Subtle changes in peptide conformation profoundly affect recognition of the non-classical MHC class I molecule HLA-E by the CD94-NKG2 natural killer cell receptors

Human leukocyte antigen (HLA)-E is a non-classical major histocompatibility complex class I molecule that binds peptides derived from the leader sequences of other HLA class I molecules. Natural killer cell recognition of these HLA-E molecules, via the CD94-NKG2 natural killer family, represents a central innate mechanism for monitoring major histocompatibility complex expression levels within a cell. The leader sequence-derived peptides bound to HLA-E exhibit very limited polymorphism, yet subtle differences affect the recognition of HLA-E by the CD94-NKG2 receptors. To better understand the basis for this peptide-specific recognition, we determined the structure of HLA-E in complex with two leader peptides, namely, HLA-Cw*07 (VMAPRALLL), which is poorly recognised by CD94-NKG2 receptors, and HLA-G*01 (VMAPRTLFL), a high-affinity ligand of CD94-NKG2 receptors. A comparison of these structures, both of which were determined to 2.5-Å resolution, revealed that allotypic variations in the bound leader sequences do not result in conformational changes in the HLA-E heavy chain, although subtle changes in the conformation of the peptide within the binding groove of HLA-E were evident. Accordingly, our data indicate that the CD94-NKG2 receptors interact with HLA-E in a manner that maximises the ability of the receptors to discriminate between subtle changes in both the sequence and conformation of peptides bound to HLA-E.     

Lack of association between human leukocyte antigen-E alleles and nasopharyngeal carcinoma in Tunisians

Nasopharyngeal carcinoma (NPC), a cancer with a remarkable geographical and worldwide ethnic distribution, has been strongly associated with human leukocyte antigen (HLA) class I genes. The presence of additional HLA risk factors has been suggested by several reports. In the present study, we analyzed the implication of HLA-E gene polymorphisms in NPC susceptibility in Tunisians, a population characterized by an intermediate incidence of NPC with specific clinical features. Peripheral blood DNA was obtained from 185 patients with NPC and 177 matched controls. Genotyping for three single-nucleotide polymorphisms, codon 83Gly/Arg, codon 157Arg/Gly, and codon 107Arg/Gly, was performed using the polymerase chain reaction method. The HLA-E*01:01 and HLA-E*01:03 were the only alleles found among Tunisians. The HLA-E*01:03 allele had a slight increase in patients with NPC (43%) compared with controls (37%), but the difference did not reach a statistical significance. Our results show the lack of association between HLA-E alleles and NPC in the Tunisian population. This is not in agreement with the previous studies, suggesting a potential implication of HLA-E gene polymorphisms in the susceptibility to NPC among populations with high-risk incidence. Our study further supports the dissimilarity of NPC between populations with different NPC incidence.     

Major histocompatibility complex variation and evolution at a single, expressed DQA locus in two genera of elephants

Genes of the vertebrate major histocompatibility complex (MHC) are crucial to defense against infectious disease, provide an important measure of functional genetic diversity, and have been implicated in mate choice and kin recognition. As a result, MHC loci have been characterized for a number of vertebrate species, especially mammals; however, elephants are a notable exception. Our study is the first to characterize patterns of genetic diversity and natural selection in the elephant MHC. We did so using DNA sequences from a single, expressed DQA locus in elephants. We characterized six alleles in 30 African elephants (Loxodonta africana) and four alleles in three Asian elephants (Elephas maximus). In addition, for two of the African alleles and three of the Asian alleles, we characterized complete coding sequences (exons 1–5) and nearly complete non-coding sequences (introns 2–4) for the class II DQA loci. Compared to DQA in other wild mammals, we found moderate polymorphism and allelic diversity and similar patterns of selection; patterns of non-synonymous and synonymous substitutions were consistent with balancing selection acting on the peptides involved in antigen binding in the second exon. In addition, balancing selection has led to strong trans-species allelism that has maintained multiple allelic lineages across both genera of extant elephants for at least 6 million years. We discuss our results in the context of MHC diversity in other mammals and patterns of evolution in elephants.     

