Apoptotic death concurrent with CD3 stimulation in primary human CD8+ T lymphocytes: a role for endogenous granzyme B.
We exposed primary CD8(+) T cells to soluble CD3 mAb plus IL-2 and limited numbers of monocytes (3%). These cells were activated but concurrently subjected to ongoing apoptosis ( approximately 25% were apoptotic from day 2 of culture). However, their costimulated CD4(+) counterparts were much less prone to apoptosis. The apoptotic signaling pathway bypassed Fas and TNFRs, and required the activity of cathepsin C, a protease which performs the proteolytic maturation of granzyme (Gr) A and GrB proenzymes within the cytolytic granules. Silencing the GrB gene by RNA interference in activated CD8(+) T cells prevented the activation of procaspase-3 and Bid, and indicated that GrB was the upstream death mediator. A GrB-specific mAb immunoprecipitated a approximately 70-kDa molecular complex from cytolytic extracts of activated CD8(+) (but not resting) T cells, that was specifically recognized by a nucleocytoplasmic protease inhibitor 9 (PI-9) specific mAb. This complex was also detected after reciprocal immunoprecipitation of PI-9. It coexisted in the cytosol with the 32-kDa form of GrB. As neither were detected in the cytosol of CD4(+) bystander T cells (which poorly synthesized GrB), and as silencing the perforin (Pf) gene had no effect in our system, endogenous GrB was likely implicated. Immunoprecipitation experiments failed to reveal Pf in the cytosol of CD8(+) T cells, and only a tiny efflux of granular GrA was detected by ELISA. We propose that some GrB is released from cytolytic granules to the cytosol of CD8(+) T lymphocytes upon CD3/TCR stimulation and escapes PI-9, thereby mediating apoptotic cell death.
Laboratoire de Greffes d'Epithéliums et Régulation de l'Activation Lymphocytaire, Unité 542, Institut National de la Santé et de la Recherche Médicale, Hôpital Paul Brousse, Villejuif, France.
Mireille
M
Laforge
Nicolas
N
Bidère
Sylvie
S
Carmona
Aurore
A
Devocelle
Bernard
B
Charpentier
Anna
A
Senik
D000906:Antibodies / Q000276:immunology; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000378:metabolism; D017252:CD3 Complex / Q000276:immunology / Q000378:metabolism*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002403:Cathepsins / Q000378:metabolism; D002457:Cell Extracts; D002478:Cells, Cultured; D003600:Cytosol / Q000276:immunology / Q000378:metabolism; D015536:Down-Regulation; D053804:Granzymes; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011485:Protein Binding; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism
D000906:Antibodies; D051023:BH3 Interacting Domain Death Agonist Protein; D017252:CD3 Complex; D002457:Cell Extracts; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D034741:RNA, Small Interfering; D015843:Serpins; D054353:Perforin; D002403:Cathepsins; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases