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(Journal of Leukocyte Biology. 2000;68:81-86.)
© 2000 by Society for Leukocyte Biology

T cell lysis of murine renal cancer: multiple signaling pathways for cell death via Fas

Thomas J. Sayers^*, Alan D. Brooks^*, Naoko Seki, Mark J. Smyth, Hideo Yagita, Bruce R. Blazar^|| and Anatoli M. Malyguine^¶

^* Intramural Research Support Program,
^¶ Clinical Services Program, SAIC-Frederick,
Laboratory of Experimental Immunology, DBS, NCI-FCRDC, Frederick, Maryland;
Cellular Cytotoxicity Laboratory, The Austin Research Institute, Heidelberg, Victoria, Australia;
Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; and
^|| Department of Pediatrics, Division of Bone Marrow Transplantation, University of Minnesota, Minneapolis

Correspondence: Dr. Thomas Sayers, SAIC-Frederick, NCI-FCRDC, Building 560, Room 31-30, Frederick, MD 21702-1201. E-mail: Sayers{at}mail.ncifcrf.gov

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Activated T cells lyse the murine renal cancer Renca. We have examined the mechanism of tumor cell lysis with the use of T cells derived from C57BL/6, BALB/c, B6.gld, and B6.Pfp^-/- mice. C57BL/6 and BALB/c T cells can lyse Renca cells through the use of both granule- and Fas ligand (FasL)-mediated pathways. However, B6.gld T cells predominantly use granule-mediated killing, whereas B6.Pfp^-/- T cells use FasL. The lysis of Renca by Pfp^-/- T cells is only partially inhibited by the caspase inhibitor ZVAD-FMK, suggesting that caspase-independent signaling is also important for Renca cell lysis. When the reactive oxygen scavenger butylated hydroxyanisole was used alone or in combination with ZVAD-FMK a substantial reduction of Renca lysis was observed. Therefore, the caspase-independent generation of reactive oxygen intermediates in Renca after Fas triggering contributes to the lysis of these cells.

Key Words: Fas ligand • cell-mediated cytotoxicity • Renca cells • kidney cancer

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Studies on the immune-mediated lysis of the murine renal cancer Renca suggest that this tumor can by lysed by both granule- and Fas ligand (FasL)-mediated lytic mechanisms [¹ ]. It is interesting that studies on effector cells from mutant or gene-targeted mice suggested that granule-mediated killing was the major lytic mechanism used by natural killer (NK) cells, whereas FasL-mediated lysis played a significant role in lysis of Renca by activated T cells. However, the relative importance of either of these lytic mechanisms to antitumor responses in vivo remains unclear. Successful immunotherapy of some experimental tumors in mice suggested that cell-mediated immunity and anti-tumor response predominantly involve granule-mediated killing, where the granule protein perforin was the critical component [^{2 3 4} ]. However, in some of these therapy models, immune responses were directed against relatively strong antigenic stimuli (like xenoantigens) provided by these tumors. In most spontaneously arising tumors, immune recognition of weaker antigenic determinants must occur. Indeed, most tumor antigens are thought to be differentiation-specific antigens, cancer/testis antigens, or single point mutations of ubiquitously expressed genes [⁵ ]. Due to the induction of self-tolerance in the immune system, the self or slightly modified self peptides derived from tumors are likely to be recognized by T cells with only an intermediate affinity for such peptides.

