the structures that interact with the T-cell receptor (TCR) leading to activation of a T-cell immune response, which in turn can active other cells of immune system. Thus, they determine which TCRs are functional which in turn indicates which peptides a T cell will respond to. Key Concepts The historical background and the genetics of MHC development are described. The expression and function of MHC class I genes is described. MHC class I molecules are expressed on virtually all nucleated cells and function to present foreign and self-antigens to the immune system. MHC class I expression is dependent on peptide binding in the peptide binding groove of the molecule. MHC class I polymorphism is important for species resistance to infectious disease. The expression and function of MHC class II genes is described. MHC class II expression is required for generation and function of CD4 T cells. Nonclassical class I molecules bind a variety of peptide and nonpeptide antigens that are commonly expressed by pathogenic organisms. . The function of other genes found within the H2 complex is described. Keywords: H2 complex; MHC; MHC restriction; class I; class II; MHC peptide association; T-cell activation

The KBF1 factor, which binds to the enhancer A located in the promoter of the mouse MHC class I gene H-2Kb, is indistinguishable from the p50 DNA binding subunit of the transcription factor NF-kappa B, which regulates a series of genes involved in immune and inflammatory responses. The KBF1/p50 factor binds as a homodimer but can also form heterodimers with the products of other members of the same family, like the c-rel and v-rel (proto)oncogenes. The dimerization domain of KBF1/p50 is contained between amino acids 201 and 367. A mutant of KBF1/p50 (delta SP), unable to bind to DNA but able to form homo- or heterodimers, has been constructed. This protein reduces or abolishes in vitro the DNA binding activity of wild-type proteins of the same family (KBF1/p50, c- and v-rel). This mutant also functions in vivo as a trans-acting dominant negative regulator: the transcriptional inducibility of the HIV long terminal repeat (which contains two potential NF-kappa B binding sites) by phorbol ester (PMA) is inhibited when it is co-transfected into CD4+ T cells with the delta SP mutant. Similarly the basal as well as TNF or IL1-induced activity of the MHC class I H-2Kb promoter can be inhibited by this mutant in two different cell lines. These results constitute the first formal demonstration that these genes are regulated by members of the rel/NF-kappa B family.

Amplification of the N-myc gene in neuroblastoma correlates with advanced stage and poor prognosis. Association of the expression between N-myc and major histocompatibility complex (MHC) class I genes in 33 neuroblastomas obtained from Japanese children was investigated. Amplification of the N-myc gene was observed in two of five cases in Stage 111, six of 11 cases in Stage IV, and one of five cases in Stage IV-S. In each case, the expression of N-myc gene was significantly increased. The expression was also increased in cases without amplification of the N-myc gene, the origin being from the suprarenal region. Expression of the MHC class I gene was significantly decreased in five of these nine with a high level of N-myc expression with amplification. These results suggest that the down-modulation of the MHC class I expression may be associated with the high level of expression and amplification of N-myc gene in the advanced stage of neuroblastoma. Cancer 67:1384-1388,1991. HE ~myc GENE is often amplified specifically in T neuroblastoma, retinobla~toma,~ and small cell lung can~er.~,~ Amplification of the N-rnvc gene in untreated patients with neuroblastoma in the advanced stage is more frequent than in the early stage and correlates with rapid tumor progression.6 We reported that amplification of the N-myc gene closely correlated not only with the advanced stage, but also with the primary site of the tumor, age at diagnosis, and histologic type.’-’ Major histocompatibility complex (MHC) class I antigens are expressed on almost all cell types, and the role of class I expression in the immunosurveillance of tumor has recently been elucidated in an analysis of several adenovirus-transformed cell lines with different metastatic potentials. Suppression of the MHC class I antigens has

Tumor cells frequently show a lack of surface class-I major histocompatibility complex (MHC) antigen expression. These molecules are key recognition structures for immune rejection of tumor cells and their absence at the surface of tumor cells could favor the progression of tumors. We have analyzed the transcriptional mechanisms that could lead to suppression of MHC-class-I expression in human tumor cell K562. The expression of MHC-class-I genes is highly controlled by regulatory factors interacting with an enhancer sequence upstream of MHC-class-I genes. In this report we show that DNA binding activity of 2 regulatory factors, KBFI and NF-kappa B, known to be essential for constitutive expression of MHC-class-I genes, is deficient in nuclear extracts from K562 cells. Induction of class-I gene expression at the surface of tumor cells by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) shows that TNF-alpha can act in synergy with IFN-gamma to induce DNA binding of both factors NF-kappa B and KBFI to the class-I gene enhancer and that this induction of transcriptional factors is correlated with enhancement of MHC-class-I mRNA transcription and cell-surface antigen expression.

Mouse Sal sarcoma cells are lethal in the autologous A/J (KkDd) host. In order to improve the immune response to the Sal tumor, Sal cells have been transfected with syngeneic MHC-class-II or allogeneic MHC-class-I genes. MHC-class-II transfectants are uniformly rejected by the autologous host and immunization with them protects against subsequent Sal challenge. The improved immunity is probably the result of enhanced generation of tumor-specific Th cells. We hypothesize that class-II+ tumor cells trigger an improved Th-cell response because they directly present Sal tumor antigens in the context of class-II molecules to Th cells, by-passing professional APC. Studies by others have demonstrated that antigen presentation requires an intracellular signal transmitted by the cytoplasmic domain of the APC class-II molecule. Sal cells expressing class-ll antigens with truncated cytoplasmic domains are as malignant as wild-type Sal cells. These experiments therefore support the role of tumor-cell class-II molecules as antigen presentation elements, and demonstrate the requirement for intact class-II molecules for tumor protection. Sal cells have also been transfected with allogeneic MHC-class-I genes. Although Kb-transfected cells are not rejected by A/J mice, Db-transfected Sal cells and Kb- plus Db-transfected cells are rejected. The Db transfectants effectively immunize A/J mice against subsequent Sal challenge. These experiments demonstrate that expression of certain allogeneic MHC-class-I genes can lead to tumor-specific immunity, and that such transfectants can protect against challenges of wild-type tumor cells. Transfection of tumor cells with syngeneic MHC-class-II or allogeneic MHC-class-I genes may therefore be a potential strategy for improving tumor-specific immunity in the autologous host.

SUMMARY The correlation between gene expression and DNA methylation is well established. In the present study we examined DNA methylation as a potential regulatory mechanism of class I histocompatibility antigen expression. We selected for study two cell clones from the same mouse tumour (GR9). One of them is H-2 class I-positive (A7) and the other negative (B9). Using five enzymes and three different probes, no rearrangement or deletion was detected in the MHC class I genes of the negative clone compared with the positive one. These results do not provide an explanation for the differences observed in the expression of class I antigens in A7 (Kd, Dd positive) and B9 (Kd, Dd negative). We then studied the DNA methylation status in both clones by Southern blot analysis. Clear differences between the Kd, Dd positive and negative clones were detected. Furthermore, both Kd and Dd were clearly expressed after culturing the H-2 class I negative clone with the demethylating agent 5-azacytidine. These results suggest that the methylation of certain MHC cytoshes is a regulatory mechanism of H-2 class I gene expression.