MAIT cell-MR1 reactivity is highly conserved across multiple divergent species.

Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells that recognize small molecule metabolites presented by major histocompatibility complex class I related protein 1 (MR1), via an αß T cell receptor (TCR). MAIT TCRs feature an essentially invariant TCR α-chain, which is highly conserved between mammals. Similarly, MR1 is the most highly conserved major histocompatibility complex-I-like molecule. This extreme conservation, including the mode of interaction between the MAIT TCR and MR1, has been shown to allow for species-mismatched reactivities unique in T cell biology, thereby allowing the use of selected species-mismatched MR1-antigen (MR1-Ag) tetramers in comparative immunology studies. However, the pattern of cross-reactivity of species-mismatched MR1-Ag tetramers in identifying MAIT cells in diverse species has not been formally assessed. We developed novel cattle and pig MR1-Ag tetramers and utilized these alongside previously developed human, mouse, and pig-tailed macaque MR1-Ag tetramers to characterize cross-species tetramer reactivities. MR1-Ag tetramers from each species identified T cell populations in distantly related species with specificity that was comparable to species-matched MR1-Ag tetramers. However, there were subtle differences in staining characteristics with practical implications for the accurate identification of MAIT cells. Pig MR1 is sufficiently conserved across species that pig MR1-Ag tetramers identified MAIT cells from the other species. However, MAIT cells in pigs were at the limits of phenotypic detection. In the absence of sheep MR1-Ag tetramers, a MAIT cell population in sheep blood was identified phenotypically, utilizing species-mismatched MR1-Ag tetramers. Collectively, our results validate the use and define the limitations of species-mismatched MR1-Ag tetramers in comparative immunology studies.

Comparative transcriptional analysis identifies genes associated with the attenuation of Theileria parva infected cells after long-term in vitro culture.

Autologous administration of attenuated Theileria parva-infected cells induces immunity to T. parva in cattle. The mechanism of attenuation, however, is largely unknown. Here, we used RNA sequencing of pathogenic and attenuated T. parva-infected T-cells to elucidate the transcriptional changes underpinning attenuation. We observed differential expression of several host genes, including TRAIL, PD-1, TGF-ß and granzymes that are known to regulate inflammation and proliferation of infected cells. Importantly, many genes linked with the attenuation of the related T. annulata-infected cells were not dysregulated in this study. Furthermore, known T. parva antigens were not dysregulated in attenuated relative to pathogenic cells, indicating that attenuation is not due to enhanced immunogenicity. Overall this study suggests that attenuation is driven by a decrease in proliferation and restoration of the inflammatory profile of T. parva-infected cells. Additionally, it provides a foundation for future mechanistic studies of the attenuation phenotype in Theileria-infected cells.

Anti-parasitic benzoxaboroles are ineffective against Theileria parva in vitro.

East Coast Fever (ECF) is a disease affecting cattle in sub-Saharan Africa, caused by the tick-borne Apicomplexan pathogen Theileria parva. The disease is a major problem for cattle farmers in affected regions and there are few methods of control, including a complex infection and treatment vaccine, expensive chemotherapy, and the more widespread tick control through acaricides. New intervention strategies are, therefore, sorely needed. Benzoxaboroles are a versatile class of boron-heterocyclic compounds with demonstrable pharmacological activity against a diverse group of pathogens, including those related to T. parva. In this study, the in vitro efficacy of three benzoxaboroles against the intracellular schizont stage of T. parva was investigated using a flow cytometry approach. Of the benzoxaboroles tested, only one showed any potency, albeit only at high concentrations, even though there is high protein sequence similarity in the CPSF3 protein target compared to other protozoan pathogen species. This finding suggests that benzoxaboroles currently of interest for the treatment of African animal trypanosomiasis, toxoplasmosis, cryptosporidiosis and malaria may not be suitable for the treatment of ECF. We conclude that testing of further benzoxaborole compounds is needed to fully determine whether any lead compounds can be identified to target T. parva.

