Major Histocompatibility Complex Haplotypes Research Articles

Association between major histocompatibility complex haplotypes and susceptibility of unvaccinated and vaccinated cattle to paratuberculosis

Bovine Johne’s disease (BJD) or paratuberculosis is caused by Mycobacterium avium spp. paratuberculosis (MAP) and is a worldwide problem among domestic and wild ruminants. While vaccines are available, natural differences in background immunity between breeds within species and between individuals within herds suggest that genetic differences may be able to be exploited in marker-assisted selection as an aid to disease control. The major histocompatibility complex (MHC) is an important component in immune recognition with considerable genetic variability. In this study, associations between the MHC and resistance to BJD were explored in dairy cattle across two herds in which some of the cattle had been vaccinated with Silirum® (n = 540 cows). A BJD susceptible animal was exposed to MAP and became infected, while a resistant animal was exposed but did not become infected. There are different ways to define both exposure and infection, with different levels of stringency, therefore many classifications of the same set of animals are possible and were included in the analysis. The polymorphic regions of major histocompatibility complex class I (MHC I) and class II (MHC II) genes were amplified from the genomic DNA by PCR and sequenced, targeting exons 2 and 3 of the classical and non-classical MHC I genes and exon 2 from the DRB3, DQA1, DQA2 + 3 and DQB MHC II genes. The frequencies of MHC I and MHC II haplotypes and alleles were determined in susceptible and resistant populations. In unvaccinated animals, seven MHC I haplotypes and seven MHC II haplotypes were associated with susceptibility while two MHC I and six MHC II haplotypes were associated with resistance (P < 0.05). In vaccinated animals, two MHC I and three MHC II haplotypes were associated with susceptibility, while one MHC I and two MHC II haplotypes were associated with resistance (P < 0.05). The alleles in significant haplotypes were also identified. Case definitions with higher stringency resulted in fewer animals being included in the analyses, but the power to detect an association was not reduced and there was an increase in strength and consistency of associations. Consistent use of stringent case definitions is likely to improve agreement in future association studies.

Antigen presentation by B cells enables epitope spreading across an MHC barrier

Circumstantial evidence suggests that B cells may instruct T cells to break tolerance. Here, to test this hypothesis, we used a murine model in which a single B cell clone precipitates an autoreactive response resembling systemic lupus erythematosus (SLE). The initiating clone did not need to enter germinal centers to precipitate epitope spreading. Rather, it localized to extrafollicular splenic bridging channels early in the response. Autoantibody produced by the initiating clone was not sufficient to drive the autoreactive response. Subsequent epitope spreading depended on antigen presentation and was compartmentalized by major histocompatibility complex (MHC). B cells carrying two MHC haplotypes could bridge the MHC barrier between B cells that did not share MHC. Thus, B cells directly relay autoreactivity between two separate compartments of MHC-restricted T cells, leading to inclusion of distinct B cell populations in germinal centers. Our findings demonstrate that B cells initiate and propagate the autoimmune response.

HLA-OLI: A new MHC class I pseudogene and HLA-Y are located on a 60 kb indel in the human MHC between HLA-W and HLA-J.

Analysis of publicly available whole-genome sequence data from the Human Pangenome Project and the 1000 Genomes Project has identified a DNA segment of approximately 60 kb in the major histocompatibility complex (MHC) between HLA-W and HLA-J that is present in some MHC haplotypes but not others. This DNA segment is largely repeat element-rich but includes the pseudogene HLA-Y, thus pinpointing the location of this pseudogene, and a new HLA class I sequence we have called HLA-OLI. HLA-OLI clusters phylogenetically with the HLA class I pseudogenes, HLA-P and HLA-W, and appears to have a similar genetic structure. The availability of whole-genome sequence data from diverse populations enables a detailed characterization of the MHC at the population level and will have implications for understanding MHC disease associations and the non-HLA MHC factors that impact unrelated hematopoietic cell transplant outcomes.