Experiments using specific T cell clones have indicated that signaling requirements for granule-mediated killing are more stringent than those required for FasL-mediated killing. Therefore, high-affinity TCR recognition of MHC-associated peptides will induce both granule and FasL-mediated lytic pathways [^{6 7 8} ]. In contrast, signaling of the same T cells with ligands of lower affinity (like self peptides) only triggers the FasL lytic pathway [⁷ , ⁸ ]. These responses seem to correlate with the Ca²⁺ influx, because rapid influx triggers granule release [⁹ ], whereas only a lower, more prolonged influx of Ca²⁺ is necessary to signal T cells to express FasL on their cell surface [¹⁰ ]. This suggests that during some antitumor responses FasL-mediated killing may be more important than has previously been assumed. Renca cells overexpressing Fas grow at much slower rates in vivo than control transfectant cells, whereas the in vitro growth rates of these cells are identical [^10a ]. In addition, recent experiments using FLIP transfectants of various tumors have shown that these tumors grow more rapidly than control transfectants in wild-type mice, but at equal rates in SCID mice [¹¹ , ¹² ]. Because FLIP only blocks lysis through death receptors, but not granule-mediated killing, these data also indicate that FasL may be important in the immune responses to these particular tumors in vivo. One requirement for FasL-mediated killing of tumor cells is the presence of the Fas receptor on the target cell surface. Furthermore, this receptor must transmit signal(s) that will ultimately result in the death of the cell. However, the signaling of cell death through the Fas receptor can occur through distinct biochemical pathways that may be cell-type specific [¹³ ]. We have determined the sensitivity of the Renca tumor to lysis by activated T cells, and have further investigated signal pathways from the Fas receptor, which may be important in determining the fate of these tumor cells.

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Mice
Specific pathogen-free BALB/c and C57BL/6 mice were obtained from the Animal Production area, National Cancer Institute, Frederick Cancer Research and Development Center (Frederick, MD). The B6Smn.C3H.FasL gld (B6.gld) and perforin-deficient (B6.Pfp^-/-) C57BL/6-Pfp^tm1Sdz mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and bred at our facility.

Tumor cell lines
The Renca tumor cell line and the A20 B lymphoma are of BALB/c origin. Cells were all maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1x nonessential amino acids, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, and 5 x 10^-5 M 2-mercaptoethanol, pH 7.4 (complete medium). The A20 lymphoma is very sensitive to Fas-mediated lysis. The d11S hybridoma cells kindly provided by Dr. P. Henkart (National Cancer Institute, National Institutes of Health, Bethesda, MD) utilize FasL to mediate their cytotoxic activity. The 2PK-3 cells, and transfectants of mouse TRAIL (mTRAIL/2PK-3) or murine FasL (mFasL/2PK-3) have previously been described [¹⁴ ].

Reagents
Mouse recombinant interferon- (IFN-; specific activity 4.7 x 10⁶ U/mg) was generously provided by Genentech (South San Francisco, CA). Mouse recombinant tumor necrosis factor (TNF-; 10⁷ U/mg) was purchased from PharMingen (San Diego, CA). Anti-mouse Fas (Jo2) monoclonal antibody, antibody to mouse CD3, and neutralizing antibody to murine TNF- were all purchased from PharMingen. The neutralizing antibodies to murine FasL (MFL-1) and murine TRAIL (N2B2) were prepared as previously described [¹⁴ ]. Soluble recombinant human Fas ligand (sFasL) was purchased from Alexis (San Diego, CA). The enzyme inhibitors Z-Val-Ala-Asp-(OMe)-CH₂F (ZVAD-FMK) and Z-Phe-Ala-(OMe)-CH₂F (ZFA-FMK) were purchased from Enzyme Systems Products (Dublin, CA). Butylated hydoxyanisole (BHA) and concanamycin A were purchased form Sigma Chemical (St. Louis, MO).