Development of lateral flow assays to detect host proteins in cattle for improved diagnosis of bovine tuberculosis.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection in cattle, is an economically devastating chronic disease for livestock worldwide. Efficient disease control measures rely on early and accurate diagnosis using the tuberculin skin test (TST) and interferon-gamma release assays (IGRAs), followed by culling of positive animals. Compromised performance of TST and IGRA, due to BCG vaccination or co-infections with non-tuberculous mycobacteria (NTM), urges improved diagnostics. Lateral flow assays (LFAs) utilizing luminescent upconverting reporter particles (UCP) for quantitative measurement of host biomarkers present an accurate but less equipment- and labor-demanding diagnostic test platform. UCP-LFAs have proven applications for human infectious diseases. Here, we report the development of UCP-LFAs for the detection of six bovine proteins (IFN-γ, IL-2, IL-6, CCL4, CXCL9, and CXCL10), which have been described by ELISA as potential biomarkers to discriminate M. bovis infected from naïve and BCG-vaccinated cattle. We show that, in line with the ELISA data, the combined PPDb-induced levels of IFN-γ, IL-2, IL-6, CCL4, and CXCL9 determined by UCP-LFAs can discriminate M. bovis challenged animals from naïve (AUC range: 0.87-1.00) and BCG-vaccinated animals (AUC range: 0.97-1.00) in this cohort. These initial findings can be used to develop a robust and user-friendly multi-biomarker test (MBT) for bTB diagnosis.

γδ TCRs Function as Innate-like Receptors in the Bovine γδ T Cell Response against Leptospira.

Leptospira serovar Hardjo are bacterial pathogens of cattle that also cause zoonotic disease in humans. Vaccine-mediated protection against Leptospira serovar Hardjo in cattle is associated with a workshop cluster 1 (WC1)+ γδ T cell response that can be recalled in vitro from PBMC by antigenic stimulation. This provides a model system in which to examine protective vaccine-induced γδ T cell responses in a γδ T cell high species. Only a small proportion (5-10%) of WC1+ γδ T cells from immunized cattle are Leptospira responders, implying that Ag specificity is determined by clonally distributed receptors. Both WC1 and TCR are known to be required for Leptospira-specific responses by bovine WC1+ γδ T cells. Through variegated expression patterns and V(D)J recombination, respectively, they have the capacity to confer Ag specificity. In this study, we develop and use a high-throughput TCR-sequencing approach to study the TCRγ and TCRδ repertoires of naive ex vivo PBMC, Leptospira-responding, and Leptospira nonresponding WC1+ γδ T cells to examine the potential role of γδ TCR in determining Ag specificity. Our results provide novel insights into the PBMC γδ TCR repertoires in cattle, demonstrating the TCRγ repertoire to be clonally stratified and essentially public, whereas the TCRδ repertoire shows much higher levels of clonal diversity and is essentially private. TCR repertoire analysis of Leptospira-responding WC1+ γδ T cells identifies no signature of TCR-mediated selection, suggesting that TCR functions largely as an innate-like receptor and does not act as a primary determinant of Ag specificity in the response to this pathogen.

Protein Levels of Pro-Inflammatory Cytokines and Chemokines as Biomarkers of Mycobacterium bovis Infection and BCG Vaccination in Cattle.

Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a globally prevalent infectious disease with significant animal welfare and economic impact. Difficulties in implementing test-and-slaughter measures in low- and middle-income countries (LMICs) and the underperformance of the current diagnostics establish a clear need to develop improved diagnostics. Adaptive immunity biomarkers other than IFNγ could be useful as suggested by various gene expression studies; however, a comprehensive assessment at the protein level is lacking. Here, we screened a range of chemokines and cytokines for their potential as biomarkers in samples from M. bovis experimentally challenged or naive animals. Although serum concentrations for most proteins were low, the pro-inflammatory markers, IL-2, CXCL-9, IP-10 and CCL4, in addition to IFNγ, were found to be significantly elevated in bovine tuberculin (PPDb)-stimulated whole blood supernatants. Further assessment of these molecules in BCG-vaccinated with or without subsequent M. bovis challenge or naive animals revealed that PPDb-specific IL-2 and IP-10, in addition to IFNγ, could discriminate naive and BCG-vaccinated from M. bovis challenged animals. Moreover, these proteins, along with CCL4, showed DIVA potential, i.e., enabling differentiation of M. bovis-infected animals from BCG-vaccinated animals. Combined analysis of cytokines and chemokines could also accurately identify M. bovis infection with strong correlations observed between PPDb-specific IFNγ, IL-2 and IP-10 levels. This provides proof of concept for utilizing multiple biomarker signatures for discrimination of animals with respect to M. bovis infection or BCG vaccination status.

