Purification and characterisation of bovine WC1+ gammadelta T lymphocytes from peripheral blood

In order to isolate and characterise resting WC1+ gammadelta T cells from cattle, we developed a protocol for purifying these cells by negative selection from peripheral blood. The purification method included five steps: separation of mononuclear cells on lymphoprep, depletion of monocytes by adherence to plasma-coated gelatin, enriching T cells on a nylon wool column, depleting CD2+ T cells by sheep red blood cells (SRBC), and finally depleting CD4+ and CD8+ T cells by the magnetic cell sorting technique (MACS). This procedure proved efficient and reproducible, and the purity of the isolated WC1+ gammadelta T cells was more than 97% as analysed by flow cytometry (FACS). Cytokines and costimulatory molecules mRNA expression was assessed by the reverse transcriptase polymerase chain reaction (RT-PCR) technique in freshly isolated resting WC1+ T cells. We found that purified uncultured WC1+ T cells express TNF-alpha, CD28, CTLA-4 and IL-2R alpha mRNA transcripts but do not express those for IL-2, IL-4, IL-6, IL-10 and IFN-gamma. The expression of CD28 and CTLA-4 transcripts on bovine WC1+ T cells indicates that these genes are evolutionarily conserved.