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.

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.

Suppression of ovine lymphocyte activation by Teladorsagia circumcincta larval excretory-secretory products.

Teladorsagia circumcincta is an important pathogenic nematode of sheep. It has been demonstrated previously that stimulation of murine T lymphocytes with excretory-secretory (ES) products derived from fourth stage larvae of T. circumcincta (Tci-L4-ES) results in de novo expression of Foxp3, a transcription factor intimately involved in regulatory T cell function. In the current study, Foxp3⁺ T cell responses in the abomasum and the effects of Tci-L4-ES on ovine peripheral blood mononuclear cells (PBMC) following T. circumcincta infection were investigated. T. circumcincta infection resulted in a significant increase in numbers of abomasal Foxp3⁺ T cells, but not an increase in the proportion of T cells expressing Foxp3. Unlike in mice, Tci-L4-ES was incapable of inducing T cell Foxp3 expression but instead suppressed mitogen-induced and antigen-specific activation and proliferation of ovine PBMC in vitro. This effect was heat labile, suggesting that it is mediated by protein(s). Suppression was associated with up-regulation of interleukin-10 (IL-10) mRNA, and specific monoclonal antibody neutralisation of IL-10 resulted in a 50% reduction in suppression, indicating involvement of the IL-10 signaling pathway. Suppression was significantly reduced in PBMC isolated from T. circumcincta infected vs. helminth-naïve lambs, and this reduction in suppression was associated with an increase in Tci-L4-ES antigen-specific T cells within the PBMC. In conclusion, we have identified a mechanism by which T. circumcincta may modulate the host adaptive immune response, potentially assisting survival of the parasite within the host. However, the impact of Tci-L4-ES-mediated lymphocyte suppression during T. circumcincta infection remains to be determined.

Successful immunization against a parasitic nematode by vaccination with recombinant proteins.

Infection of humans and livestock with parasitic nematodes can have devastating effects on health and production, affecting food security in both developed and developing regions. Despite decades of research, the development of recombinant sub-unit vaccines against these pathogens has been largely unsuccessful. We have developed a strategy to identify protective antigens from Teladorsagia circumcincta, the major pathogen causing parasitic gastroenteritis in small ruminants in temperate regions, by studying IgA responses directed at proteins specific to post-infective larvae. Antigens were also selected on the basis of their potential immunomodulatory role at the host/parasite interface. Recombinant versions of eight molecules identified by immunoproteomics, homology with vaccine candidates in other nematodes and/or with potential immunoregulatory activities, were therefore administered to sheep in a single vaccine formulation. The vaccine was administered three times with Quil A adjuvant and the animals subsequently subjected to a repeated challenge infection designed to mimic field conditions. Levels of protection in the vaccinates were compared to those obtained in sheep administered with Quil A alone. The trial was performed on two occasions. In both trials, vaccinates had significantly lower mean fecal worm egg counts (FWECs) over the sampling period, with a mean reduction in egg output of 70% (Trial 1) and 58% (Trial 2). During the period of peak worm egg shedding, vaccinates shed 92% and 73% fewer eggs than did controls in Trials 1 and 2, respectively. At post mortem, vaccinates had 75% (Trial 1) and 56% (Trial 2) lower adult nematode burdens than the controls. These levels of protection are the highest observed in any system using a nematode recombinant sub-unit vaccine in the definitive ruminant host and indicate that control of parasitic helminths via vaccination with recombinant subunit vaccine cocktails is indeed an alternative option in the face of multi-drug resistance.