Proliferative unresponsiveness of T-HA cells induced by immobilized anti-CD3 is only after IL-2 but not IL-15 conditioning the consequence of cell death.

(A) Immobilized anti-CD3 does not induce proliferation of T-HA cells. T-HA cells were treated for 48 hours with IL-2 (10 ng/mL, solid bars) or IL-15 (1 ng/mL, open bars), harvested and stimulated with antigen in the presence of irradiated splenocytes (APC) or immobilized anti-CD3 mAb (10 μg/mL). Medium and APC alone were added as controls. Proliferation was measured by [³H]thymidine incorporation after 72 hours in culture. Data shown are the mean cpm of triplicate cultures ± SD. (B) IL-15 protects against anti-CD3-induced cell death. Viable T-HA cells (4 × 10⁴), treated for 48 hours with IL-2 (solid bars) or IL-15 (open bars), were incubated on 96-well plates coated with anti-CD3 mAb (10 μg/mL). Anti-CD3-induced death was measured after 24 and 48 hours by PI uptake and flow cytometry. Cultures containing cytokine but no anti-CD3 mAb were used as controls. Results shown represent 3 pooled wells and are representative for several similar experiments. (C) Anti-CD3-induced death is partially inhibited by anti-Fas plus anti-FasL. T-HA cells treated with IL-2 (10 ng/mL) were seeded on plates coated with anti-CD3 in the absence or presence of anti-Fas (5 μg/mL) and anti-FasL (2.5 μg/mL) mAb. Cell death was determined after 24 hours as described in panel B. (D) IL-2 and IL-15 do not differentially influence CD3 expression. IL-2-conditioned (black lines) or IL-15-conditioned (gray lines) T-HA cells were labeled with anti-CD3 mAb and FITC-conjugated antihamster IgG (thick lines) or with the detecting antibody alone (thin lines). Fluorescence was measured by flow cytometry. Imm indicates immobilized.