This study reports the presence of two distinct MHC class II β genes in the Antarctic icefish Chionodraco hamatus, belonging to the classical (ChhaDAB) and nonclassical (ChhaDBB) evolutionary lineages. By the application of targeted sequencing approach, a remarkable molecular diversity in the exon 2 sequence of the highly expressed gene ChhaDAB has been observed, resulting in an estimate of 92 different variants translated in 87 different peptides from 54 analysed icefish individuals. A highly conservative estimate, based on a 95% sequence identity threshold clustering, translate this variability in 41 different peptide clusters belonging to four different clades and showing the signature of different kinds of selection. In stark contrast, the poorly expressed ChhaDBB gene displayed a very low level of molecular diversity within exon 2, in agreement with expectations for a nonclassical MHC class II β gene.

The major histocompatibility complex (MHC) genes are the most polymorphic genes in vertebrates, and their proteins play a critical role in adaptive immunity for defense against a variety of pathogens. MHC diversity was lost in many species after experiencing a decline in size. To understand the variation and evolution of MHC genes in the Siberian ibex, Capra sibirica, which has undergone a population decline, we analyzed the variation of the second exon of MHC class II DRB genes in samples collected from five geographic localities in Xinjiang, China, that belong to three diverged mitochondrial clades. Consequently, we identified a total of 26 putative functional alleles (PFAs) with 260 bp in length from 43 individuals, and found one (for 27 individuals) to three (for 5 individuals) PFAs per individual, indicating the presence of one or two DRB loci per haploid genome. The Casi-DRB1*16 was the most frequently occurring PFA, Casi-DRB1*22 was found in only seven individuals, 14 PFAs occurred once, 7 PFAs twice, implying high frequency of rare PFAs. Interestingly, more than half (15) of the PFAs were specific to clade I, only two and three PFAs were specific to clades II and III, respectively. So, we assume that the polygamy and sexual segregation nature of this species likely contributed to the allelic diversity of DRB genes. Genetic diversity indices showed that PFAs of clade II were lower in nucleotide, amino acid, and supertype diversity compared to those of the other two clades. The pattern of allele sharing and FST values between the three clades was to some extent in agreement with the pattern observed in mitochondrial DNA divergence. In addition, recombination analyses revealed no evidence for significant signatures of recombination events. Alleles shared by clades III and the other two clades diverged 6 million years ago, and systematic neighbor grids showed Trans-species polymorphism. Together with the PAML and MEME analyses, the results indicated that the DRB gene in C. sibirica evolved under balancing and positive selection. However, by comparison, it can be clearly seen that different populations were under different selective pressures. Our results are valuable in understanding the diversity and evolution of the DRB gene in a mountain living C. sibirica and in making decisions on future long-term protection strategies.

In natural populations, allelic diversity of the major histocompatibility complex (MHC) is commonly interpreted as resulting from positive selection in varying spatiotemporal pathogenic landscapes. Composite pathogenic landscape data are, however, rarely available. We studied the spatial distribution of allelic diversity at two MHC class II loci (DQA, DQB) in hares, Lepus capensis, along a steep ecological gradient in North Africa and tested the role of climatic parameters for the spatial distribution of DQA and DQB proteins. Climatic parameters were considered to reflect to some extent pathogenic landscape variation. We investigated historical and contemporary forces that have shaped the variability at both genes, and tested for differential selective pressure across the ecological gradient by comparing allelic variation at MHC and neutral loci. We found positive selection on both MHC loci and significantly decreasing diversity from North to South Tunisia. Our multinomial linear models revealed significant effects of geographical positions that were correlated with mean annual temperature and precipitation on the occurrence of protein variants, but no effects of co-occurring DQA or DQB proteins, respectively. Diversifying selection, recombination, adaptation to local pathogenic landscapes (supposedly reflected by climate parameters) and neutral demographic processes have shaped the observed MHC diversity and differentiation patterns.

