In Vivo Rejection of Tumor Cells Dependent on CD8 Cells That Kill

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In vivo rejection of tumor cells dependent on CD8 cells that kill independently of perforin and FasL Sung-Hyung Lee,1 Erez Bar-Haim,1 Arthur Machlenkin,1 Ofir Goldberger,1 Ilan Volovitz,1 Ezra Vadai,1 Esther Tzehoval,1 and Lea Eisenbach1 1Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

Perforin/granzyme B- and Fas/FasL-mediated killing pathways are the main effector mechanisms of CTL and NK cells in antitumor immune responses. In this study, we investigated the relative role of these two lytic mechanisms in protection of the host from tumor progression, as well as spontaneous metastasis, using the D122 Lewis lung carcinoma and its gene-modified cells. Utilizing perforin knockout mice (B6-PKO) and Fas and FasL mutant (B6-MRL and B6-Smn) mice, we found that perforin expression in the host plays a crucial function in the prevention of metastasis. However, local tumor rejection of an H-2Kb and B7-1 transfectant, 39.5-B7 cells, was not dependent either on perforin or Fas/FasL expression in vivo. In addition, CTL lysis of 39.5-B7 cells was independent of perforin and Fas/FasL interactions in 18-hour in vitro assays. We also confirmed that CD8 T-cells were responsible for rejecting 39.5-B7 local tumors, yet cytokines, TNF-a and gIFN were not involved in tumor rejection in vivo. Furthermore, blocking assays using caspase inhibitors (zVAD-fmk, zIETD-fmk and zLEHD-fmk) showed that, whereas caspase activation was partially required to induce 39.5-B7 lysis mediated by the perforin-dependent pathway, 39.5-B7 lysis by CTLs through the perforin-independent mechanism required caspase activation. Thus, these results suggested that perforin, Fas/FasL, gIFN and TNF-a independent lytic mechanisms, mediated by CD8 T cells, have a crucial role in rejection of 39.5-B7 cells in vivo. Caspase activation is a pre requisite for apoptosis of targets by CTLs Cancer Gene Therapy (2004) 11, 237–248. doi:10.1038/sj.cgt.7700678 Published online 23 January 2004

