Decreased expression of Fas and AICD in CD4⁺ T cells by reduced Cyclon expression. (A) Expression levels of Cyclon mRNA in activated CD4⁺ T cells from WT and Cyclon^+/EN mice. (B) Surface expression of Fas on activated CD4⁺ T cells from WT and Cyclon^+/EN mice. CD4⁺ T cells sorted magnetically were activated with anti-CD3 Ab (2 μg/mL) and anti-CD28 Ab (2 μg/mL) for 3 days and cultured in the presence of IL-2 for 1 day, and then stained with PE-conjugated anti-Fas Ab. (C) Expression of Fas mRNA in activated CD4⁺ T cells from WT and Cyclon^+/EN mice. (D) Decreased AICD of CD4⁺ T cells from Cyclon^+/EN mice. CD4⁺ T cells sorted magnetically were activated with anti-CD3 Ab (2 μg/mL) and anti-CD28 Ab (2 μg/mL) for 3 days. Live cells were purified by density centrifugation and restimulated with different concentrations of anti-CD3 in the presence of IL-2 (5 ng/mL). Cells were incubated for 2 days, and viability was examined by PI staining and flow cytometry. (E) Decreased FasL-induced cell death of CD4⁺ T cells from Cyclon^+/EN mice. CD4⁺ T cells were activated with anti-CD3 Ab (2 μg/mL) and anti-CD28 Ab (2 μg/mL) for 3 days. Live cells were purified by density centrifugation and restimulated with different concentrations of FasL in the presence of IL-2 (5 ng/mL). Cells were incubated for 24 hours, and viability was examined by PI staining and flow cytometry.