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The leukocyte immunoglobulin–like receptor (LILR)B3 and LILRA6 genes encode homologous myeloid inhibitory and activating orphan receptors, respectively. Both genes exhibit a strikingly high level of polymorphism at the amino acid level and LILRA6 (but not LILRB3) displays copy number variation (CNV). Although multiple alleles have been reported for both genes, limited data is available on frequencies of these alleles among humans. We have sequenced LILRB3/A6 exons encoding signal peptides and ectodomains in 91 healthy blood donors of European descent who carry one or two copies of LILRA6 per diploid genome. Analysis of haplotypes among individuals with two LILRA6 copies, representing the majority in this cohort (N = 86), shows that common LILRB3 and LILRA6 alleles encode some distinct amino acid sequences in homologous regions of the receptors, which could potentially impact their respective functions differentially. Comparison of sequences in individuals with one vs. two copies of LILRA6 supports non-allelic homologous recombination between LILRB3 and LILRA6 as a mechanism for generating LILRA6 CNV and LILRB3 diversity. These data characterize LILRB3/LILRA6 genetic variation in more detail than previously described and underscore the need to determine their ligands.

Shotgun microbiome sequencing analysis presents several challenges to accurately and consistently depict sample composition and functional potential. Here we present a two-part framework – JAMS (Just a Microbiology System) – whereby with raw fastq files and metadata as input, meaningful analysis within a sample and between a sample can be performed with ease for either shotgun or 16S sequences. JAMS is the first package to provide seamless deconvolution of functions into their taxonomic contributors. We validated our JAMS framework on two human gut shotgun metagenome test datasets against the popular tool MetaPhlAn 4. We further demonstrate the application of the JAMS package, particularly the plotting functions, on a mouse shotgun dataset.

The cathelin‐related antimicrobial peptide CRAMP protects the mouse colon from inflammation, inflammation‐associated carcinogenesis, and disrupted microbiome balance, as shown in systemic Cnlp −/− mice (also known as Camp −/− mice). However, the mechanistic basis for the role and the cellular source of CRAMP in colon pathophysiology are ill defined. This study, using either epithelial or myeloid conditional Cnlp −/− mice, demonstrated that epithelial cell‐derived CRAMP played a major role in supporting normal development of colon crypts, mucus production, and repair of injured mucosa. On the other hand, myeloid cell‐derived CRAMP potently supported colon epithelial resistance to bacterial invasion during acute inflammation with exacerbated mucosal damage and higher rate of mouse mortality. Therefore, a well concerted cooperation of epithelial‐ and myeloid‐derived CRAMP is essential for colon mucosal homeostasis. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

The physiological homeostasis of gut mucosal barrier is maintained by both genetic and environmental factors and its impairment leads to pathogenesis such as inflammatory bowel disease. A cytokine like molecule, FAM3D (mouse Fam3D), is highly expressed in mouse gastrointestinal tract. Here, we demonstrate that deficiency in Fam3D is associated with impaired integrity of colonic mucosa, increased epithelial hyper-proliferation, reduced anti-microbial peptide production and increased sensitivity to chemically induced colitis associated with high incidence of cancer. Pretreatment of Fam3D−/− mice with antibiotics significantly reduces the severity of chemically induced colitis and wild type (WT) mice co-housed with Fam3D−/− mice phenocopy Fam3D-deficiency showing increased sensitivity to colitis and skewed composition of fecal microbiota. An initial equilibrium of microbiota in cohoused WT and Fam3D−/− mice is followed by an increasing divergence of the bacterial composition after separation. These results demonstrate the essential role of Fam3D in colon homeostasis, protection against inflammation associated cancer and normal microbiota composition., The cytokine like protein FAM3D (Fam3D in mice) is highly expressed in the digestive tract with unknown role in colon pathophysiology. Here, by using gene deficient mice, the authors show that Fam3D is critically involved in colon homeostasis, host defense against colitis-associated carcinogenesis, and the balance of microbiota.

Background: The success of unrelated haemopoietic cell transplantation (HCT) is limited by graft-versus-host disease (GVHD), which is the main post-transplantation challenge when HLA-matched donors are unavailable. A sequence dimorphism in exon 1 of HLA-B gives rise to leader peptides containing methionine (Met; M) or threonine (Thr; T), which differentially influence natural killer and T-cell alloresponses. The main aim of the study was to evaluate the role of the leader dimorphism in GVHD after HLA-B-mismatched unrelated HCT. Methods: We did a retrospective cohort study of 33 982 patients who received an unrelated HCT done in Australia, Europe, Japan, North America, and the UK between Jan 1, 1988, and Dec 31, 2016. Data were contributed by participants of the International Histocompatibility Working Group in Hematopoietic Cell Transplantation. All cases were included and there were no exclusion criteria. Multivariate regression models were used to assess risks associated with HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 mismatching. Among the 33 982 transplantations, the risks of GVHD associated with HLA-B M and T leaders were established in 17 100 (50·3%) HLA-matched and 1457 (4·3%) single HLA-B-mismatched transplantations using multivariate regression models. Leader frequencies were defined in 2 004 742 BeTheMatch US registry donors. Findings: Between Jan 20, 2017, and March 11, 2019, we assessed 33 982 HCTs using multivariate regression models for the role of HLA mismatching on outcome. Median follow-up was 1841 days (IQR 909–2963). Mortality and GVHD increased with increasing numbers of HLA mismatches. A single HLA-B mismatch increased grade 3–4 acute GVHD (odds ratio [OR] 1·89, 95% CI 1·53–2·33; p

Human immunoglobulin G (IgG) molecules, IgG1, IgG2 and IgG3, exhibit substantial inter-individual variation in their constant heavy chain regions, as discovered by serological methods. This polymorphism is encoded by theIGHG1,IGHG2, andIGHG3genes and may influence antibody function. We sequenced the coding fragments of these genes in 95 European Americans, 94 African Americans, and 94 Black South Africans. Striking differences were observed between the population groups, including extremely low amino acid sequence variation in IGHG1 among South Africans, and higher IGHG2 and IGHG3 diversity in individuals of African descent compared to individuals of European descent. Molecular definition of the loci illustrates a greater level of allelic polymorphism than previously described, including the presence of common IGHG2 and IGHG3 variants that were indistinguishable serologically. Comparison of our data with the 1000 Genome Project sequences indicates overall agreement between the datasets, although some inaccuracies in the 1000 Genomes Project are likely. These data represent the most comprehensive analysis of IGHG polymorphisms across major populations, which can now be applied to deciphering their functional impact.

