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.

Next generation sequencing of transcribed genes in ruminant γδ T cell populations.

Bovine γδ T cells are distinguished by expression of WC1, hybrid pattern recognition receptors and co-receptors to the T cell receptor (TCR), or their absence. WC1 molecules bind pathogens and the ability of γδ T cells to respond to pathogens largely correlates with their expression of particular WC1 genes. Following activation, the TCR and WC1 molecules co-localize and knocking down WC1 abrogates the ability of WC1-expressing γδ T cells to respond to antigen. It is known that these two major populations, WC1+ and WC1-, differ in their TCR gene expression and previous studies showed other differences using semi-quantitative RT-PCR and serial analysis of gene expression. Differences in genes expressed would influence the functional outcome when WC1+ vs. WC1- γδ T cells respond to pathogens. To identify unique aspects of their transcriptome, here we performed RNA-Seq of flow cytometrically sorted bovine WC1+ and WC1- γδ T cells and compared them to all mononuclear cells in blood. The greatest differences in gene expression were found between γδ T cells and other mononuclear cells and included those involved in lymphocyte activation and effector processes. Only minor differences occurred between ex vivo WC1+ vs. WC1- γδ T cells with those gene products being involved in cell adhesion and chemotaxis. After culturing cells from primed animals with Leptospira antigens major difference in the transcriptome was evident, with over 600 genes significantly differentially expressed including those focused on cytokine signaling. Unexpectedly, antigen-responding and non-responding populations of WC1+ γδ T cells had few differences in their transcriptomes outside of cytotoxic factors although they had more WC1-1, WC1-2 and WC1-13 transcripts. Through differential gene expression we were able to define properties of ex vivo and stimulated WC1+ cells which will be useful in understanding their functional biology.

Special features of γδ T cells in ruminants.

Ruminant γδ T cells were discovered in the mid-1980's shortly after a novel T cell receptor (TCR) gene from murine cells was described in 1984 and the murine TCRγ gene locus in 1985. It was possible to identify γδ T cell populations early in ruminants because they represent a large proportion of the peripheral blood mononuclear cells (PBMC). This null cell population, γδ T cells, was designated as such by its non-reactivity with monoclonal antibodies (mAb) against ovine and bovine CD4, CD8 and surface immunoglobulin (Ig). γδ T cells are non-conventional T cells known as innate-like cells capable of using both TCR as well as other types of receptor systems including pattern recognition receptors (PRR) and natural killer receptors (NKR). Bovine γδ T cells have been shown to respond to stimulation through toll-like receptors, NOD, and NKG2D as well as to cytokines alone, protein and non-protein antigens through their TCR, and to pathogen-infected host cells. The two main populations of γδ T cells are distinguished by the presence or absence of the hybrid co-receptor/PRR known as WC1 or T19. These two populations not only differ by their proportional representation in various tissues and organs but also by their migration into inflamed tissues. The WC1+ cells are found in the blood, skin and spleen while the WC1- γδ T cells predominate in the gut, mammary gland and uterus. In ruminants, γδ T cells may produce IFNγ, IL-17, IL-10 and TGFß, have cytotoxic activity and memory responses. The expression of particular WC1 family members controls the response to particular pathogens and correlates with differences in cytokine responses. The comparison of the WC1 gene families in cattle, sheep and goats is discussed relative to other multigenic arrays that differentiate γδ T cells by function in humans and mice.

The WC1 γδ T cell pathogen receptor of ruminants is preserved in the genome of ancient extinct auroch.

The work reported here investigated the γδ T cell-specific cell surface receptor known as workshop cluster 1 (WC1) in the extinct Auroch and compared the gene sequences to those in modern cattle breeds. These molecules function as hybrid pattern recognition receptors (PRR), binders of microbial pathogens, and as signaling co-receptors of the T cell antigen receptor (TCR), directing the immune responses by γδ T cell subsets. Sequences in the Auroch genome included both WC1.1 and WC1.2-like a-patterned scavenger receptor cytsteine-rich (SRCR) domains as well as the more conserved b, c, d, and e-patterned SRCR domains. While there was much sequence homology with bovine WC1 genes, there are also unique Auroch genes based on the signature a1 SRCR domain sequences that are used to identify individual WC1 genes. There was also conservation of genes coding for Type I and II intracytoplasmic endodomains although no evidence for gene sequences for Type III endodomains or the extracellular 6 SRCR domains that are associated with this endodomain. This particular WC1 molecule has been proposed to represent the most ancient WC1, and thus, it is particularly interesting that it is apparently missing in the Auroch genome although it could be due to incomplete sequencing. Overall, the results suggest that while WC1 genes have been preserved from Ancient Auroch to modern cattle, they may have co-evolved perhaps as a result of differing pathogen or environmental antigen exposure.

Gene characterization and expression of the γδ T cell co-receptor WC1 in sheep.

Sheep are known to express the hybrid co-receptor/pattern recognition receptor WC1 on their γδ T cells but details of the ovine WC1 multigenic array and gene expression were unknown. Annotation of the sheep genome assembly (Oar_rambouillet_v1.0) yielded 15 complete and 42 partial WC1 genes predicted to code for six different protein structures. RT-PCR amplification of the most distal scavenger receptor cysteine rich (SRCR) domain known as a1, which serves as the gene signature, from genomic and cDNA templates verified the majority of annotated genes. As for cattle and goats, sheep a1 domain sequences included WC1.1 and WC1.2 types. A unique ovine gene, WC1-16, had multiple SRCR a-pattern domains in tandem similar to one found in goats. Intracytoplasmic domains of WC1 transcripts had splice variants that may affect signal transduction. The larger number of WC1 genes in sheep and differences in structures and splice variants relative to cattle could have implications in expression patterns and engagement of γδ T cells by pathogens or vaccine constructs.

Special features of γδ T cells in ruminants

Ruminant γδ T cells were discovered in the mid-1980′s shortly after a novel T cell receptor (TCR) gene from murine cells was described in 1984 and the murine TCRγ gene locus in 1985. It was possible to identify γδ T cell populations early in ruminants because they represent a large proportion of the peripheral blood mononuclear cells (PBMC). This null cell population, γδ T cells, was designated as such by its non-reactivity with monoclonal antibodies (mAb) against ovine and bovine CD4, CD8 and surface immunoglobulin (Ig). γδ T cells are non-conventional T cells known as innate-like cells capable of using both TCR as well as other types of receptor systems including pattern recognition receptors (PRR) and natural killer receptors (NKR). Bovine γδ T cells have been shown to respond to stimulation through toll-like receptors, NOD, and NKG2D as well as to cytokines alone, protein and non-protein antigens through their TCR, and to pathogen-infected host cells. The two main populations of γδ T cells are distinguished by the presence or absence of the hybrid co-receptor/PRR known as WC1 or T19. These two populations not only differ by their proportional representation in various tissues and organs but also by their migration into inflamed tissues. The WC1+ cells are found in the blood, skin and spleen while the WC1− γδ T cells predominate in the gut, mammary gland and uterus. In ruminants, γδ T cells may produce IFNγ, IL-17, IL-10 and TGFβ, have cytotoxic activity and memory responses. The expression of particular WC1 family members controls the response to particular pathogens and correlates with differences in cytokine responses. The comparison of the WC1 gene families in cattle, sheep and goats is discussed relative to other multigenic arrays that differentiate γδ T cells by function in humans and mice.