Special features of γδ T cells in ruminants

The major functions of γδ T cells in mammals overlap with those of αβ T cells but differ in that γδ T cells are rapid responders and see different types of antigens. While γδ T cells have been shown to be a major population of circulating lymphocytes in artiodactyl species such as cattle, sheep, and pigs, less is known about these cells in goats, an important agricultural species. We have recently shown that WC1, a γδ T cell-specific family of hybrid pattern recognition receptors/co-receptors, is a multigenic family in goats expanded beyond what occurs in cattle. This study was conducted to address some of the limitations of previous studies in determining the proportions of γδ T cells, WC1+ γδ T cells as well as the WC1.1+ and WC1.2+ subpopulations in blood and to evaluate their responses to various pathogens. Previously, the proportion of caprine γδ T cells was determined using a monoclonal antibody (mAb) 86D that we show here does not react with all γδ T cells thereby underestimating their contribution to the lymphocyte population. Using a mAb reactive with the TCRδ constant region we found the proportion of γδ T cells in blood was not significantly less than that of either CD4 or CD8 T cells and did not decrease with age after 6 months. γδ T cells that expressed WC1 ranged from ~20 to 85% of the total γδ T cells. Less than half of those were classified as WC1.1+ or WC1.2+ by mAb staining thus indicating a third major WC1+ population. We found that naive γδ T cells proliferated in cultures of PBMC stimulated with antigens of Leptospira or Mycobacterium avium paratuberculosis (MAP) more than they did in control medium cultures or in those stimulated with M. bovis BCG antigens and that the responding γδ T cells included both WC1+ and WC1- cells. In ex vivo PMA/ionomycin-stimulated cultures of WC1- γδ T cells but not WC1+ cells produced both IL-17 and IFNγ. In longterm cultures with Leptospira or MAP both WC1- and WC1+ cells proliferated but only WC1- γδ T cells produced IL-17. In conclusion, goats have a substantial number of WC1- and WC1+ γδ T cells in PBMC that do not decrease with animal age after 6 months; both populations respond to bacterial antigens as naive cells but in these cultures only the WC1- γδ cells produc IL-17 and IFNγ .

Goat γδ T cell subpopulations defined by WC1 expression, responses to pathogens and cytokine production

γδ T cells constitute a major portion of lymphocytes in the blood of both ruminants and swine. Subpopulations of swine γδ T cells have been distinguished by CD2 and CD8α expression. However, it was not clear if they have distinct expression profiles of their T-cell receptor (TCR) or WC1 genes. Identifying receptor expression will contribute to understanding the functional differences between these subpopulations and their contributions to immune protection. Here, we annotated three genomic assemblies of the swine TCRγ gene locus finding four gene cassettes containing C, J and V genes, although some haplotypes carried a null TRGC gene (TRGC4). Genes in the TRGC1 cassette were homologs of bovine TRGC5 cassette while the others were not homologous to bovine genes. Here we evaluated three principal populations of γδ T cells (CD2+/SWC5-, CD2-/SWC5+, and CD2-/SWC5-). Both CD2− subpopulations transcribed WC1 co-receptor genes, albeit with different patterns of gene expression but CD2+ cells did not. All subpopulations transcribed TCR genes from all four cassettes, although there were differences in expression levels. Finally, the CD2+ and CD2− γδ T-cell populations differed in their representation in various organs and tissues, presumably at least partially reflective of different ligand specificities for their receptors.

Subpopulations of swine γδ T cells defined by TCRγ and WC1 gene expression.

The WC1 cell surface family of molecules function as hybrid gamma delta (γδ) TCR co-receptors, augmenting cellular responses when cross-linked with the TCR, and as pattern recognition receptors, binding pathogens. It is known that following activation, key tyrosines are phosphorylated in the intracytoplasmic domains of WC1 molecules and that the cells fail to respond when WC1 is knocked down or, as shown here, when physically separated from the TCR. Based on these results we hypothesized that the colocalization of WC1 and TCR will occur following cellular activation thereby allowing signaling to ensue. We evaluated the spatio-temporal dynamics of their interaction using imaging flow cytometry and stochastic optical reconstruction microscopy. We found that in quiescent γδ T cells both WC1 and TCR existed in separate and spatially stable protein domains (protein islands) but after activation using Leptospira, our model system, that they concatenated. The association between WC1 and TCR was close enough for fluorescence resonance energy transfer. Prior to concatenating with the WC1 co-receptor, γδ T cells had clustering of TCR-CD3 complexes and exclusion of CD45. γδ T cells may individually express more than one variant of the WC1 family of molecules and we found that individual WC1 variants are clustered in separate protein islands in quiescent cells. However, the islands containing different variants merged following cell activation and before merging with the TCR islands. While WC1 was previously shown to bind Leptospira in solution, here we showed that Leptospira bound WC1 proteins on the surface of γδ T cells and that this could be blocked by anti-WC1 antibodies. In conclusion, γδ TCR, WC1 and Leptospira interact directly on the γδ T cell surface, further supporting the role of WC1 in γδ T cell pathogen recognition and cellular activation.

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Gamma Delta TCR and the WC1 Co-Receptor Interactions in Response to Leptospira Using Imaging Flow Cytometry and STORM

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.

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

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.

Julie McLaughlin

Papers

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