The MHC continues to have the most disease-associations compared to other regions of the human genome, even in the genome-wide association study (GWAS) and single nucleotide polymorphism (SNP) era. Analysis of non-coding variation and their impact on the level of expression of HLA allotypes has shed new light on the potential mechanisms underlying HLA disease associations and alloreactivity in transplantation. Next-generation sequencing (NGS) technology has the capability of delineating the phase of variants in the HLA antigen-recognition site (ARS) with non-coding regulatory polymorphisms. These relationships are critical for understanding the qualitative and quantitative implications of HLA gene diversity. This article summarizes current understanding of non-coding region variation of HLA loci, the consequences of regulatory variation on HLA expression, the role for evolution in shaping lineage-specific expression, and the impact of HLA expression on disease susceptibility and transplantation outcomes. A role for phased sequencing methods for the MHC, and perspectives for future directions in basic and applied immunogenetic studies of the MHC are presented.

An efficacious human immunodeficiency virus (HIV) vaccine will likely require induction of both mucosal and systemic immune responses. We compared the immunogenicity and protective efficacy of two mucosal/systemic vaccine regimens and investigated their effects on the rectal microbiome. Rhesus macaques were primed twice mucosally with replication-competent adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus (SIV) recombinants and boosted twice intramuscularly with ALVAC-SIV recombinant plus SIV gp120 protein or with DNA for SIV genes and rhesus interleukin-12 plus SIV gp120 protein. Controls received empty Ad5hr vector and alum adjuvant only. Both regimens elicited strong, comparable mucosal and systemic cellular and humoral immunity. Prevaccination rectal microbiomes of males and females differed and significantly changed over the course of immunization, most strongly in females after Ad5hr immunizations. Following repeated low-dose intrarectal SIV challenges, both vaccine groups exhibited modestly but significantly reduced acute viremia. Male and female controls exhibited similar acute viral loads; however, vaccinated females, but not males, exhibited lower levels of acute viremia, compared to same-sex controls. Few differences in adaptive immune responses were observed between the sexes. Striking differences in correlations of the rectal microbiome of males and females with acute viremia and immune responses associated with protection were seen and point to effects of the microbiome on vaccine-induced immunity and viremia control. Our study clearly demonstrates direct effects of a mucosal SIV vaccine regimen on the rectal microbiome and validates our previously reported SIV vaccine-induced sex bias. Sex and the microbiome are critical factors that should not be overlooked in vaccine design and evaluation. IMPORTANCE Differences in HIV pathogenesis between males and females, including immunity postinfection, have been well documented, as have steroid hormone effects on the microbiome, which is known to influence mucosal immune responses. Few studies have applied this knowledge to vaccine trials. We investigated two SIV vaccine regimens combining mucosal priming immunizations and systemic protein boosting. We again report a vaccine-induced sex bias, with female rhesus macaques but not males displaying significantly reduced acute viremia. The vaccine regimens, especially the mucosal primes, significantly altered the rectal microbiome. The greatest effects were in females. Striking differences between female and male macaques in correlations of prevalent rectal bacteria with viral loads and potentially protective immune responses were observed. Effects of the microbiome on vaccine-induced immunity and viremia control require further study by microbiome transfer. However, the findings presented highlight the critical importance of considering effects of sex and the microbiome in vaccine design and evaluation.

Significance HLA class I molecules bind antigenic peptides and present them on the cell surface to cytotoxic T cells to initiate immune responses. The peptide selection process occurs intracellularly with the aid of a molecule called tapasin. HLA class I molecules are highly variable, which influences their structural characteristics and the level of tapasin involvement in peptide selection. We measured tapasin dependence levels of nearly 100 HLA variants and found that the level of tapasin dependence negatively correlates with the number of peptides that the HLA class I molecule presents to T cells, thereby affecting breadth of the immune response. Analysis of HLA genotypes in HIV cohorts reveals that greater tapasin independence associates with slower disease progression and lower viral load., Human leukocyte antigen (HLA) class I allotypes vary in their ability to present peptides in the absence of tapasin, an essential component of the peptide loading complex. We quantified tapasin dependence of all allotypes that are common in European and African Americans (n = 97), which revealed a broad continuum of values. Ex vivo examination of cytotoxic T cell responses to the entire HIV-1 proteome from infected subjects indicates that tapasin-dependent allotypes present a more limited set of distinct peptides than do tapasin-independent allotypes, data supported by computational predictions. This suggests that variation in tapasin dependence may impact the strength of the immune responses by altering peptide repertoire size. In support of this model, we observed that individuals carrying HLA class I genotypes characterized by greater tapasin independence progress more slowly to AIDS and maintain lower viral loads, presumably due to increased breadth of peptide presentation. Thus, tapasin dependence level, like HLA zygosity, may serve as a means to restrict or expand breadth of the HLA-I peptide repertoire across humans, ultimately influencing immune responses to pathogens and vaccines.

International audience; Severe aplastic anemia (SAA) is a rare disorder characterized by hypoplastic bone marrow and progressive pancytopenia. The etiology of acquired SAA is not understood but is likely related to abnormal immune responses and environmental exposures. We conducted a genome-wide association study of individuals with SAA genetically matched to healthy controls in discovery (359 cases, 1,396 controls) and validation sets (175 cases, 1,059 controls). Combined analyses identified linked SNPs in distinct blocks within the major histocompatibility complex on 6p21. The top SNP encodes p.Met76Val in the P4 binding pocket of the HLA class II gene HLA-DPB1 (rs1042151A>G, odds ratio [OR] 1.75, 95% confidence interval [CI] 1.50–2.03, p = 1.94 × 10−13) and was associated with HLA-DP cell surface expression in healthy individuals (p = 2.04 × 10−6). Phylogenetic analyses indicate that Val76 is not monophyletic and likely occurs in conjunction with different HLA-DP binding groove conformations. Imputation of HLA-DPB1 alleles revealed increased risk of SAA associated with Val76-encoding alleles DPB1∗03:01, (OR 1.66, p = 1.52 × 10−7), DPB1∗10:01 (OR 2.12, p = 0.0003), and DPB1∗01:01 (OR 1.60, p = 0.0008). A second SNP near HLA-B, rs28367832G>A, reached genome-wide significance (OR 1.49, 95% CI 1.22–1.78, p = 7.27 × 10−9) in combined analyses; the association remained significant after excluding cases with clonal copy-neutral loss-of-heterozygosity affecting class I HLA genes (8.6% of cases and 0% of controls). SNPs in the HLA class II gene HLA-DPB1 and possibly class I (HLA-B) are associated with SAA. The replacement of Met76 to Val76 in certain HLA-DPB1 alleles might influence risk of SAA through mechanisms involving DP peptide binding specificity, expression, and/or other factors affecting DP function.

