Materials and methods

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

Animals and cell lines

B10.D2 mice (Olac, Bicester, UK) were kept under specific pathogen free conditions and were used at the age of 6–8 weeks. All animal experiments were performed according to the standards required by the UKCCCR Guidelines. Eb is a chemically induced T cell lymphoma (L5178Y/E) of DBA/2 mice. ESb-MP is a spontaneous high metastatic adhesion variant of Eb and arose most likely after fusion of Eb cells with a host macrophage (Larizza et al, 1984). Jurkat is a human leukaemia T cell line, BL60 is a human leukaemia B cell line and U937 is a human promonocytic cell line. Cell lines were maintained in 5% CO₂ at 37°C in RPMI-1640 containing 10% FCS (both Life Technologies, Eggenstein, Germany). In some experiments, V6⁺ T cells were primed by injecting 5 10⁴ ESb-MP tumour cells in PBS into the ear pinna of B10.D2 mice. Animals were sacrificed 9 days after injection.

Abs and other reagents

The rat-anti-mouse V6 mAb was used as culture supernatant (clone 44-22-1) (Payne et al, 1988). The anti-APO-1 antibody (Trauth et al, 1989) is directed against human (hu) CD95. Hu TRAIL-R2-Fc, hu TNF-R2-Fc and hu CD95-Fc were used in the blocking experiments (Walczak et al, 1997, 1999). Leucine Zipper (LZ)-TRAIL, TNF- and LZ-CD95L were used for induction of apoptosis (Walczak et al, 1999). All human reagents are also reactive to murine cells (Walczak et al, 1999). Human IgG was purchased from Sigma (Deisenhofen, Germany). Recombinant (rec.) human IFN- was obtained from Biomol (Hamburg, Germany). Annexin V-FITC was obtained from R&D systems (Wiesbaden, Germany). The following caspase inhibitors were purchased from Bachem (Heidelberg, Germany): ZVAD-fmk (Z-Val-Ala-DL-Asp-fluoromethylketone) and IETD-CHO (Ac-Ile-Glu-Thr-Asp-aldehyde (pseudo acid). JC1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazol-carbocyanine iodide) was from Molecular Probes (Eugene, OR, USA). Concanamycin A and granzyme B inhibitor (Z-AAD-CMK) were obtained from Calbiochem (Bad Soden, Germany). To evaluate apoptotic effects of nitric oxide (NO), glycerol trinitrate (GTN; Merck; Darmstadt, Germany) was used.

Isolation of V6⁺ T cells via magnetic beads

V6⁺ T cells were isolated as previously described (Müerköster et al, 1998). Spleen cells from B10.D2 mice were incubated with rat-anti-V6 mAbs and with magnetic beads-conjugated anti-rat IgG isotype Abs (Dynal, Hamburg, Germany). Cells were separated via a Dynal-magnet. The purity of the positively selected V6⁺ T cells was >88%. In some experiments, CD4⁺ or CD8⁺ T cells were isolated by depletion of CD8⁺ or CD4⁺ T cells by Dynabeads-conjugated anti-mouse CD8 or Dynabeads-conjugated anti-mouse CD4 (both Dynal) respectively. Then CD4⁺V6⁺ and CD8⁺V6⁺ T cells were isolated as previously described (Müerköster et al, 1998). After isolation, CD8⁺V6⁺ T cell population contained 0.4% CD4⁺ cells and the population of CD4⁺V6⁺ T cells 0.3% CD8⁺ cells.

Mixed lymphocyte tumour cell culture for induction of cytotoxic actitivity

Isolated V6⁺ T cells from normal or immunised B10.D2 mice were incubated with 100 Gy -irradiated (Gammacell 1000, Ottawa, Canada) V6 negative spleen cells (used as APC) and ESb-MP or Eb tumour cells for 4 days. Cytotoxic acticity of in vitro stimulated V6⁺ T cells were tested either by a ⁵¹Cr-release-assay or by FACS analysis.

⁵¹Cr-release-assay

ESb-MP and Eb target cells were labelled with 0.2 Ci ⁵¹Cr sodium chromate (Amersham, Braunschweig, Germany) in RPMI-1640 medium with 30% FCS for 90 min at 37°C. V6⁺ effector T cells were incubated with target cells (effector : target ratio 10 : 1) in 96-well round bottom plates (Renner, Dannstadt, Germany) for 4 h at 37°C. Radioactivity released in supernatants was measured by a -counter (LKB-Wallac, Freiburg, Germany) as described (Schirrmacher et al, 2000). In all experiments, an effector : target ratio of 10 : 1 was used. This ratio has been previously shown to be an optimal (Schirrmacher et al, 2000).