Cytotoxicity assays
Renca or A20 cells that had been incubated overnight in the presence or absence of various cytokines were labeled with ¹¹¹indium-labeled 8-hydroxyquinolone (¹¹¹In-oxine; Medi-Physics, Silver Spring, MD) as previously described [¹ ]. Briefly 1 x 10⁶ target cells were incubated with 10 µCi of ¹¹¹In-oxine for 30 min at room temperature. Cells were then washed twice in complete medium, and labeled cells (1 x 10⁴) were then incubated for 18 h at 37°C in the presence or absence of various antibodies or cells in a final volume of 200 µL. The d11S cells (FasL-positive hybridoma) or T cells were added at various effector-to-target ratios. Different concentrations of anti-Fas antibody (Jo2) or isotype control antibody were added alone or in the presence of P815 cells (1 x 10⁵) to promote antibody cross-linking. This method of efficiently cross-linking antibodies on target cells was kindly provided by Dr. H. Kojima (National Institute of Arthritis and Infectious Diseases, National Institutes of Health, Bethesda, MD). Controls were always run with P815 cells in the absence of antibodies. In the absence of cross-linking the Jo2 antibody does not trigger lysis of Renca but acts as a blocking antibody. sFasL was added at various concentrations. In experiments where inhibitors were used, the caspase inhibitors ZVAD-FMK or control ZFA-FMK (Enzyme System Products, Dublin, CA) were added to target cells at 4x final concentrations (final concentration in assay, 50 µM) in medium (50 µL). In a similar manner, target cells were also incubated with 4x final concentration of BHA (final concentration 100 µM). Targets plus inhibitors were then immediately added to U-bottomed microtiter plates and left for 3 h to allow target cells to adhere. Effector cells were then added at various ratios. For concanamycin treatment of effector cells, the effectors were preincubated with concanamycin A (100 nM) for 2 h before addition of the target cells. After overnight incubation at 37°C, supernatants were harvested and counted on a gamma counter. Specific killing (% cytotoxicity) was calculated as [(experimental release - spontaneous release)/(maximal release - spontaneous release)] x 100. All groups were run in triplicate, and standard deviations were calculated for all groups. Student’s t test was used to determine the significance of cytotoxicity differences between groups.

Activation of T cells
Activated murine T cells were prepared as previously described [¹ ]. Briefly, resting mouse lymph node cells were cultured in 5 µg/mL concanamycin A for 72 h, incubated with 10 mg/mL -methylmannoside (Sigma) for 30 min at 37°C, washed, and incubated with 100 U/mL of interleukin-2 (IL-2) for a further 48–72 h. These cells were used as activated T cells in cytotoxicity assays and were >93% CD3⁺ <2% CD3^-, DX5⁺ from all strains of mice used. Activated T cells were used as effectors in the presence of an antibody to mouse CD3 (PharMingen) at 1 µg/mL to promote cross-linking of the T cell receptor. Appropriate control antibody was added at the same concentration.

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Fas antibodies block T cell killing
In the presence of activated T cells stimulated through anti-CD3, Renca target cells are efficiently lysed in an 18-h indium-release assay. To evaluate the contribution of granule and FasL-mediated lytic mechanisms wild-type, gld, and Pfp^-/- T effector cells were used in cytotoxicity assays. Also, the effect of an antibody against murine Fas (Jo2 antibody) was determined. This antibody to murine Fas can act as a blocking antibody in the absence of cross-linking (data not shown). Blocking using the Fas antibody Jo2 (10 µg/mL) significantly reduced (P < 0.005) killing by wild-type C57BL/6, BALB/c T cells, as well as by Pfp^-/- T cells and the hybridoma dllS (Fig. 1 ). It is interesting that no blocking of killing by gld T cells was seen in the presence of this antibody. This suggests, as might be expected, that FasL-mediated killing is much reduced in cells from gld mice. However, gld T cells do show potent lytic activity against Renca, particularly after 5–6 days stimulation in vitro. The basis of this lytic activity was further investigated.

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Figure 1. Activated T cell lysis of Renca target cells. Renca cells were labeled with 111In-oxine and effector T cells from C57BL/6, BALB/c, B6.gld, and Pfp-/- mice or the hybridoma cells d11S were added at an effector-to-target ratio of 20:1 in the presence of Ab to CD3 at 1 µg/mL, and in the presence (open bars) or absence (filled bars) of a blocking antibody to Fas at 10 µg/mL. After 18 h cell supernatants were harvested and counted.

Granule-mediated Renca lysis
To assess the contribution of granule-mediated killing of Renca by T cells, cytotoxic assays were carried out in the presence of the perforin inhibitor concanamycin A. Concanamycin A had particularly pronounced effects on inhibiting lysis by gld T cells (Fig. 2 ). Substantial inhibition (25–50%) was also seen on lysis mediated by C57BL/6 and BALB/c T cells (P < 0.005). By contrast, there was no effect on Renca killing by Pfp^-/- T cells or the dllS hybridoma. This suggests that the effects of this inhibitor are probably specific for granule-mediated killing. Granule-mediated lysis can usually occur with a more rapid kinetics of lysis than FasL-mediated killing. However, using Renca targets, very little lysis of targets was seen in 4- to 6-h assays. Even if granule-mediated killing was the predominant mechanism of lysis as is the case with gld T cells (data not shown).

