Figure 5
Figure 5. Cyclon positively regulates AICD through up-regulation of Fas expression. (A) Increased AICD of T cells by transgenic expression of Cyclon. CD4+ or CD8+ T cells sorted magnetically were activated with anti-CD3 Ab (2 μg/mL) and anti-CD28 Ab (2 μg/mL) for 2 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 (left panel, CD4+ T cells) or 3 days (right panel, CD8+ T cells), and viability was examined by PI staining and flow cytometry. Indicated are representative data using WT and Cyclon-Tg littermates from 4 independent experiments. (B) Transgenic expression of Cyclon enhances Fas expression. CD4+ or CD8+ T cells were activated with anti-CD3 Ab (2 μg/mL) and anti-CD28 Ab (2 μg/mL) for 2 days. Live cells were purified by density centrifugation and restimulated with anti-CD3 Ab (1 μg/mL) in the presence of IL-2 (5 ng/mL). Cells were incubated for 16 hours and stained with PE-conjugated anti-Fas Ab. (C) Transgenic expression of Cyclon enhances Fas expression on IL-2Rα− CD4+ T cells. CD4+ T cells sorted magnetically were activated with anti-CD3 Ab (2 μg/mL) and anti-CD28 Ab (2 μg/mL) in the presence of IL-15 (100 ng/mL) for 2 days. Cells were stained with PE-conjugated anti-Fas Ab. (D) Increased cell death by FasL stimulation of T cells by transgenic expression of Cyclon. CD4+ or CD8+ T cells were activated with anti-CD3 Ab (2 μg/mL) and anti-CD28 Ab (2 μg/mL) for 2 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. Indicated are representative data using WT and Cyclon-Tg littermates from 4 independent experiments. (E) Cyclon transgenic expression fails to suppress splenomegaly and lymphadenopathy in Fas− IL-2Rα− mice. Spleens and numbers of splenocytes and inguinal lymph nodes from 7-week-old Fas+ IL-2Rα+, Fas− IL-2Rα−, and Cyclon-Tg Fas− IL-2Rα− mice are shown.

Cyclon positively regulates AICD through up-regulation of Fas expression. (A) Increased AICD of T cells by transgenic expression of Cyclon. CD4+ or CD8+ T cells sorted magnetically were activated with anti-CD3 Ab (2 μg/mL) and anti-CD28 Ab (2 μg/mL) for 2 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 (left panel, CD4+ T cells) or 3 days (right panel, CD8+ T cells), and viability was examined by PI staining and flow cytometry. Indicated are representative data using WT and Cyclon-Tg littermates from 4 independent experiments. (B) Transgenic expression of Cyclon enhances Fas expression. CD4+ or CD8+ T cells were activated with anti-CD3 Ab (2 μg/mL) and anti-CD28 Ab (2 μg/mL) for 2 days. Live cells were purified by density centrifugation and restimulated with anti-CD3 Ab (1 μg/mL) in the presence of IL-2 (5 ng/mL). Cells were incubated for 16 hours and stained with PE-conjugated anti-Fas Ab. (C) Transgenic expression of Cyclon enhances Fas expression on IL-2Rα− CD4+ T cells. CD4+ T cells sorted magnetically were activated with anti-CD3 Ab (2 μg/mL) and anti-CD28 Ab (2 μg/mL) in the presence of IL-15 (100 ng/mL) for 2 days. Cells were stained with PE-conjugated anti-Fas Ab. (D) Increased cell death by FasL stimulation of T cells by transgenic expression of Cyclon. CD4+ or CD8+ T cells were activated with anti-CD3 Ab (2 μg/mL) and anti-CD28 Ab (2 μg/mL) for 2 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. Indicated are representative data using WT and Cyclon-Tg littermates from 4 independent experiments. (E) Cyclon transgenic expression fails to suppress splenomegaly and lymphadenopathy in Fas− IL-2Rα− mice. Spleens and numbers of splenocytes and inguinal lymph nodes from 7-week-old Fas+ IL-2Rα+, Fas− IL-2Rα−, and Cyclon-Tg Fas− IL-2Rα− mice are shown.

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