FIGURE 5.

T cells stimulated strongly through the TCR are resistant to Treg-mediated suppression, and this lack of suppression correlates with phosphorylation of Stat3. CD4⁺CD25^– T cells and CD4⁺CD25^high Tregs were isolated from peripheral blood of healthy donors. A, Cells were cultured with allogeneic APCs in a MLR or with anti-CD3/anti-CD28 Abs (both at 1 μg/ml) for 6 d, and proliferation is indicated by incorporation of [³H] during the final 16 h of culture. B and C, Cells were cultured for 6 d with 1 μg/ml anti-CD28 and varying concentrations of anti-CD3 Abs, as indicated. B, Proliferation was assessed by [³H] incorporation during the final 16 h of culture. Results show the mean of eight (A) or three (B) separate experiments ± SEM. p Values have been adjusted using the Bonferroni method. C, Protein extracts were prepared on day 6 of coculture and probed with Abs specific for pStat1, pStat3, total Stat1, or total Stat3. The immunoblot shown is representative of three separate experiments. D, Cells were stimulated with either allogeneic APCs, anti-CD3/anti-CD28 Abs (both at 1 μg/ml), or rhIL-6 (10 ng/ml) as indicated. Cells cultured with APCs were back-sorted from the coculture at 48 h. Cells were lysed at indicated time points and probed with Abs specific for pStat3 or total Stat3. Arrows indicate differential pStat3 levels in cells cultured with anti-CD3/anti-CD28, compared with those stimulated with allogeneic APCs. The immunoblot shown is representative of two separate experiments. E, Cells were stimulated with 1 μg/ml anti-CD3/anti-CD28 and lysed at indicated time points and then probed with Abs specific for pStat3, pStat1, total Stat3, or total Stat1. The immunoblot shown is representative of three separate experiments. F, Cells were cultured with anti-CD3/anti-CD28 Abs (both at 1 μg/ml) and indicated concentrations of Stattic V. Proliferation was assessed by [³H] incorporation during the final 16 h of a 7-d culture. Results show the mean of three separate experiments ± SEM.