Determination of cytotoxicity via flow cytometry

Effector cells (V6⁺ T cells) were incubated with target ESb-MP cells (10 : 1 ratio) in 6-well plates for different periods of time (10–240 min). To determine which death receptors could be involved in the induction of apoptosis, ESb-MP cells were treated with 1 or 10 g ml^-1 LZ-TRAIL, 1 g ml^-1 LZ-CD95L, TNF- or 10 g ml^-1 control IgG for 4 h without or with 24 h pre-incubation with IFN-. As a control for induction of apoptosis, either ESb-MP cells were incubated with 0.5 mM GTN for 24 h or Jurkat cells were treated with 1 g ml^-1 anti-APO-1/Protein A (10 ng ml^-1, Sigma) or 1 g ml^-1 LZ-CD95L, BL60 cells were treated with 1 g ml^-1 LZ-TRAIL and U937 cells were treated with TNF- overnight to induce apoptosis. To block apoptosis in ESb-MP cells, the following reagents were used: hu TRAIL-R2-Fc (10 g ml^-1), hu CD95-Fc (10 g ml^-1), hu TNF-R2-Fc (10 g ml^-1), hu IgG1 as isotype control (10 g ml^-1), Concanamycin A (10 nM), granzyme B inhibitor (20 M), ZVAD-fmk (25 M) and IETD-CHO (25 M). Treatment of effector and target cells with the respective reagent started 15–60 min before the co-incubation. As a positive control, 1–30 g ml^-1 CD95-Fc, TNF-R2-Fc and TRAIL-R2-Fc were used to block apoptosis in Jurkat, BL60 and U937 cells. After removal of effector cells, ESb-MP cells were washed, detached from the plates, and cytotoxicity was determined by staining with 1 g ml^-1 propidium iodide (PI) which was added to the cells 5 min before measurement.

Target cell apoptosis

Apoptosis was assessed either by Annexin V/PI staining (Koopman et al, 1994) or by determining DNA-fragmentation (Nicoletti et al, 1991). After co-incubation with effector cells, 5 10⁵ ESb-MP target cells were either resuspended in binding buffer (R&D systems), 50 ng ml^-1 Annexin V and 1 g ml^-1 PI or treated with a hypotonic solution (0.1% sodium citrate, 0.1% Triton X-100) containing 50 g ml^-1 PI overnight. Jurkat cells were incubated in the hypotonic solution containing PI and DNA-fragmentation was measured as described above. Target cell death and apoptosis were analyzed by flow cytometry using a FACScan analyzer with CellQuest software (BD Bioscience, Heidelberg, Germany).

Cytofluorometric analysis of mitochondrial transmembrane potential (_m)

_m was measured with JC1 as described (Salvioli et al, 1997). JC1 is a cyanine dye which accumulates in the mitochondrial matrix under the influence of the _m. In the presence of a high _m, JC1 forms aggregates which have characteristic absorption and emission spectra so that cells are detectable as FL1^- and FL2⁺. 5 10⁵ ESb-MP tumour cells were treated with 5 g ml^-1 JC1 for 20 min at 4°C followed by FACScan analysis. As a control, Jurkat cells were treated with 1 g ml^-1 anti-APO-1 mAb crosslinked by 10 ng ml^-1 Protein A for 12 h followed by incubation with JC1 as described above.

Detection of perforin in V6⁺ T cells

Isolated V6⁺ T cells were stimulated either with ESb-MP or with Eb cells and APCs or were left without any stimulation. After 4 days supernatants were taken and centrifuged by 500 r.p.m. for 5 min. The pellet containing V6⁺ T cells were washed with PBS and centrifuged on siliconised glass slides (Sigma). After drying, slides were fixed in acetone for 10 min at room temperature followed by washing in PBS. To avoid nonspecific binding, slides were treated with 1% normal goat serum for 15 min followed by incubation with the rat-anti-mouse perforin mAb (Alexis, Grünberg, Germany) for 45 min. After washing, slides were treated with a goat-anti-rat antibody conjugated with alkaline phosphatase (AP). Then slides were washed with water, counterstained with hemalaun (Sigma) and mounted with glycerol-gelatin (Merck). The substrate for the development of AP consisted of 6.3 l 5% Neufuchsin (Sigma) or 2 mg Fast Blue (Sigma) in 16 l 4% sodium nitrite (Fluka, Buchs, Switzerland), 2 mg naphthol-As-Bi-phosphate (Sigma) in 20 l N,N-dimethylformamide (Merck) and 3 ml of 0.05 M l^-1 Tris-HCl-buffer, pH 8.7 containing 1 mM levamisole (Sigma). As negative control either the first antibody was omitted or an isotype matched control antibody was used. Both stainings revealed no staining.

Results

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

Activated V6⁺ T cells kill vSAG7⁺ ESb-MP tumour cells by induction of apoptosis

V6⁺ T cells, stimulated for 4 days with tumour cells and spleen cells as APC were able to kill vSAG7⁺ ESb-MP tumour cells while vSAG7^- Eb tumour cells were hardly lysed (Figure 1A). Experiments with the separation of V6⁺ T cells into CD4⁺ and CD8⁺ T cells revealed that both T cell subpopulations induced cell death in ESb-MP target cells (Figure 1B).

Figure 1.

Cytotoxicity towards vSAG7⁺ tumour cells generated by stimulated V6⁺ T cells in vitro. (A) V6⁺ T cells were isolated from B10.D2 mice which were either immunised with ESb-MP cells or remained untreated (1° in vivo). Then, V6⁺ T cells were stimulated for 4 days either with irradiated vSAG7^- Eb or with vSAG7⁺ ESb-MP cells and APC (2° in vitro) and tested for cytotoxicity towards Eb or ESb-MP target cells (effector : target ratio 10 : 1) in a 4 h ⁵¹Cr-release assay. The meanstandard deviation of triplicates of two independent experiments are shown. (B) CD4⁺V6⁺, CD8⁺V6⁺ and total V6⁺ T cells were isolated from non-immunised B10.D2 mice, purified and stimulated with irradiated ESb-MP and APCs for 4 days. Then effector cells were co-cultured with target ESb-MP cells for 2 h, and tumour cell lysis was determined by PI staining and FACScan analysis. The meanstandard deviation from 2–4 independent experiments are shown.

