Toll-like receptors (TLRs) and T cell effector function

The aim of the project was to identify whether cattle T cell subsets express toll-like receptors (TLRs), and if so whether there was any functional consequence of this when they were stimulated with TLR-ligands. CD4+ T cells, CD8+ T cells, and γδ T cells were studied in the context of whole PBMC or as isolated cells. To develop rabbit Tregs as a source of cells to study expression of toll-like receptors (TLRs), putative rabbit regulatory T cells (Tregs) were developed as cultured cells from MLN as part of an ongoing project and to inform generation of cattle Treg development for this study. Culture of rabbit MLN cells resulted in detection of CD4+CD25hiFOXP3+ (putative nTregs) in about one fifth of the total cells. The cultured rabbit Tregs did not exhibit any immunosuppressive effect on autologous MLN (Mesenteric lymph node) cells in a suppression assy. This and the complexity of this method meant that the cattle Treg work was not initiated for this study. The main part of the project involved screening of 10 bovine toll-like receptors (TLRs 1-10) on cattle blood samples (PBMC fraction and T cell subsets) plus investigation of their expression using RT-qPCR to detect transcripts in the T cell subsets. TLRs were variably expressed in the T cell subsets from the samples. With regard to function measured by proliferation in response to stimulation with TLR agonist (ligand), CFSE (Carboxy-fluorescein Succinimidyl Ester)-labelled PBMCs were stimulated with TLR1-9 agonists and each CFSE-labelled T cell subset identified by flow cytometry and proliferation based on the CFSE profile for each subset within the whole PBMC sample. No significant proliferation was recorded in activated PBMCs while T cell subsets showed variation in responses with CD4 T cells generally most effective followed by CD8+ T cells and least proliferation was seen with γδ T cells. The functional role of expressed TLRs stimulated with ligands and Con-A or antiCD3 were also assessed by expression or not of 7 cytokines (CXCL-8, IFNα, IFNγ, TNFα, TGFβ, IL-4, and IL-10) by specific ELISA. However, the results showed variation between different cattle blood samples. T cells generally activated PBMCs producing CXCL-8 while the isolated T cell subsets showed a more selective secretion. No IFN α expression was found. IFNγ was secreted in a limited amount in one PBMC animal and some γδ T cell samples rather than CD4 or CD8 T cells. TNF-α was produced by one PBMC sample stimulated with TLR2 agonist. The T cell subsets did not record any TNF-α secretion. There was no real functional link between the CFSE proliferation assay and the cytokine expression results. In conclusion, TLRs are expressed on cattle T cell subsets but with variable activation outcomes after stimulation of the cells with TLR ligands