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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) of L1210 (A and E), L1210.Fas (B), A1.1 (C), and TA3 (D) target cells induced by in vitro– derived alloreactive Tc cells. Splenocytes from B6 (•), gzmA−/− (○), gzmB−/− (□), gzmA×B−/− (▪), and perforin−/− (*) mice (pools of two spleens  per mouse strain, A–D; (E) spleens of four individual gzmA×B−/− (▪), one B6 mouse (•), and one perforin−/− mouse (*) were activated in primary  MLC (A–D, E, top) or secondary MLC (E, bottom), and tested for cytolytic (top) and nucleolytic (bottom) activities for the indicated time periods. All values are the mean lysis of triplicate samples at three e/t values given. SEM never exceeded 3%.
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Figure 2: 51Cr-release (top) and 125I-DNA release (bottom) of L1210 (A and E), L1210.Fas (B), A1.1 (C), and TA3 (D) target cells induced by in vitro– derived alloreactive Tc cells. Splenocytes from B6 (•), gzmA−/− (○), gzmB−/− (□), gzmA×B−/− (▪), and perforin−/− (*) mice (pools of two spleens per mouse strain, A–D; (E) spleens of four individual gzmA×B−/− (▪), one B6 mouse (•), and one perforin−/− mouse (*) were activated in primary MLC (A–D, E, top) or secondary MLC (E, bottom), and tested for cytolytic (top) and nucleolytic (bottom) activities for the indicated time periods. All values are the mean lysis of triplicate samples at three e/t values given. SEM never exceeded 3%.

Mentions: In vitro–derived H-2d–reactive Tc cells from gzmA−/−, gzmB−/−, gzmA×B−/−, perforin−/−, and B6 mice were tested for their ability to induce 51Cr-release in various target cell populations in 2–10-h cytotoxicity assays. Representative experiments are shown in Fig. 2, A–E. Tc cells from all gzm ko mice, including gzmA×B−/−, expressed cytolytic activities with kinetics and levels comparable to those seen with B6 Tc, when tested on either L1210.3, L1210.Fas, A1.1, or TA3 lymphoma target cells (Fig. 2, A–D, top panels) and P815 mastocytoma targets (data not shown). In addition, Tc cells from gzmA−/−, gzmB−/−, gzmA×B−/−, and B6 also lysed L1210.Fas target cells in the presence of EGTA-Mg2+ to the same extent, when tested at 10 h, indicating the involvement of the Fas pathway (1). Tc cells from perforin−/− mice did not express any cytolytic activity on the respective target cells with the exception that 51Cr-release was observed with L1210.Fas target cells in a 10-h assay, which was reduced in the presence of EGTA-Mg2+. As slight variations in lytic activity of gzmA×B−/− T cells occurred between individual sets of experiments, we tested the cytolytic activity of alloreactive Tc cell populations from eight (four male, four female) individual gzmA×B−/− mice and compared them with B6 alloreactive Tc cells in a primary and secondary MLC (Fig. 2 E, only shown for male mice). No differences in lytic activity could be discerned. Similar results were obtained with in vivo–derived H-2d reactive splenocytes from two individual mice of B6, gzmA−/−, gzmB−/−, gzmA×B−/−, and perforin−/− strains (Fig. 3). This was true for both target cells, L1210.3 and L1210.Fas, with the exception of one gzmA×B−/− mouse, which showed reduced cytolytic activity on both target cells. However, in vivo–generated alloreactive Tc cells from perforin−/− mice lysed L1210.Fas (Fig. 3) and P815 (data not shown) targets at levels comparable to those from B6 and gzm−/− mice. Lysis in the presence of EGTA-Mg2+ was only marginally reduced. The phenotype of the in vitro– and ex vivo–derived cytolytic cells from all mutant and B6 mice was CD4−CD8+ and no cytolytic activity was observed on H-2–matched EL4 target cells (data not shown). The differences observed in the cytolytic activity of in vitro– versus ex vivo–derived alloreactive Tc cells from perforin−/− mice may reflect differential regulation of Fas ligand and Fas expression during ex vivo induction and in vitro culture.