Identification and Phenotype of MAIT Cells in Cattle and Their Response to Bacterial Infections.

Mucosal-associated invariant T (MAIT) cells are a population of innate-like T cells that utilize a semi-invariant T cell receptor (TCR) α chain and are restricted by the highly conserved antigen presenting molecule MR1. MR1 presents microbial riboflavin biosynthesis derived metabolites produced by bacteria and fungi. Consistent with their ability to sense ligands derived from bacterial sources, MAIT cells have been associated with the immune response to a variety of bacterial infections, such as Mycobacterium spp., Salmonella spp. and Escherichia coli. To date, MAIT cells have been studied in humans, non-human primates and mice. However, they have only been putatively identified in cattle by PCR based methods; no phenotypic or functional analyses have been performed. Here, we identified a MAIT cell population in cattle utilizing MR1 tetramers and high-throughput TCR sequencing. Phenotypic analysis of cattle MAIT cells revealed features highly analogous to those of MAIT cells in humans and mice, including expression of an orthologous TRAV1-TRAJ33 TCR α chain, an effector memory phenotype irrespective of tissue localization, and expression of the transcription factors PLZF and EOMES. We determined the frequency of MAIT cells in peripheral blood and multiple tissues, finding that cattle MAIT cells are enriched in mucosal tissues as well as in the mesenteric lymph node. Cattle MAIT cells were responsive to stimulation by 5-OP-RU and riboflavin biosynthesis competent bacteria in vitro. Furthermore, MAIT cells in milk increased in frequency in cows with mastitis. Following challenge with virulent Mycobacterium bovis, a causative agent of bovine tuberculosis and a zoonosis, peripheral blood MAIT cells expressed higher levels of perforin. Thus, MAIT cells are implicated in the immune response to two major bacterial infections in cattle. These data suggest that MAIT cells are functionally highly conserved and that cattle are an excellent large animal model to study the role of MAIT cells in important zoonotic infections.

Bovine T cell receptors and γδ WC1 co-receptor transcriptome analysis during the first month of life.

Here we evaluated neonatal transcription of α, ß, γ and δ TCR and the γδ T cell co-receptor family WC1 in peripheral blood mononuclear cells. A previous report showed a rapid and global shift in transcription of immunoglobulin genes in neonatal calves during the first month after birth but this was not found here for the T cell genes. Transcription frequency of genes within TRAV subgroups correlated with the number of members, indicating a stochastic choice. In contrast, of the approximately 60 TRDV genes those in two of eleven TRDV1 clades and TRDVb3 were transcribed significantly more than the others while those in only one TRBV subgroup were. Transcription of genes in the TRGV5-containing cassette predominated among TRGV genes as a result of their exclusive usage by the WC1+ γδ T cells with a preference for transcription of two of four TRGV genes in that cassette. Finally, we report no large differences in transcription frequencies among the 13 WC1 genes.

Identification of epitopes recognised by mucosal CD4(+) T-cell populations from cattle experimentally colonised with Escherichia coli O157:H7.