The minor chicken class I gene BF1 is deleted between short imperfect direct repeats in the B14 and typical B15 major histocompatibility complex (MHC) haplotypes

The chicken major histocompatibility complex (MHC, also known as the BF-BL region of the B locus) is notably small and simple with few genes, most of which are involved in antigen processing and presentation. There are two classical class I genes, of which only BF2 is well and systemically expressed as the major ligand for cytotoxic T lymphocytes (CTLs). The other class I gene, BF1, is believed to be primarily a natural killer (NK) cell ligand. Among most standard chicken MHC haplotypes examined in detail, BF1 is expressed tenfold less than BF2 at the RNA level due to defects in the promoter or in a splice site. However, in the B14 and typical B15 haplotypes, BF1 RNA was not detected, and here, we show that a deletion between imperfect 32 nucleotide direct repeats has removed the BF1 gene entirely. The phenotypic effects of not having a BF1 gene (particularly on resistance to infectious pathogens) have not been systematically explored, but such deletions between short direct repeats are also found in some BF1 promoters and in the 5′ untranslated region (5′UTR) of some BG genes found in the BG region of the B locus. Despite the opposite transcriptional orientation of homologous genes in the chicken MHC, which might prevent the loss of key genes from a minimal essential MHC, it appears that small direct repeats can still lead to deletion.

Social and paternal female choice for male MHC genes in golden snub-nosed monkeys (Rhinopithecus roxellana).

Genes of the major histocompatibility complex (MHC) play important roles in vertebrate immunocompetence. MHC genes thus offer females indirect benefits to mate choice through the production of offspring of an optimal MHC genotype. Females may choose males with specific MHC haplotypes, dissimilar MHC genotypes, MHC heterozygous males or MHC-diverse males. We tested these four alternatives for both female social and paternal choice in wild golden snub-nosed monkeys (Rhinopithecus roxellana) by examining overall genetic variability (via microsatellites) and four MHC-genes (DRB1, DRB2, DQA1 and DQB1). Monte Carlo randomization tests showed that MHC dissimilarity was favoured for social choice (males to which females were socially affiliated) and intermediate MHC dissimilarity was favoured in paternal choice (fathers of offspring). No evidence of inbreeding avoidance was found for either social or paternal mates. We found that MHC heterozygotes, higher microsatellite multilocus heterozygosity and higher microsatellites diversity were favoured for social mates, and higher microsatellite diversity was favoured for paternal mates. Independent of male age, we found that the formation of male-female social pairings is significantly predicted by compatibility based on the sharing of MHC haplotypes. However, we found no evidence of independent genetic effects on the duration of male-female social pairings, male social status (achieving OMU leader male status or not), the number of females with which individual leader males paired, the likelihood of potential male-female pairings producing offspring, or whether males fathered offspring or not. Overall, our findings suggest different genetic factors are involved in social and paternal choice in R. roxellana.

Complete sequences of six major histocompatibility complex haplotypes, including all the major MHC class II structures.

Accurate and comprehensive immunogenetic reference panels are key to the successful implementation of population-scale immunogenomics. The 5Mbp Major Histocompatibility Complex (MHC) is the most polymorphic region of the human genome and associated with multiple immune-mediated diseases, transplant matching and therapy responses. Analysis of MHC genetic variation is severely complicated by complex patterns of sequence variation, linkage disequilibrium and a lack of fully resolved MHC reference haplotypes, increasing the risk of spurious findings on analyzing this medically important region. Integrating Illumina, ultra-long Nanopore, and PacBio HiFi sequencing as well as bespoke bioinformatics, we completed five of the alternative MHC reference haplotypes of the current (GRCh38/hg38) build of the human reference genome and added one other. The six assembled MHC haplotypes encompass the DR1 and DR4 haplotype structures in addition to the previously completed DR2 and DR3, as well as six distinct classes of the structurally variable C4 region. Analysis of the assembled haplotypes showed that MHC class II sequence structures, including repeat element positions, are generally conserved within the DR haplotype supergroups, and that sequence diversity peaks in three regions around HLA-A, HLA-B+C, and the HLA class II genes. Demonstrating the potential for improved short-read analysis, the number of proper read pairs recruited to the MHC was found to be increased by 0.06%-0.49% in a 1000 Genomes Project read remapping experiment with seven diverse samples. Furthermore, the assembled haplotypes can serve as references for the community and provide the basis of a structurally accurate genotyping graph of the complete MHC region.