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Figure 2. Effect of concanamycin A on Renca lysis. Renca cells treated overnight with IFN- and TNF- (A) or A20 cells (B) were labeled with 111In-oxine and effector cells T cells from C57BL/6, BALB/c, B6.gld, and Pfp-/- mice or the hybridoma cells d11S were added at an effector-to-target cell ratio of 20:1 in the presence of Ab to CD3 at 1 µg/mL and in the presence of concanamycin A or its absence as described in Materials and Methods. After 18 h cell supernatants were harvested and counted.

Effect of caspase inhibitors on lysis of Renca
The triggering of apoptosis via receptors for FasL, TNF, and TRAIL usually involves signaling through initiator caspases [¹⁵ , ¹⁶ ]. Therefore, in the majority of cells, caspase inhibition will block death receptor-mediated lysis. We used the general caspase inhibitor ZVAD-FMK to block caspases in target cells, and compared using Renca to that of the FasL-sensitive cell line A2O (Fig. 3 ). No effect of ZVAD-FMK was seen for gld T cell killing of either Renca or A2O. Because granule-mediated lysis is not usually blocked by caspase inhibitors, this further reinforces the assumption that killing by gld T cells is mostly granule-mediated. It was surprising that, although ZVAD-FMK could completely block killing of A2O cells by either Pfp^-/- T cells or dllS hybridoma cells (P < 0.005), Renca cells were still significantly lysed in the presence of ZVAD-FMK. Therefore, in the presence of ZVAD-FMK some reduction of killing of Renca by Pfp^-/- T cells was usually observed (ranging from 10 to 40% reduction). Nonetheless, a substantial amount of lysis still occurred even in the presence of high concentrations of this inhibitor. Killing of Renca by dllS cells was usually more sensitive to inhibition by ZVAD-FMK than killing by Pfp^-/- T cells. This suggested that at least part of the killing of Renca cells by Pfp^-/- T cells was caspase-independent, whereas killing of A2O was completely caspase dependent.

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Figure 3. Effect of caspase inhibitors on lysis of Renca cells. Renca cells treated overnight with IFN- and TNF- (A) or A20 cells (B) were labeled with 111In-oxine and effector cells T cells from C57BL/6, BALB/c, B6.gld, and Pfp-/- mice or the hybridoma cells d11S were added at an effector-to-target cell ratio of 20:1 in the presence of Ab to CD3 at 1 µg/mL and in the presence of ZVAD-FMK (filled bars) or control ZFA-FMK (open bars) treatment of target cells as described in Materials and Methods. After 18 h cell supernatants were harvested and counted.

Lysis of Renca by TNF-family members
There are reports that TNF- can kill certain tumor targets via a necrotic pathway that is caspase independent [¹⁷ , ¹⁸ ]. Therefore we assessed the ability of other TNF-family proteins to lyse Renca. However, Renca cells are extremely resistant to exogenous TNF even at very high concentrations of up to 10,000 U/mL. Furthermore, neutralizing antibodies to TNF- did not reduce killing by activated T cells (data not shown). This makes it unlikely that TNF- produced by Pfp^-/- T cells is of major importance in Renca lysis. Recently much interest has focused on lytic effects mediated by the TNF-family member TRAIL. In initial experiments using recombinant human TRAIL from two independent sources, no lytic effects on Renca were seen (data not shown). However, transfected 2PK-3 cells expressing murine TRAIL protein (mTRAIL/2PK-3) efficiently lysed Renca cells (Fig. 4 ). In addition, TRAIL killing was not modulated by cytokine treatment of the Renca targets. In contrast, the transfectants expressing murine FasL (mFasL/2PK-3) only lysed cytokine-treated Renca. Fas expression is dramatically increased in Renca by prior treatment with IFN- and TNF-, which results in increased sensitivity to Fas-mediated lysis. In contrast, endogenous levels of expression of TRAIL receptors by Renca must be sufficient to trigger cell death. To further dissect the relative roles of FasL and TRAIL in lysis of Renca by T cells, appropriate neutralizing antibodies were added to cytotoxicity assays. Somewhat surprisingly, neutralizing antibodies to FasL completely blocked killing of Renca by Pfp^-/- T cells (P < 0.005), whereas neutralizing antibodies to TRAIL were without significant effect (Fig. 5 ). Therefore, under the conditions we employed, TRAIL played no role in T cell-mediated killing. Also, FasL seems to account for all the killing by Pfp^-/- T cells unless the neutralizing antibody to FasL cross-reacts with another unidentified molecule important in lysis of Renca cells.

