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.

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.

Marek’s disease virus (MDV), an alphaherpesvirus of poultry, causes Marek’s disease and is characterized by visceral CD4⁺TCRαβ⁺ 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.

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.

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.