CRISPR-based targeted haplotype-resolved assembly of a megabase region

Constructing high-quality haplotype-resolved genome assemblies has substantially improved the ability to detect and characterize genetic variants. A targeted approach providing readily access to the rich information from haplotype-resolved genome assemblies will be appealing to groups of basic researchers and medical scientists focused on specific genomic regions. Here, using the 4.5 megabase, notoriously difficult-to-assemble major histocompatibility complex (MHC) region as an example, we demonstrated an approach to construct haplotype-resolved assembly of the targeted genomic region with the CRISPR-based enrichment. Compared to the results from haplotype-resolved genome assembly, our targeted approach achieved comparable completeness and accuracy with reduced computing complexity, sequencing cost, as well as the amount of starting materials. Moreover, using the targeted assembled personal MHC haplotypes as the reference both improves the quantification accuracy for sequencing data and enables allele-specific functional genomics analyses of the MHC region. Given its highly efficient use of resources, our approach can greatly facilitate population genetic studies of targeted regions, and may pave a new way to elucidate the molecular mechanisms in disease etiology.

Induction of antigen-specific tolerance by hepatic AAV immunotherapy regardless of T cell epitope usage or mouse strain background

Invivo induction of antigen (Ag)-specific regulatory Tcells (Treg) is considered the holy grail of therapeutic strategies for restoring tolerance in autoimmunity. Unfortunately, in the autoimmune disease multiple sclerosis, an effective and durable therapy targeting the diverse repertoire of emerging Ags without compromising the patient's natural immunity has remained elusive. To address this deficiency, we have developed an Ag-specific adeno-associated virus (AAV) immunotherapy that will restore tolerance in a Treg-dependent manner. Using multiple strains of mice with different genetic and immunological backgrounds, we demonstrate that a liver directed AAV vector expressing a single transgene can prevent experimental autoimmune encephalomyelitis from developing and effectively mitigate pre-existing or established disease that was induced by one or more auto-reactive myelin oligodendrocyte glycoprotein-derived peptides. Overall, the results suggests that AAV can efficiently restore Ag-specific immune tolerance to an immunogenic protein that is neither restricted by the major histocompatibility complex haplotype, nor by the specific antigenic epitope(s) presented. These findings may pave the way for developing a comprehensive Ag-specific immunotherapy that does not require prior knowledge of the specific immunogenic epitopes and that may prove to be universally applicable to all MS patients, and adaptable for other autoimmune diseases.

Long-read assembly of major histocompatibility complex and killer cell immunoglobulin-like receptor genome regions in cynomolgus macaque

BackgroundThe major histocompatibility complex (MHC) and the killer cell immunoglobulin-like receptors (KIR) are key regulators of immune responses. The cynomolgus macaque, an Old World monkey species, can be applied as an important preclinical model for studying human diseases, including coronavirus disease 2019 (COVID-19). Several MHC-KIR combinations have been associated with either a poor or good prognosis. Therefore, macaques with a well-characterized immunogenetic profile may improve drug evaluation and speed up vaccine development. At present, a complete overview of the MHC and KIR haplotype organizations in cynomolgus macaques is lacking, and characterization by conventional techniques is hampered by the extensive expansion of the macaque MHC-B region that complicates the discrimination between genes and alleles.MethodsWe assembled complete MHC and KIR genomic regions of cynomolgus macaque using third-generation long-read sequencing approach. We identified functional Mafa-B loci at the transcriptome level using locus-specific amplification in a cohort of 33 Vietnamese cynomolgus macaques.ResultsThis is the first physical mapping of complete MHC and KIR gene regions in a Vietnamese cynomolgus macaque. Furthermore, we identified four functional Mafa-B loci (B2, B3, B5, and B6) and showed that alleles of the Mafa-I*01, -B*056, -B*034, and -B*001 functional lineages, respectively, are highly frequent in the Vietnamese cynomolgus macaque population.ConclusionThe insights into the MHC and KIR haplotype organizations and the level of diversity may refine the selection of animals with specific genetic markers for future medical research.