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Figure 4. Lysis of Renca by TRAIL and FasL Renca cells (A) or Renca cells treated overnight with IFN- and TNF- (B). Cells were labeled with 111In-oxine and the cell line 2PK-3 or transfectants mTRAIL/2PK-3 or mFasL/2PK-3 were added as effector cells at an effector-to-target cell ratio of 10:1. Assays were performed in the presence of the N2B2 neutralizing antibody to mTRAIL, the MFL-1 neutralizing antibody to mFasL, or media at antibody concentrations of 10 µg/mL. After 18 h cell supernatants were harvested and counted.

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Figure 5. Effect of neutralizing antibodies to TRAIL and FasL on T cell lysis. Effector cells T cells from B6.Pfp-/- mice at effector-to-target ratios of 20:1 or 2:1 were added to Renca cells treated overnight with IFN- and TNF- (A), or A20 cells (B) labeled with 111In-oxine in the presence or absence of neutralizing antibody to mFasL, MFL-1 at 10 or 1 µg/mL, or neutralizing antibody to mTRAIL, N2B2 at 10 µg/mL. After 18 h cell supernatants were harvested and counted.

Alternative caspase-independent pathways in Renca lysis?
Inhibitor studies with ZVAD-FMK showed that lysis of Renca by Pfp^-/- T cells was only partially dependent on caspases. Therefore, we hypothesized that Pfp^-/- T cells could also trigger Renca lysis by using a signaling pathway that was caspase-independent. It has previously been reported that TNF lysis of L929 cells can proceed via a caspase-independent necrotic pathway mediated by the generation of reactive oxygen intermediates [¹⁸ ]. This necrotic pathway could be blocked by the oxygen radical scavengers like BHA, so we determined the effects of BHA as Renca killing. As seen in Figure 6 , which is a combination of data from two independent experiments, Renca killing by Pfp^-/- T cells was blocked by BHA alone at both effector-to-target ratios, whereas ZVAD-FMK had no effect on lysis. Also, the combination of ZVAD-FMK and BHA was not significantly more effective than BHA alone. Inhibition by BHA alone was highly significant (P < 0.005) at the lowest effector-to-target ratio. This suggests that reactive oxygen intermediates generated in Renca cells after Fas signaling contribute to the lysis of these tumor cells.

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Figure 6. Effects of ZVAD-FMK and BHA on Renca lysis. B6.Pfp-/- T cells in the presence of 1 µg/mL antibody to CD3 were used as effector cells at 20:1 or 2:1 ratios. Renca target cells treated overnight with IFN- and TNF- were labeled 111In-oxine. Target cells were then treated with ZFA-FMK, ZVAD-FMK alone, BHA alone, or ZVAD-FMK plus BHA as described in Materials and Methods. After 18 h cell supernatants were harvested and counted. The data presented are averaged from two independent experiments.