The major histocompatibility complex (MHC) is a multigene family responsible for pathogen detection, and initiation of adaptive immune responses. Duplication, natural selection, recombination, and their resulting high functional genetic diversity spread across several duplicated loci are the main hallmarks of the MHC. Although these features were described in several jawed vertebrate lineages, a detailed MHC IIβ characterization at the population level is still lacking for chondrichthyans (chimaeras, rays and sharks), i.e. the most basal lineage to possess an MHC-based adaptive immune system. We used the small-spotted catshark (Scyliorhinus canicula, Carcharhiniformes) as a case-study species to characterize MHC IIβ diversity using complementary molecular tools, including publicly available genome and transcriptome datasets, and a newly developed high-throughput Illumina sequencing protocol. We identified three MHC IIβ loci within the same genomic region, all of which are expressed in different tissues. Genetic screening of the exon 2 in 41 individuals of S. canicula from a single population revealed high levels of sequence diversity, evidence for positive selection, and footprints of recombination. Moreover, the results also suggest the presence of copy number variation in MHC IIβ genes. Thus, the small-spotted catshark exhibits characteristics of functional MHC IIβ genes typically observed in other jawed vertebrates.

A paradigm shifting study demonstrated that induction of MHC class E and II-restricted CD8+ T cells was associated with the clearance of SIV infection in rhesus macaques. Another recent study highlighted the presence of HIV-1-specific class II-restricted CD8+ T cells in HIV-1 patients who naturally control infection (virus controllers; VCs). However, questions regarding class II-restricted CD8+ T cells ontogeny, distribution across different HIV-1 disease states and their role in viral control remain unclear. In this study, we investigated the distribution and anti-viral properties of HLA-DRB1*0701 and DQB1*0501 class II-restricted CD8+ T cells in different HIV-1 patient cohorts; and whether class II-restricted CD8+ T cells represent a unique T cell subset. We show that memory class II-restricted CD8+ T cell responses were more often detectable in VCs than in chronically infected patients, but not in healthy seronegative donors. We also demonstrate that VC CD8+ T cells inhibit virus replication in both a class I- and class II-dependent manner, and that in two VC patients the class II-restricted CD8+ T cells with an anti-viral gene signature expressed both CD4+ and CD8+ T cell lineage-specific genes. These data demonstrated that anti-viral memory class II-restricted CD8+ T cells with hybrid CD4+ and CD8+ features are present during natural HIV-1 infection.

The multi-subunit CCR4 (carbon catabolite repressor 4)-NOT (Negative on TATA) complex serves as a central coordinator of all different steps of eukaryotic gene expression. Here we performed a systematic and comparative analysis of cells where the CCR4-NOT subunits CNOT1, CNOT2 or CNOT3 were individually downregulated using doxycycline-inducible shRNAs. Microarray experiments showed that downregulation of either CNOT subunit resulted in elevated expression of major histocompatibility complex class II (MHC II) genes which are found in a gene cluster on chromosome 6. Increased expression of MHC II genes after knock-down or knock-out of either CNOT subunit was seen in a variety of cell systems and also in naïve macrophages from CNOT3 conditional knock-out mice. CNOT2-mediated repression of MHC II genes occurred also in the absence of the master regulator class II transactivator (CIITA) and did not cause detectable changes of the chromatin structure at the chromosomal MHC II locus. CNOT2 downregulation resulted in an increased de novo transcription of mRNAs whereas tethering of CNOT2 to a regulatory region governing MHC II expression resulted in diminished transcription. These results expand the known repertoire of CCR4-NOT members for immune regulation and identify CNOT proteins as a novel group of corepressors restricting class II expression.

Little is known about the major histocompatibility complex (MHC) in the genome of Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) (YFP) or other cetaceans. In this study, a high-quality YFP bacterial artificial chromosome (BAC) library was constructed. We then determined the organization and characterization of YFP MHC class II region by screening the BAC library, followed by sequencing and assembly of positive BAC clones. The YFP MHC class II region consists of two segregated contigs (218,725 bp and 328,435 bp respectively) that include only eight expressed MHC class II genes, three pseudo MHC genes and twelve non-MHC genes. The YFP has fewer MHC class II genes than ruminants, showing locus reduction in DRB, DQA, DQB, and loss of DY. In addition, phylogenic and evolutionary analyses indicated that the DRB, DQA and DQB genes might have undergone birth-and-death evolution, whereas the DQB gene might have evolved under positive selection in cetaceans. These findings provide an essential foundation for future work, such as estimating MHC genetic variation in the YFP or other cetaceans. This work is the first report on the MHC class II region in cetaceans and offers valuable information for understanding the evolution of MHC genome in cetaceans.