Marek’s disease virus-specific T cells proliferate, express antiviral cytokines but have impaired degranulation response

The major histocompatibility complex (MHC) haplotype is one of the major determinants of genetic resistance and susceptibility of chickens to Marek’s disease (MD) which is caused by an oncogenic herpesvirus; Marek’s disease virus (MDV). To determine differential functional abilities of T cells associated with resistance and susceptibility to MD, we identified immunodominant CD4+TCRvβ1 T cell epitopes within the pp38 antigen of MDV in B19 and B21 MHC haplotype chickens using an ex vivo ELISPOT assay for chicken IFN-gamma. These novel pp38 peptides were used to characterize differential functional abilities of T cells as associated with resistance and susceptibility to MD. The results demonstrated an upregulation of cytokines (IL-2, IL-4, IL-10) and lymphocyte lysis-related genes (perforin and granzyme B) in an antigen specific manner using RT-PCR. In the MD-resistant chickens (B21 MHC haplotype), antigen-specific and non-specific response was highly skewed towards Th2 response as defined by higher levels of IL-4 expression as well as lymphocyte lysis-related genes compared to that in the MD-susceptible chicken line (B19 MHC haplotype). Using CD107a degranulation assay, the results showed that MDV infection impairs cytotoxic function of T cells regardless of their genetic background. Taken together, the data demonstrate an association between type of T cell response to pp38 and resistance to the disease and will shed light on our understanding of immune response to this oncogenic herpesvirus and failure to induce sterile immunity.

Hands-on immunology: Engaging learners of all ages through tactile teaching tools.

Ensuring the public has a fundamental understanding of human–microbe interactions, immune responses, and vaccines is a critical challenge in the midst of a pandemic. These topics are commonly taught in undergraduate- and graduate-level microbiology and immunology courses; however, creating engaging methods of teaching these complex concepts to students of all ages is necessary to keep younger students interested when science seems hard. Building on the Tactile Teaching Tools with Guided Inquiry Learning (TTT-GIL) method we used to create an interactive lac operon molecular puzzle, we report here two TTT-GIL activities designed to engage diverse learners from middle schoolers to masters students in exploring molecular interactions within the immune system. By pairing physical models with structured activities built on the constructivist framework of Process-Oriented Guided Inquiry Learning (POGIL), TTT-GIL activities guide learners through their interaction with the model, using the Learning Cycle to facilitate construction of new concepts. Moreover, TTT-GIL activities are designed utilizing Universal Design for Learning (UDL) principles to include all learners through multiple means of engagement, representation, and action. The TTT-GIL activities reported here include a web-enhanced activity designed to teach concepts related to antibody–epitope binding and specificity to deaf and hard-of-hearing middle and high school students in a remote setting and a team-based activity that simulates the evolution of the Major Histocompatibility Complex (MHC) haplotype of a population exposed to pathogens. These activities incorporate TTT-GIL to engage learners in the exploration of fundamental immunology concepts and can be adapted for use with learners of different levels and educational backgrounds.

