Large scale transcriptional analysis of MHC class I haplotype diversity in sheep.

Domestic sheep (Ovis aries) have been an important component of livestock agricultural production for thousands of years. Preserving genetic diversity within livestock populations maintains a capacity to respond to changing environments and rapidly evolving pathogens. MHC genetic diversity can influence immune functionality at individual and population levels. Here, we focus on defining functional MHC class I haplotype diversity in a large cohort of Scottish Blackface sheep pre-selected for high levels of MHC class II DRB1 diversity. Using high-throughput amplicon sequencing with three independent sets of barcoded primers we identified 134 MHC class I transcripts within 38 haplotypes. Haplotypes were identified with between two and six MHC class I genes, plus variable numbers of conserved sequences with very low read frequencies. One or two highly transcribed transcripts dominate each haplotype indicative of two highly polymorphic, classical MHC class I genes. Additional clusters of medium, low, and very low expressed transcripts are described, indicative of lower transcribed classical, non-classical and genes whose function remains to be determined.

High-Resolution Genotyping of Expressed Equine MHC Reveals a Highly Complex MHC Structure.

The Major Histocompatibility Complex (MHC) genes play a key role in a number of biological processes, most notably in immunological responses. The MHCI and MHCII genes incorporate a complex set of highly polymorphic and polygenic series of genes, which, due to the technical limitations of previously available technologies, have only been partially characterized in non-model but economically important species such as the horse. The advent of high-throughput sequencing platforms has provided new opportunities to develop methods to generate high-resolution sequencing data on a large scale and apply them to the analysis of complex gene sets such as the MHC. In this study, we developed and applied a MiSeq-based approach for the combined analysis of the expressed MHCI and MHCII repertoires in cohorts of Thoroughbred, Icelandic, and Norwegian Fjord Horses. The approach enabled us to generate comprehensive MHCI/II data for all of the individuals (n = 168) included in the study, identifying 152 and 117 novel MHCI and MHCII sequences, respectively. There was limited overlap in MHCI and MHCII haplotypes between the Thoroughbred and the Icelandic/Norwegian Fjord horses, showcasing the variation in MHC repertoire between genetically divergent breeds, and it can be inferred that there is much more MHC diversity in the global horse population. This study provided novel insights into the structure of the expressed equine MHC repertoire and highlighted unique features of the MHC in horses.

High throughput analysis of MHC class I and class II diversity of Zambian indigenous cattle populations.

The classical MHC class I and class II molecules play key roles in determining the antigenic-specificity of CD8+ and CD4+ T-cell responses-as such characterisation of the repertoire of MHCI and MHCII allelic diversity is fundamental to our ability to understand, and potentially, exploit how genetic diversity influences the outcome of immune responses. Cattle remain one of the most economically livestock species, with particular importance to many small-holder farmers in low-and-middle income countries (LMICs). However, our knowledge of MHC (BoLA) diversity in the indigenous breeds that form the mainstay of cattle populations in many LMICs remains very limited. In this study we develop a MiSeq-based platform to enable the rapid analysis of BoLA-DQA and BoLA-DQB, and combine this with similar platforms to analyse BoLA-I and BoLA-DRB repertoires, to study a large cohort of cattle (~800 animals) representing the 3 major indigenous breeds (Angoni, Barotse, Tonga) in Zambia. The data presented confirms the capacity of this high-throughput and high-resolution approach to provide a full characterisation of the MHCI-MHCII genotypes of cattle for which little previous MHC sequence data has been obtained. The cattle in Zambia were found to express a diverse range of MHCI, MHCII and extended MHCI-MHCII haplotypes. The combined MHCI-MHCII genotyping now possible opens new opportunities to rapidly expand our knowledge of MHC diversity in cattle that could find applications in a related translational disciplines such as vaccine development.

High throughput analysis of MHC-I and MHC-DR diversity of Brazilian cattle populations.

The major histocompatibility complex (MHC) contains many genes that play key roles in initiating and regulating immune responses. This includes the polymorphic MHCI and MHCII genes that present epitopes to CD8+ and CD4+ T-cells, respectively. Consequently, the characterisation of the repertoire of MHC genes is an important component of improving our understanding of the genetic variation that determines the outcomes of immune responses. In cattle, MHC (BoLA) research has predominantly focused on Holstein-Friesian animals (as the most economically important breed globally), although the development of high-throughput approaches has allowed the BoLA-DRB3 repertoire to be studied in a greater variety of breeds. In a previous study we reported on the development of a MiSeq-based method to enable high-throughput and high-resolution analysis of bovine MHCI repertoires. Herein, we report on the expansion of this methodology to incorporate analysis of the BoLA-DRB3 and its application to analyse MHC diversity in a large cohort of cattle from Brazil (>500 animals), including representatives from the three major Bos indicus breeds present in Brazil - Guzerat, Gir and Nelore. This large-scale description of paired MHCI-DRB3 repertoires in Bos indicus cattle has identified a small number of novel DRB3 alleles, a large number of novel MHCI alleles and haplotypes, and provided novel insights into MHCI-MHCII association - further expanding our knowledge of bovine MHC diversity.

