Purification and characterisation of bovine WC1(+) gamma delta T lymphocytes from peripheral blood

In order to isolate and characterise resting WC1(+) gamma delta T cells fro m cattle, we developed a protocol for purifying these cells by negative sel ection from peripheral blood. The purification method included five steps: separation of mononuclear cells on lymphoprep, depletion of monocytes by ad herence to plasma-coated gelatin, enriching T cells on a nylon wool column, depleting CD2(+) T cells by sheep red blood cells (SRBC), and finally depl eting CD4(+) and CD8(+) T cells by the magnetic cell sorting technique (MAC S). This procedure proved efficient and reproducible, and the purity of the isolated WCl+ gamma delta T cells was more than 97% as analysed by flow cy tometry (FACS). Cytokines and costimulatory molecules mRNA expression was a ssessed by the reverse transcriptase polymerase chain reaction (RT-PCR) tec hnique in freshly isolated resting WC1(+) T cells. We found that purified u ncultured WC1(+) T cells express TNF-alpha, CD28, CTLA-4 and IL-2R alpha mR NA 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 ce lls indicates that these genes are evolutionarily conserved.