Genomic variation in the untranslated region (UTR) has been shown to influence HLA class I expression level and associate with disease outcomes. Sequencing of the 3′UTR of common HLA-A alleles indicated the presence of two polyadenylation signals (PAS). The proximal PAS is conserved, whereas the distal PAS is disrupted within certain alleles by sequence variants. Using 3′RACE, we confirmed expression of two distinct forms of the HLA-A 3′UTR based on use of either the proximal or the distal PAS, which differ in length by 100 bp. Specific HLA-A alleles varied in the usage of the proximal versus distal PAS, with some alleles using only the proximal PAS, and others using both the proximal and distal PAS to differing degrees. We show that the short and the long 3′UTR produced similar mRNA expression levels. However, the long 3′UTR conferred lower luciferase activity as compared with the short form, indicating translation inhibition of the long 3′UTR. RNA affinity pull-down followed by mass spectrometry analysis as well as RNA coimmunoprecipitation indicated differential binding of Syncrip to the long versus short 3′UTR. Depletion of Syncrip by small interfering RNA increased surface expression of an HLA-A allotype that uses primarily the long 3′UTR, whereas an allotype expressing only the short form was unaffected. Furthermore, specific blocking of the proximal 3′UTR reduced surface expression without decreasing mRNA expression. These data demonstrate HLA-A allele-specific variation in PAS usage, which modulates their cell surface expression posttranscriptionally.

Polymorphisms located within the MHC have been linked to many disease outcomes by mechanisms not yet fully understood in most cases. Variants located within untranslated regions of HLA genes are involved in allele-specific expression and may therefore underlie some of these disease associations. We determined sequences extending nearly 2 kb upstream of the transcription start site for 68 alleles from 57 major lineages of classical HLA class I genes. The nucleotide diversity within this promoter segment roughly follows that seen within the coding regions, with HLA-B showing the highest (∼1.9%), followed by HLA-A (∼1.8%), and HLA-C showing the lowest diversity (∼0.9%). Despite its greater diversity, HLA-B mRNA expression levels determined in 178 European Americans do not vary in an allele- or lineage-specific manner, unlike the differential expression levels of HLA-A or HLA-C reported previously. Close proximity of promoter sequences in phylogenetic trees is roughly reflected by similarity of expression pattern for most HLA-A and -C loci. Although promoter sequence divergence might impact promoter activity, we observed no clear link between the phylogenetic structures as represented by pairwise nucleotide differences in the promoter regions with estimated differences in mRNA expression levels for the classical class I loci. Further, no pair of class I loci showed coordinated expression levels, suggesting that distinct mechanisms across loci determine their expression level under nonstimulated conditions. These data serve as a foundation for more in-depth analysis of the functional consequences of promoter region variation within the classical HLA class I loci.

In this minireview, we highlight the contributions of the late Austin L. Hughes to two areas of molecular evolution: the role of positive (Darwinian) selection, and the impact of gene duplications during genome evolution.

Autoimmune uveitis is a T cell driven, intraocular inflammation that affects the neuroretina and can lead to blindness. In a mouse model of spontaneous autoimmune uveitis (R161H), retina-specific T cells are primed in the gut through their TCR and trigger disease (PMID: 26287682). To support the relevance to human disease, we examined development of uveitis and its association with bacterial taxa in gnotobiotic R161H mice harboring human flora. Specifically, fecal samples from three healthy human donors were inoculated into germ-free R161H mice and their wild type (WT) littermates in separate isolators. Fecal pellets from reconstituted mice and their offspring were collected periodically and subjected to 16S amplicon sequencing. Uveitis scores were determined in R161H offspring by histology at termination of the experiment. Our results show that healthy human gut commensals can support uveitis in R161H mice, but to a lesser extent than native mouse flora from the NIH facility. Diversity analyses indicate that human flora-reconstituted mice retained a distinct but simplified gut microbial community compared to the original sample, and R161H mice tended to have a more complex microbiome profile than WT littermates. Among R161H offspring, mice with high disease scores appeared to harbor more diverse gut flora than those with low scores. Differentially abundant bacterial taxa were identified between mice with different disease scores, which may associate with intensity of autoreactive T cell activation and development of autoimmunity. R161H associated with human gut flora could provide a more translatable platform than mouse flora to explore microbial candidates and products that may modulate autoreactive T cells in uveitis.

Transplantation of hematopoietic cells from unrelated donors can cure blood disorders but carries a significant risk of acute graft-versus-host disease (GVHD). The risk is higher when the recipient and donor are HLA-DPB1-mismatched, but the mechanisms leading to GVHD are unknown. The HLA-DPB1 regulatory region variant rs9277534 is associated with HLA-DPB1 expression. We tested the hypothesis that the GVHD risk correlates with the rs9277534 allele linked to the mismatched HLA-DPB1 in the recipient.We genotyped rs9277534 in 3505 persons to define rs9277534-DPB1 haplotypes. Among 1441 recipients of transplants from HLA-A,B,C,DRB1,DQB1-matched unrelated donors with only one HLA-DPB1 mismatch, linkage of the rs9277534 A and G alleles to the mismatched HLA-DPB1 was determined. HLA-DPB1 expression was assessed by means of a quantitative polymerase-chain-reaction assay. The risk of acute GVHD among recipients whose mismatched HLA-DPB1 allele was linked to rs9277534G (high expression) was compared with the risk among recipients whose mismatched HLA-DPB1 allele was linked to rs9277534A (low expression).The mean HLA-DPB1 expression was lower with rs9277534A than with rs9277534G. Among recipients of transplants from donors with rs9277534A-linked HLA-DPB1, the risk of acute GVHD was higher for recipients with rs9277534G-linked HLA-DPB1 mismatches than for recipients with rs9277534A-linked HLA-DPB1 mismatches (hazard ratio, 1.54; 95% confidence interval [CI], 1.25 to 1.89; P0.001), as was the risk of death due to causes other than disease recurrence (hazard ratio, 1.25; 95% CI, 1.00 to 1.57; P=0.05).The risk of GVHD associated with HLA-DPB1 mismatching was influenced by the HLA-DPB1 rs9277534 expression marker. Among recipients of HLA-DPB1-mismatched transplants from donors with the low-expression allele, recipients with the high-expression allele had a high risk of GVHD. (Funded by the National Institutes of Health and others.).

MHC class I expression levels influence the strength of immune responses and represent another variable in determining outcome to disease beyond peptide binding alone. Identification of the HLA loci that vary in allelic expression levels and delineating the mechanism responsible for expression variation may provide the opportunity to modify their expression therapeutically. We have examined the expression levels of allelic lineages at the HLA-A locus in a sample of 216 European Americans using a real-time polymerase chain reaction assay, which amplifies all HLA-A lineages specifically with equal efficiency, and observed a gradient of expression that associates with HLA-A allelic lineage (R = 0.6, P = 5 × 10(-25)). DNA methylation of the HLA-A gene appears to contribute to the variation in HLA-A mRNA expression levels, as a significant inverse correlation was observed between HLA-A mRNA expression levels in untreated cells and the degree to which expression is increased after treatment of the cells with a DNA methyltransferase inhibitor (R = 0.6, P = 2.8 × 10(-6)). Further, deep-sequencing and immunoprecipitation assays revealed allelic lineage-specific methylation patterns within the HLA-A promoter region where increased DNA methylation levels correlated significantly with reduced HLA-A expression levels (R = 0.89, P = 3.7 × 10(-9)). These data demonstrate HLA-A allelic lineage-specific variation in expression levels, and DNA methylation as a likely factor in contributing to this variation.

