Figure 4.

Autoproteolysis-resistant full-length casp8 rescues proliferative defect of casp8^−/− T cells. (a) Reconstitution of casp8^−/− T cells with a cleavage site casp8 mutant (C8-D387A) restores proliferation. Mitogenically activated primary casp8^−/− T cells were retrovirally transduced with empty vector pMit, casp8 wild type (C8-Wt), or a processing mutant C8-D387A. As in Figure 2, Thy1.1 expression was monitored by flow cytometry and fold change relative to day 3 plotted. Experiment was repeated with three casp8^−/− mice and results represent the mean ± S.E.M. (b) Transduction efficiency was assessed by casp8 immunobloting of lysates from T cells expressing the above constructs for 7 days. As a control, wild-type T cells transduced with empty pMit were also loaded. Membrane probed with α-tubulin as a loading control. (c) Casp8 processing mutant D387A rescues the defective accumulation of activated casp8^−/− T cells. Cycling rate and accumulation profiles of Thy1.1⁺ cells, expressing pMit, C8-Wt, or C8-D387A, were evaluated by CFSE staining and cytometry. (d) Percentage of Annexin-V positive cells after transduction of the various casp8 viruses in activated casp8^−/− T cells was monitored by flow cytometry and plotted as fold change relative to day 4. Mean ± S.E.M. from three independent experiments plotted for each data point