Full figure and legend (15K)

CTLs directed to conventional antigens consisting of peptide/MHC complexes kill their target cells via apoptosis (Henkart, 1994; Kägi et al, 1994). To determine whether vSAG7 reactive V6⁺ T cells are able to induce apoptosis, we assessed the death of vSAG7⁺ ESb-MP cells after co-incubation with stimulated V6⁺ T cells by two different methods. Figure 2A shows the kinetics of apoptosis measured by Annexin V staining/PI staining. After 10 min, 21% of the ESb-MP cells were Annexin V⁺ indicating that they were in the early phase of apoptosis. Additionally, at this time point, 26% were Annexin V⁺/PI⁺, a phenotype specific for the late stage of apoptosis. The maximal amount of apoptotic cells (63%) was detected after 20 min and remained at this level until 240 min of coincubation. Changes in the forward/side scatter which are also typical for apoptosis started as well after 10 min of coincubation of effector and target cells (data not shown). When measuring oligonucleosomal DNA-fragmentation, 45% of apoptotic ESb-MP cells were observed after 10 min of coculture (Figure 2B). The maximal level of apoptotic cells was reached after 30 min. Taken together, these observations reveal that V6⁺ T cells are able to rapidly induce apoptosis in vSAG7⁺ ESb-MP tumour cells.

Figure 2.

Stimulated V6⁺ T cells induce apoptosis in vSAG7⁺ ESb-MP tumour cells. In vitro stimulated V6⁺ T cells were co-incubated with ESb-MP cells in a ratio of 10 : 1 for different periods of time and stained either with Annexin V-FITC and 1 g ml^-1 PI (A) or were treated with a hypotonic solution containing 50 g ml^-1 PI overnight to determine DNA-fragmentation (B). Untreated cells showed no changes in the amount of apoptotic cells during coincubation. Apoptosis was determined by FACScan analysis. One representative experiment out of four (A) or three (B) independent experiments is shown.

Full figure and legend (8K)

Induction of apoptosis in ESb-MP cells is caspase-independent and does not involve mitochondrial damage

Caspases are known as main effector enzymes responsible for the initiation of DNA-fragmentation and the typical morphological changes in apoptosis (Henkart, 1994; Krammer, 1998). To analyse the involvement of caspases in this rapid vSAG7-mediated tumour cell lysis, we added either ZVAD-fmk (a broad spectrum caspase inhibitor) or IETD-CHO (a specific inhibitor of caspase 8) to the coculture of V6⁺ T cells and ESb-MP tumour cells. As shown in Figure 3, ZVAD-fmk and IETD-CHO blocked almost completely the death of Jurkat cells which were treated with anti-CD95 mAbs. In addition, ZVAD-fmk also inhibited NO-mediated apoptosis in ESb-MP cells. In contrast, ZVAD-fmk and IETD-CHO did not influence significantly the V6⁺ T cell mediated apoptosis in ESb-MP cells. Thus V6⁺ T cells were able to induce apoptosis in vSAG7⁺ ESb-MP tumour cells in a caspase-independent manner.

Figure 3.

V6⁺ T cell induced apoptosis in ESb-MP cells is caspase-independent. Stimulated V6⁺ T cells were coincubated with ESb-MP tumour cells (effector : target ratio 10 : 1) for 4 h or treated with 0.5 mM GTN (NO). Jurkat cells were stimulated with an anti-APO-1 (CD95) mAb/Protein A for 12 h. In some cultures, 25 M ZVAD-fmk or 25 M IETD-CHO were added. As a control, ESb-MP cells and Jurkat cells were cultured without V6⁺ T cells and reagents. Cell death was determined by PI staining and FACScan analysis. The meanstandard deviation from three independent experiments are shown.

Full figure and legend (7K)

The dye JC1 is known as a marker for changes in the _m which can be associated with apoptosis (Salvioli et al, 1997). To evaluate whether mitochondria are involved in the induction of vSAG7 mediated apoptosis in ESb-MP cells, we performed JC1 staining of ESb-MP cells after co-incubation with V6⁺ T cells for different periods of time. In cells with intact mitochondria and high _m, JC1 forms so called J-aggregates that are associated with a large shift in emission (590 nm=Fl 1). In Jurkat cells which were treated with anti-CD95 mAbs for 12 h the involvement of mitochondria could be clearly detected (Figure 4). In contrast no changes in _m were observed during 4 h co-culture of V6⁺ T cells with ESb-MP tumour cells (Figure 4). Staining with 3,3'-dihexyloxacarbocyanine iodide (DiOC₆) and dihydroethidine (HE), which are used to determine changes in _m and the production of reactive oxygen species (ROS), respectively (Ushmorov et al, 1999) also showed no mitochondrial alterations during V6⁺ T cell-mediated apoptosis in ESb-MP tumour cells (data not shown). In contrast, NO, another inducer of apoptosis in these cells, stimulated mitochondrial changes (data not shown).

Figure 4.

V6⁺ T cell-induced apoptosis in ESb-MP cells does not involve changes in _m. ESb-MP cells remained untreated (light line) or were cocultured with stimulated V6⁺ T cells in a effector : target ratio of 10 : 1 for 4 h (dark line). As a control, Jurkat cells were left untreated (light line) or treated with anti-APO-1 mAb/Protein A for 12 h (dark line). Afterwards, ESb-MP cells and Jurkat cells were either analysed for DNA-fragmentation (FL3) or for changes in _m by JC1 staining (FL1). Upon incubation of Jurkat cells with anti-APO-1 mAb, FL2 did not significantly change, whereas FL1, which indicates the formation of dye monomer, increased due to the reduction of _m. This increase is taken as a measure for the loss of _m. One representative experiment out of three is shown.

Full figure and legend (33K)

TRAIL, CD95L and TNF- are not involved in the induction of apoptosis in ESb-MP cells

Several death receptors and their respective ligands have been shown to be involved in the induction of apoptosis (Kägi et al, 1994; Wiley et al, 1995). The most common are TRAIL-R1+2/TRAIL, CD95/CD95L and TNF-R1+2/TNF. We tested whether ESb-MP cells were sensitive towards the apoptosis-inducing potential of CD95L, TRAIL and TNF-. Figure 5A shows the positive controls: CD95L was able to induce apoptosis in Jurkat cells, TRAIL induced apoptosis in BL60 and TNF- induced apoptosis in U937 cells. In each cell line, apoptosis could be blocked using soluble Fc-fusion proteins against the respective ligands. In contrast, ESb-MP tumour cells were not sensitive towards any of these ligands, even after IFN- stimulation for 24 h since cytotoxicity was not increased in comparison to untreated ESb-MP cells (Figure 5B). Furthermore, blocking with TRAIL-R2-Fc, CD95-Fc and TNF-R2-Fc did not significantly affect cell lysis of ESb-MP target cells by stimulated V6⁺ T cells (Figure 5C). Treatment with the isotype matched Ig control increased slightly but not significantly the number of apoptotic ESb-MP cells. We conclude that V6⁺ T cells induce apoptosis in ESb-MP tumour cells independently of CD95L, TRAIL and TNF-.

Figure 5.

ESb-MP cells are resistent towards TRAIL-, CD95L- or TNF- induced apoptosis. (A) Jurkat cells were treated with 1 g ml^-1 CD95L, BL60 cells were cultured with 1 g ml^-1 TRAIL and U937 cells were incubated with 1 g ml^-1 TNF- for 12 h. Apoptosis was blocked by 1–30 g ml^-1 CD95-Fc, TRAIL-R2-Fc or TNF-R2-Fc. One representative experiment out of three is shown. (B) ESb-MP cells were incubated with 10 nM recombinant IFN- for 24 h in RPMI medium supplemented with 10% FCS followed by treatment with 1 or 10 g ml^-1 TRAIL, 1 g ml^-1 CD95L, 1 or 10 g ml^-1 TNF-. As a control, ESb-MP cells remained untreated (ESb-MP). (C) ESb-MP cells were cultured for 4 h alone, with V6⁺ T cells (V6), V6⁺ T cells and IgG (10 g ml^-1), with V6⁺ T cells and TRAIL-R2-Fc (10 g ml^-1), V6⁺ T cells and CD95-Fc (10 g ml^-1) or with V6⁺ T cells and TNF-R2-Fc (10 g ml^-1). In (B) and (C) the effector : target cell ratio was 10 : 1. The death of ESb-MP and Jurkat cells was assessed by PI staining and FACScan analysis. The meanstandard deviation from three independent experiments are shown.

Full figure and legend (18K)

V6⁺ T cells induce cytotoxicity in ESb-MP cells through perforin/granzyme B

Perforin/granzymes are able to induce apoptosis in a death receptor-independent manner (Kägi et al, 1994; Trapani et al, 1998; Pinkoski et al, 2000). Figure 6A shows that V6⁺ T cells stimulated with vSAG7⁺ ESb-MP cells for 4 days expressed perforin while no expression of perforin was found in V6⁺ T cells from control cultures without stimulation or stimulated with vSAG7^- Eb cells. Figure 6B shows the effects of treatment of V6⁺ T effector cells and ESb-MP target cells with Concanamycin A, an inhibitor of perforin or of treatment with an inhibitor of granzyme B or a combination of both. Concanamycin A alone did not inhibit target cell lysis while treatment with the granzyme B inhibitor led to a significant reduction of apoptosis. Treatment with both inhibitors almost completely prevented the lysis of ESb-MP cells (from 35 to 5% apoptosis, Figure 6B). Together, these data indicate that vSAG7 stimulated expression of perforin in V6⁺ T cells. These activated cells induced apoptosis in ESb-MP tumour cells via released perforin and granzyme B.

Figure 6.

Apoptosis of ESb-MP cells is mediated by perforin and granzyme B. (A) Shows perforin expression in V6⁺ T cells. V6⁺ T cells were stimulated with vSAG7^- Eb or vSAG7⁺ ESb-MP cells and APCs for 4 days. Then unstimulated (control) or stimulated V6⁺ T cells were stained with an anti perforin mAb. Cells were counterstained with hemalaun. Membrane staining indicates positively labelled cells. Black dots are magnetic beads which were used for the purification and could not be removed after 4 days of stimulation. Magnification 400. (B) ESb-MP cells were cultured either alone (ESb-MP) or with stimulated V6⁺ T cells at an effector : target ratio of 10 : 1. Separate groups of effector cells were treated either with 10 nM Concanamycin A (Concan.A), 20 M granzyme B inhibitor (Z-AAD-CMK) or with a combination of both inhibitors (V6+Concan.A+GranzB.Inh.) for 15 min before coincubation with target cells for 4 h. Incubation of ESb-MP cells alone with Concanamycin A and granzyme B inhibitor revealed no significant difference in comparison to the ESb-MP control. Apoptosis was determined by PI incorporation and FACScan analysis. The meanstandard deviation from three independent experiments are shown.

Full figure and legend (34K)

Discussion

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

We have recently identified a tumour associated endogenous vSAG7 as a new target antigen for allogeneic GvL reactivity in a murine tumour model (Schirrmacher et al, 2000). In DBA/2 mice, vSAG7 behaves as a strong tolerogen leading to central deletion of SAG-reactive V6⁺ T cells during thymic maturation. While the DBA/2 derived Eb T lymphoma cells did not express this SAG, the spontaneous metastatic variant ESb and its adhesion variant ESb-MP expressed endogenous mouse mammary tumour provirus (Mtv)- typical intracisternal A-particles (IAP), Mtv7 orf/SAG message and vSAG7 protein at the cell surface (Schirrmacher et al, 2000). MMTV and IAPs have been suggested to be associated with tumorigenicity and progression towards increased malignancy (Schirrmacher et al, 1998). We previously reported that the allogeneic MHC identical mouse strain B10.D2 in contrast to DBA/2 was able to strongly reject ESb and ESb-MP tumours and that vSAG7 reactive V6⁺ T cells were involved in this GvL reactivity (Schirrmacher et al, 2000).

In this study, we investigated the mechanism of tumour cell destruction in vitro by vSAG7 reactive V6⁺ T cells. We show here that V6⁺ T cells kill vSAG7⁺ tumour cells via release of perforin and granzyme B. Interestingly, this form of cell death is independent of the known caspases and occurs without the involvement of mitochondria. It has been previously reported that vSAG7 can induce IFN- production by specifically primed CD8⁺ T cells but fails to trigger cytotoxicity (Herrmann et al, 1992). However, in contrast to Herrmann et al (1992), we find that vSAG7 stimulates V6⁺ T cells to express perforin and to kill vSAG7⁺ target cells. This kill is SAG-specific and occurs in several tumour cells that express this endogenous viral superantigen (Schirrmacher et al, 2000). Interestingly, this SAG specific CTL activity is mediated via both CD8⁺V6⁺ and CD4⁺V6⁺ T cells.

Bacterial SAG have been shown to trigger CD8⁺ T cell clones that are specific for other antigens (e.g. influenza virus peptides), but in these systems the exerted cytotoxicity was mediated via the CD95/CD95L system (Fuller and Braciale, 1998). Sundstedt et al (1998) showed that injection of staphylococcal enterotoxin A into perforin-deficient mice led to less depletion of B-cells than in control mice. This depletion was due to the release of perforin by CD8⁺ T cells. In GvL and GvH reactivity, a role for both systems (granule exocytosis as well as CD95/CD95L) was reported (Miwa et al, 1999; Yasukawa et al, 2000). It was thus of interest to elucidate the mechanism by which allogeneic V6⁺ T cells kill vSAG7⁺ tumour cells.

TNF- and CD95L play a crucial role in the activation induced cell death (AICD) of mature T cells (Dhein et al, 1995; Zheng et al, 1995) whereas TRAIL has been shown to induce apoptosis mainly in tumour cells (Wiley et al, 1995; Walczak et al, 1999) but also in normal human hepatocytes, astrocytes and keratinocytes (Jo et al, 2000). ESb-MP tumour cells were not sensitive towards any of these death ligands and blocking of these pathways during coincubation of effector and tumour cells did not influence the cytotoxic reactivity of V6⁺ T cells.

The perforin/granzyme system is involved in the cytotoxic reactivity of CD8⁺ T cells (Kägi et al, 1994; Pinkoski et al, 2000). Recently, perforin has been shown to play a role in AICD of T cells in an allogeneic transplantation model (Spaner et al, 1999). In our model, V6⁺ T cells showed a high perforin expression after 4 days of in vitro stimulation. The lysis of ESb-MP tumour cells was almost completely inhibited by the perforin (concanamycin A) and granzyme B inhibitors. Interestingly, treatment with granzyme B inhibitor but not with concanamycin A alone inhibited target cell lysis. This might be due to the fact that membrane damage induced by perforin does not directly cause target cell death, but facilitates the entry of granzymes into the cell (Shresta et al, 1999; Trapani et al, 1998). Granzyme B, however, can also enter the cell via the cation-independent mannose 6-phosphate/insulin-like growth factor receptor (Motyka et al, 2000), which might explain our observations.

The vSAG7 mediated tumour cell lysis was very fast. This is in accordance with other observations demonstrating that granzyme B, in contrast to granzyme A, is responsible for a rapid cytotoxic effect (Drénou et al, 1999). We cannot exclude that granzyme A might also be active in ESb-MP cells, since application of the granzyme B inhibitor alone did not completely block apoptosis. However, there is evidence that granzyme A and B do not synergize but act independently of each other (Shresta et al, 1999).

The requirement of caspases as the main effector enzymes in the initiation of the apoptotic cascade is still a matter of debate (Krammer, 1998; Pinkoski et al, 2000). Several recent reports indicate that induction of apoptosis and subsequent DNA-fragmentation do not neccessarily involve the activation of caspases (Beresford et al, 1999; Drénou et al, 1999). vSAG7 specific V6⁺ T cells could kill ESb-MP tumour cells independently of caspases since blocking with ZVAD-fmk and IETD-CHO did not lead to an inhibition of apoptosis in ESb-MP cells. In contrast, NO-induced apoptosis in these tumour cells required caspase activity. Beresford et al (1999) showed that cytolysis mediated by granzyme B was caspase independent whereas DNA-fragmentation was caspase-dependent. In contrast, in our model blocking with caspase inhibitors influenced neither cytolysis nor DNA-fragmentation (data not shown).

The question arises how granzyme B may lead to DNA cleavage without activation of caspases. It was reported that granzyme B directly cleaves caspase substrates such as poly (ADP-ribose) polymerase, the catalytic subunit of DNA-dependent protein kinases and NuMA to bring about apoptotic changes in cells (Froelich et al, 1996; Andrade et al, 1998). Moreover, granzyme B induced the processing of DFF45/ICAD in a caspase-independent fashion resulting in DNase activation and DNA fragmentation (Thomas et al, 2000). In addition, granule-mediated cell death can proceed independently of caspases through a non-nuclear pathway (Sarin et al, 1997). These pathways seemed to be important for cells that are infected by viruses which are able to postpone cell death by caspase inhibitors like crmA (Zhou et al, 1997). This might explain why we find this kind of cell death in ESb-MP tumour cells in which virus-like particles and proviral genes are expressed (Müerköster et al, 1998).

The involvement of mitochondria in apoptosis was described for different apoptotic agents (Kroemer and Reed, 2000). We reported previously that NO-mediated apoptosis in human leukaemia cells is associated with mitochondrial damage (i.e., a degradation of major mitochondrial lipid cardiolipin and cytochrome c release into the cytosol) followed by an activation of caspase-9 and caspase-3 (Ushmorov et al, 1999). Furthermore, we found that NO produced by activated Kupffer cells during adoptive immunotherapy with GvL-reactive T cells contributed to apoptosis in metastatic ESb-MP tumour cells in the liver (Müerköster et al, 2000). We showed also that this cell death can be induced by a mitochondria-dependent mechanism driven by CD40-CD40L interactions (Müerköster et al, 2000). In contrast, vSAG7 mediated apoptosis in ESb-MP cells occurred independently of mitochondria since staining with JC1, DiOC₆ and HE revealed no mitochondrial damage.

In conclusion, we show that vSAG7 specific T cells rapidly induce apoptosis in vSAG7⁺ tumour cells via perforin and granzyme B which occurs without involvement of mitochondria and independently of caspases. This CTL activation leads to increased tumour resistance and can be used for cellular therapy and to break immunological tumour tolerance (Schirrmacher et al, 2000). These results support the importance of granule-mediated CTL lysis of tumour cells together with apoptosis induced by NO releasing host macrophages and endothelial cells for an effective antitumour response in vivo during adoptive immunotherapy.