A third of African Americans with sporadic focal segmental glomerulosclerosis (FSGS) or HIV-associated nephropathy (HIVAN) do not carry APOL1 renal risk genotypes. This raises the possibility that other APOL1 variants may contribute to kidney disease. To address this question, we sequenced all APOL1 exons in 1, 437 Americans of African and European decent, including 464 patients with biopsy-proven FSGS/HIVAN. Testing for association with 33 common and rare variants with FSGS/HIVAN revealed no association independent of strong recessive G1 and G2 effects. Seeking additional variants that might have been under selection by pathogens and could represent candidates for kidney disease risk, we also sequenced an additional 1, 112 individuals representing 53 global populations. Except for G1 and G2, none of the 7 common codon-altering variants showed evidence of selection or could restore lysis against trypanosomes causing human African trypanosomiasis. Thus, only APOL1 G1 and G2 confer renal risk and other common and rare APOL1 missense variants, including the archaic G3 haplotype, do not contribute to sporadic FSGS and HIVAN in the United States population. Hence, in most potential clinical or screening applications, our study suggests that sequencing APOL1 exons is unlikely to bring additional information compared to genotyping only APOL1 G1 and G2 risk alleles.

Introduction. Acquired severe aplastic anemia (SAA) is a life-threatening disorder characterized by severe progressive pancytopenia and hypocellular bone marrow. The etiology of acquired SAA is not understood but believed to be related to abnormal immune responses to environmental exposures. We conducted a genome-wide association study (GWAS) to identify common germline variants associated with SAA. Methods. We identified 895 patients with SAA who underwent related or unrelated hematopoietic cell transplant (HCT) with clinical data and pre-HCT blood samples available in the Center for International Blood and Marrow Transplant Research (CIBMTR) database and biorepository. Pre-HCT DNA was extracted from blood of patients with SAA and genome-wide genotyping was conducted using the Illumina OmniExpress array. We excluded 93 inherited bone marrow failure cases. The SAA cases were grouped cases into discovery and validation sets based on time of batch sample receipt. Analyses were limited to patients of European ancestry based on principal component analyses to minimize the potential effect of population stratification. Controls were genomically matched and selected from previously scanned cancer-free subjects at the Cancer Genomics Research Laboratory, Division of Cancer Epidemiology and Genetics, NCI. The final analysis included 534 acquired SAA cases (359 in the discovery set and 175 in the validation set), and 2,455 controls (1,396 in the discovery set, and 1,059 in the validation set). Results. Patients with SAA in this study received HCT between 1989-2015 at a median age of 21 years, 56% were male, and the median time between SAA diagnosis and HCT was 11 months. Strong genome-wide association signals were identified across the human leukocyte antigen (HLA) genes encoded at the major histocompatibility complex (MHC) on chromosome 6p21 (Figure 1). The top SNP was located in the P4 binding pocket of the HLA class II gene HLA-DPB1(rs1042151A>G, p.Met105Val, pooled-odds ratio [OR]=1.75, 95% confidence interval [CI]=1.50-2.03, p=1.94x10-13). The expression of HLA-DP in CD19+ cells from 175 healthy donors was significantly different by rs1042151 A>G genotype (p=2.04x10-6) (Figure 2). A second SNP near HLA-B, rs28367832G>A, also reached genome-wide significance (pooled-OR=1.49, 95% CI=1.22-1.78, p=7.27x10-9). Copy-number variant analysis and next generation sequencing also identified somatic, clonal copy-neutral loss-of-heterozygosity affecting class I HLA genes in 8.6% of the SAA cases and none of the controls. Conclusion. This SAA GWAS identified strong association signals between common germline genetic variants in HLA class I and II genes and SAA. The main SNP is associated with changes in HLA-DP expression suggesting a key role for this locus in SAA etiology. This study adds further evidence to the connection between SAA and immune dysregulation. Disclosures Cerhan: Janssen: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding; Celgene: Research Funding. Lee:Incyte: Research Funding; Syndax: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Kadmon: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; AstraZeneca: Research Funding.

Autoimmune uveitis, thought to be driven by retina-specific T cells, is a major cause of blindness. Using a model of experimental autoimmune uveitis (EAU), we showed that long-term elimination of commensals by oral antibiotic treatment (ABX) did not affect susceptibility to EAU, but others reported that short-term ABX was protective. To reconcile these contradictory findings, we studied whether duration of ABX differentially affected EAU development, gut microbiota and host-immune responses. B10.RIII mice were treated orally with a broad-spectrum antibiotic cocktail, starting 12-weeks (long-term) or 1-week (short-term) before IRBP immunization for EAU induction, and continuing until the end of the experiment. Short-term ABX delayed the onset of EAU, whereas long-term ABX showed a pattern of EAU kinetics and severity similar to untreated SPF controls. Metagenomic analyses revealed ABX duration-dependent depletion of gut microbial communities. Interestingly, ABX also resulted in progressive depletion of CD4+ and CD4+CD8+ T cells from the gut intraepithelial lymphocyte (IEL) compartment. Notably, these IELs showed some cytotoxic activity in vitro, and are thought to help preserve gut mucosal barrier integrity. We propose that microbiota play dual roles in uveitis development: they provide a stimulus for uveitogenic effector cells, but also maintain a “regulatory” IEL population, whose progressive loss reverses the temporary protection seen with short-term ABX. Further understanding of the role of IELs in the microbiota-gut-eye axis may lead to novel treatments for autoimmunity through manipulation of the commensal microbiome and gut epithelium.

Commensal bacteria are critical for physiological functions in the gut, and dysbiosis in the gut may cause diseases. In this article, we report that mice deficient in cathelin-related antimicrobial peptide (CRAMP) were defective in the development of colon mucosa and highly sensitive to dextran sulfate sodium (DSS)-elicited colitis, as well as azoxymethane-mediated carcinogenesis. Pretreatment of CRAMP−/− mice with antibiotics markedly reduced the severity of DSS-induced colitis, suggesting CRAMP as a limiting factor on dysbiosis in the colon. This was supported by observations that wild-type (WT) mice cohoused with CRAMP−/− mice became highly sensitive to DSS-induced colitis, and the composition of fecal microbiota was skewed by CRAMP deficiency. In particular, several bacterial species that are typically found in oral microbiota, such as Mogibacterium neglectum, Desulfovibrio piger, and Desulfomicrobium orale, were increased in feces of CRAMP−/− mice and were transferred to WT mice during cohousing. When littermates of CRAMP+/− parents were examined, the composition of the fecal microbiota of WT pups and heterozygous parents was similar. In contrast, although the difference in fecal microbiota between CRAMP−/− and WT pups was small early on after weaning and single mouse housing, there was an increasing divergence with prolonged single housing. These results indicate that CRAMP is critical in maintaining colon microbiota balance and supports mucosal homeostasis, anti-inflammatory responses, and protection from carcinogenesis.

