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Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic
1Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada.
Abstract:
Apoptosis induced by T cell receptor (TCR) triggering in T lymphocytes involves activation of cysteine proteases of the caspase family through their proteolytic processing. Caspase-3 cleavage was also reported during T cell stimulation in the absence of apoptosis, although the physiological relevance of this response remains unclear. We show here that the caspase inhibitor benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD) blocks proliferation, major histocompatibility complex class II expression, and blastic transformation during stimulation of peripheral blood lymphocytes. Moreover, T cell activation triggers the selective processing and activation of downstream caspases (caspase-3, -6, and -7), but not caspase-1, -2, or -4, as demonstrated even in intact cells using a cell-permeable fluorescent substrate. Caspase-3 processing occurs in different T cell subsets (CD4(+), CD8(+), CD45RA(+), and CD45RO(+)), and in activated B lymphocytes. The pathway leading to caspase activation involves death receptors and caspase-8, which is also processed after TCR triggering, but not caspase-9, which remains as a proenzyme. Most importantly, caspase activity results in a selective substrate specificity, since poly(ADP-ribose) polymerase (PARP), lamin B, and Wee1 kinase, but not DNA fragmentation factor (DFF45) or replication factor C (RFC140), are processed. Caspase and substrate processing occur in nonapoptotic lymphocytes. Thus, caspase activation is an early and physiological response in viable, stimulated lymphocytes, and appears to be involved in early steps of lymphocyte activation.
Insights
T cell activation triggers caspase processing in viable lymphocytes, impacting proliferation and MHC class II expression. This caspase activity is a physiological response involved in early lymphocyte activation steps.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- T cell receptor (TCR) triggering in T lymphocytes can induce apoptosis via caspase activation.
- Caspase-3 cleavage has been observed during T cell stimulation without apoptosis, but its physiological role is unclear.
Purpose of the Study:
- To investigate the role and physiological relevance of caspase activation during T cell activation.
- To determine the specific caspases involved and their substrates in non-apoptotic T cell stimulation.
Main Methods:
- Inhibition of caspases using benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD).
- Assessment of T cell proliferation, MHC class II expression, and blastic transformation.
- Detection of caspase processing and activation in intact cells using fluorescent substrates.
- Analysis of caspase involvement in different T cell subsets and B lymphocytes.
- Investigation of upstream pathways involving death receptors and caspase-8.
Main Results:
- zVAD inhibited T cell proliferation, MHC class II expression, and blastic transformation.
- T cell activation selectively processed and activated downstream caspases (caspase-3, -6, -7), but not caspase-1, -2, or -4.
- Caspase-3 processing occurred in various T cell subsets and activated B lymphocytes.
- The pathway involved death receptors and caspase-8, but not caspase-9.
- Specific substrates like PARP, lamin B, and Wee1 kinase were processed, while DFF45 and RFC140 were not.
- Caspase and substrate processing occurred in non-apoptotic lymphocytes.
Conclusions:
- Caspase activation is an early, physiological response in viable, stimulated lymphocytes.
- This caspase activity appears to be involved in the early stages of lymphocyte activation.
- The findings clarify the role of caspases beyond apoptosis in T cell signaling.