γδ T cells have similar TCR threshold but enhanced ERK1/2 phosphorylation comparing to αβ T cells.

<p>(A) Percentages of BrdU⁺ proliferative cells are higher in the γδ T cell subset <i>in vivo</i>. Left: representative plots of BrdU⁺ populations (circled) in αβ and γδ T cells. Right: Graph of percentages of BrdU⁺ cells in αβ and γδ T cells. C57BL/6J mice received single dose of BrdU (1 mg) via intraperitoneal injection, and splenocytes were isolated 3 days later. BrdU⁺ cells were identified by fluorescently labeled anti-BrdU antibody and flow cytometry. Results were averaged from 8 mice, and shown in mean ± SEM. *** P<0.001. (B) Top: Splenocytes from C57BL/6J mice were labeled with CFSE (2.5 µM), and then stimulated with 0–10 µg/ml plate-bound αCD3 and 1 µg/ml soluble αCD28 for 24 hours. CFSE content in cells was measured by flow cytometry and graphed against αCD3 concentrations. Results were averaged from 5 mice, and shown in mean ± SEM. Lower: Log scaling of αCD3 concentration against % of maximum response was used to calculate EC₅₀. (C) CD3 expression in γδ T cells was higher than in αβ T cells under no or lower stimulation, but decreased more dramatically with stronger stimulation. The experimental condition was the same as in (B). (D) Western blotting shows high levels of phosphorylated ERK1/2 in γδ T cells, relative to αβ T cells, at baseline. αβ and γδ T cells were isolated from the same set of C57BL/6J mice via FACS sorting. Cells were either untreated, or incubated with αCD3 and αCD28 antibodies (20 µg/ml each) for 5 minutes and then with anti-hamster IgG (20 µg/ml) for additional 2 minutes at 37°C. Cells were lyzed, separated by SDS-PAGE, and immunoblotted with indicated antibodies. The image was representative of 3 independent experiments. (E) Inhibition of ERK1/2 significantly lowers proliferation in γδ T cells <i>in vitro</i>. Splenocytes from C57BL/6J mice were labeled with CFSE (2.5 µM), and then stimulated with 1 µg/ml plate-bound αCD3 and 1 µg/ml soluble αCD28 for 24 hours, in the absence (vehicle) or presence of 10 µM and 25 µM U0126. CFSE dilution in cells was measured by flow cytometry, and used to calculate cell proliferation. Results were averaged from 7–8 mice, and shown in mean ± SEM. ** P<0.01; *** P<0.001; ns not statistically significant.