The mammalian Major Histocompatibility Complex (MHC) region contains several gene families characterized by highly polymorphic loci with extensive nucleotide diversity, copy number variation of paralogous genes, and long repetitive sequences. This structural complexity has made it difficult to construct a reliable reference sequence of the horse MHC region. In this study, we used long-read single molecule, real-time (SMRT) sequencing technology from Pacific Biosciences (PacBio) to sequence eight Bacterial Artificial Chromosome (BAC) clones spanning the horse MHC class II region. The final assembly resulted in a 1,165,328 bp continuous gap free sequence with 35 manually curated genomic loci of which 23 were considered to be functional and 12 to be pseudogenes. In comparison to the MHC class II region in other mammals, the corresponding region in horse shows extraordinary copy number variation and different relative location and directionality of the Eqca-DRB, -DQA, -DQB and -DOB loci. This is the first long-read sequence assembly of the horse MHC class II region with rigorous manual gene annotation, and it will serve as an important resource for association studies of immune-mediated equine diseases and for evolutionary analysis of genetic diversity in this region.

EBV-associated gastric adenocarcinomas (EBVaGCs) often exhibit better clinical outcomes than EBV negative gastric cancers (GCs), which could be related to their consistent expression of foreign viral antigens. Antigen-presenting cells (APCs) present peptide antigens in the context of the class-II major histocompatibility complex (MHC-II). During inflammatory conditions, epithelial cells express MHC-II and function as accessory APCs. Utilizing RNA-seq data from nearly 400 GC patients, we determined the impact of EBV-status on expression of MHC-II components, genes involved in their regulation, and T-cell co-stimulation. Virtually all MHC-II genes were significantly upregulated in EBVaGCs compared to normal tissues, or other GC subtypes. Genes involved in antigen presentation were also significantly upregulated in EBVaGCs, as were the key MHC-II transcriptional regulators CIITA and RFX5. This was unexpected as the EBV encoded BZLF1 protein can repress CIITA transcription and is expressed in many EBVaGCs. Furthermore, MHC-II upregulation was strongly correlated with elevated intratumoral levels of interferon-gamma. In addition, expression of co-stimulatory molecules involved in T-cell activation and survival was also significantly increased in EBVaGCs. Thus, gastric adenocarcinoma cells may functionally contribute to the highly immunogenic tumor microenvironment observed in EBVaGCs via a previously unappreciated role in interferon-induced antigen presentation.

Chinese alligator (Alligator sinensis) is an endangered freshwater crocodilian endemic to China, which experienced a severe bottleneck about 30 years ago. In this study, we developed locus-specific primers to investigate the polymorphism of 3 major histocompatibility complex (MHC) loci in 3 Chinese alligator populations, in combination with 6 neutral microsatellite markers as a contrast. We found the genetic trace for the bottleneck effect on the endangered Chinese alligator: the low allelic diversity (2 alleles at each locus), the low nucleotide substitution rate (no more than 0.009) at all sites, the deviation from Hardy-Weinberg Equilibrium/heterozygote deficiency, and the significant Tajima's D values, indicating the MHC class I and class II loci being at different stages of bottleneck. We also obtained 3 pieces of evidence for balancing selection on this severely bottlenecked reptile: an obvious excess of nonsynonymous substitutions over synonymous at the antigen-binding positions, the mean synonymous substitution rate of MHC exons significantly higher than mean nucleotide substitution rate of introns, and the differentiation coefficient F ST of MHC loci significantly lower than that of microsatellite loci. Consequently, we emphasize that the Chinese alligator holds a pretty low adaptive ability and requires scientific conservation strategies to ensure the long-term population development.

