Effect of rATG on expression of CD25 and FOXP3, apoptosis and cytokine production. (A-B) CD4⁺CD25⁻ T cells were untreated, incubated with 1, 10, or 100 μg/mL of rabbit antithymocyte globulin (rATG), rabbit immunoglobulin G (rIgG; 10 μg/mL), or anti-CD3/28–coated beads in the presence of IL-2 and analyzed for expression of CD25 or FOXP3 over 10 days. Percentage of FOXP3⁺ at day 3: rIgG, 0.38 ± 0.09; rATG 10 μg/mL, 4.52 ± 0.98; rATG 100 μg/μL, 12.02 ± 0.96 (P < .001; n = 10). (C) Apoptosis in cultures of CD4⁺CD25⁻ T cells incubated with 10 or 100 μg/mL of rATG, or anti-CD3/28–coated beads in the presence of IL-2 for 5 days was evaluated by staining for annexin V. The average percentage of FOXP3⁺ annexin V⁺ cells (% FOXP3⁺ annexin V⁺/% FOXP3⁺ annexin V⁺ + FOXP3⁺ annexin V⁻) is as follows: anti-CD3/28, 14.81 ± 2.69; rATG 10 μg/mL, 13.21 ± 0.66; rATG 100 μg/mL, 22.37 ± 3.6. The average % annexin V⁺ cells in the FOXP3⁻ gate is as follows: anti-CD3/28, 9.95 ± 2.79; rATG 10 μg/mL, 9.03 ± 1.07; rATG 100 μg/mL, 17.12 ± 5.23. n = 3; P = not significant (ns). (D) CD4⁺CD25⁻ T cells were incubated with rabbit IgG (100 μg/mL), anti-CD3/28–coated beads, or rATG (100 μg/mL) in the presence of IL-2 and after 3 days cells were unstimulated or restimulated with PMA and ionomycin (Io) and analyzed for expression of IFN-γ, IL-2, IL-10, and FOXP3. The average percentage of cytokine-producing cells in the FOXP3⁺ gate is as follows: IL-2: anti-CD3/28, 89.2 ± 1.6; rATG 100 μg/mL, 92.7 ± 1.6; IFN-γ: anti-CD3/28, 94.1 ± 1.5; rATG 100 μg/mL, 90.8 ± 3.0, n = 6; IL-10: anti-CD3/28, 4.2 ± 0.9; rATG 100 μg/mL, 8.23 ± 2.2 (P = ns). Data are representative of 10 (A-B), 3 (C), and 6 (D, IL-2 and IFN-γ) and 3 (D, IL-10) experiments with different donors.