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In recent years much progress has been made in elucidating the molecular events that occur after engagement of the Fas receptor [¹⁹ ]. It is thought that trimerization or oligomerization of Fas occurs. This process results in the formation of the death-induced signaling complex or DISC [²⁰ ] via recruitment of molecules like the adapter protein FADD and FLICE or caspase 8 [¹⁵ , ¹⁶ , ²¹ , ²² ]. Caspase 8 is then activated by autocatalysis to trigger further downstream events that result in target cell apoptosis. However, in human cell lines at least two distinct molecular pathways are triggered after caspase 8 activation [²³ ], and the relative importance of each pathway is cell-type-specific. Therefore in Type I cells there is formation of high levels of the DISC, which results in substantial activation of caspase 8. Activated caspase 8 can then directly trigger the caspase cascade of enzymes resulting in apoptosis. This Type I killing is largely independent of mitochondria and is not affected by the presence of the anti-apoptotic protein bcl-2. In contrast, in Type II cells there is a lower level of DISC formation and caspase 8 activation. Rather than directly activating the caspase cascade, caspase 8 cleaves the cytosolic protein bid [²⁴ , ²⁵ ]. Cleaved bid is thought to interact with mitochondria inducing the release of cytochrome and activation of caspase 9. Because caspase 8 signaling is critical to both pathways, cell death mediated through Fas is blocked by caspase inhibitors [²³ ]. However, death signaling via TNF receptor family members is not caspase-dependent in all cells. Therefore, the TNF-mediated lysis of L929 cells is independent of caspase signaling and proceeds with a necrotic phenotype in the presence of caspase inhibitors [¹⁸ ]. Furthermore, activated oxygen intermediates play an important role in this death pathway and cells undergoing death in this manner have been termed Type III cells [²⁶ ]. The data we obtained with lysis of Renca by Pfp^-/- T cells is also consistent with a contribution of a Type III mechanism in the death of these cells. A caspase-independent pathway involved in Fas signaling would be consistent with recent observations from other groups using different target cells. Vercammen et al. demonstrated that L929 cells transfected with human Fas could be triggered to die by agonist antibodies to human Fas in the presence of caspase inhibitors like ZVAD [²⁶ ]. Indeed, in these L929 cells, caspase inhibitors promoted rather than inhibited death. This pathway also seemed to involve reactive oxygen intermediates because lysis of these L929 cells was only significantly inhibited by combinations of ZVAD-FMK and BHA. Kawahara et al., using a Jurkat-derived cell (JB-6), reported that the induction of dimerization of transfected FADD resulted in a necrotic form of cell death that was not blocked by caspase inhibitors [²⁷ ]. This suggested that FADD could be involved in cell death signaling through a caspase-independent pathway. It is interesting that inhibitory effects of bcl-2 on cell death were not noted in either of these reports. Our studies show that a caspase-independent signaling pathway can occur in non-transfected cells. It seems likely that the lysis of Renca cells by activated T cells can be triggered by both caspase-dependent and caspase-independent signaling pathways, and the cells in which both pathways are operating may therefore be more sensitive to T cell-mediated lysis.

There are, however, some inhibitory effects of ZVAD-FMK on killing of Renca by Pfp^-/- T cells, although inhibition is somewhat variable and never complete. One of the most likely explanations for this observation is that the Renca cell line is heterogeneous. Therefore a Type III mechanism could contribute in amplifying death pathways in some Renca cells but not others. Indeed we have isolated individual clones from the Renca cell line where lysis by Pfp^-/- T cells is totally blocked by ZVAD-FMK, so killing is completely caspase dependent (data not shown). Therefore, a minority of clones in the Renca population are only lysed by caspase-dependent pathways. The proportion of these clones in the general Renca population would therefore determine the effectiveness of inhibition by ZVAD-FMK. In conclusion, after the engagement of Fas on Renca by FasL on activated T cells, multiple signaling pathways can occur. In Renca cells caspase-independent signaling is also important in determining survival or death of these cells. In contrast, signaling in A20 cells is completely caspase-dependent. A number of issues still need to be resolved. The components involved in the caspase-independent signaling pathway are as yet unidentified. Furthermore a role for the mitochondria in this pathway is likely, but unproven. In addition, the threshold for activation of these various pathways on the interaction of Fas with more physiological levels of FasL is unknown. We are currently further investigating these important questions.

 
This project was funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under contract number N01-C0-56000. The authors are grateful to Dr. J. Ortaldo and Dr. R. Wiltrout for their suggestions and critical reading of the manuscript. The technical assistance of John Wine, Eric Derby, and Vasavi Reddy is much appreciated. We thank Susan Charbonneau and Joyce Vincent for help in the preparation of the manuscript.

By acceptance of this article, the publisher or recipient acknowledges the right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.

Animal care was provided in accordance with the procedures outlined in A Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, 1985).

Received December 20, 1999; revised February 24, 2000; accepted February 25, 2000.

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