MHC diversity and the association to nematode parasitism in the yellow-necked mouse (Apodemus flavicollis)

In vertebrates, the genes of the major histocompatibility complex (MHC) are among the most debated candidates accounting for co-evolutionary processes of host-parasite interaction at the molecular level. The exceptionally high allelic polymorphism found in MHC loci is believed to be maintained by pathogen-driven selection, mediated either through heterozygous advantage or rare allele advantage (= frequency dependent selection). While investigations under natural conditions are still very rare, studies on humans or mice under laboratory conditions revealed support for both hypotheses. We investigated nematode burden and allelic diversity of a functional important MHC class II gene (DRB exon2) in free-ranging yellow-necked mice (Apodemus flavicollis). Twenty-seven distinct Apfl-DRB alleles were detected in 146 individuals with high levels of amino acid sequence divergence, especially at the antigen binding sites (ABS), indicating selection processes acting on this locus. Heterozygosity had no influence on the infection status (being infected or not), the number of different nematode infections (NNI) or the intensity of infection, measured as the individual faecal egg count (FEC). However, significant associations of specific Apfl-DRB alleles to both nematode susceptibility and resistance were found, for all nematodes as well as in separate analyses of the two most common nematodes. Apodemus flavicollis individuals carrying the alleles Apfl-DRB*5 or Apfl-DRB*15 revealed significantly higher FEC than individuals with other alleles. In contrast, the allele Apfl-DRB*23 showed a significant association to low FEC of the most common nematode. Thus, our results provide evidence for pathogen-driven selection acting through rare allele advantage under natural conditions.     

Plasma paraoxonase polymorphism: a new enzyme assay, population, family, biochemical, and linkage studies.

Plasma paraoxonase hydrolyzes paraoxon, the principal metabolite of the insecticide parathione. A genetic polymorphism for enzyme activity has been previously demonstrated. We describe a new assay based on the differential inhibition by EDTA of plasma paraoxonase from persons with the high-activity allele (PX*H) that suggests a trimodality of activity levels in population studies. The gene frequency of the low activity allele (PX*L) in 531 Seattle blood donors of European origin was .7207. Family studies were consistent with codominant autosomal inheritance of two alleles, PX*L (low) and PX*H (high), coding for products with different activity levels. Biochemical measurements of sera from presumed homozygotes for the two different alleles revealed minor physicochemical differences suggestive of a structural difference between the allelic products. No evidence for linkage of the paraoxonase locus with any of 19 polymorphic markers would be detected.     

Peptide-presenting similarities among functionally distant HLA-B27 subtypes revealed by alloreactive T lymphocytes of unusual specificity.

Functional dissection of HLA-B27 subtypes using alloreactive or B27-restricted CTL has shown that the structurally related B*2704 and B*2706 are the most distant subtypes relative to the prototype B*2705. In particular, previous studies have failed to find anti-B*2705 CTL cross-reacting with B*2704 or B*2706. Such failure can be accounted for by the drastic effect on T cell recognition of the change at residue 152 in both subtypes relative to B*2705, as established with site-directed mutants. B*2704 and B*2706 are also related in ethnic distribution, as they are restricted to Orientals, jointly being the predominant HLA-B27 subtypes in this population. As far as it is known, there are no differences relative to B*2705 in their linkage to ankylosing spondylitis. In our study, 5 of 13 examined anti-B*2705 limiting dilution CTL lines from a particular HLA-B27- individual were shown to crossreact with B*2704, B*2706 or both. The monoclonal nature of this cross-reaction was established by cold target competition analysis. This result demonstrates that the apparent differences in T cell antigenicity among anti-B27 subtypes are strongly influenced by the responder individual, as the spectrum of clonal specificities in anti-B27 responses may show significant differences among unrelated responders. Fine specificity differences among the cross-reactive CTL allowed unambiguous functional distinction between B*2704 and B*2706. The molecular basis of such cross-reactivity was examined by correlating CTL reaction patterns with the structure of both subtypes, which differ only by two residues located in the beta-pleated sheet bottom of the peptide binding site, and with site-directed mutants mimicking HLA-B27 subtype polymorphism. The results suggest that: 1) distinct peptides are involved in the allospecific epitopes recognized by the various crossreactive CTL, and 2) B*2704, B*2706, and B*2705 differ in their peptide-presenting specificity, but can present some identical or structurally similar peptides.     

Gametophytic self-incompatibility in Lycium parishii (Solanaceae): allelic diversity, genealogical structure, and patterns of molecular evolution at the S-RNase locus

We characterized allelic diversity at the locus controlling self-incompatibility (SI) for a population of Lycium parishii (Solanaceae) from Organ Pipe National Monument, Arizona. Twenty-four partial sequences of S-RNase alleles were recovered from 25 individuals. Estimates of allelic diversity range from 23 to 27 alleles and, consistent with expectations for SI, individuals are heterozygous. We compare S-RNase diversity, patterns of molecular evolution, and the genealogical structure of alleles from L. parishii to a previously studied population of its congener L. andersonii. Gametophytic SI is well characterized for Solanaceae and although balancing selection is hypothesized to be responsible for high levels of allelic divergence, the pattern of selection varies depending on the portion of the gene considered. Site-specific models investigating patterns of selection for L. parishii and L. andersonii indicate that positive selection occurs in those regions of the S-RNase gene hypothesized as important to the recognition response, whereas positive selection was not detected for any position within regions previously characterized as conserved. A 10-species genealogy including S-RNases from a pair of congeners from each of five genera in Solanaceae reveals extensive transgeneric evolution of L. parishii S-RNases. Further, within Lycium, the Dn/Ds ratios for pairs of closely related alleles for intraspecific versus interspecific comparisons were not significantly different, suggesting that the S-RNase diversity recovered in these two species was present prior to the speciation event separating them. Despite this, two S-RNases from L. parishii are identical to two previously reported alleles for L. andersonii, suggesting gene flow between these species.     

Nonallelic interactions between het-c and a polymorphic locus, pin-c, are essential for nonself recognition and programmed cell death in Neurospora crassa

Nonself recognition in filamentous fungi is conferred by genetic differences at het (heterokaryon incompatibility) loci. When individuals that differ in het specificity undergo hyphal fusion, the heterokaryon undergoes a programmed cell death reaction or is highly unstable. In Neurospora crassa, three allelic specificities at the het-c locus are conferred by a highly polymorphic domain. This domain shows trans-species polymorphisms indicative of balancing selection, consistent with the role of het loci in nonself recognition. We determined that a locus closely linked to het-c, called pin-c (partner for incompatibility with het-c) was required for het-c nonself recognition and heterokaryon incompatibility (HI). The pin-c alleles in isolates that differ in het-c specificity were extremely polymorphic. Heterokaryon and transformation tests showed that nonself recognition was mediated by synergistic nonallelic interactions between het-c and pin-c, while allelic interactions at het-c increased the severity of the HI phenotype. The pin-c locus encodes a protein containing a HET domain; predicted proteins containing HET domains are frequent in filamentous ascomycete genomes. These data suggest that nonallelic interactions may be important in nonself recognition in filamentous fungi and that proteins containing a HET domain may be a key factor in these interactions.     

Residue 81 confers a restricted C-terminal peptide binding motif in HLA-B*44:09

Knowledge about the magnitude of individual polymorphism is a critical part in understanding the complexity of comprehensive mismatching. HLA-B*44:09 differs from the highly frequent HLA-B*44:02 allele by amino acid exchanges at residues 77, 80, 81, 82 and 83. We aimed to identify the magnitude of these mismatches on the features of HLA-B*44:09 bound peptides since residues 77, 80 and 81 comprise part of the F pocket which determines sequence specificity at the pΩ position of the peptide. Using soluble HLA technology we determined >200 individual (nonduplicate) self-peptides from HLA-B*44:09 and compared their features with that of the published peptide features of HLA-B*44:02. Both alleles illustrate an anchor motif of E at p2. In contrast to the C-terminal peptide binding motif of B*44:02 (W, F, Y or L), B*44:09-derived peptides are restricted predominantly to L or F. The source of peptides for both alleles is identical (LCL 721.221 cells) allowing us to identify 23 shared peptides. The majority of these peptides however contained the restricted B*44:09 anchor motif of F or L at the pΩ position. Molecular modelling based on the B*44:02 structure highlights that the differences of the C-terminal peptide anchor between both alleles can be explained primarily by the B*44:02(81Ala)?>?B*44:09(81Leu) polymorphism which restricts the size of the amino acid that can be accommodated in the F pocket of B*44:09. These results highlight that every amino acid substitution has an impact of certain magnitude on the alleles function and demonstrate how surrounding residues orchestrate peptide specificity.     