Integral Use of Immunopeptidomics and Immunoinformatics for the Characterization of Antigen Presentation and Rational Identification of BoLA-DR-Presented Peptides and Epitopes.

MHC peptide binding and presentation is the most selective event defining the landscape of T cell epitopes. Consequently, understanding the diversity of MHC alleles in a given population and the parameters that define the set of ligands that can be bound and presented by each of these alleles (the immunopeptidome) has an enormous impact on our capacity to predict and manipulate the potential of protein Ags to elicit functional T cell responses. Liquid chromatography-mass spectrometry analysis of MHC-eluted ligand data has proven to be a powerful technique for identifying such peptidomes, and methods integrating such data for prediction of Ag presentation have reached a high level of accuracy for both MHC class I and class II. In this study, we demonstrate how these techniques and prediction methods can be readily extended to the bovine leukocyte Ag class II DR locus (BoLA-DR). BoLA-DR binding motifs were characterized by eluted ligand data derived from bovine cell lines expressing a range of DRB3 alleles prevalent in Holstein-Friesian populations. The model generated (NetBoLAIIpan, available as a Web server at www.cbs.dtu.dk/services/NetBoLAIIpan) was shown to have unprecedented predictive power to identify known BoLA-DR-restricted CD4 epitopes. In summary, the results demonstrate the power of an integrated approach combining advanced mass spectrometry peptidomics with immunoinformatics for characterization of the BoLA-DR Ag presentation system and provide a prediction tool that can be used to assist in rational evaluation and selection of bovine CD4 T cell epitopes.

Whole-Genome Sequence Data Suggest Environmental Adaptation of Ethiopian Sheep Populations.

Great progress has been made over recent years in the identification of selection signatures in the genomes of livestock species. This work has primarily been carried out in commercial breeds for which the dominant selection pressures are associated with artificial selection. As agriculture and food security are likely to be strongly affected by climate change, a better understanding of environment-imposed selection on agricultural species is warranted. Ethiopia is an ideal setting to investigate environmental adaptation in livestock due to its wide variation in geo-climatic characteristics and the extensive genetic and phenotypic variation of its livestock. Here, we identified over three million single nucleotide variants across 12 Ethiopian sheep populations and applied landscape genomics approaches to investigate the association between these variants and environmental variables. Our results suggest that environmental adaptation for precipitation-related variables is stronger than that related to altitude or temperature, consistent with large-scale meta-analyses of selection pressure across species. The set of genes showing association with environmental variables was enriched for genes highly expressed in human blood and nerve tissues. There was also evidence of enrichment for genes associated with high-altitude adaptation although no strong association was identified with hypoxia-inducible-factor (HIF) genes. One of the strongest altitude-related signals was for a collagen gene, consistent with previous studies of high-altitude adaptation. Several altitude-associated genes also showed evidence of adaptation with temperature, suggesting a relationship between responses to these environmental factors. These results provide a foundation to investigate further the effects of climatic variables on small ruminant populations.

Transcriptional Response of Ovine Lung to Infection with Jaagsiekte Sheep Retrovirus.

Jaagsiekte sheep retrovirus (JSRV) is the etiologic agent of ovine pulmonary adenocarcinoma (OPA), a neoplastic lung disease of sheep. OPA is an important economic and welfare issue for sheep farmers and a valuable naturally occurring animal model for human lung adenocarcinoma. Here, we used RNA sequencing to study the transcriptional response of ovine lung tissue to infection by JSRV. We identified 1,971 ovine genes differentially expressed in JSRV-infected lung compared to noninfected lung, including many genes with roles in carcinogenesis and immunomodulation. The differential expression of selected genes was confirmed using immunohistochemistry and reverse transcription-quantitative PCR. A key finding was the activation of anterior gradient 2, yes-associated protein 1, and amphiregulin in OPA tumor cells, indicating a role for this oncogenic pathway in OPA. In addition, there was differential expression of genes related to innate immunity, including genes encoding cytokines, chemokines, and complement system proteins. In contrast, there was little evidence for the upregulation of genes involved in T-cell immunity. Many genes related to macrophage function were also differentially expressed, reflecting the increased abundance of these cells in OPA-affected lung tissue. Comparison of the genes differentially regulated in OPA with the transcriptional changes occurring in human lung cancer revealed important similarities and differences between OPA and human lung adenocarcinoma. This study provides valuable new information on the pathogenesis of OPA and strengthens the use of this naturally occurring animal model for human lung adenocarcinoma.IMPORTANCE Ovine pulmonary adenocarcinoma is a chronic respiratory disease of sheep caused by jaagsiekte sheep retrovirus (JSRV). OPA is a significant economic problem for sheep farmers in many countries and is a valuable animal model for some forms of human lung cancer. Here, we examined the changes in host gene expression that occur in the lung in response to JSRV infection. We identified a large number of genes with altered expression in infected lung, including factors with roles in cancer and immune system function. We also compared the data from OPA to previously published data from human lung adenocarcinoma and found a large degree of overlap in the genes that were dysregulated. The results of this study provide exciting new avenues for future studies of OPA and may have comparative relevance for understanding human lung cancer.