Polymorphisms located within the MHC have been linked to many disease outcomes by mechanisms not yet fully understood in most cases. Variants located within untranslated regions of HLA genes are involved in allele specific expression and may therefore underlie some of these disease associations. We determined sequences extending nearly 2kb upstream of the transcription start site for 68 alleles from 57 major lineages of classical HLA class I genes. The nucleotide diversity within this promoter segment roughly follows that seen within the coding regions, with HLA-B showing the highest (~1.9%), followed by HLA–A (~1.8%), and HLA–C showing the lowest diversity (~0.9%). In spite of its greater diversity, HLA-B mRNA expression levels determined in 178 European Americans do not vary in an allele- or lineage-specific manner, unlike the differential expression levels of HLA-A or HLA-C reported previously. Close proximity of promoter sequences in phylogenetic trees is roughly reflected by similarity of expression pattern for most HLA-A and -C loci. While promoter sequence divergence might impact promoter activity, we observed no clear link between the phylogenetic structure as represented by pairwise nucleotide differences in the promoter regions with estimated differences in mRNA expression levels for the classical class I loci. Further, no pair of class I loci showed coordinated expression levels, suggesting that distinct mechanisms across loci determine their expression level under non-stimulated conditions. These data serve as a foundation for more in depth analysis of the functional consequences of promoter region variation within the classical HLA class I loci.

Hepatitis C virus (HCV) infection is the most common blood borne infection in the U.S. with estimates of 4 million HCV-infected individuals in the U.S. and 170 million worldwide1. The majority (70%–80%) of HCV infections persist and about 30% of individuals with persistent infection develop chronic liver disease, including cirrhosis and hepatocellular carcinoma2. Epidemiological, viral, and host factors have been associated with the differences in HCV clearance or persistence and studies have demonstrated that a strong host immune response against HCV favors viral clearance3,4. Thus, variation in genes involved in the immune response may contribute to the ability to clear the virus. In a recent genome-wide association study, a single nucleotide polymorphism (rs12979860) 3kb upstream of the IL28B gene, which encodes the type III interferon IFN-λ3, was shown to associate strongly with more than a 2-fold difference in response to HCV drug treatment5. To determine the potential effect of rs12979860 variation on outcome to HCV infection in a natural history setting, we genotyped this variant in HCV cohorts comprised of individuals who spontaneously cleared the virus (N = 388) or had persistent infection (N = 620). We show that the C/C genotype strongly enhances resolution of HCV infection amongst individuals of both European and African ancestry (European: OR = 0.38, p = 10−7; African: OR = 0.32, p = 10−4; combined: OR = 0.33, p

Tripartite motif (TRIM) proteins are composed of RING, B-box 2, and coiled coil domains. Some TRIM proteins, such as TRIM5α, also possess a carboxy-terminal B30.2(SPRY) domain and localize to cytoplasmic bodies. TRIM5α has recently been shown to mediate innate intracellular resistance to retroviruses, an activity dependent on the integrity of the B30.2 domain, in particular primate species. An examination of the sequences of several TRIM proteins related to TRIM5 revealed the existence of four variable regions (v1, v2, v3, and v4) in the B30.2 domain. Species-specific variation in TRIM5α was analyzed by amplifying, cloning, and sequencing nonhuman primate TRIM5 orthologs. Lineage-specific expansion and sequential duplication occurred in the TRIM5α B30.2 v1 region in Old World primates and in v3 in New World monkeys. We observed substitution patterns indicative of selection bordering these particular B30.2 domain variable elements. These results suggest that occasional, complex changes were incorporated into the TRIM5α B30.2 domain at discrete time points during the evolution of primates. Some of these time points correspond to periods during which primates were exposed to retroviral infections, based on the appearance of particular endogenous retroviruses in primate genomes. The results are consistent with a role for TRIM5α in innate immunity against retroviruses.

The effective sizes of ancestral populations and species divergence times of six primate species (humans, chimpanzees, gorillas, orangutans, and representatives of Old World monkeys and New World monkeys) are estimated by applying the two-species maximum likelihood (ML) method to intron sequences of 20 different loci. Examination of rate heterogeneity of nucleotide substitutions and intragenic recombination identifies five outrageous loci (ODC1, GHR, HBE, INS, and HBG). The estimated ancestral polymorphism ranges from 0.21 to 0.96% at major divergences in primate evolution. One exceptionally low polymorphism occurs when African and Asian apes diverged. However, taking into consideration the possible short generation times in primate ancestors, it is concluded that the ancestral population size in the primate lineage was no smaller than that of extant humans. Furthermore, under the assumption of 6 million years (myr) divergence between humans and chimpanzees, the divergence time of humans from gorillas, orangutans. Old World monkeys, and New World monkeys is estimated as 7.2, 18, 34, and 65 myr ago, respectively, which are generally older than traditional estimates. Beside the intron sequences, three other data sets of orthologous sequences are used between the human and the chimpanzee comparison. The ML application to these data sets including 58,156 random BAC end sequences (BES) shows that the nucleotide substitution rate is as low as 0.6-0.8 x 10(-9) per site per year and the extent of ancestral polymorphism is 0.33-0.51%. With such a low substitution rate and short generation time, the relatively high extent of polymorphism suggests a fairly large effective population size in the ancestral lineage common to humans and chimpanzees.

In the zebrafish, Danio rerio, and other teleosts, the class I and class II loci of the major histocompatibility complex ( Mhc) reside on different chromosomes. To shed light on the events that might have generated this difference from tetrapods, in which these two types of loci are clustered in a single chromosomal region, the organization of the class II loci in linkage group 8 of the zebrafish was determined by the characterization of contigs of PAC clones. Three contigs were defined: DAB, DCB, and DBB. The 350-kb-long DAB contig contained only four genes: DDB, DAB, SLC7A4, and DAA. The 150-kb-long DCB contig contained the DCB, DCA, and fz10 genes at an undetermined distance from the DAB contig. And the 120-kb-long DBB contig comprised the DBB gene presumably in another linkage group. The low gene density of the linkage group 8 contigs, contrasting with the high gene density of the zebrafish class I region, and the close association with genes [ SLC7A4 coding for an amino acid transporter, and fz10 (frizzled 10) coding for a receptor of the WNT glycoprotein] that are not linked with the tetrapod Mhc, is interpreted to mean that the separation of the class II from class I loci in teleosts occurred by translocation rather than by genomic or chromosomal duplication.