Major Histocompatibility Complex Variation and Haplotype Associated Survival in Response to Experimental Infection of Two Bd-GPL Strains Along a Latitudinal Gradient

While both innate and adaptive immune system mechanisms have been implicated in resistance against the chytrid fungus Batrachochytrium dendrobatidis (Bd), studies on the role of specific MHC haplotypes on Bd infection are rare. Here, we studied variation in MHC Class IIB loci in the common toad Bufo bufo along a latitudinal gradient across Sweden. In general, Swedish toad populations had few MHC Class IIB haplotypes and MHC diversity declined from south (13 haplotypes) to the north (four haplotypes). The low diversity may compromise the ability of northern populations to fight emerging disease, such as Bd. In a laboratory experiment, we infected newly metamorphosed toads with two strains of the Global Pandemic Lineage of the fungus (Bd-GPL) and compared survival with sham controls. Bd-infected toads had lower survival compared to controls. Moreover, survival was dependent on the Bd-strain and northern toads had lower Bd-mediated survival than southern individuals. MHC diversity was lower in northern toads. All northern experimental animals were monomorphic for a single MHC haplotype, whereas we found seven different haplotypes in southern experimental animals. In southern toads, survival was dependent on both Bd-strain and MHC haplotype suggesting differential infection dynamics depending on both Bd-strain and host immune system characteristics.

MHCtools - an R package for MHC high-throughput sequencing data: Genotyping, haplotype and supertype inference, and downstream genetic analyses in non-model organisms.

The major histocompatibility complex (MHC) plays a central role in the vertebrate adaptive immune system and has been of long‐term interest in evolutionary biology. While several protocols have been developed for MHC genotyping, there is a lack of transparent and standardized tools for downstream analysis of MHC data. Here, we present the r package mhctools and demonstrate the use of its functions to (i) assist accurate MHC genotyping from high‐throughput amplicon‐sequencing data, (ii) infer functional MHC supertypes using bootstrapped clustering analysis, (iii) identify segregating MHC haplotypes from family data, and (iv) analyse functional and genetic distances between MHC sequences. We employed mhctools to analyse MHC class I (MHC‐I) amplicon data of 559 great reed warblers (Acrocephalus arundinaceus). We identified 390 MHC‐I alleles which clustered into 14 functional supertypes. A phylogenetic analysis and analyses of positive selection suggested that the MHC‐I alleles belong to several distinct functional groups. We furthermore identified 107 segregating haplotypes among 116 families, and found substantial variation in diversity with 4–21 MHC‐I alleles and 3–13 MHC‐I supertypes per haplotype. Finally, we show that the great reed warbler haplotypes harboured combinations of MHC‐I supertypes with greater functional divergence than observed in simulated populations of possible haplotypes, a result that is in accordance with the divergent allele advantage hypothesis. Our study demonstrates the power of mhctools to support genotyping and analysis of MHC in non‐model species, which we hope will encourage broad implementation among researchers in MHC genetics and evolution.

Abstract 1387: High-dimensional analysis of T cell responses to mutant neoantigens and nonmutant antigens in mouse models of melanoma