The major histocompatibility complex (MHC) plays an important role in immune response. Avian MHCs are not well characterized, only reporting highly compact Galliformes MHCs and extensively fragmented zebra finch MHC. We report the first genomic structure of an endangered Pelecaniformes (crested ibis) MHC containing 54 genes in three regions spanning ~500 kb. In contrast to the loose BG (26 loci within 265 kb) and Class I (11 within 150) genomic structures, the Core Region is condensed (17 within 85). Furthermore, this Region exhibits a COL11A2 gene, followed by four tandem MHC class II αβ dyads retaining two suites of anciently duplicated "αβ" lineages. Thus, the crested ibis MHC structure is entirely different from the known avian MHC architectures but similar to that of mammalian MHCs, suggesting that the fundamental structure of ancestral avian class II MHCs should be "COL11A2-IIαβ1-IIαβ2." The gene structures, residue characteristics, and expression levels of the five class I genes reveal inter-locus functional divergence. However, phylogenetic analysis indicates that these five genes generate a well-supported intra-species clade, showing evidence for recent duplications. Our analyses suggest dramatic structural variation among avian MHC lineages, help elucidate avian MHC evolution, and provide a foundation for future conservation studies.

Aleutian disease (AD) is a multi-systemic infectious disease in American mink (Neogale vison) caused by Aleutian mink disease virus (AMDV). This study aimed to identify candidate regions and genes underlying selection for response against AMDV using whole-genome sequence (WGS) data. Three case-control selection signatures studies were conducted between animals (N = 85) producing high versus low antibody levels against AMDV, grouped by counter immunoelectrophoresis (CIEP) test and two enzyme-linked immunosorbent assays (ELISA). Within each study, selection signals were detected using fixation index (FST) and nucleotide diversity (θπ ratios), and validated by cross-population extended haplotype homozygosity (XP-EHH) test. Within- and between-studies overlapping results were then evaluated. Within-studies overlapping results indicated novel candidate genes related to immune and cellular responses (e.g., TAP2, RAB32), respiratory system function (e.g., SPEF2, R3HCC1L), and reproduction system function (e.g., HSF2, CFAP206) in other species. Between-studies overlapping results identified three large segments under strong selection pressure, including two on chromosome 1 (chr1:88,770-98,281 kb and chr1:114,133-120,473) and one on chromosome 6 (chr6:37,953-44,279 kb). Within regions with strong signals, we found novel candidate genes involved in immune and cellular responses (e.g., homologous MHC class II genes, ITPR3, VPS52) in other species. Our study brings new insights into candidate regions and genes controlling AD response.

Human papillomavirus (HPV) is the most common sexually transmitted infection in the world and the main cause of cervical cancer. Nowadays, the virus-like particles (VLPs) based on L1 proteins have been considered as the best candidate for vaccine development against HPV infections. Two commercial HPV (Gardasil and Cervarix) are available. These HPV VLP vaccines induce genotype-limited protection. The major impediments such as economic barriers especially gaps in financing obstructed the optimal delivery of vaccines in developing countries. Thus, many efforts are underway to develop the next generation of vaccines against other types of high-risk HPV. In this study, we developed DNA constructs (based on L1 and L2 genes) that were potentially immunogenic and highly conserved among the high-risk HPV types. The framework of analysis include (1) B-cell epitope mapping, (2) T-cell epitope mapping (i.e., CD4+ and CD8+ T cells), (3) allergenicity assessment, (4) tap transport and proteasomal cleavage, (5) population coverage, (6) global and template-based docking, and (7) data collection, analysis, and design of the L1 and L2 DNA constructs. Our data indicated the 8-epitope candidates for helper T-cell and CTL in L1 and L2 sequences. For the L1 and L2 constructs, combination of these peptides in a single universal vaccine could involve all world population by the rate of 95.55% and 96.33%, respectively. In vitro studies showed high expression rates of multiepitope L1 (~57.86%) and L2 (~68.42%) DNA constructs in HEK-293T cells. Moreover, in vivo studies indicated that the combination of L1 and L2 DNA constructs without any adjuvant or delivery system induced effective immune responses, and protected mice against C3 tumor cells (the percentage of tumor-free mice: ~66.67%). Thus, the designed L1 and L2 DNA constructs would represent promising applications for HPV vaccine development.