The jawless Agnatha (lampreys and hagfishes) represent the phylogenetically oldest order of vertebrates that are believed to lack the adaptive immune system of jawed vertebrates. In order to search for molecular markers specific for cellular components of the adaptive immune system in lampreys, we used the polymerase chain reaction (PCR) to identify genes for transcription factors of the Ikaros family in genomic DNA and cDNA libraries from two species of lampreys, Petromyzon marinus and Lampetra fluviatilis. The mammalian Ikaros-like family of transcription factors consists of five members, Ikaros, Helios, Aiolos, Eos and Pegasus, of which the first three appear to be essential for lymphocyte development. Two different Ikaros-like genes, named IKLF1 and IKLF2, were identified in lamprey. They both have the conserved exon-intron structure of seven exons and show alternative splicing like their counterparts in jawed vertebrates. The genes code for predicted proteins of 589 and 513 amino acid residues, respectively. The proteins contain six highly conserved zinc finger motifs that are 83-91% identical to the mammalian members of the Ikaros-like family. The remaining parts of the sequences are, however, mostly unalignable. Phylogenetic analysis based on the alignable segments of the sequences does not identify the orthologous gene in jawed vertebrates but rather shows equidistance of the lamprey Ikaros-like factors to each other and to Ikaros, Helios, Aiolos and Eos. Expression studies by reverse transcription (RT)-PCR and in situ hybridization (ISH), however, provide evidence for moderate expression in presumed lymphoid tissues like the gut epithelium and for high levels of expression in the gonads, especially in the ovary.

Life-threatening graft-versus-host disease (GVHD) limits the use of HLA-C-mismatched unrelated donors in transplantation. Clinicians lack criteria for donor selection when HLA-C-mismatched donors are a patient's only option for cure. We examined the role for HLA-C expression levels to identify permissible HLA-C mismatches. The median fluorescence intensity, a proxy of HLA-C expression, was assigned to each HLA-C allotype in 1975 patients and their HLA-C-mismatched unrelated transplant donors. The association of outcome with the level of expression of patients' and donors' HLA-C allotypes was evaluated in multivariable models. Increasing expression level of the patient's mismatched HLA-C allotype was associated with increased risks of grades III to IV acute GVHD, nonrelapse mortality, and mortality. Increasing expression level among HLA-C mismatches with residue 116 or residue 77/80 mismatching was associated with increased nonrelapse mortality. The immunogenicity of HLA-C mismatches in unrelated donor transplantation is influenced by the expression level of the patient's mismatched HLA-C allotype. HLA-C expression levels provide new information on mismatches that should be avoided and extend understanding of HLA-C-mediated immune responses in human disease.

Molecular phylogenetic analysis using genes coding for ribosomal RNA and proteins suggests that trypanosomes are monophyletic. Salivarian trypanosomes showing antigenic variation of the variant surface glycoprotein (VSG) diverged from non-Salivarian trypanosomes some 200-300 million years ago. Representatives of the non-Salivarian group, the mammalian parasite, Trypanosoma cruzi, and the fresh-water fish trypanosome, T. carassii, are characterised by surfaces dominated by carbohydrate-rich mucin-like glycoproteins, which are not subject to antigenetic variation. It is suggested that this latter surface structure is typical for non-Salivarian trypanosomes as well as members of the other Kinetoplastid suborder, the Bodonina. This would imply that at some point in time in the evolution of the Salivaria the highly abundant and comparatively poorly immunogenetic mucin-like molecules must have been replaced for equally abundant but highly immunogenic VSG-like molecules. While the selective advantage for such a unique transition is difficult to imagine, the subsequent diversification of VSG genes/molecules may have been comparatively straightforward because even the most limited form of antigenic variation would have extended the duration of infection in the vertebrate and thus would have increased the chance for transfer to the vector.

Darwin's finches comprise a group of 15 species endemic to the Galápagos (14 species) and Cocos (1 species) Islands in the Pacific Ocean. The group is monophyletic and originated from an ancestral species that reached the Galápagos Archipelago from Central or South America. Descendants of this ancestor on the Archipelago then colonized Cocos Island. In the present study, we used sequences of two mitochondrial (mt) DNA segments (922 bp of the cytochrome b gene and 1,082 bp of the control region), as well as two nuclear markers (830 bp of numt2, consisting of 140 bp of mtDNA control region and 690 bp of flanking nuclear DNA; and 740 bp of numt3, consisting of 420 bp of mt cytochrome b sequence flanked by 320 bp of nuclear DNA) to identify the species group most closely related to the Darwin's finches. To this end, we analyzed the sequences of 28 species representing the main groups (tribes) of the family Fringillidae, as well as 2 outgroup species and 13 species of Darwin's finches. In addition, we used mtDNA cytochrome b sequences of some 180 additional Fringillidae species from the database for phylogeny reconstruction by maximum-parsimony, maximum-likelihood, minimum-evolution, and neighbor-joining methods. The study identifies the grassquit genus Tiaris, and specifically the species Tiaris obscura, as the nearest living relative of Darwin's finches among the species surveyed. Darwin's finches diverged from the Tiaris group shortly after the various extant species of Tiaris diverged from one another. The initial adaptive radiation of the Tiaris group apparently occurred on the Caribbean islands and then spread to Central and South America, from where the ancestors of Darwin's finches departed for the Galápagos Islands approximately 2.3 MYA, at the time of the dramatic climatic changes associated with the closure of the Panamanian isthmus and the onset of Pleistocene glaciation.

A systematic survey of six intergenic regions flanking the human HLA-B locus in eight haplotypes reveals the regions to be up to 20 times more polymorphic than the reported average degree of human neutral polymorphism. Furthermore, the extent of polymorphism is directly related to the proximity to the HLA-B locus. Apparently linkage to HLA-B locus alleles, which are under balancing selection, maintains the neutral polymorphism of adjacent regions. For these linked polymorphisms to persist, recombination in the 200-kb interval from HLA-B to TNF must occur at a low frequency. The high degree of polymorphism found distal to HLA-B suggests that recombination is uncommon on both sides of the HLA-B locus. The least-squares estimate is 0.15% per megabase with an estimated range from 0.02 to 0.54%. These findings place strong restrictions on possible recombinational mechanisms for the generation of diversity at the HLA-B.

It is generally accepted that living jawless vertebrates (lampreys and hagfishes) lack the capability of mounting an adaptive immune response. At the same time, however, there are reports describing histological evidence for the presence in agnathan tissues of lymphocytes, the key players in adaptive immunity. The question therefore arises whether the cells identified morphologically as lymphocytes are true lymphocytes in terms of their genetic developmental program. In this study, evidence is provided that the lampreys express a member of the purine box 1 ( PU .1 )/spleen focus-forming virus integration B ( Spi-B ) gene family known to be critically and specifically involved in the differentiation of lymphocytes in jawed vertebrates. The lamprey gene is expressed in the lymphocyte-like cells of the digestive tract and inexplicably also in the ovary.

