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Original Article

^a Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, Montréal, Québec H2W 1R7, Canada
^b Département de Microbiologie et d'Immunologie, Université de Montréal, Montréal, Québec H3C 3J7, Canada
^c Department of Microbiology and Immunology, McGill University, Montréal, Québec H3A 2B4, Canada
^d Department of Experimental Medicine, McGill University, Montréal, Québec H3A 2B4, Canada
Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, 110 avenue des Pins Ouest, Montréal, Québec H2W 1R7, Canada.514-987-5711514-987-5550

sekalyr{at}ircm.qc.ca

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.

Key Words: T cell receptor • proliferation • apoptosis • protease • inhibitor

Abbreviations used in this paper: AV, annexin V; Cdc2, cell division control 2; DFF45, the 45-kd subunit of DNA fragmentation factor; Extra-HRP, HRP–conjugated extravidin; FADD, Fas-associated death domain protein; HRP, horseradish peroxidase; IAP, inhibitor of apoptosis; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; RFC140, the 140-kD subunit of replication factor C; SAC, Staphylococcus aureus Cowan cell extracts; zVAD, benzyloxycarbonyl (Cbz)-Val-Ala-Asp(OMe)-fluoromethylketone.

A. Alam and L.Y. Cohen contributed equally to this work.

© 1999 The Rockefeller University Press

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