Vaccines targeting enterohaemorrhagic Escherichia coli (EHEC) O157:H7 shedding in cattle are only partially protective. The correlates of protection of these vaccines are unknown, but it is probable that they reduce bacterial adherence at the mucosal surface via the induction of blocking antibodies. Recent studies have indicated a role for cellular immunity in cattle during colonisation, providing an impetus to understand the bacterial epitopes recognised during this response. This study mapped the epitopes of 16 EHEC O157:H7 proteins recognised by rectal lymph node CD4(+) T-cells from calves colonised with Shiga toxin producing EHEC O157:H7 strains. 20 CD4(+) T-cell epitopes specific to E. coli from 7 of the proteins were identified. The highly conserved N-terminal region of Intimin, including the signal peptide, was consistently recognised by mucosal CD4(+) T-cell populations from multiple animals of different major histocompatibility complex class II haplotypes. These T-cell epitopes are missing from many Intimin constructs used in published vaccine trials, but are relatively conserved across a range of EHEC serotypes, offering the potential to develop cross protective vaccines. Antibodies recognising H7 flagellin have been consistently identified in colonised calves; however CD4(+) T-cell epitopes from H7 flagellin were not identified in this study, suggesting that H7 flagellin may act as a T-cell independent antigen. This is the first time that the epitopes recognised by CD4(+) T-cells following colonisation with an attaching and effacing pathogen have been characterised in any species. The findings have implications for the design of antigens used in the next generation of EHEC O157:H7 vaccines.

CD8 T-cell responses against the immunodominant Theileria parva peptide Tp249-59 are composed of two distinct populations specific for overlapping 11-mer and 10-mer epitopes.

Immunity against Theileria parva is associated with CD8 T-cell responses that exhibit immunodominance, focusing the response against limited numbers of epitopes. As candidates for inclusion in vaccines, characterization of responses against immunodominant epitopes is a key component in novel vaccine development. We have previously demonstrated that the Tp249-59 and Tp1214-224 epitopes dominate CD8 T-cell responses in BoLA-A10 and BoLA-18 MHC I homozygous animals, respectively. In this study, peptide-MHC I tetramers for these epitopes, and a subdominant BoLA-A10-restricted epitope (Tp298-106 ), were generated to facilitate accurate and rapid enumeration of epitope-specific CD8 T cells. During validation of these tetramers a substantial proportion of Tp249-59 -reactive T cells failed to bind the tetramer, suggesting that this population was heterogeneous with respect to the recognized epitope. We demonstrate that Tp250-59 represents a distinct epitope and that tetramers produced with Tp50-59 and Tp49-59 show no cross-reactivity. The Tp249-59 and Tp250-59 epitopes use different serine residues as the N-terminal anchor for binding to the presenting MHC I molecule. Molecular dynamic modelling predicts that the two peptide-MHC I complexes adopt structurally different conformations and Tcell receptor ß sequence analysis showed that Tp249-59 and Tp250-59 are recognized by non-overlapping T-cell receptor repertoires. Together these data demonstrate that although differing by only a single residue, Tp249-59 and Tp250-59 epitopes form distinct ligands for T-cell receptor recognition. Tetramer analysis of T. parva-specific CD8 T-cell lines confirmed the immunodominance of Tp1214-224 in BoLA-A18 animals and showed in BoLA-A10 animals that the Tp249-59 epitope response was generally more dominant than the Tp250-59 response and confirmed that the Tp298-106 response was subdominant.

Haematopoietic depletion in vaccine-induced neonatal pancytopenia depends on both the titre and specificity of alloantibody and levels of MHC I expression.