Abstract Effective cancer immunotherapy, such as immune checkpoint therapy (ICT) [e.g., anti-CTLA-4 and anti-PD-1/PD-L1], is dependent on T cell recognition of tumor antigens presented on major histocompatibility complex (MHC) and leads to durable responses in certain cancer patients. Recent advances have facilitated identification of tumor mutant neoantigens and led to efforts to develop effective personalized neoantigen cancer vaccines. However, little is known about how expression of multiple neoantigens with variable peptide-MHC (pMHC) binding or TCR affinity/avidity affects T cell responses to both discrete neoantigens and shared non-mutant antigens, and if the localization of T cells with distinctive pMHC specificity differs within the tumor. To address this, we genetically introduced multiple combinations of MHC-I and MHC-II neoantigens in the poorly immunogenic Brafv600e Pten-/- Cdkn2a-/- YUMM1.7 (Y1.7) mouse melanoma line that contains a paucity of neoantigens. We expressed neoantigens previously found in a mouse sarcoma line of the same MHC haplotype as Y1.7. The MHC-I neoantigens used were formed by G1254V mutation in Laminin alpha subunit-4 (Lama4) and A506T mutation in the Asparagine-linked glycosylation 8 (Alg8) and the MHC-II neoantigen was formed by A710T mutation in Integrin beta-1 (Itgb1). We generated Y1.7 lines expressing minigenes for mLama4, mAlg8, or mLama4 + mAlg8 MHC-I neoantigens. All three lines also expressed the mItgb1 MHC-II neoantigen. Whereas ICT-treated mice bearing the parental Y1.7 melanoma line showed rapid tumor growth, modified Y1.7 lines expressing mLama4 and/or mAlg8 plus mItgb1 were rendered sensitive to ICT. Tumors expressing mLama4 and/or mAlg8 contained mLama4- and/or mAlg8-specific CD8+ T cells, respectively. Preliminary analysis revealed expression of either MHC-I neoantigen alters the frequency of CD8+ T cells specific for non-mutant endogenous antigens in Y1.7. Interestingly, therapeutic synthetic long-peptide (SLP) vaccines incorporating mLama4 and/or mAlg8 neoantigens plus poly I:C induced tumor rejection comparable to ICT. Moreover, SLP vaccines amplified intratumoral mLama4- and mAlg8-specific CD8+ T cells, as well as in tumor-draining lymph nodes, where these neoantigen-specific CD8+ T cells were found to express IFN-γ. During either vaccination or ICT, CD8+ T cell response to mLama4 was higher than that to mAlg8 when both neoantigens were co-expressed. Altogether, these melanoma models will enable us to determine whether expression of one or more MHC-I neoantigens influences CD8+ T cell responses to both distinct neoantigens and non-mutant antigens (e.g., gp100, Trp2) during ICT and SLP vaccination. Furthermore, high-dimensional analyses approaches including scRNA-seq. and CODEX imaging are ongoing and will facilitate development of improved immunotherapies and may help uncover novel biomarkers of response. Citation Format: Sunita Keshari, Charmelle D. Williams, Elizabeth D. Sanchez, Alexander S. Shavkunov, Matthew M. Gubin. High-dimensional analysis of T cell responses to mutant neoantigens and nonmutant antigens in mouse models of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1387.

The Diverse Major Histocompatibility Complex Haplotypes of a Common Commercial Chicken Line and Their Effect on Marek's Disease Virus Pathogenesis and Tumorigenesis.

The major histocompatibility complex (MHC) is crucial for appropriate immune responses against invading pathogens. Chickens possess a single predominantly-expressed class I molecule with strong associations between disease resistance and MHC haplotype. For Marek’s disease virus (MDV) infections of chickens, the MHC haplotype is one of the major determinants of genetic resistance and susceptibility. VALO specific pathogen free (SPF) chickens are widely used in biomedical research and vaccine production. While valuable findings originate from MDV infections of VALO SPF chickens, their MHC haplotypes and associated disease resistance remained elusive. In this study, we used several typing systems to show that VALO SPF chickens possess MHC haplotypes that include B9, B9:02, B15, B19 and B21 at various frequencies. Moreover, we associate the MHC haplotypes to MDV-induced disease and lymphoma formation and found that B15 homozygotes had the lowest tumor incidence while B21 homozygotes had the lowest number of organs with tumors. Finally, we found transmission at variable levels to all contact birds except B15/B21 heterozygotes. These data have immediate implications for the use of VALO SPF chickens and eggs in the life sciences and add another piece to the puzzle of the chicken MHC complex and its role in infections with this oncogenic herpesvirus.

