Proliferation of CD4⁺ and CD8⁺ T lymphocytes in response to anti-CD3. (A) Proliferation of CD4⁺25⁺cells compared with CD4⁺25^-and CD8⁺ cells. T cells (5 × 10⁴/well) were incubated with 1 × 10⁵ irradiated APCs and 1 μg/mL anti-CD3. Proliferation was measured by ³H-thymidine incorporation during the last 12 hours of a 72-hour culture. Results are the mean ± SD of triplicate wells. (B) Effect of CD4⁺25⁺ T_REGs on the proliferation of CD8⁺ cells in response to anti-CD3. Two days after the intraperitoneal administration of 2 mg CY, CD4⁺25⁺ T_REGs were separated from spleens of treated and control mice. CD8⁺ cells (5 × 10⁴) from healthy mice were incubated with 1 × 10⁵ irradiated APCs from healthy mice and 1 μg/mL anti-CD3 along with either no T, 5 × 10⁴, 5 × 10⁴ T_REGs from untreated mice, or 5 × 10⁴ T_REGs from CY-treated mice. Proliferation was measured by ³H-thymidine incorporation during the last 12 hours of a 72-hour culture. Results are the mean ± SD of triplicate wells. Data are representative of 3 experiments. (C) Effect of CD4⁺25⁺ T_REGs on the proliferation of CD4⁺25^- cells in response to anti-CD3. Two days after the intraperitoneal administration of 2 mg CY, CD4⁺25⁺ T_REGs were separated from spleens of treated and control mice. CD4⁺25^- cells (5 × 10⁴) from healthy mice were incubated with 1 × 10⁵ irradiated APCs from healthy mice and 1 μg/mL anti-CD3 along with either no T_REGs, 5 × 10⁴ ,5 × 10⁴ T_REGs from untreated mice, or 5 × 10⁴ T_REGs from CY-treated mice. Proliferation was measured by ³H-thymidine incorporation during the last 12 hours of a 72-hour culture. Results are the mean ± SD of triplicate wells. Data are representative of 3 experiments. (D) Effect of CD4⁺25⁺ T_REGs on the proliferation of CD8⁺ cells in response to anti-CD3. Ten days after the intraperitoneal administration of 2 mg CY, CD4⁺25⁺ T_REGs were separated from spleens of treated and control mice. CD8⁺ cells (5 × 10⁴) from healthy mice were incubated with 1 × 10⁵ irradiated APCs from healthy mice and 1 μg/mL anti-CD3 along with either no T_REGs 5 × 10⁴ T_REGs from untreated mice, or 5 × 10⁴ T_REGs from CY-treated mice. Proliferation was measured by ³H-thymidine incorporation during the last 12 hours of a 72-hour culture. Results are the mean ± SD of triplicate wells. Data are representative of 3 experiments. (E) Effect of CD4⁺25⁺ T_REGs on the proliferation of CD4⁺25^- cells in response to anti-CD3. Two days after the intraperitoneal administration of 2 mg CY, CD4⁺25⁺ T_REGs were separated from spleens of treated and control mice. CD4⁺25^- cells (5 × 10⁴) from healthy mice were incubated with 1 × 10⁵ irradiated APCs from healthy mice and 1 μg/mL anti-CD3 along with either no T_REGs 5 × 10⁴ T_REGs from untreated mice, or 5 × 10⁴ T_REGs from CY-treated mice. Proliferation was measured by ³H-thymidine incorporation during the last 12 hours of a 72-hour culture. Results are the mean ± SD of triplicate wells. Data are representative of 3 experiments.