Bovine Neonatal Pancytopenia (BNP) is a disease of calves characterised by haematopoietic depletion, mediated by ingestion of alloantibodies in colostrum. It has been linked epidemiologically to vaccination of the dams of affected calves with a particular vaccine (Pregsure) containing a novel adjuvant. Evidence suggests that BNP-alloantibodies are directed against MHC I molecules, induced by contaminant bovine cellular material from Madin-Darby Bovine Kidney (MDBK) cells used in the vaccine's production. We aimed to investigate the specificity of BNP-alloantibody for bovine MHC I alleles, particularly those expressed by MDBK cells, and whether depletion of particular cell types is due to differential MHC I expression levels. A complement-mediated cytotoxicity assay was used to assess functional serum alloantibody titres in BNP-dams, Pregsure-vaccinated dams with healthy calves, cows vaccinated with an alternative product and unvaccinated controls. Alloantibody specificity was investigated using transfected mouse lines expressing the individual MHC I alleles identified from MDBK cells and MHC I-defined bovine leukocyte lines. All BNP-dams and 50% of Pregsure-vaccinated cows were shown to have MDBK-MHC I specific alloantibodies, which cross-reacted to varying degrees with other MHC I genotypes. MHC I expression levels on different blood cell types, assessed by flow cytometry, were found to correlate with levels of alloantibody-mediated damage in vitro and in vivo. Alloantibody-killed bone marrow cells were shown to express higher levels of MHC I than undamaged cells. The results provide evidence that MHC I-specific alloantibodies play a dominant role in the pathogenesis of BNP. Haematopoietic depletion was shown to be dependent on the titre and specificity of alloantibody produced by individual cows and the density of surface MHC I expression by different cell types. Collectively, the results support the hypothesis that MHC I molecules originating from MDBK cells used in vaccine production, coupled with a powerful adjuvant, are responsible for the generation of pathogenic alloantibodies.

Genomic analysis offers insights into the evolution of the bovine TRA/TRD locus.

BACKGROUND: The TRA/TRD locus contains the genes for V(D)J somatic rearrangement of TRA and TRD chains expressed by αß and γδ T cells respectively. Previous studies have demonstrated that the bovine TRA/TRD locus contains an exceptionally large number of TRAV/TRDV genes. In this study we combine genomic and transcript analysis to provide insights into the evolutionary development of the bovine TRA/TRD locus and the remarkable TRAV/TRDV gene repertoire. RESULTS: Annotation of the UMD3.1 assembly identified 371 TRAV/TRDV genes (distributed in 42 subgroups), 3 TRDJ, 6 TRDD, 62 TRAJ and single TRAC and TRDC genes, most of which were located within a 3.5 Mb region of chromosome 10. Most of the TRAV/TRDV subgroups have multiple members and several have undergone dramatic expansion, most notably TRDV1 (60 genes). Wide variation in the proportion of pseudogenes within individual subgroups, suggest that differential 'birth' and 'death' rates have been used to form a functional bovine TRAV/TRDV repertoire which is phylogenetically distinct from that of humans and mice. The expansion of the bovine TRAV/TRDV gene repertoire has predominantly been achieved through a complex series of homology unit (regions of DNA containing multiple gene) replications. Frequent co-localisation within homology units of genes from subgroups with low and high pseudogene proportions suggest that replication of homology units driven by evolutionary selection for the former may have led to a 'collateral' expansion of the latter. Transcript analysis was used to define the TRAV/TRDV subgroups available for recombination of TRA and TRD chains and demonstrated preferential usage of different subgroups by the expressed TRA and TRD repertoires, indicating that TRA and TRD selection have had distinct impacts on the evolution of the TRAV/TRDV repertoire. CONCLUSION: Both TRA and TRD selection have contributed to the evolution of the bovine TRAV/TRDV repertoire. However, our data suggest that due to homology unit duplication TRD selection for TRDV1 subgroup expansion may have substantially contributed to the genomic expansion of several TRAV subgroups. Such data demonstrate how integration of genomic and transcript data can provide a more nuanced appreciation of the evolutionary dynamics that have led to the dramatically expanded bovine TRAV/TRDV repertoire.