Characterizing the diversity of MHC conserved extended haplotypes using families from the United Arab Emirates

Aside from its anthropological relevance, the characterization of the allele frequencies of genes in the human Major Histocompatibility Complex (MHC) and the combination of these alleles that make up MHC conserved extended haplotypes (CEHs) is necessary for histocompatibility matching in transplantation as well as mapping disease association loci. The structure and content of the MHC region in Middle Eastern populations remain poorly characterized, posing challenges when establishing disease association studies in ethnic groups that inhabit the region and reducing the capacity to translate genetic research into clinical practice. This study was conceived to address a gap of knowledge, aiming to characterize CEHs in the United Arab Emirates (UAE) population through segregation analysis of high-resolution, pedigree-phased, MHC haplotypes derived from 41 families. Twenty per cent (20.5%) of the total haplotype pool derived from this study cohort were identified as putative CEHs in the UAE population. These consisted of CEHs that have been previously detected in other ethnic groups, including the South Asian CEH 8.2 [HLA- C*07:02-B*08:01-DRB1*03:01-DQA1*05:01-DQB1*02:01 (H.F. 0.094)] and the common East Asian CEH 58.1 [HLA- C*03:02-B*58:01-DRB1*03:01- DQA1*05:01-DQB1*02:01 (H.F. 0.024)]. Additionally, three novel CEHs were identified in the current cohort, including HLA- C*15:02-B*40:06-DRB1*16:02-DQB1*05:02 (H.F. 0.035), HLA- C*16:02-B*51:01-DRB1*16:01-DQA1*01:02-DQB1*05:02 (H.F. 0.029), and HLA- C*03:02-B*58:01-DRB1*16:01-DQA1*01:02-DQB1*05:02 (H.F. 0.024). Overall, the results indicate a substantial gene flow with neighbouring ethnic groups in the contemporary UAE population including South Asian, East Asian, African, and European populations. Importantly, alleles and haplotypes that have been previously associated with autoimmune diseases (e.g., Type 1 Diabetes) were also present. In this regard, this study emphasizes that an appreciation for ethnic differences can provide insights into subpopulation-specific disease-related polymorphisms, which has remained a difficult endeavour.

Preclinical evaluation of a precision medicine approach to DNA vaccination in type 1 diabetes

Antigen-specific immunotherapy involves the delivery of self-antigens as proteins or peptides (or using nucleic acids encoding them) to reestablish tolerance. The Endotope platform supports the optimal presentation of endogenously expressed epitopes on appropriate major histocompatibility complex (MHC) class I and II molecules. Using specific epitopes that are disease-relevant (including neoepitopes and mimotopes) and restricted to the subject’s MHC haplotypes provides a more focused and tailored way of targeting autoreactive T cells. We evaluated the efficacy of an Endotope DNA vaccine tailored to the nonobese diabetic (NOD) mouse in parallel to one expressing the Proinsulin protein, a central autoantigen in NOD mice, and assessed the influence of several parameters (e.g., route, dosing frequency, disease stage) on diabetes prevention. Secretion of encoded peptides and intradermal delivery of DNA offered more effective disease prevention. Long-term weekly treatments were needed to achieve protection that can persist after discontinuation, likely mediated by regulatory T cells induced by at least one epitope. Although epitopes were presented for at least 2 wk, weekly treatments were needed, at least initially, to achieve significant protection. While Endotope and Proinsulin DNA vaccines were effective at both the prediabetic normoglycemic and dysglycemic stages of disease, Proinsulin provided better protection in the latter stage, particularly in animals with slower progression of disease, and Endotope limited insulitis the most in the earlier stage. Thus, our data support the possibility of applying a precision medicine approach based on tailored epitopes for the treatment of tissue-specific autoimmune diseases with DNA vaccines.

The Path to Conserved Extended Haplotypes: Megabase-Length Haplotypes at High Population Frequency.

