rATG-treated Tconv cells do not acquire suppressive capacity. (A-B) Responder (R) CD4⁺CD25⁻ T cells were stimulated with soluble anti-CD3 (1 μg/mL) and APCs and cocultured with the indicated ratio of putative “suppressor” (S) CD4⁺CD25⁻ T cells that had been incubated with different concentrations of rATG, rIgG (10 μg/mL), or anti-CD3/CD28–coated beads for either (A) 3 or (B) 10 days. Suppression was assessed by measuring the amount of [³H]-thymidine incorporation in the final 16 hours of a 4-day culture period. Left panels represent results from an individual experiment. Right panels represent the average % suppression at a 1:1 ratio from 7 independent experiments with different donors. Percentage suppression at day 3 with a 1:2 ratio (Treg/responder): −143.78 ± 32.30, untreated; −117.74 ± 40.87, treated with rIgG; −189.34 ± 78.0, with anti-CD3/28 beads; −178.74 ± 42.42, with 1 μg/mL rATG; −70.69 ± 21.76, with 10 μg/mL rATG; 15.17 ± 8.22, with 100 μg/mL rATG (P = ns); and 92.4 ± 7.5, with ex vivo Tregs; 73.41 ± 8.44, with Tregs treated with 10 μg/mL rATG (P = ns compared with ex vivo Tregs). (C) To further examine the suppressive capacity of CD4⁺CD25⁻ T cells exposed to 100 μg of rATG, suppression was assessed at different time points by adding [³H]-thymidine after 1, 2, or 3 days. Assays were harvested 16 hours after [³H]-thymidine addition. Results are representative of % suppression. Representative data from 3 independent experiments with different donors are depicted.