References

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

1. Andrade F, Roy S, Nicholson D, Thornberry N, Rosen A, Casciola-Rosen L (1998) Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis. Immunity 8: 451–460 | Article | PubMed | ISI | ChemPort |
2. Beresford PJ, Xia Z, Greenberg AH, Liebermann J (1999) Granzyme A loading induces rapid cytolysis and a novel form of DNA damage independently of caspase activation. Immunity 10: 585–594 | Article | PubMed | ISI | ChemPort |
3. Beutner U, Frankel WN, Cote MS, Coffin JM, Huber BT (1992) Mls-1 is encoded by the long terminal repeat open reading frame of the mouse mammary tumor provirus Mtv-7. Proc Natl Acad Sci USA 89: 5432–5436 | PubMed |
4. Choi Y, Marrack P, Kappler JW (1996) Structural analysis of a mouse mammary tumor virus superantigen. J Exp Med 175: 847–852
5. Dhein J, Walczak H, Bäumler C, Debatin KM, Krammer PH (1995) Autocrine T-cell suicide mediated by APO-1 (Fas/CD95). Nature (Lond.) 373: 438–441
6. Drénou B, Blanchereau V, Burgess DH, Fauchet R, Charron DJ, Mooney NA (1999) A caspase-independent pathway of MHC Class II antigen-mediated apoptosis of human B lymphocytes. J Immunol 163: 4115–4124 | PubMed | ISI | ChemPort |
7. Ebnet K, Chluba-de Tapia J, Hurtenbach U, Kramer MD, Simon MM (1991) In vivo-primed mouse T cells selectively express T cell-specific serine proteinase-1 and proteinase-like molecules granzyme B and C. Int Immunol 3: 9–19 | PubMed | ChemPort |
8. Froelich CJ, Hanna WL, Poirier GG, Duriez PJ, D'Amours D, Salvesen GS, Alnemri ES, Earnshaw WC, Shah GM (1996) Granzyme B/perforin-mediated apoptosis of Jurkat cells results in cleavage of poly(ADP-ribose) polymerase to the 89-kDa apoptotic fragment and less abundant 64-kDa fragment. Biochem Biophys Res Commun 227: 658–665 | Article | PubMed | ISI | ChemPort |
9. Fuller CL, Braciale VL (1998) Selective induction of CD8+ cytotoxic T lymphocytes effector function by Staphylococcus Enterotoxin B. J Immunol 161: 5179–5186 | PubMed |
10. Henkart PA (1994) Lymphocyte-mediated cytotoxicity: two pathways and multiple effector molecules. Immunity 1: 343–346 | Article | PubMed | ISI | ChemPort |
11. Herrmann T, Waander GA, Chvatchko Y, MacDonald HR (1992) The viral superantigen Mls-1^a induces interferon- secretion by specifically primed CD8+ cells but fails to trigger cytotoxicity. Eur J Immunol 22: 2789–2793 | PubMed |
12. Jo M, Kim TH, Seol DW, Esplen JE, Dorko K, Billiar TR, Strom SC (2000) Apoptosis induced in normal human hepatocytes by tumor necrosis factor-related apoptosis-inducing ligand. Nature Med 6: 564–567 | Article |
13. Kägi D, Vignaux F, Ledermann B, Bürki K, Depraetere V, Nagata S, Hengartner H, Golstein P (1994) Fas and perforin pathways as major mechanisms of T-cell mediated cytotoxicity. Science 265: 528–530 | Article | PubMed | ISI | ChemPort |
14. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84: 1415–1420 | PubMed | ISI | ChemPort |
15. Krammer PH (1998) CD95 (Apo-1/Fas)-mediated apoptosis: Live and let die. Adv Immunol 71: 163–207
16. Kroemer G, Reed JC (2000) Mitchondrial control of cell death. Nature Med 6: 513–519 | Article |
17. Larizza L, Schirrmacher V, Pflüger E (1984) Acquisition of high metastatic capacity after in vitro fusion of a non-metastatic tumor line with a bone-marrow derived macrophage. J Exp Med 160: 1579–1584 | Article | PubMed | ISI | ChemPort |
18. Mackinnon S, Papadopoulos EB, Carabasi MH, Reich L, Collins NH, Boulad F, Castro-Malaspina H, Childs BH, Gillio AP, Kernan NA (1995) Adoptive immunotherapy evaluating escalating doses of donor leukocytes for relapse of chronic myeloid leukemia after bone marrow transplantation: separation of graft-versus-leukemia responses from graft-versus-host disease. Blood 86: 1261–1268 | PubMed | ISI | ChemPort |
19. Miwa K, Hashimoto H, Yatomi T, Nakamura N, Nagata S, Suda T (1999) Therapeutic effect of an anti-Fas ligand mAb on lethal graft-versus-host disease. Int Immunol 11: 925–931 | Article | PubMed | ISI | ChemPort |
20. Motyka B, Korbutt G, Pinkowski MJ, Heilbein JA, Caputo A, Hobman M, Barry M, Shostak I, Sawchuk T, Holmes CFB, Gauldie J, Bleackley RC (2000) Mannose 6-Phosphate/Insulin-like Growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis. Cell 103: 491–500 | Article | PubMed | ISI | ChemPort |
21. Müerköster S, Wachowski O, Zerban H, Schirrmacher V, Umansky V, Rocha M (1998) Graft-versus-leukemia reactivity involves cluster formation between superantigen-reactive donor T lymphocytes and host macrophages. Clin Cancer Res 4: 3095–3106 | PubMed | ISI |
22. Müerköster S, Laman JD, Rocha M, Umansky V, Schirrmacher V (2000) Functional and in situ evidence for nitric oxide production driven by CD40-CD40L interactions in graft-versus-leukemia reactivity. Clin Cancer Res 6: 1988–1996 | PubMed |
23. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Method 139: 271–279 | Article |
24. Payne J, Huber BT, Cannon NA, Schneider R, Schilham MW, Acha-Orbea H, Mac-Donald HR, Hengartner H (1988) Two monoclonal rat antibodies with specificity for the -chain variable region V6 of the murine T-cell receptor. Proc Natl Acad Sci USA 855: 7695–7698