Current interest in Foxp3+ T-regulatory (Treg) cells as therapeutic targets in transplantation is largely focused on their harvesting pre-transplant, expansion and infusion post-transplantation. An alternate strategy of pharmacologic modulation of Treg function using histone/protein deacetylase inhibitors (HDACi) may allow more titratable and longer-term dosing. However, the effects of broadly acting HDACi vary, such that HDAC isoform-selective targeting is likely required. We report data from mice with constitutive or conditional deletion of HDAC11 within Foxp3+ Treg cells, and their use, along with small molecule HDAC11 inhibitors, in allograft models. Global HDAC11 deletion had no effect on health or development, and compared to WT controls, Foxp3+ Tregs lacking HDAC11 showed increased suppressive function, and increased expression of Foxp3 and TGF-β. Likewise, compared to WT recipients, conditional deletion of HDAC11 within Tregs led to long-term survival of fully MHC-mismatched cardiac allografts, and prevented development of transplant arteriosclerosis in an MHC class II-mismatched allograft model. The translational significance of HDAC11 targeting was shown by the ability of an HDAC11i to promote long-term allograft allografts in fully MHC-disparate strains. These data are powerful stimuli for the further development and testing of HDAC11-selective pharmacologic inhibitors, and may ultimately provide new therapies for transplantation and autoimmune diseases.

Brain death is characterized by a generalized inflammatory response that results in multiorgan damage. This process is mainly mediated through cytokines, which amplify graft immunogenicity. We investigated the immunological response in a brain death liver donor model and analysed the effects of thalidomide, a drug with powerful immunomodulatory properties. Brain death was induced in male Lewis rats. We studied three groups: Control (sham-operated rats in which trepanation was performed without inserting the balloon catheter), BD (rats subjected to brain death by increasing intracranial pressure) and BD + Thalid (BD rats receiving thalidomide after brain death). After 6 h, serum levels of AST, ALT, LDH, and ALP as well as systemic and hepatic levels of TNF-α, IL1-β, IL-6, and IL-10 were analysed. We also determined the mRNA expression of MHC Class I and Class II, NF-κB, and macrophage infiltration. NF-κB was also examined by electrophoretic mobility shift assay. Thalidomide treatment significantly reduced serum levels of hepatic enzymes and TNF-α, IL-1-β, and IL-6. These cytokines were evaluated at either the mRNA expression or protein level in liver tissue. In addition, thalidomide administration resulted in a significant reduction in macrophages, MHC Class I and Class II, and NF-κB activation. This study reveals that thalidomide significantly inhibited the immunologic response and graft immunogenicity, possibly through suppression of NF-κB activation.

Human metapneumovirus (HMPV) is a leading cause of acute lower respiratory tract illness in children and adults. Repeated infections are common and can be severe in young, elderly, and immunocompromised persons due to short-lived protective humoral immunity. In turn, few protective T cell epitopes have been identified in humans. Thus, we infected transgenic mice expressing the common human HLA MHC-I allele B*07:02 (HLA-B7) with HMPV and screened a robust library of overlapping and computationally predicted HLA-B7 binding peptides. Six HLA-B7-restricted CD8+ T cell epitopes were identified using ELISPOT screening in the F, M, and N proteins, with M195-203 (M195) eliciting the strongest responses. MHC-tetramer flow cytometric staining confirmed HLA-B7 epitope-specific CD8+ T cells migrated to lungs and spleen of HMPV-immune mice. Immunization with pooled HLA-B7-restricted peptides reduced viral titer and protected mice from virulent infection. Finally, we confirmed that CD8+ T cells from HLA-B7 positive humans also recognize the identified epitopes. These results enable identification of HMPV-specific CD8+ T cells in humans and help to inform future HMPV vaccine design.

While the principles of classical antigen presentation via MHC class II are well-established, the mechanisms for the many routes of cross-presentation by which endogenous antigens become associated with class II molecules are not fully understood. We have recently demonstrated that the single amino acid polymorphism HLA-DPβ84Gly (DP84Gly) is critical to abrogate class II invariant chain associated peptide (CLIP) region-mediated binding of invariant chain (Ii) to DP, allowing endoplasmic reticulum (ER)-resident endogenous antigens to constitutively associate with DP84Gly such as DP4. In this study, we demonstrate that both the CLIP and N-terminal non-CLIP Ii regions cooperatively generate an Ii conformation that cannot associate with DP84Gly via the CLIP region. We also demonstrate the ability of DP4 to efficiently process and present antigens encoded in place of CLIP in a chimeric Ii, regardless of wild type Ii and HLA-DM expression. These data highlight the complex interplay between DP polymorphisms and the multiple Ii regions that cooperatively regulate this association, ultimately controlling the presentation of endogenous antigens on DP molecules. These results may also offer a mechanistic explanation for recent studies identifying the differential effects between DP84Gly and DP84Asp as clinically relevant in human disease.