Strain-dependent cellular immune responses in cattle following Escherichia coli O157:H7 colonization.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic diarrhea and potentially fatal renal failure in humans. Ruminants are considered to be the primary reservoir for human infection. Vaccines that reduce shedding in cattle are only partially protective, and their underlying protective mechanisms are unknown. Studies investigating the response of cattle to colonization generally focus on humoral immunity, leaving the role of cellular immunity unclear. To inform future vaccine development, we studied the cellular immune responses of cattle during EHEC O157:H7 colonization. Calves were challenged either with a phage type 21/28 (PT21/28) strain possessing the Shiga toxin 2a (Stx2a) and Stx2c genes or with a PT32 strain possessing the Stx2c gene only. T-helper cell-associated transcripts at the terminal rectum were analyzed by reverse transcription-quantitative PCR (RT-qPCR). Induction of gamma interferon (IFN-γ) and T-bet was observed with peak expression of both genes at 7 days in PT32-challenged calves, while upregulation was delayed, peaking at 21 days, in PT21/28-challenged calves. Cells isolated from gastrointestinal lymph nodes demonstrated antigen-specific proliferation and IFN-γ release in response to type III secreted proteins (T3SPs); however, responsiveness was suppressed in cells isolated from PT32-challenged calves. Lymph node cells showed increased expression of the proliferation marker Ki67 in CD4(+) T cells from PT21/28-challenged calves, NK cells from PT32-challenged calves, and CD8(+) and γδ T cells from both PT21/28- and PT32-challenged calves following ex vivo restimulation with T3SPs. This study demonstrates that cattle mount cellular immune responses during colonization with EHEC O157:H7, the temporality of which is strain dependent, with further evidence of strain-specific immunomodulation.

NKp46+ CD3+ cells: a novel nonconventional T cell subset in cattle exhibiting both NK cell and T cell features.

The NKp46 receptor demonstrates a high degree of lineage specificity, being expressed almost exclusively in NK cells. Previous studies have demonstrated NKp46 expression by T cells, but NKp46+ CD3+ cells are rare and almost universally associated with NKp46 acquisition by T cells following stimulation. In this study we demonstrate the existence of a population of NKp46+ CD3+ cells resident in normal bovine PBMCs that includes cells of both the αß TCR+ and γδ TCR+ lineages and is present at a frequency of 0.1-1.7%. NKp46+ CD3+ cells express transcripts for a broad repertoire of both NKRs and TCRs and also the CD3ζ, DAP10, and FcεR1γ but not DAP12 adaptor proteins. In vitro functional analysis of NKp46+ CD3+ cells confirm that NKp46, CD16, and CD3 signaling pathways are all functionally competent and capable of mediating/redirecting cytolysis. However, only CD3 cross-ligation elicits IFN-γ release. NKp46+ CD3+ cells exhibit cytotoxic activity against autologous Theileria parva-infected cells in vitro, and during in vivo challenge with this parasite an expansion of NKp46+ CD3+ cells was observed in some animals, indicating the cells have the potential to act as an anti-pathogen effector population. The results in this study identify and describe a novel nonconventional NKp46+ CD3+ T cell subset that is phenotypically and functionally distinct from conventional NK and T cells. The ability to exploit both NKRs and TCRs suggests these cells may fill a functional niche at the interface of innate and adaptive immune responses.

Escape from CD8+ T cell response by natural variants of an immunodominant epitope from Theileria parva is predominantly due to loss of TCR recognition.

Polymorphism of immunodominant CD8(+) T cell epitopes can facilitate escape from immune recognition of pathogens, leading to strain-specific immunity. In this study, we examined the TCR ß-chain (TRB) diversity of the CD8(+) T cell responses of cattle against two immunodominant epitopes from Theileria parva (Tp1(214-224) and Tp2(49-59)) and investigated the role of TCR recognition and MHC binding in determining differential recognition of a series of natural variants of the highly polymorphic Tp2(49-59) epitope by CD8(+) T cell clones of defined TRB genotype. Our results show that both Tp1(214-224) and Tp2(49-59) elicited CD8(+) T cell responses using diverse TRB repertoires that showed a high level of stability following repeated pathogenic challenge over a 3-y period. Analysis of single-alanine substituted versions of the Tp2(49-59) peptide demonstrated that Tp2(49-59)-specific clonotypes had a broad range of fine specificities for the epitope. Despite this diversity, all natural variants exhibited partial or total escape from immune recognition, which was predominantly due to abrogation of TCR recognition, with mutation resulting in loss of the lysine residue at P8, playing a particularly dominant role in escape. The levels of heterozygosity in individual Tp2(49-59) residues correlated closely with loss of immune recognition, suggesting that immune selection has contributed to epitope polymorphism.