25. Pinkoski MJ, Heilbein JA, Barry M, Bleackley RC (2000) Nuclear translocation of granzyme B in target cell apoptosis. Cell Death Diff 7: 17–24 | Article |
26. Salvioli S, Ardizzoni A, Franceschi C, Cossarizza A (1997) JC-1, but not DiOC₆(3) or rhodamine 123, is a reliable fluorescent probe to assess changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett 411: 77–82 | Article | PubMed | ISI | ChemPort |
27. Sarin A, Williams MS, Alexander-Miller MA, Berzofsky JA, Zacharchuk CM, Henkart PA (1997) Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases. Immunity 6: 209–215 | Article | PubMed | ISI | ChemPort |
28. Schirrmacher V, Beckhove P, Krüger A, Rocha M, Umansky V, Fichtner KP, Hull WE, Zangemeister-Wittke U, Griesbach A, Jurianz K, von Hoegen P (1995) Effective immune rejection of advanced metastasized cancer. Int J Oncol 6: 505–521
29. Schirrmacher V, Beutner U, Bucur M, Umansky V, Rocha M, von Hoegen P (1998) Loss of endogenous mouse mammary tumor virus superantigen increases tumor resistance. J Immunol 161: 563–570 | PubMed |
30. Schirrmacher V, Müerköster S, Bucur M, Umansky V, Rocha M (2000) Breaking tolerance to a tumor-associated viral superantigen as a basis for graft versus leukemia (GvL) reactivity. Int J Cancer 87: 695–706 | Article | PubMed |
31. Shresta S, Graubert TA, Thomas DA, Raptis SZ, Ley TJ (1999) Granzyme A initiates an alternative pathway for granule-mediated apoptosis. Immunity 10: 595–605 | Article | PubMed | ISI | ChemPort |
32. Slavin S, Nagler A, Naparstek E, Kapelushnik Y, Aker M, Cividalli G, Varadi G, Kirschbaum M, Ackerstein A, Samuel S, Amar A, Brautbar C, Ben-Tal O, Eldor A, Or R (1998) Nonmyeloablative stem cell transplantation and cell therapy as an alternative to conventional bone marrow transplantation with lethal cytoreduction for the treatment of malignant and nonmalignant hematologic diseases. Blood 91: 756–765 | PubMed | ISI | ChemPort |
33. Spaner D, Raju K, Rabinovich B, Miller RG (1999) A role for perforin in activation-induced T cell death in vivo: Increased expansion of allogeneic perforin-deficient T cells in SCID mice. J Immunol 162: 1192–1199 | PubMed | ISI | ChemPort |
34. Speiser DE, Schneider R, Hengartner H, MacDonald HR, Zinkernagel RM (1989) Clonal deletion of self-reactive T cells in irradiation bone marrow chimeras and neonatally tolerant mice. Evidence for intercellular transfer of Mls-1^a. J Exp Med 170: 559–600 | PubMed |
35. Sundstedt A, Grundstrom S, Dohlsten M (1998) T cell- and perforin-dependent depletion of B cells in vivo by staphylococcal enterotoxin A. Immunology 95: 76–82 | Article | PubMed |
36. Thomas DA, Du C, Xu M, Wang X, Ley TJ (2000) DFF45/ICAD can be directly processed by granzyme B during the induction of apoptosis. Immunity 12: 621–632 | Article | PubMed | ISI | ChemPort |
37. Trapani JA, Jans PJ, Smyth MJ, Froelich CJ, Williams EA, Sutton VR, Jans DA (1998) Perforin-dependent nuclear entry of granzyme B precedes apoptosis, and is not a consequence of nuclear membrane dysfunction. Cell Death Diff 5: 488–496 | Article |
38. Trauth BC, Klas C, Peters AMJ, Matzku S, Möller P, Falk W, Debatin KM, Krammer PH (1989) Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science 245: 301–305 | Article | PubMed | ISI | ChemPort |
39. Ushmorov A, Ratter F, Lehmann V, Dröge W, Schirrmacher V, Umansky V (1999) Nitric oxide-induced apoptosis in human leukemic lines requires mitochondrial degradation and cytochrome C release. Blood 93: 2342–2352 | PubMed | ISI | ChemPort |
40. Walczak H, Degli-Esposti MA, Johnson RS, Smolak PJ, Waugh JY, Boiani N, Timour TS, Gerhart MJ, Schooley KA, Smith KA, Goodwin RG, Rauch CT (1997) TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J 16: 5386–5397 | Article | PubMed | ISI | ChemPort |
41. Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH (1999) Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nature Med 5: 155–162
42. Wiley SR, Schooley K, Smolak PJ, Din WS, Huang C-P, Nicholl JK, Sutherland GR, Smith TD, Rauch CT, Smith CA (1995) Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3: 673–682 | Article | PubMed | ISI | ChemPort |
43. Yasukawa M, Ohminami H, Kasahara Y, Ishida Y, Fujita S (2000) Granule exocytosis, and not the Fas/Fas ligand system, is the main pathway of cytoxicity mediated by alloantigen-specific CD4+ as well as CD8+ cytotoxic T lymphocytes in humans. Blood 95: 2352–2355 | PubMed | ChemPort |
44. Zheng L, Fisher G, Miller RE, Peschon J, Lynch DH, Lenardo MJ (1995) Induction of apoptosis in mature T cells by tumor necrosis factor. Nature 377: 348–351 | Article | PubMed | ISI | ChemPort |
45. Zhou Q, Snipas S, Orth K, Muzio M, Dixit VM, Salvesen GS (1997) Target protease specificity of the viral serpin CrmA. Analysis of five caspases. J Biol Chem 272: 7797–7800 | Article | PubMed | ISI | ChemPort |

Acknowledgements

• Materials and methods
• Results
• Discussion
• References
• Acknowledgements
• Figures and Tables

We thank Dr T Sebens for critical reading of the manuscript and helpful discussions. We also thank H Stahl and D Suess for the production of recombinant proteins. Grant sponsor: Dr Mildred Scheel Stiftung (project number 10-0980).