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Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation.

Balaji KN, Schaschke N, Machleidt W, Catalfamo M, Henkart PA - J. Exp. Med. (2002)

Bottom Line: Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering.Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B.Degranulated CTLs are surface biotinylated by the cathepsin B-specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Building 10, Bethesda, MD 20892, USA.

ABSTRACT
The granule exocytosis cytotoxicity pathway is the major molecular mechanism for cytotoxic T lymphocyte (CTL) and natural killer (NK) cytotoxicity, but the question of how these cytotoxic lymphocytes avoid self-destruction after secreting perforin has remained unresolved. We show that CTL and NK cells die within a few hours if they are triggered to degranulate in the presence of nontoxic thiol cathepsin protease inhibitors. The potent activity of the impermeant, highly cathepsin B-specific membrane inhibitors CA074 and NS-196 strongly implicates extracellular cathepsin B. CTL suicide in the presence of cathepsin inhibitors requires the granule exocytosis cytotoxicity pathway, as it is normal with CTLs from gld mice, but does not occur in CTLs from perforin knockout mice. Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering. Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B. Degranulated CTLs are surface biotinylated by the cathepsin B-specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B. These experiments support a model in which granule-derived surface cathepsin B provides self-protection for degranulating cytotoxic lymphocytes.

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TCR cross-linking rapidly increases CTL surface expression of cathepsin B, but not cathepsin L. (B and D) CD8+ cloned human CTL were incubated on surface-bound anti-CD3, or (A and C) isotype IgG for 2 h at 37°C. (A and B) Cells were then stained with anti-cathepsin B or (C and D) with anti-cathepsin L antibody followed by FITC-anti–mouse IgG (heavy lines). Dashed lines show staining by normal IgG controls.
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fig4: TCR cross-linking rapidly increases CTL surface expression of cathepsin B, but not cathepsin L. (B and D) CD8+ cloned human CTL were incubated on surface-bound anti-CD3, or (A and C) isotype IgG for 2 h at 37°C. (A and B) Cells were then stained with anti-cathepsin B or (C and D) with anti-cathepsin L antibody followed by FITC-anti–mouse IgG (heavy lines). Dashed lines show staining by normal IgG controls.

Mentions: Using flow cytometry, cells from a human CD8+ CTL clone were examined for surface expression of cathepsin B after a degranulation stimulus. Fig. 4 shows that nonstimulated CTL did not express significant levels of surface cathepsin B, although it was minimally detectable in some experiments. However, within 2 h after exposure to plate-bound anti-CD3, cathepsin B was clearly increased with a homogenous peak of positive cells. In contrast, cathepsin L showed weak surface expression on unstimulated CTL, which was not increased after CD3 cross-linking.


Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation.

Balaji KN, Schaschke N, Machleidt W, Catalfamo M, Henkart PA - J. Exp. Med. (2002)

TCR cross-linking rapidly increases CTL surface expression of cathepsin B, but not cathepsin L. (B and D) CD8+ cloned human CTL were incubated on surface-bound anti-CD3, or (A and C) isotype IgG for 2 h at 37°C. (A and B) Cells were then stained with anti-cathepsin B or (C and D) with anti-cathepsin L antibody followed by FITC-anti–mouse IgG (heavy lines). Dashed lines show staining by normal IgG controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196055&req=5

fig4: TCR cross-linking rapidly increases CTL surface expression of cathepsin B, but not cathepsin L. (B and D) CD8+ cloned human CTL were incubated on surface-bound anti-CD3, or (A and C) isotype IgG for 2 h at 37°C. (A and B) Cells were then stained with anti-cathepsin B or (C and D) with anti-cathepsin L antibody followed by FITC-anti–mouse IgG (heavy lines). Dashed lines show staining by normal IgG controls.
Mentions: Using flow cytometry, cells from a human CD8+ CTL clone were examined for surface expression of cathepsin B after a degranulation stimulus. Fig. 4 shows that nonstimulated CTL did not express significant levels of surface cathepsin B, although it was minimally detectable in some experiments. However, within 2 h after exposure to plate-bound anti-CD3, cathepsin B was clearly increased with a homogenous peak of positive cells. In contrast, cathepsin L showed weak surface expression on unstimulated CTL, which was not increased after CD3 cross-linking.

Bottom Line: Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering.Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B.Degranulated CTLs are surface biotinylated by the cathepsin B-specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B.

View Article: PubMed Central - PubMed

Affiliation: Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Building 10, Bethesda, MD 20892, USA.

ABSTRACT
The granule exocytosis cytotoxicity pathway is the major molecular mechanism for cytotoxic T lymphocyte (CTL) and natural killer (NK) cytotoxicity, but the question of how these cytotoxic lymphocytes avoid self-destruction after secreting perforin has remained unresolved. We show that CTL and NK cells die within a few hours if they are triggered to degranulate in the presence of nontoxic thiol cathepsin protease inhibitors. The potent activity of the impermeant, highly cathepsin B-specific membrane inhibitors CA074 and NS-196 strongly implicates extracellular cathepsin B. CTL suicide in the presence of cathepsin inhibitors requires the granule exocytosis cytotoxicity pathway, as it is normal with CTLs from gld mice, but does not occur in CTLs from perforin knockout mice. Flow cytometry shows that CTLs express low to undetectable levels of cathepsin B on their surface before degranulation, with a substantial rapid increase after T cell receptor triggering. Surface cathepsin B eluted from live CTL after degranulation by calcium chelation is the single chain processed form of active cathepsin B. Degranulated CTLs are surface biotinylated by the cathepsin B-specific affinity reagent NS-196, which exclusively labels immunoreactive cathepsin B. These experiments support a model in which granule-derived surface cathepsin B provides self-protection for degranulating cytotoxic lymphocytes.

Show MeSH
Related in: MedlinePlus