In both Old World and New World monkeys Mhc-DRB sequences have been found which resemble human DRB1*03 and DRB3 genes in their second exon. The resemblance is shared sequence motifs and clustering of the genes or the encoded proteins in phylogenetic trees. This similarity could be due to common ancestry, convergence at the molecular level, or chance. To test which of these three explanations applies, we sequenced segments of New World monkey and macaque genes which encompass the entire second exon and large parts of both flanking introns. The test strongly supports the monophyly of New World monkey DRB intron sequences. The phylogenies of introns 1 and 2 from DRB1*03-like and DRB3-like genes are congruent, but both are incongruent with the exon 2-based phylogeny. The matching of intron 1- and intron 2-based phylogenies with each other suggests that reciprocal recombination has not played a major role in exon 2 evolution. Statistical comparisons of exon 2 from different DRB1*03 and DRB3 lineages indicate that it was neither gene conversion (descent), nor chance, but molecular convergence that has shaped their characteristic motifs. The demonstration of convergence in anthropoid Mhc-DRB genes has implications for the classification, age, and mechanism of generation of DRB allelic lineages.

A distinctive feature of essential major histocompatibility complex (Mhc) loci is their polymorphism characterized by large genetic distances between alleles and long persistence times of allelic lineages. Since the lineages often span several successive speciations, we investigated the behavior of the Mhc alleles during or close to the speciation phase. We sequenced exon 2 of the class II B locus 4 from 232 East African cichlid fishes representing 32 related species. The divergence times of the (sub)species ranged from 6,000 to 8.4 million years. Two types of evolutionary analysis were used to elucidate the pattern of exon 2 sequence divergence. First, phylogenetic methods were applied to reconstruct the most likely evolutionary pathways leading from the last common ancestor of the set to the extant sequences, and to assess the probable mechanisms involved in allelic diversification. Second, pairwise comparisons of sequences were carried out to detect differences seemingly incompatible with origin by nonparallel point mutations. The analysis revealed point mutations to be the most important mechanism behind allelic divergences, with recombination playing only an auxiliary part. Comparison of sequences from related species revealed evidence of random allelic (lineage) losses apparently associated with speciation. Sharing of identical alleles could be demonstrated between species that diverged 2 million years ago. The phylogeny of the exon was incongruent with that of the flanking introns, indicating either a high degree of convergent evolution at the peptide-binding region-encoding sites, or intron homogenization.

The human acetyl-CoA acetyltransferase 2 gene, ACAT2, codes for a thiolase, an enzyme involved in lipid metabolism. The human T-complex protein 1 gene, TCP1, encodes a molecular chaperone of the chaperonin family. The two genes overlap by their 3′-untranslated regions, their coding sequences being located on opposite DNA strands in a tail-to-tail orientation. To find out how the overlap might have arisen in evolution, the homologous genes of the zebrafish, the African toad, caiman, platypus, opossum, and wallaby were identified. In each species, standard or long polymerase chain reactions were used to determine whether the ACAT2 and TCP1 homologs are closely linked and, if so, whether they overlap. The results reveal that the overlap apparently arose during the transition from therapsid reptiles to mammals and has been retained for >200 million years. Part of the overlapping untranslated region shows remarkable sequence conservation. The overlap presumably arose during the chromosomal rearrangement that brought the two unrelated and previously separated genes together. One or both of the transposed genes found by chance signals that are necessary for the processing of their transcripts to be present on the noncoding strand of the partner gene.

Darwin’s finches comprise a group of passerine birds first collected by Charles Darwin during his visit to the Galápagos Archipelago. The group, a textbook example of adaptive radiation (the diversification of a founding population into an array of species differentially adapted to diverse environmental niches), encompasses 14 currently recognized species, of which 13 live on the Galápagos Islands and one on the Cocos Island in the Pacific Ocean. Although Darwin’s finches have been studied extensively by morphologists, ecologists, and ethologists, their phylogenetic relationships remain uncertain. Here, sequences of two mtDNA segments, the cytochrome b and the control region, have been used to infer the evolutionary history of the group. The data reveal the Darwin’s finches to be a monophyletic group with the warbler finch being the species closest to the founding stock, followed by the vegetarian finch, and then by two sister groups, the ground and the tree finches. The Cocos finch is related to the tree finches of the Galápagos Islands. The traditional classification of ground finches into six species and tree finches into five species is not reflected in the molecular data. In these two groups, ancestral polymorphisms have not, as yet, been sorted out among the cross-hybridizing species.

We report the cDNA sequences for the DMA and DMB family of Mhc genes of the gray short-tailed opossum. Until now DM sequences were available only in eutherian mammals. The marsupial sequences indicate that both members of the family are old and probably diverged from other classical class II families about the time of the radiation of jawed vertebrates some 450 million years ago. We examine the evolutionary rates of equivalent sets of classical and nonclassical genes to check for rate heterogeneity. We find the alpha-1 domain of the DR genes to be untypically conservative in its evolutionary mode. The DM genes appear to evolve at rates typical of other class II genes, indicating that their placement at the root of class II gene evolutionary trees may be justified.

▪ Abstract Trans-species polymorphism (TSP) is the occurrence of similar alleles in related species. Excluding instances in which the similarity arose by convergent evolution, TSP is generated by the passage of alleles from ancestral to descendant species. Closely related, recently diverged species, such as those of the Lake Victoria cichlid flock, may share neutral alleles, but long-lasting TSPs occur only in genetic systems evolving under balancing selection. Two such systems have been studied extensively, the major histocompatibility complex (Mhc) of jawed vertebrates and the self-incompatibility (SI) system of flowering plants. Allelic lineages that diverged many millions of years ago and passed through numerous speciation events have been described in both systems. The lineages may differ at up to 50% of their coding sites, both synonymous and nonsynonymous. The differences arise by the process of incorporation of mutations, which is different from the process of fixation. TSP, on the one hand, complicates phylogenetic analysis, but on the other, it is a useful tool for the study of speciation.

Chronic infections with trypanosomes dwelling extracellularly in the blood and tissues of their hosts are observed in all vertebrate classes. We present here a molecular phylogenetic reconstruction of trypanosome evolution based on nucleotide sequences of small subunit rRNA genes. The evolutionary tree suggests an ancient split into one branch containing all Salivarian trypanosomes and a branch containing all non-Salivarian lineages. The latter branch splits into a clade containing bird, reptilian and Stercorarian trypanosomes infecting mammals and a clade with a branch of fish trypanosomes and a branch of reptilian/amphibian lineages. The branching order of the non-Salivarian trypanosomes supports host-parasite cospeciation scenarios, but also suggests host switches, e.g. between bird and reptilian trypanosomes. The tree is discussed in relation to the modes of adaptation that allow trypanosomes to infect immunocompetent vertebrates. Most importantly, the early divergence of the Salivarian lineages suggests that the presence of a dense proteinaceous surface coat that is subject to antigenic variation is a unique invention of this group of parasites.

