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In vitro- and ex vivo-derived cytolytic leukocytes from granzyme A x B double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells.

Simon MM, Hausmann M, Tran T, Ebnet K, Tschopp J, ThaHla R, Müllbacher A - J. Exp. Med. (1997)

Bottom Line: Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains unclear.In gzmAxB-/- mice, Tc/NK-mediated target cell DNA fragmentation was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2-4 h), but not long-term (4-10 h), nucleolytic assays.This suggests that gzmA and B are critical for Tc/NK granule- mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely to perforin and independent of both proteases.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Immunbiology, Freiburg, Germany. simon@immunbiol.mpg.de

ABSTRACT
Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains unclear. In this study, we compare the potential of Tc and natural killer (NK) cells of mice deficient in both gzmA and B (gzmAxB-/-) with those from single knockout mice deficient in gzmA (-/-), gzmB (-/-), or perforin (-/-) to induce nuclear damage and lysis in target cells. With the exception of perforin-/-, all in vitro- and ex vivo-derived Tc and NK cell populations from the mutant strains induced 51Cr-release in target cells at levels and with kinetics similar to those of normal mice. This contrasts with their capacity to induce apoptotic nuclear damage in target cells. In gzmAxB-/- mice, Tc/NK-mediated target cell DNA fragmentation was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2-4 h), but not long-term (4-10 h), nucleolytic assays. This suggests that gzmA and B are critical for Tc/NK granule- mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely to perforin and independent of both proteases.

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51Cr-release (top)  and 125I-DNA release (bottom)  from YAC-1 target cells by ex  vivo–derived NK cells from mutant or B6 mice. B6 (•),  gzmA−/− (○), gzmB−/− (□),  gzmA×B−/− (▪), and perforin−/−  (*) mice were either treated with  poly I:C (20 h) or infected with  SFV (2 d) and their splenocytes  (poly I:C, pool of two spleens;  SFV, pool of three spleens) were  tested for cytolytic (top) or nucleolytic (bottom) activities on  YAC-1 target cells. Assay times  were 2 and 4 h. All values are the  mean lysis of triplicate samples at  three or four e/t values given.  SEM never exceeded 3%.
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Figure 5: 51Cr-release (top) and 125I-DNA release (bottom) from YAC-1 target cells by ex vivo–derived NK cells from mutant or B6 mice. B6 (•), gzmA−/− (○), gzmB−/− (□), gzmA×B−/− (▪), and perforin−/− (*) mice were either treated with poly I:C (20 h) or infected with SFV (2 d) and their splenocytes (poly I:C, pool of two spleens; SFV, pool of three spleens) were tested for cytolytic (top) or nucleolytic (bottom) activities on YAC-1 target cells. Assay times were 2 and 4 h. All values are the mean lysis of triplicate samples at three or four e/t values given. SEM never exceeded 3%.

Mentions: To assess the cytolytic activities of ex vivo–derived NK cell populations of mutant and B6 mice, recipients were treated with either poly I:C (29) or SFV (30). Splenocytes were tested for lytic activity on 51Cr-labeled YAC-1 target cells. Similar levels of cytolytic activity were observed with effector cell populations from gzmA−/−, gzmB−/−, gzmA× B−/−, and B6 mice, when assayed between 2 and 4 h (Fig. 5, top). The occasional reduction in the activity of NK cells from gzm single or double ko mice as compared to B6 mice to induce 51Cr-release was neither significant nor reproducible in three additional experiments (data not shown). As shown in previous studies (2), marginal or no NK cell– mediated lytic activity was obtained with splenocytes from perforin−/− mice.


In vitro- and ex vivo-derived cytolytic leukocytes from granzyme A x B double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells.

Simon MM, Hausmann M, Tran T, Ebnet K, Tschopp J, ThaHla R, Müllbacher A - J. Exp. Med. (1997)

51Cr-release (top)  and 125I-DNA release (bottom)  from YAC-1 target cells by ex  vivo–derived NK cells from mutant or B6 mice. B6 (•),  gzmA−/− (○), gzmB−/− (□),  gzmA×B−/− (▪), and perforin−/−  (*) mice were either treated with  poly I:C (20 h) or infected with  SFV (2 d) and their splenocytes  (poly I:C, pool of two spleens;  SFV, pool of three spleens) were  tested for cytolytic (top) or nucleolytic (bottom) activities on  YAC-1 target cells. Assay times  were 2 and 4 h. All values are the  mean lysis of triplicate samples at  three or four e/t values given.  SEM never exceeded 3%.
© Copyright Policy
Related In: Results  -  Collection

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Figure 5: 51Cr-release (top) and 125I-DNA release (bottom) from YAC-1 target cells by ex vivo–derived NK cells from mutant or B6 mice. B6 (•), gzmA−/− (○), gzmB−/− (□), gzmA×B−/− (▪), and perforin−/− (*) mice were either treated with poly I:C (20 h) or infected with SFV (2 d) and their splenocytes (poly I:C, pool of two spleens; SFV, pool of three spleens) were tested for cytolytic (top) or nucleolytic (bottom) activities on YAC-1 target cells. Assay times were 2 and 4 h. All values are the mean lysis of triplicate samples at three or four e/t values given. SEM never exceeded 3%.
Mentions: To assess the cytolytic activities of ex vivo–derived NK cell populations of mutant and B6 mice, recipients were treated with either poly I:C (29) or SFV (30). Splenocytes were tested for lytic activity on 51Cr-labeled YAC-1 target cells. Similar levels of cytolytic activity were observed with effector cell populations from gzmA−/−, gzmB−/−, gzmA× B−/−, and B6 mice, when assayed between 2 and 4 h (Fig. 5, top). The occasional reduction in the activity of NK cells from gzm single or double ko mice as compared to B6 mice to induce 51Cr-release was neither significant nor reproducible in three additional experiments (data not shown). As shown in previous studies (2), marginal or no NK cell– mediated lytic activity was obtained with splenocytes from perforin−/− mice.

Bottom Line: Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains unclear.In gzmAxB-/- mice, Tc/NK-mediated target cell DNA fragmentation was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2-4 h), but not long-term (4-10 h), nucleolytic assays.This suggests that gzmA and B are critical for Tc/NK granule- mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely to perforin and independent of both proteases.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Immunbiology, Freiburg, Germany. simon@immunbiol.mpg.de

ABSTRACT
Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains unclear. In this study, we compare the potential of Tc and natural killer (NK) cells of mice deficient in both gzmA and B (gzmAxB-/-) with those from single knockout mice deficient in gzmA (-/-), gzmB (-/-), or perforin (-/-) to induce nuclear damage and lysis in target cells. With the exception of perforin-/-, all in vitro- and ex vivo-derived Tc and NK cell populations from the mutant strains induced 51Cr-release in target cells at levels and with kinetics similar to those of normal mice. This contrasts with their capacity to induce apoptotic nuclear damage in target cells. In gzmAxB-/- mice, Tc/NK-mediated target cell DNA fragmentation was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2-4 h), but not long-term (4-10 h), nucleolytic assays. This suggests that gzmA and B are critical for Tc/NK granule- mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely to perforin and independent of both proteases.

Show MeSH
Related in: MedlinePlus