Figure 7.

Signaling through TNF-RII is not sufficient to decrease Bcl-2 levels, but TNF-α signaling contributes to this effect. High avidity CTLs were stimulated by transfer to soluble D^d coated wells pulsed with optimal (0.0005 μM) or supraoptimal (50 μM) I10 peptide in either the presence or absence of anti–TNF-α. After 6 h, cells were transferred to either untreated wells or wells coated with anti–TNF-RII and incubated for a further 22–24 h under their initial media conditions. CTLs were then harvested and solubilized with 0.5% Triton X-100 lysis buffer containing protease inhibitors. Lysates were analyzed by Western blot analysis for determination of Bcl-2 concentrations. Actin was used as a control to ensure equivalent loading among the samples. Anti–TNF-α blocked the decrease in Bcl-2 that occurred after high avidity CTLs were stimulated with high peptide–MHC determinant density. Substrate-bound anti–TNF-RII caused no decrease in the levels of Bcl-2, either in the presence or in the absence of anti– TNF-α, indicating that these conditions were not equivalent to stimulation through the TCR by supraoptimal levels of peptide–MHC.