Monoclonal antibodies detecting discrete epitopes of human perforin

Geisberg, Mark; Trapani, Joseph A.; Dupont, Bo

doi:10.1111/j.1399-0039.1990.tb01788.x

Cited by 9 publications

(4 citation statements)

References 28 publications

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“…Western Blotting-Western blotting was performed essentially as described previously (18). The mAbs used were 2C5, a mouse IgG2a that reacts specifically with human and rat granzyme B (18), and PB2, a mouse IgG1 reacting with human perforin (19). An antiserum reactive with human BiP was purchased (StressGen, Victoria, Canada).…”

Section: Methodsmentioning

confidence: 99%

Localization of Granzyme B in the Nucleus

Trapani¹,

Browne²,

Smyth³

et al. 1996

Journal of Biological Chemistry

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One mechanism used by cytotoxic T cells and natural killer cells to kill target cells involves synergy between the pore-forming protein, perforin, and a serine protease termed granzyme B, both constituents of the cytoplasmic granules of cytolytic lymphocytes. Exposing susceptible cells to perforin and granzyme B results in apoptosis, the morphological consequences of which are most clearly seen in the nucleus. It is conventionally accepted that perforin acts by perforating the target cell membrane; however, the site and mode of action of granzyme B are unknown. We have addressed this issue using Western blotting, proteolytic assays, and confocal laser scanning microscopy to demonstrate that purified human granzyme B can be taken up in large amounts and bound within nuclei. By contrast, perforin and nongranzyme serine proteases did not undergo nuclear uptake. Both unglycosylated human granzyme B (26 kDa) and that bearing high mannose glycosylation (32 kDa) were internalized and bound within nuclei, but forms greater than 32 kDa with complex carbohydrate addition were excluded. The uptake of granzymes was not dependent on net charge, as nuclei absorbed similar quantities of granzyme B at neutral pH and through a range of basic pHs but did not take up other very basic serine proteases such as the mouse mast cell protease 5. Confocal laser scanning microscopy indicated nuclear and nucleolar accumulation of fluoresceinated granzyme B by isolated nuclei. Measurement of the kinetics of nuclear import using an in vitro nuclear transport assay indicated maximal levels of nuclear accumulation of granzyme about 2.5-fold above those in the cytoplasm and nucleolar accumulation a further 3-4-fold higher. Nuclear and nucleolar accumulation were exceedingly rapid, reaching half-maximal levels within 3.3 and 7.5 min, respectively, implying that nuclear accumulation probably occurs prior to transport to the nucleolus. Our observations may provide a mechanism explaining how aspartate-specific cell death proteases access the nuclear substrate poly(ADP-ribose) polymerase, the cleavage of which is an early event in apoptosis.

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Section: Methodsmentioning

confidence: 99%

Localization of Granzyme B in the Nucleus

Trapani¹,

Browne²,

Smyth³

et al. 1996

Journal of Biological Chemistry

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“…An antibody against perforin (PFP; IgG2b), was kindly provided by Professor Bo Dupont (Laboratory of Human Immunogenetics, Sloan-Kettering Cancer Center, New York; ref. 9). Another cytotoxic granule protein was identified by the antibody TIA-1 (IgG1; Coulter Clone, Luton, UK).…”

Section: Methodsmentioning

confidence: 99%

The specific recognition by macrophages of CD8+,CD45RO+ T cells undergoing apoptosis: a mechanism for T cell clearance during resolution of viral infections.

Akbar

Savill

Gombert

et al. 1994

The Journal of Experimental Medicine

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Summal~During viral infections, CD8 +,CD45RO + T populations expand. These primed cells express abundant levels of cytoplasmic granules that contain perforin and TIA-L Recent work has suggested that the majority of this CD8 + population downregulates Bcl-2 protein expression and is destined to undergo apoptosis. In this study we have investigated the elimination of these apoptotic CD8 § T cells by both human monocyte-derived and murine bone marrow macrophages. We have found that these phagocytes recognize and ingest both apoptotic CD8 + and CD4 + T cells using an Otv33 (vitronectin receptor)/CD36/thrombospondin recognition system, with the same receptors being used in the recognition of apoptotic neutrophils. These data provide new evidence for a mechanism that enables the clearance of greatly increased populations of CD8 + effector cells which are found during viral infections. This enables cellular homeostasis to occur in the host upon resolution of viral diseases in vivo.

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“…Antibodies-Antibodies used in Western blotting and immunohistochemistry included several mAbs made by our laboratory: 2C5 (antigzm B) (19), 4H10 (anti-gzm M) (20), PB2 (anti-perforin) (21), and 7D8 (anti-PI-9) (22). In addition, mAb GB7 (anti-gzm B) was purchased from Serotec.…”

Section: Methodsmentioning

confidence: 99%

Discordant Regulation of Granzyme H and Granzyme B Expression in Human Lymphocytes

Sedelies

Sayers

Edwards

et al. 2004

Journal of Biological Chemistry

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We analyzed the expression of granzyme H in human blood leukocytes, using a novel monoclonal antibody raised against recombinant granzyme H. 33-kDa granzyme H was easily detected in unfractionated peripheral blood mononuclear cells, due to its high constitutive expression in CD3 ؊ CD56 ؉ natural killer (NK) cells, whereas granzyme B was less abundant. The NK lymphoma cell lines, YT and Lopez, also expressed high granzyme H levels. Unstimulated CD4؉ and particularly CD8 ؉ T cells expressed far lower levels of granzyme H than NK cells, and various agents that classically induce T cell activation, proliferation, and enhanced granzyme B expression failed to induce granzyme H expression in T cells. Also, granzyme H was not detected in NK T cells, monocytes, or neutrophils. There was a good correlation between mRNA and protein expression in cells that synthesize both granzymes B and H, suggesting that gzmH gene transcription is regulated similarly to gzmB. Overall, our data indicate that although the gzmB and gzmH genes are tightly linked, expression of the proteins is quite discordant in T and NK cells. The finding that granzyme H is frequently more abundant than granzyme B in NK cells is consistent with a role for granzyme H in complementing the pro-apoptotic function of granzyme B in human NK cells.

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Monoclonal antibodies detecting discrete epitopes of human perforin

Cited by 9 publications

References 28 publications

Localization of Granzyme B in the Nucleus

Localization of Granzyme B in the Nucleus

The specific recognition by macrophages of CD8+,CD45RO+ T cells undergoing apoptosis: a mechanism for T cell clearance during resolution of viral infections.

Discordant Regulation of Granzyme H and Granzyme B Expression in Human Lymphocytes

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