HLA Allele E*01:01 Is Associated with a Reduced Risk of EBV-Related Classical Hodgkin Lymphoma Independently of HLA-A*01/*02

Background

An inefficient immune response against Epstein-Barr virus (EBV) infection is related to the pathogenesis of a subgroup of classical Hodgkin lymphomas (cHL). Some EBV immune-evasion mechanisms target HLA presentation, including the non-classical HLA-E molecule. HLA-E can be recognized by T cells via the TCR, and it also regulates natural killer (NK) cell signaling through the inhibitory CD94/NKG2A receptor. Some evidences indicate that EBV-infected B-cells promote the proliferation of NK subsets bearing CD94/NKG2A, suggesting a relevant function of these cells in EBV control. Variations in CD94/NKG2A-HLA-E interactions could affect NK cell-mediated immunity and, consequently, play a role in EBV-driven transformation and lymphomagenesis. The two most common HLA-E alleles, E*01:01 and E*01:03, differ by a single amino acid change that modifies the molecule function. We hypothesized that the functional differences in these variants might participate in the pathogenicity of EBV.

Aim

We studied two series of cHL patients, both with EBV-positive and-negative cases, and a cohort of unrelated controls, to assess the impact of HLA-E variants on EBV-related cHL susceptibility.

Results

We found that the genotypes with at least one copy of E*01:01 (i.e., E*01:01 homozygous and heterozygous) were underrepresented among cHL patients from both series compared to controls (72.6% and 71.6% vs 83%, p = 0.001). After stratification by EBV status, we found low rates of E*01:01-carriers mainly among EBV-positive cases (67.6%). These reduced frequencies are seen independently of other factors such as age, gender, HLA-A*01 and HLA-A*02, HLA alleles positively and negatively associated with the disease (adjusted OR = 0.4, p = 0.001). Furthermore, alleles from both HLA loci exert a cumulative effect on EBV-associated cHL susceptibility.

Conclusions

These results indicate that E*01:01 is a novel protective genetic factor in EBV-associated cHL and support a role for HLA-E recognition on the control of EBV infection and lymphomagenesis.     

Polymorphism, natural selection, and structural modeling of class Ia MHC in the African clawed frog (Xenopus laevis)

In the African clawed frog (Xenopus laevis), two deeply divergent allelic lineages of multiple genes of the class I MHC region have been discovered. For the MHC class I UAA locus, functional differences and the molecular basis for lineages maintenance are unknown. Alleles of linked class I region genes also exhibit strong disequilibrium with specific MHC alleles, but the underlying cause is not clear. We use MHC class Ia sequence data to estimate substitution rates and investigate structural differences between allelic lineages from protein models. Results indicate the operation of natural selection, and differences in the steric properties in the F pocket of the peptide-binding region among lineages. Variability in this pocket likely enables allelic lineages to bind very different sets of peptides and to interact differently with MHC chaperones in the endoplasmic reticulum. These results constitute evidence of the molecular evolutionary basis for 1) the maintenance of allelic lineages, 2) functional differences among lineages, and 3) strong linkage disequilibrium of allelic variants of class I region genes in X. laevis.Electronic Supplementary Material Supplementary material is available for this article at     

Shared evolutionary origin of major histocompatibility complex polymorphism in sympatric lemurs

Genes of the major histocompatibility complex (MHC) play a central role in adaptive immune responses of vertebrates. They exhibit remarkable polymorphism, often crossing species boundaries with similar alleles or allelic motifs shared across species. This pattern may reflect parallel parasite‐mediated selective pressures, either favouring the long maintenance of ancestral MHC allelic lineages across successive speciation events by balancing selection (“trans‐species polymorphism”), or alternatively favouring the independent emergence of functionally similar alleles post‐speciation via convergent evolution. Here, we investigate the origins of MHC similarity across several species of dwarf and mouse lemurs (Cheirogaleidae). We examined MHC class II variation in two highly polymorphic loci (DRB, DQB) and evaluated the overlap of gut–parasite communities in four sympatric lemurs. We tested for parasite‐MHC associations across species to determine whether similar parasite pressures may select for similar MHC alleles in different species. Next, we integrated our MHC data with those previously obtained from other Cheirogaleidae to investigate the relative contribution of convergent evolution and co‐ancestry to shared MHC polymorphism by contrasting patterns of codon usage at functional vs. neutral sites. Our results indicate that parasites shared across species may select for functionally similar MHC alleles, implying that the dynamics of MHC‐parasite co‐evolution should be envisaged at the community level. We further show that balancing selection maintaining trans‐species polymorphism, rather than convergent evolution, is the primary mechanism explaining shared MHC sequence motifs between species that diverged up to 30 million years ago.     