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) of L1210 (A and E), L1210.Fas (B), A1.1 (C), and TA3 (D) target cells induced by in vitro– derived alloreactive Tc cells. Splenocytes from B6 (•), gzmA−/− (○), gzmB−/− (□), gzmA×B−/− (▪), and perforin−/− (*) mice (pools of two spleens  per mouse strain, A–D; (E) spleens of four individual gzmA×B−/− (▪), one B6 mouse (•), and one perforin−/− mouse (*) were activated in primary  MLC (A–D, E, top) or secondary MLC (E, bottom), and tested for cytolytic (top) and nucleolytic (bottom) activities for the indicated time periods. All values are the mean lysis of triplicate samples at three e/t values given. SEM never exceeded 3%.
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Related In: Results  -  Collection

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Figure 2: 51Cr-release (top) and 125I-DNA release (bottom) of L1210 (A and E), L1210.Fas (B), A1.1 (C), and TA3 (D) target cells induced by in vitro– derived alloreactive Tc cells. Splenocytes from B6 (•), gzmA−/− (○), gzmB−/− (□), gzmA×B−/− (▪), and perforin−/− (*) mice (pools of two spleens per mouse strain, A–D; (E) spleens of four individual gzmA×B−/− (▪), one B6 mouse (•), and one perforin−/− mouse (*) were activated in primary MLC (A–D, E, top) or secondary MLC (E, bottom), and tested for cytolytic (top) and nucleolytic (bottom) activities for the indicated time periods. All values are the mean lysis of triplicate samples at three e/t values given. SEM never exceeded 3%.
Mentions: In vitro–derived H-2d–reactive Tc cells from gzmA−/−, gzmB−/−, gzmA×B−/−, perforin−/−, and B6 mice were tested for their ability to induce 51Cr-release in various target cell populations in 2–10-h cytotoxicity assays. Representative experiments are shown in Fig. 2, A–E. Tc cells from all gzm ko mice, including gzmA×B−/−, expressed cytolytic activities with kinetics and levels comparable to those seen with B6 Tc, when tested on either L1210.3, L1210.Fas, A1.1, or TA3 lymphoma target cells (Fig. 2, A–D, top panels) and P815 mastocytoma targets (data not shown). In addition, Tc cells from gzmA−/−, gzmB−/−, gzmA×B−/−, and B6 also lysed L1210.Fas target cells in the presence of EGTA-Mg2+ to the same extent, when tested at 10 h, indicating the involvement of the Fas pathway (1). Tc cells from perforin−/− mice did not express any cytolytic activity on the respective target cells with the exception that 51Cr-release was observed with L1210.Fas target cells in a 10-h assay, which was reduced in the presence of EGTA-Mg2+. As slight variations in lytic activity of gzmA×B−/− T cells occurred between individual sets of experiments, we tested the cytolytic activity of alloreactive Tc cell populations from eight (four male, four female) individual gzmA×B−/− mice and compared them with B6 alloreactive Tc cells in a primary and secondary MLC (Fig. 2 E, only shown for male mice). No differences in lytic activity could be discerned. Similar results were obtained with in vivo–derived H-2d reactive splenocytes from two individual mice of B6, gzmA−/−, gzmB−/−, gzmA×B−/−, and perforin−/− strains (Fig. 3). This was true for both target cells, L1210.3 and L1210.Fas, with the exception of one gzmA×B−/− mouse, which showed reduced cytolytic activity on both target cells. However, in vivo–generated alloreactive Tc cells from perforin−/− mice lysed L1210.Fas (Fig. 3) and P815 (data not shown) targets at levels comparable to those from B6 and gzm−/− mice. Lysis in the presence of EGTA-Mg2+ was only marginally reduced. The phenotype of the in vitro– and ex vivo–derived cytolytic cells from all mutant and B6 mice was CD4−CD8+ and no cytolytic activity was observed on H-2–matched EL4 target cells (data not shown). The differences observed in the cytolytic activity of in vitro– versus ex vivo–derived alloreactive Tc cells from perforin−/− mice may reflect differential regulation of Fas ligand and Fas expression during ex vivo induction and in vitro culture.

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