Results presented in this report show that caspase activation after TCR triggering is a physiological, tightly regulated, and early response that appears to be required for efficient T cell activation. Indeed, the selective processing of caspase-3, -6, -7, and -8 was detected within 24 h after anti-CD3 stimulation of peripheral blood lymphocytes. Caspase processing occurred in various T and B cell subsets, and was found in proliferating and nonapoptotic lymphocytes. Activation of caspases was confirmed through binding of caspase-3–processed forms to a specific substrate, and by showing that a cell-permeable substrate was cleaved in intact, activated lymphocytes. Importantly, activation of the caspase cascade was associated to restricted substrate specificity, with cleavage of PARP and Wee1 being observed while two other substrates, DFF45 and RFC140, remained unaffected. Caspase processing after T cell stimulation correlated with a defective lymphocyte activation in the presence of the caspase inhibitor zVAD, suggesting that caspase activity could be involved in some early steps of lymphocyte activation.
Death receptors such as TNFR and Fas can act as costimulatory molecules and enhance T cell proliferation 252627
. We have also observed that a soluble form of TNFR significantly inhibited T cell proliferation induced by anti-CD3 antibody (our unpublished results). Cross-linking of these death receptors triggers the FADD-dependent recruitment of caspase-8, which is processed and can directly mediate the activation of caspase-3, -6, and -7 by proteolytic cleavage 3637
. Our results show that caspase-8 is processed after T cell stimulation (as early as 16 h after TCR cross-linking), and activates caspase-3, -6, and –7, leading to a selective cleavage of their substrates. Interestingly, addition of the caspase-8 inhibitor zIETD during T cell stimulation inhibited >60% of the proliferation (data not shown), which further confirms that caspase-8 activity is involved in cell cycle entry of activated lymphocytes. The fact that activation of the whole caspase cascade occurs in proliferating, viable (AV−
) cells indicates that caspases could be the executioners in the costimulatory function of members of the TNFR family. In support of this hypothesis, impaired T cell proliferation is also observed in FADD-deficient and in FADD dominant negative transgenic mice 202122
, suggesting a potential role for caspases in the early events leading to cell division. Caspase processing in stimulated cells appears to be mediated mostly through the activation of caspase-8 associated to death receptor. The other pathway of downstream caspase activation involves caspase-9, which is triggered after the release in the cytosol of mitochondrial cytochrome c 3839
. However, we have found that in stimulated T cells this caspase remains as a proenzyme.
Bcl-2 and other antiapoptotic members of the family have been shown to block caspase activation after several apoptotic stimuli 43
. However, Bcl-2 is also endowed with the ability to influence cell cycle progression. Indeed, Bcl-2 overexpression in transgenic mice was reported to decrease cell cycle entry of primary lymphocytes 23
. Transfection of Bcl-2 into NIH 3T3 cells also delayed their reentry into cell cycle after serum withdrawal, and a tyrosine 28 mutant in the NH2
-terminal BH4 domain exhibited dominant negative effects over the wild-type Bcl-2 24
. Whether the effect of Bcl-2 on cell cycle progression also involves regulation of caspase activity remains to be addressed. However, these data associated with our results showing the inhibitory effect of zVAD on DNA synthesis suggest that caspases (along with their upstream regulators FADD and Bcl-2) may be involved in cell cycle entry during lymphocyte activation.
Results presented in this study clearly demonstrate a selective processing of caspase substrates. PARP, which is involved in DNA repair 44
, is probably processed in apoptotic cells to allow for DNA fragmentation after DFF45 cleavage. Of note, PARP expression and activity increase after T cell stimulation 41
. Therefore, the 85-kD fragment of PARP probably conserves an enzymatic activity that mediates an unknown function in proliferating cells. Our results also demonstrate the selective processing of Wee1. This tyrosine kinase is a negative regulator of Cdc2 45
, a cyclin-dependent kinase required for the G2
/M transition during cell cycle, as well as for Fas-mediated apoptosis in immortalized cell lines 13
. Wee1 is a critical component of the G2
/M cell cycle checkpoint machinery, and mediates cell cycle arrest after DNA damage by phosphorylation of Cdc2 45
. Therefore, cleavage of Wee1 in proliferating lymphocytes could lead to its inactivation, thus allowing cell cycle progression. Of note, Wee1 processing by caspases during apoptosis in Jurkat cells correlates with a 20-fold decrease in Wee1 activity and an increase in Cdc2 activity 13
. The nuclear protein lamin B is considered as a caspase-6 substrate 7
. Our results show that in fresh PBMCs, two forms of 66 and 45 kD can be identified by Western blot using an anti–lamin B antibody, and at least two fragments of 35 and 28 kD are produced after T cell activation. Appearance of these products correlates with a decrease in the amount of the 45-kD form of lamin B, and is inhibited by addition of zVAD ( A). Although the significance of this cleavage in activated lymphocytes is unclear, it confirms the activation of caspase-6, which generates the 28-kD fragment after a cleavage at the consensus sequence VEID 7
In our experiments, DFF45 is present in resting as well as in proliferating cells, whereas RFC140 is upregulated after T cell stimulation. Since caspase-mediated DFF45 processing leads to DNA fragmentation, one could easily predict that it would not be cleaved in activated living cells. Indeed, we were unable to detect DFF45 cleavage in AV−
cells after T cell activation, although the 14-kD fragment generated by caspase-3 cleavage at the second site was observed in lysate from activated lymphocytes after anti-Fas treatment. The lack of RFC140 cleavage in proliferating T cells was also critical, since processing of RFC140 by caspases leads to inactivation of the DNA replication machinery and cell cycle arrest at the G2
/M boundary 11
. This selective substrate processing could explain why T lymphocytes survive and proliferate although the caspase cascade including caspase-8, -3, -6, and -7 is activated after TCR triggering. To explain this selective caspase specificity in stimulated lymphocytes, several hypotheses can be proposed. Endogenous caspase inhibitors, such as the inhibitor of apoptosis (IAP) family members, could selectively inhibit the cleavage of specific substrates in stimulated cells 46
. Supporting this hypothesis, the expression of thymic IAP, a murine homologue of survivin, is upregulated within 24 h of stimulation in splenic T cells 47
, and this member of the IAP family was shown to inhibit already processed caspase-3, -7, and -9 46
. Association of thymic IAP to caspases could have a differential effect on the cleavage of their substrates, depending on the affinity of the caspase for these substrates or their accessibility. Alternatively, some caspase substrates involved in cell survival could be protected against cleavage by phosphorylation near the caspase cleavage site. This mechanism was recently demonstrated for presenilin-2. Caspase-mediated cleavage of this molecule was abrogated by Ser327
phosphorylation inside the consensus cleavage site DSYD↓S 48
In summary, activation of the caspase cascade in nonapoptotic lymphocytes, together with the impairment of T cell activation by caspase inhibitors, indicates that these proteases may play an important role during T cell stimulation, and caspases may be added to the growing list of molecules that are involved in cell death and proliferation. However, elucidation of the function of caspase processing in activated lymphocytes will require further experiments and will provide new insights into the biological function of caspases, as well as the regulation of T cell activation.