Characterization of naturally processed and presented peptides associated with bovine major histocompatibility complex (BoLA) class II DR molecules

Differential regulation of genetic resistance to infectious disease may partially be explained by variation in the binding affinity and the repertoire of pathogen-derived antigenic peptides associated with major histocompatibility complex (MHC) molecules. In this study, we investigated characteristics of peptides that bind to the bovine MHC allele BoLA-DRB3*2703, which is associated with occurrence of clinical mastitis in Holstein dairy cattle, and assigned a putative peptide-binding motif to this allele. This was achieved by in vitro expression of allele *2703 as well as a control allele, BoLA-DRB3*1201 which is present at high frequency in Holsteins. Transfected cell lines alone (for allele *1201) or in combination with blood mononuclear cells from an animal homozygous for allele *2703 were used as the source of naturally processed and presented peptides. Subsequent to elution of peptides from BoLA-DR+ cells, their sequences were determined by electrospray ionization mass spectrometry. Eluted peptides were between 13 and 20 amino acids long and the majority were in sets of overlapping sequences. These peptides were derived from intra- and extracellular proteins, as well as foreign proteins present in the culture medium. Some peptides had originated from molecular chaperones present in the endoplasmic reticulum, such as ER-60 and GRP78, pointing to some degree of overlap and cross-sampling between MHC class I and class II antigen presentation pathways. Consistent with reports of human and mouse MHC class II-associated peptides, putative peptide-binding motifs could be assigned to alleles *2703 and *1201, comprising a hydrophobic or an aromatic residue at relative position 1, a hydrophobic residue at position 4 and a small residue at position 6 of the eluted peptides. These findings provide the foundation for future studies of molecular mechanisms of MHC-disease associations of cattle.     

Acid phosphatase, adenosine deaminase and esterase D polymorphisms in the Spanish Basque population.

The 3 red-cell polymorphic systems acid phosphatase (ACP), adenosine deaminase (ADA) and esterase D (ESD) have been studied in a random sample of 1,112 individuals from the Basque country: The allelic frequencies obtained were ACP*A = 0.275, ACP*B = 0.718 and ACP*C = 0.007; ADA*2 = 0.021, and, ESD*2 = 0.066. The allelic frequencies have been compared with those of other Basque and other European populations. In comparison with Basques, significant differences were detected only for ACP, whereas as regards other Europeans significant differences were obtained with practically all the populations compared for the 3 genetic systems studied. The low values of the less frequent alleles, especially that for the ACP*C allele which is the lowest reported in Europe, are noteworthy.     

Interactions between NKG2x immunoreceptors and HLA-E ligands display overlapping affinities and thermodynamics

The NKG2x/CD94 family of C-type lectin-like immunoreceptors (x = A, B, C, E, and H) mediates surveillance of MHC class Ia cell surface expression, often dysregulated during infection or tumorigenesis, by recognizing the MHC class Ib protein HLA-E that specifically presents peptides derived from class Ia leader sequences. In this study, we determine the affinities and interaction thermodynamics between three NKG2x/CD94 receptors (NKG2A, NKG2C, and NKG2E) and complexes of HLA-E with four representative peptides. Inhibitory NKG2A/CD94 and activating NKG2E/CD94 receptors bind HLA-E with indistinguishable affinities, but with significantly higher affinities than the activating NKG2C/CD94 receptor. Despite minor sequence differences, the peptide presented by HLA-E significantly influenced the affinities; HLA-E allelic differences had no effect. These results reveal important constraints on the integration of opposing activating and inhibitory signals driving NK cell effector functions.     

Identification of two new alleles, IGHV3-23*04 and IGHJ6*04, and the complete sequence of the IGHV3-h pseudogene in the human immunoglobulin locus and their prevalences in Danish Caucasians

More than 100 variable (V), 27 diversity (D), and six joining (J) genes are encoded in the human heavy chain locus, and many of these genes exists in different allelic forms. The number of genes and the allelic differences help to create diversity in the immunoglobulin receptors, a key feature of the adaptive immune system. We here report the identification of two novel and seemingly functional alleles of human heavy chain genes. The variable IGHV3-23*04 allele is found with an allele frequency of 0.21 amongst Danish Caucasians, whereas the novel joining IGHJ6*04 allele is rare (allele frequency 0.02). We also report the full sequence of IGHV3-h. The gene exists in two allelic forms but is only found in 58% of the Danish Caucasians studied. The methionine translation initiation codon is mutated, ATG→AAG, and we therefore propose that the gene is a pseudogene incapable of being translated.     