Cichlid fishes of the East African Rift Valley lakes constitute an important model of adaptive radiation. Explosive speciation in the Great Lakes, in some cases as recently as 12 400 years ago, generated large species flocks that have been the focus of evolutionary studies for some time. The studies have, however, been hampered by the paucity of biochemical markers for phylogenetic reconstruction. Here, we describe a set of markers which should help to alleviate this problem. They are the class I genes of the major histocompatibility complex. We provide evidence for the existence of at least 17 class I loci in cichlid fishes, and for extensive polymorphism of three of these loci. Since the polymorphism has a trans-species character, it will be possible to use it in investigating the founding events of the individual species. The sequences of the cichlid class I fishes support the monophyly of actinopterygian fish on the one hand, and of tetrapods on the other.

A key assumption of many allopatric speciation models is that evolution in peripheral or isolated populations is facilitated by drastic reductions in population size. Population bottlenecks are believed to lead to rapid changes in gene frequencies through genetic drift, to facilitate rapid emergence of novel phenotypes, and to enhance reproductive isolation via genetic revolutions. For such effects to occur, founding populations must be very small, and remain small for some time after founding. This assumption has, however, rarely been tested in nature. One approach is to exploit the polymorphism of the major histocompatibility complex ( Mhc ) to obtain information about the founding population. Here, we use the Mhc polymorphism to estimate the size of the founding population of Darwin9s finches in the Galapagos Archipelago. The results indicate that the population could not have been smaller than 30 individuals.

The evolution of the major histocompatibility complex (Mhc) has been studied to understand the origin of the immune system, of which it constitutes an essential part. In the present study, the Mhc is used to shed light on questions regarding the origin of introns and the phylogeny of fishes. The organization of the coding (exon) and non-coding (intron) regions of both class I and class II major histocompatibility complex (Mhc) genes is highly conserved in all vertebrate classes; the only variation observed until now is in the number of exons encoding the membrane-anchoring part. Moreover, there is a good correspondence between the exon-intron organization at the DNA level and the division into structurally and functionally defined domains at the protein level. Here we describe the first major exception to this uniformity. The immunoglobulin-like domain of the class II $\beta $-chains in perch-like fishes (Percomorpha) is not encoded in one exon, as it is in all other vertebrates studied thus far, but in two exons. The length of the extra intron varies from gene to gene and from species to species, but is generally less than 200 base pairs (b.p.). Only one of the sequenced introns is about 500 b.p. long. In some of the genes, the intron contains a hexamer repeat. The repeat is present in the transcript at the site at which the intron interrupts exon 3 in the genomic DNA. The intron may therefore have arisen by repeated tandem duplication of this sequence. The intron is present in representatives of the Percomorpha and Atherinomorpha (silverside-like fishes) and absent in non-acanthopterygian fishes (and other vertebrates) tested thus far. The intron's distribution supports the monophyly of the Acanthopterigii (ray-finned fishes). In the perch-like fishes, the intron is present in different class II B loci, supporting the notion that these loci originated by repeated duplications from a single ancestral gene.

Over a period of some 20 years, Werner Eugen Mayer played a significant role in establishing a framework for molecular studies of Mhc genes in multiple vertebrates. His work largely concerned gene isolation, sequencing, and related bioinformatic analyses both for the Mhc and for immune system genes of about 200 species, ranging from apes, monkeys, rodents, and marsupials, through to birds, bony fishes, and lampreys. In addition to his exploration of diverse Mhc genes, Werner is remembered for playing a critical role in the development of two important insights into the evolution of immune systems. His was among the first published DNA sequence-based descriptions of trans-species evolution of Mhc alleles, including the first description of the long-lived polymorphisms shared by humans and chimpanzees. This research opened the way for using Mhc polymorphisms in demographic analyses. The second important insight in which he played a prominent role involved the characterization of immune cells and their expressed genes in the lamprey, a jawless vertebrate. His findings helped to indicate the considerable degree to which extant immune mechanisms were co-opted in the creation of the adaptive immune system of jawed vertebrates.

The major histocompatibility complex (MHC) polymorphism is marked by the existence of allelic lineages that are extremely old, having been passed from one species to another in an evolutionary line of descent. Each species has several of these lineages and many of their more recent derivatives, the actual alleles. The lineages are separated by large genetic distances and are characterized by the presence of short sequence motifs which, at the protein level, have remained virtually unaltered for over 40 million years. Several explanations for the mhc polymorphism have been proposed. We argue that the only one consistent with the entire body of knowledge about the MHC is an explanation based on the immune response to parasites. Furthermore, we propose that parasites coevolving with their hosts have had a major influence on MHC polymorphism, whereas parasites that switched hosts recently and became very virulent have had little effect. The latter category includes micro- and macroparasites responsible for the major human infectious diseases. This hypothesis explains why no convincing association between human leucocyte antigen ( HLA ) alleles and resistance to infectious disease can thus far be documented, and indicates the direction in which the search for such associations should be taken.

The major histocompatibility complex (MHC) plays a cardinal role in the defense of vertebrates against parasites and other pathogens. In some genes there are extensive and ancient polymorphisms that have passed from ancestral to descendant species and are shared among contemporary species. The polymorphism at the DRB1 locus, represented by 58 known alleles in humans, has existed for at least 30 million years and is shared by humans, apes, and other primates. The coalescence theory of populations genetics leads to the conclusion that the DRB1 polymorphism requires that the population ancestral to modern humans has maintained a mean effective size of 100,000 individuals over the 30-million-year persistence of this polymorphism. We explore the possibility of occasional population bottlenecks and conclude that the ancestral population could not have at any time consisted of fewer than several thousand individuals. The MHC polymorphisms exclude the theory claiming, on the basis of mitochondrial DNA polymorphisms, that a constriction down to one or few women occurred in Africa, at the transition from archaic to anatomically modern humans, some 200,000 years ago. The data are consistent with, but do not provide specific support for, the claim that human populations throughout the World were at that time replaced by populations migrating from Africa. The MHC and other molecular polymorphisms are consistent with a "multiregional" theory of Pleistocene human evolution that proposes regional continuity of human populations since the time of migrations of Homo erectus to the present, with distinctive regional selective pressures and occasional migrations between populations.

Long persistence of allelic lineages, prevalence of nonsynonymous over synonymous substitutions in the peptide-binding region (PBR), and deviation from neutrality of the expected gene identity parameter F all indicate indirectly that balancing selection is operating at functional major histocompatibility complex (MHC) loci. Direct demonstrations of the existence of balancing selection at MHC loci are, however, either lacking or not fully convincing. To define the conditions under which balancing selection could be demonstrated, we estimated its intensity from the mean number of nonsynonymous substitutions, KB, at the PBR and the mutation rate mu. We compared the five available methods for estimating KB by computer simulation and chose the most reliable ones for estimation of selection intensity. For the human MHC, the selection coefficients of the HLA-A, -B, -C, -DRB1, -DQB1, -DQA1, and -DPB1 loci are 0.015, 0.042, 0.0026, 0.019, 0.0085, 0.0028, and 0.0007, respectively. This low selection intensity places severe restrictions on the possibility of measuring selection directly in vertebrate populations.