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, 18 (1), 90-8

Enzymatically Active Single Chain caspase-8 Maintains T-cell Survival During Clonal Expansion

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Enzymatically Active Single Chain caspase-8 Maintains T-cell Survival During Clonal Expansion

S Leverrier et al. Cell Death Differ.

Abstract

The extrinsic, or death receptor, pathway integrates apoptotic signals through the protease caspase-8 (casp8). Beyond cell death regulation, non-apoptotic functions of casp8 include its essential requirement for hematopoiesis and lymphocyte clonal expansion, and tempering of autophagy in T cells. However, the mechanistic basis for the control of these disparate cellular processes remains elusive. Here, we show that casp8-deficient T-cell survival was rescued by enzymatically active, but not inactive, casp8-expressing retroviruses. The casp8 catalytic induction in proliferating T cell occurred independent of extrinsic and intrinsic apoptotic-signaling cascades and did not induce casp8 proteolytic processing. Using a biotinylated probe selectively targeting enzymatically active caspases, catalytically active full-length casp8 was found in vivo in dividing T cells. A casp8 D387A processing mutant was able to rescue casp8-deficient T-cell proliferation, validating that casp8 self-processing is not required for its non-apoptotic function(s). Finally, casp8 activity was highest in CD8(+) T cells, the most rapidly proliferating subset. These results show that the catalytically competent form of casp8 is required for rapid T-cell proliferation in response to TCR ligation, but that processing of the caspase is only necessary to promote apoptosis.

Figures

Figure 1
Figure 1
Induction of IETDase activity in non-apoptotic-activated T cells depends on FADD. (a) IETDase induction in mitogenically stimulated T cells is diminished in FADDdd versus wild-type (Wt) T cells. Wt, FADDdd, or casp8−/− T cells were activated with plate-bound anti-CD3 (0.5 μg/ml) plus anti-CD28 (200 ng/ml) for the indicated times. IETDase (a) and DEVDase (c) activity were measured using a synthetic substrate IETD-AFC or DEVD-AFC, respectively, as described in the Materials and methods. (b) Lack of casp8 processing in non-apoptotic-activated T cells. At each time point and for each genotype, a fraction of cells obtained in (a) was processed for casp8 immunoprobing; position of the 55 kDa full-length casp8 is indicated. (d) A fraction of cells was stained with Annexin-V-APC plus 7AAD and the percentages of Annexin-V positive cells were plotted. These experiments were repeated in four mice per genotype and data are expressed as the mean ± S.E.M.
Figure 2
Figure 2
Reconstitution of casp8 catalytic activity in casp8−/− T cells restores proliferation. (a) Rescue of casp8−/− T cells with catalytically active, but not catalytically inactive casp8. After stimulation with anti-CD3 (0.5 μg/ml) plus anti-CD28 (200 ng/ml) for 24 h, primary casp8−/− T cells were transduced with retroviral supernatants (sups) containing casp8 wild type (C8-Wt) or a catalytically inactive mutant of casp8, C8-CA. Expression of these constructs, as well as the empty vector pMit, was monitored by assessing Thy1.1 expression by flow cytometry. The fold change in Thy1.1 expression from 3 to 7 days relative to day 3 was plotted. Data represent the mean ± S.E.M. of three independent experiments with each vector. (b) Catalytically active casp8 rescues the defective survival of activated casp8−/− T cells. Cycling rate and accumulation profiles of Thy1.1+ cells, expressing pMit, C8-Wt, or C8-CA, were analyzed by CFSE staining. Representative flow cytometry traces of CFSE dilution histograms are shown; samples were collected for equivalent times
Figure 3
Figure 3
Full-length casp8 is enzymatically active in proliferating T cells. (a) Casp8 fingerprinting assay reveals active pro-casp8 in mitogen-stimulated T cells. Activated caspase labeling was performed in vivo before lysis using a biotinylated probe, bEVD, with purified mouse primary T cell following (a) activation for 36 h with anti-CD3 plus anti-CD28, or (b) incubation of activated T cells with plate-bound anti-Fas (5 μg/ml) for an additional 6 h. Lysates were separated using 2DGE; first dimension based on pI (pH 4–7 range) and second dimension based on molecular weight. Membranes were probed with anti-casp8, anti-casp3, and with an Avidin:Biotinylated-HRP enzyme complex. Immunoblots shown are representative of three independent experiments
Figure 4
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
Figure 5
Figure 5
Differential activation of casp8 in mitogenically stimulated CD8 versus CD4 T cells. (a) CD8+, but not CD4+, T cells exhibit significant IETDase activity after TCR stimulation. CD8+ or CD4+ T-cells subsets from Wt and FADDdd mice were magnetically purified using biotin anti-CD8 or biotin anti-CD4, respectively, and then streptavidin microbeads. After stimulation for the indicated times with anti-CD3 plus anti-CD28, IETDase catalytic activity was measured using the fluorogenic synthetic probe IETD-AFC. Experiments replicated using three mice per genotype; bar charts represent the mean ± S.E.M. (b) Rescue of casp8−/− T cells with wild-type casp8 is greater in the CD8+ subset. CD8+ and CD4+ T cells were stimulated (anti-CD3 plus anti-CD28) and transduced with retroviral supernatants-containing casp8 wild type (C8-Wt) or the empty vector pMit as in Figure 2a. Expression of these vectors was monitored by assessing Thy1.1 expression using flow cytometry. The fold change in Thy1.1 expression from 3 to 7 days relative to day 3 was plotted. Data represent the mean ± S.E.M. of three independent experiments with each vector and each cellular subset. (c) Similar induction of casp8 and c-FLIP expression in CD4+ versus CD8+ T cells. Western blots of naive or activated CD4+ and CD8+ T cells, from either Wt or FADDdd mice, were probed with anti-casp8 or anti-c-FLIP; blots were stripped and reprobed with anti-β-actin as a loading control. Numbers indicate the signal quantification made using ImageJ software. Normalization was performed using β-actin signal

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