Fig. 4.

Vγ9Vδ1 and Vγ9Vδ2 T cells respond to different antigens. (a) Both the Vγ9Vδ1 (upper panels) and Vγ9Vδ2 (lower panels) T cell clones were shown to express high levels of TCR by FACS. Clones were stained with PE-conjugated anti-human Vγ9 mAb clone B3·1 (all panels), FITC-conjugated anti-human Vδ1 mAb clone TS8·2 (left panels) and FITC-conjugated anti-human Vδ2 mAb clone B6·1 (right panels). (b) Both clones can be activated through their TCR by anti-CD3 antibody. Clones were stimulated with anti-CD3 antibody (10 µg/ml) for 3 min, washed once with PBS, and lysed, before separation by SDS-PAGE alongside unstimulated controls. Proteins bearing phosphorylated tyrosine residues were revealed by immunoblotting with anti-phosphotyrosine antibody clone 4G10 as described [38]. The lysates of 10⁶ cells per lane were loaded as follows: Vγ9Vδ1 T lymphocytes before (lane 1) and after (lane 2) stimulation; Vγ9Vδ2 T lymphocytes before (lane 3) and after (lane 4) stimulation. (c) Vγ9Vδ2, but not Vγ9Vδ1 T cells, are able to recognize alkylphosphate, alkylamine and nBP antigens. MIP1β release from 5 × 10⁴ clonal T cells as described in the Materials and Methods section is shown in response to 1·5 µg/ml anti-CD3 antibody clone UCHT-1, 10 µm IPP, 10 mm secbutylamine and 1 µm risedronate. (d) IFNγ ELISpot assay with a titration of potential antigens. 1000 Vγ9Vδ2 T cells were used with 25 000 EBV transformed B cells (Sparky line) per well as antigen presenting cells. The Vγ9Vδ1 clone failed to activate in response to any of these antigens (data not shown).