This minireview describes the history of the conceptual development of conserved extended haplotypes (CEHs): megabase-length haplotypes that exist at high (≥0.5%) population frequency. My career began in internal medicine, shifted to pediatrics, and clinical practice changed to research. My research interest was initially in hematology: on plasma proteins, their metabolism, synthesis, and function. This narrowed to a focus on proteins of the human complement system, their role in immunity and their genetics, beginning with polymorphism and deficiency of C3. My group identified genetic polymorphisms and/or inherited deficiencies of C2, C4, C6, and C8. After defining glycine-rich beta glycoprotein as factor B (Bf) in the properdin system, we found that the genes for Bf (CFB), C2, C4A, and C4B were inherited as a single haplotypic unit which we named the “complotype.” Complotypes are located within the major histocompatibility complex (MHC) between HLA-B and HLA-DRB1 and are designated (in arbitrary order) by their CFB, C2, C4A, and C4B types. Pedigree analysis revealed long stretches (several megabases) of apparently fixed DNA within the MHC that we referred to as “extended haplotypes” (later as “CEHs”). About 10 to 12 common CEHs constitute at least 25 – 30% of MHC haplotypes among European Caucasian populations. These CEHs contain virtually all the most common markers of MHC-associated diseases. In the case of type 1 diabetes, we have proposed a purely genetic and epigenetic model (with a small number of Mendelian recessive disease genes) that explains all the puzzling features of the disease, including its rising incidence.

Segregation Analysis of Genotyped and Family-Phased, Long Range MHC Classical Class I and Class II Haplotypes in 5 Families With Type 1 Diabetes Proband in the United Arab Emirates.

The classical Human Leucocyte Antigen (HLA) class II haplotypes of the Major Histocompatibility Complex (MHC) that are associated with type 1 diabetes (T1D) were identified in five families from the United Arab Emirates (UAE). Segregation analyses were performed on these 5 families with the disease, 3 with one child and 2 with 2 children diagnosed with T1D. Three HLA-DR4 haplotypes were identified: HLA- DRB1∗04:01:01-DQB1∗03:02:01:01; HLA- DRB1∗04:02:01- DQB1∗03:02:01; and HLA -DRB1∗04:05:01-DQB1∗02:02:01:02. All have previously been identified to be associated with T1D in studies of the Arabian population. In the 10 parents from the 5 families, 9 had at least one HLA-DR4 and HLA-DR3 haplotype which potentially increases the risk of T1D. Of these 9 parents, 3 were heterozygous for HLA-DR4/HLA-DR3 and one was homozygous for HLA-DR3. Two haplotypes that were identified here extend to the HLA class I region were previously designated AH8.2 (HLA -A∗26-B∗08-DRB1∗03) and AH50.2 (HLA -C∗06-B∗50-DRB1∗03:01-DQ∗02) and associated with diabetes in neighboring North Indian populations. This study provides examples of MHC haplotype analysis in pedigrees to improve our understanding of the genetics of T1D in the understudied population of the UAE.

MHC class IIa haplotypes derived by high-throughput SNP screening in an isolated sheep population.

Investigating the current evolutionary processes acting on a highly polymorphic gene region, such as the major histocompatibility complex (MHC), requires extensive population data for both genotypes and phenotypes. The MHC consists of several tightly linked loci with both allelic and gene content variation, making it challenging to genotype. Eight class IIa haplotypes have previously been identified in the Soay sheep (Ovis aries) of St. Kilda using Sanger sequencing and cloning, but no single locus is representative of all haplotypes. Here, we exploit the closed nature of the island population of Soay sheep and its limited haplotypic variation to identify a panel of SNPs that enable imputation of MHC haplotypes. We compared MHC class IIa haplotypes determined by Sanger sequence-based genotyping of 135 individuals to their SNP profiles generated using the Ovine Infinium HD BeadChip. A panel of 11 SNPs could reliably determine MHC diplotypes, and two additional SNPs within the DQA1 gene enabled detection of a recombinant haplotype affecting only the SNPs downstream of the expressed genes. The panel of 13 SNPs was genotyped in 5951 Soay sheep, of which 5349 passed quality control. Using the Soay sheep pedigree, we were able to trace the origin and inheritance of the recombinant SNP haplotype. This SNP-based method has enabled the rapid generation of locus-specific MHC genotypes for large numbers of Soay sheep. This volume of high-quality genotypes in a well-characterized population of free-living sheep will be valuable for investigating the mechanisms maintaining diversity at the MHC.