Differentially expressed genes during spontaneous lytic switch of Marek's disease virus in lymphoblastoid cell lines determined by global gene expression profiling.

Marek's disease virus (MDV), an alphaherpesvirus of poultry, causes Marek's disease and is characterized by visceral CD4+TCRαß+ T-cell lymphomas in susceptible hosts. Immortal cell lines harbouring the viral genome have been generated from ex vivo cultures of MD tumours. As readily available sources of large numbers of cells, MDV-transformed lymphoblastoid cell lines (LCLs) are extremely valuable for studies of virus-host interaction. While the viral genome in most cells is held in a latent state, minor populations of cells display spontaneous reactivation identifiable by the expression of lytic viral genes. Spontaneous reactivation in these cells presents an opportunity to investigate the biological processes involved in the virus reactivation. For detailed characterization of the molecular events associated with reactivation, we used two lymphoblastoid cell lines derived from lymphomas induced by pRB1B-UL47eGFP, a recombinant MDV engineered to express enhanced green fluorescent protein (EGFP) fused with the UL47. We used fluorescence-activated cell sorting to purify the low-frequency EGFP-positive cells with a spontaneously activating viral genome from the majority EGFP-negative cells and analysed their gene expression profiles by RNA-seq using Illumina HiSeq2500. Ingenuity pathway analysis on more than 2000 differentially expressed genes between the lytically infected (EGFP-positive) and latently infected (EGFP-negative) cell populations identified the biological pathways involved in the reactivation. Virus-reactivating cells exhibited differential expression of a significant number of viral genes, with hierarchical differences in expression levels. Downregulation of a number of host genes including those directly involved in T-cell activation, such as CD3, CD28, ICOS and phospholipase C, was also noticed in the LCL undergoing lytic switch.

Activation of gga-miR-155 by reticuloendotheliosis virus T strain and its contribution to transformation.

The v-rel oncoprotein encoded by reticuloendotheliosis virus T strain (Rev-T) is a member of the rel/NF-κB family of transcription factors capable of transformation of primary chicken spleen and bone marrow cells. Rapid transformation of avian haematopoietic cells by v-rel occurs through a process of deregulation of multiple protein-encoding genes through its direct effect on their promoters. More recently, upregulation of oncogenic miR-155 and its precursor pre-miR-155 was demonstrated in both Rev-T-infected chicken embryo fibroblast cultures and Rev-T-induced B-cell lymphomas. Through electrophoresis mobility shift assay and reporter analysis on the gga-miR-155 promoter, we showed that the v-rel-induced miR-155 overexpression occurred by the direct binding to one of the putative NF-κB binding sites. Using the v-rel-induced transformation model on chicken embryonic splenocyte cultures, we could demonstrate a dynamic increase in miR-155 levels during the transformation. Transcriptome profiles of lymphoid cells transformed by v-rel showed upregulation of miR-155 accompanied by downregulation of a number of putative miR-155 targets such as Pu.1 and CEBPß. We also showed that v-rel could rescue the suppression of miR-155 expression observed in Marek's disease virus (MDV)-transformed cell lines, where its functional viral homologue MDV-miR-M4 is overexpressed. Demonstration of gene expression changes affecting major molecular pathways, including organismal injury and cancer in avian macrophages transfected with synthetic mature miR-155, underlines its potential direct role in transformation. Our study suggests that v-rel-induced transformation involves a complex set of events mediated by the direct activation of NF-κB targets, together with inhibitory effects on microRNA targets.

Rapid identification of bovine MHCI haplotypes in genetically divergent cattle populations using next-generation sequencing.

The major histocompatibility complex (MHC) region contains many genes that are key regulators of both innate and adaptive immunity including the polymorphic MHCI and MHCII genes. Consequently, the characterisation of the repertoire of MHC genes is critical to understanding the variation that determines the nature of immune responses. Our current knowledge of the bovine MHCI repertoire is limited with only the Holstein-Friesian breed having been studied in any depth. Traditional methods of MHCI genotyping are of low resolution and laborious and this has been a major impediment to a more comprehensive analysis of the MHCI repertoire of other cattle breeds. Next-generation sequencing (NGS) technologies have been used to enable high throughput and much higher resolution MHCI typing in a number of species. In this study we have developed a MiSeq platform approach and requisite bioinformatics pipeline to facilitate typing of bovine MHCI repertoires. The method was validated initially on a cohort of Holstein-Friesian animals and then demonstrated to enable characterisation of MHCI repertoires in African cattle breeds, for which there was limited or no available data. During the course of these studies we identified >140 novel classical MHCI genes and defined 62 novel MHCI haplotypes, dramatically expanding the known bovine MHCI repertoire.