Tetherin/BST-2 is a host restriction factor that inhibits retrovirus release from infected cells in vitro by tethering nascent virions to the plasma membrane. However, contradictory data exists on whether Tetherin inhibits acute retrovirus infection in vivo. Previously, we reported that Tetherin-mediated inhibition of Friend retrovirus (FV) replication at 2 weeks post-infection correlated with stronger natural killer, CD4+ T and CD8+ T cell responses. Here, we further investigated the role of Tetherin in counteracting retrovirus replication in vivo. FV infection levels were similar between wild-type (WT) and Tetherin KO mice at 3 to 7 days post-infection despite removal of a potent restriction factor, Apobec3/Rfv3. However, during this phase of acute infection, Tetherin enhanced myeloid dendritic cell (DC) function. DCs from infected, but not uninfected, WT mice expressed significantly higher MHC class II and the co-stimulatory molecule CD80 compared to Tetherin KO DCs. Tetherin-associated DC activation during acute FV infection correlated with stronger NK cell responses. Furthermore, Tetherin+ DCs from FV-infected mice more strongly stimulated FV-specific CD4+ T cells ex vivo compared to Tetherin KO DCs. The results link the antiretroviral and immunomodulatory activity of Tetherin in vivo to improved DC activation and MHC class II antigen presentation.

While it is now broadly accepted that inter-individual variation in the outcomes of host-pathogen interactions is at least partially genetically controlled, host immunogenetic characteristics are rarely investigated in wildlife epidemiological studies. Furthermore, most immunogenetic studies in the wild focused solely on the major histocompatibility complex (MHC) diversity despite it accounts for only a fraction of the genetic variation in pathogen resistance. Here, we investigated immunogenetic diversity of the Alpine ibex (Capra ibex) population of the Bargy massif, reservoir of a virulent outbreak of brucellosis. We analysed the polymorphism and associations with disease resistance of the MHC Class II Drb gene and several non-MHC genes (Toll-like receptor genes, Slc11A1) involved in the innate immune response to Brucella in domestic ungulates. We found a very low neutral genetic diversity and a unique MHC Drb haplotype in this population founded few decades ago from a small number of individuals. By contrast, other immunity-related genes have maintained polymorphism and some showed significant associations with the brucellosis infection status hence suggesting a predominant role of pathogen-mediated selection in their recent evolutionary trajectory. Our results highlight the need to monitor immunogenetic variation in wildlife epidemiological studies and to look beyond the MHC.

The major histocompatibility complex (MHC) is highly polymorphic and plays a central role in the vertebrate immune system. Despite its functional consistency, the MHC genomic structure differs substantially among organisms. In birds, the MHCs of Galliformes and the Japanese crested ibis (Pelecaniformes) are well-characterized, but information about other avian MHCs remains scarce. The Oriental stork (Ciconia boyciana, order Ciconiiformes) is a large endangered migrant. The current Japanese population of this bird originates from a few founders; thus, understanding the genetic diversity among them is critical for effective population management. We report the structure and polymorphisms in C. boyciana MHC. One contig (approximately 128 kb) was assembled by screening of lambda phage genomic library and its complete sequence was determined, revealing a gene order of COL11A2, two copies of MHC-IIA/IIB pairs, BRD2, DMA/B1/B2, MHC-I, TAP1/2, and two copies each of pseudo MHC-I and TNXB. This structure was highly similar to that of the Japanese crested ibis, but largely different from that of Galliformes, at both the terminal regions. Genotyping of the MHC-II region detected 10 haplotypes among the six founders. These results provide valuable insights for future studies on the evolution of the avian MHCs and for conservation of C. boyciana.