Figure 5.

IAP antagonists target cIAP-1 and cIAP-2 in human T cells. (A and B) Immunoblots using the indicated antibodies on lysates from human CD4⁺ T cells isolated using magnetic beads. (A) Total lysates from CD4⁺ T cells were stimulated with 10 µg/ml anti-CD3 and 2 µg/ml anti-CD28 and exposed to IAP antagonists (M1, M2, and M3), a control compound (C1), or vehicle (−) for 2 h. All compounds were used at 500 nM. (B) Total cell lysates or nuclear (nuc.) lysates from human CD4⁺ T cells. Cells were either stimulated as in A (top) or left unstimulated (bottom) for 24 h in the presence of vehicle, M1, or control compound, which were used at 500 nM. (C and D) Human CD4⁺ T cells were isolated and stably transfected with lentiviral vectors encoding short hairpin RNAs against cIAP-1, cIAP-2, or nontargeting control (scramble [scr]). Three constructs (KD1–KD3) were used per gene. (C) Immunoblot using the indicated antibodies on lysates from primary CD4⁺ T cells expressing the indicated short hairpin RNA constructs; quantification of knockdown is included below. (D) 2 × 10⁴ cells were stimulated for 72 h. 20 U/ml recombinant human IL-2 was added to all cultures. (E, left) Immunoblot using the indicated antibodies on total lysates from human CD4⁺ T cells stimulated with anti-CD3/CD28; after 24 h, cells were exposed to 5 µg/ml anti-GITR antibodies as indicated. (right) IFN-γ production from GITR-stimulated cells. (A–E) Cytokines were measured by ELISA. Error bars represent SEM. Results are representative of at least two independent experiments.