Promiscuous Recognition of a Trypanosoma cruzi CD8+ T Cell Epitope among HLA-A2, HLA-A24 and HLA-A1 Supertypes in Chagasic Patients

Background

TcTLE is a nonamer peptide from Trypanosoma cruzi KMP-11 protein that is conserved among different parasite strains and that is presented by different HLA-A molecules from the A2 supertype. Because peptides presented by several major histocompatibility complex (MHC) supertypes are potential targets for immunotherapy, the aim of this study was to determine whether MHC molecules other than the A2 supertype present the TcTLE peptide.

Methodology/Principal Findings

From 36 HLA-A2-negative chagasic patients, the HLA-A genotypes of twenty-eight patients with CD8⁺ T cells that recognized the TcTLE peptide using tetramer (twenty) or functional (eight) assays, were determined. SSP-PCR was used to identify the A locus and the allelic variants. Flow cytometry was used to analyze the frequency of TcTLE-specific CD8⁺ T cells, and their functional activity (IFN-γ, TNFα, IL-2, perforin, granzyme and CD107a/b production) was induced by exposure to the TcTLE peptide. All patients tested had TcTLE-specific CD8⁺ T cells with frequencies ranging from 0.07–0.37%. Interestingly, seven of the twenty-eight patients had HLA-A homozygous alleles: A*24 (5 patients), A*23 (1 patient) and A*01 (1 patient), which belong to the A24 and A1 supertypes. In the remaining 21 patients with HLA-A heterozygous alleles, the most prominent alleles were A24 and A68. The most common allele sub-type was A*2402 (sixteen patients), which belongs to the A24 supertype, followed by A*6802 (six patients) from the A2 supertype. Additionally, the A*3002/A*3201 alleles from the A1 supertype were detected in one patient. All patients presented CD8⁺ T cells producing at least one cytokine after TcTLE peptide stimulation.

Conclusion/Significance

These results show that TcTLE is a promiscuous peptide that is presented by the A24 and A1 supertypes, in addition to the A2 supertype, suggesting its potential as a target for immunotherapy.     

Multiple parasites mediate balancing selection at two MHC class II genes in the fossorial water vole: insights from multivariate analyses and population genetics

We investigated the factors mediating selection acting on two MHC class II genes (DQA and DRB) in water vole (Arvicola scherman) natural populations in the French Jura Mountains. Population genetics showed significant homogeneity in allelic frequencies at the DQA1 locus as opposed to neutral markers (nine microsatellites), indicating balancing selection acting on this gene. Moreover, almost exhaustive screening for parasites, including gastrointestinal helminths, brain coccidia and antibodies against viruses responsible for zoonoses, was carried out. We applied a co-inertia approach to the genetic and parasitological data sets to avoid statistical problems related to multiple testing. Two alleles, Arte-DRB-11 and Arte-DRB-15, displayed antagonistic associations with the nematode Trichuris arvicolae, revealing the potential parasite-mediated selection acting on DRB locus. Selection mechanisms acting on the two MHC class II genes thus appeared different. Moreover, overdominance as balancing selection mechanism was showed highly unlikely in this system.     

Selective pressures on MHC class II genes in the guppy (Poecilia reticulata) as inferred by hierarchical analysis of population structure

Genes of the major histocompatibility complex, which are the most polymorphic of all vertebrate genes, are a pre‐eminent system for the study of selective pressures that arise from host–pathogen interactions. Balancing selection capable of maintaining high polymorphism should lead to the homogenization of MHC allele frequencies among populations, but there is some evidence to suggest that diversifying selection also operates on the MHC. However, the pattern of population structure observed at MHC loci is likely to depend on the spatial and/or temporal scale examined. Here, we investigated selection acting on MHC genes at different geographic scales using Venezuelan guppy populations inhabiting four regions. We found a significant correlation between MHC and microsatellite allelic richness across populations, which suggests the role of genetic drift in shaping MHC diversity. However, compared to microsatellites, more MHC variation was explained by differences between populations within larger geographic regions and less by the differences between the regions. Furthermore, among proximate populations, variation in MHC allele frequencies was significantly higher compared to microsatellites, indicating that selection acting on MHC may increase population structure at small spatial scales. However, in populations that have significantly diverged at neutral markers, the population‐genetic signature of diversifying selection may be eradicated in the long term by that of balancing selection, which acts to preserve rare alleles and thus maintain a common pool of MHC alleles.