Keywords: CTL; NK; apoptosis; cytotoxicity; tumor immunity

ytotoxic T cells (CTLs) and natural killer (NK) cells addition, besides granule-mediated killing, interaction of Care the major effectors in tumor surveillance. CTL death receptors, such as Fas on the target cells with their have to recognize their target cells by associating their T- ligand, FasL on CTL can also induce apoptosis by cell receptor (TCR) with MHC/peptide complexes on the activation of cystein proteinase,5 the caspase cascades.10 target cells as signal 1. Besides this signal 1, costimulation Recently, a new member of the TNF family, TNF-related signal interactions between receptors, for example CD28 apoptosis-inducing ligand (TRAIL) was shown to play an on CTL and their ligand, B7-1 (CD80) on antigen- important role in antitumor effects of immune cells. presenting cells (APCs) can provide additional signals to Although caspase activation is essentially required to fully activate T cells. These second signals can modulate transmit apoptotic signals,11,12 certain target cells can also the production of various lymphokines/cytokines, such as be induced to undergo apoptosis by caspase-independent IL-2,1 enhance survival of activated T cells2 and regulate lytic mechanisms.13,14 However, the relative importance cell-cycle progression. These activated CTL3 can prolif- of perforin and Fas/FasL-mediated cytolytic functions erate vigorously and then differentiate into effector cells has not been well demonstrated in vivo. to eliminate target cells. It is known that perforin/ Antitumor vaccines strategies broadly rely on using granzyme and Fas/FasL-mediated killing pathways are tumor-associated antigens and their peptides, or gene- the main mechanisms of effectors to eliminate target cells, modified (GM-CSF, IL-2, IL-12, etc) tumor cells.15,16 Our like transformed cells.3–5 In the absence of these cytolytic group has reported that transfection of H2-Kb into Lewis functions, gIFN and TNF-a are also responsible for the lung carcinoma derived D122 cells, which are low antitumor effects of immunocytes.6–8 immunogenic and high metastatic, abolished their meta- Granules containing the pore-forming protein perforin static phenotype. In addition, immunization with H-2Kb and granzymes, which are serine proteinases, are released transfectants provoked protective immunity mediated from CTL to target cells that undergo apoptosis.9 In through CD8 T cells against parental D122 tumor cells, indicating that CTL were the main effectors that protect 17 Received September 24, 2003. the host from metastasis. Addresss correspondence and reprint requests to: Dr Lea Eisenbach, In the present study, we tested how perforin and Fas/ Department of Immunology, Weizmann Institute of Science, Rehovot FasL expression by the host affects tumorigenicity and 76100, Israel. E-mail: [email protected] immunogenicity of parental D122, the H-2Kb transfectant Tumor rejection by CTL independent of perforin/FasL S-H Lee et al 238 (39.5.1.11) and the B7-1-expressing Kb transfectant (39.5- SD) were used (stock conc. 50 mM) in CTL inhibition B7) in order to study their ability to elicit antitumor assays. immune responses and their susceptibility to various killing mechanisms of CTL and NK cells. We found that RT-PCR analysis CD8 CTL played a major role in rejecting 39.5-B7 local Total RNA from tumor cells were isolated using the tumors. 39.5-B7 cell lysis by CTL was induced by TRIREAGENT (Molecular Research Center, Cincinnati, mechanisms that are dependent on caspases activation, OH, USA) according to the manufacturer’s instructions. but not mediated through perforin, Fas/FasL or cytokine Reverse transcription was performed from 5 mg of total cytotoxicity. RNA with an oligo d(T) primer. cDNA corresponding to 500 ng of total RNA was amplified under standard condition. For amplification of mouse Fas receptor Materials and methods (mFas), TRAIL receptor (DR5, Accession No. Mice af176833) and GAPDH, primer sequences and annealing 0 tm1sdz lpr temperature (Ta) were used as follows: (1) mFas : 5 -GAG C57BL/6-Pfp (PKO), B6-MRL-Fas (B6-lpr/lpr) 0 0 gld GCC CAT TTT GCT GTC AA-3 (forward) and 5 -ATC and B6-Smn.C3H-Fasl (B6-gld/gld) mice were all 0 TAA GGT TCT GCG ACA TTC-3 (reverse : Ta ¼ 601C), purchased from The Jackson Laboratory (Bar Harbor, (2) TRAIL receptor (DR5) : 50-AAC CCA GCC CAT ME) and confirmed in impairment of perforin gene, Fas AAT CGT CC-30 (forward) and 50-ACA GAG CAA and FasL mutation by RT-PCR based on published data 0 4,18,19 CCA TTG CCT CC-3 (reverse : Ta ¼ 571C), 3) GAPDH : (Data not shown). Mice used for the experiments 50-CCA TCA ACG ACC CCT TCA TTG AC-30 were generally 8–12 weeks old and were maintained in the (forward) and 50-GGA TGA CCT TGC CCA CAG animal facility of the Weizmann Institute of Science. 0 CCT TG-3 (reserve : Ta ¼ 601C)

Tumor cells FACS analyses The highly metastatic and low immunogenic D122 clone Parental D122 and the transfectants were washed in of the C57BL/6-derived Lewis lung carcinoma, H-2Kb b phosphate-buffered saline (PBS) and incubated for transfected D122 cells (39.5. 1.11), H-2K and B7- 30 minutes at 41C with FITC-labeled mAbs, anti-mFas 1(CD80) transfected clone 39.5-B7 were used in these (Jo2), H-2Kb, H-2Db and B7-1(CD80). FITC-labeled experiments. Murine gIFN transduced D122 clones, isotype control antibodies (Jackson Immuno Research) NAT.A (nonsecretor of gIFN), DIF-2 (high secretor of 20 were used as negative controls. Cells were then washed gIFN), human IL-6 transfected D122 clone, DIL6-7, once with PBS containing 0.1% sodium azide and 1% human IL-2 transduced D122, DIL2-102, murine IL-12 BSA. After washing, the stained cells were analyzed by transfectant BD9, murine GM-CSF D122 transfectant, FACScan (Becton Dickinson, San Jose, CA) and the data DG4, were used in apoptotic assays or RT-PCR experi- through the CellQuest software (Becton Dickinson). ments.21 The cells were maintained in DMEM supplemented with 10% heat-inactivated fetal bovine serum Cell Killing assays (FBS: GIBCO BRL, Paisley, Scotland), 50 mg/ml gentamicin, 10 mM sodium pyruvate and nonessential amino Human FasL transfected D122 cells (39.5-B7-FasL7) and acid. Murine Fas receptor transfected L1210 cells (L1210- parental cells (39.5-B7) were distributed into flat bottom fas), which were kindly provided by Dr Zelig Eshhar 96-well plates. These cells were admixed at different ratios 35 (Weizmann Institute of Science), and Jurkat T-cell with L-[ S]methionine parental D122, transfectants and lymphoma and A20 a murine B-cell lymphoma were used L1210-fas target cells (1 Â 105cells/ml), and incubated for as positive controls of apoptotic assays or RT-PCR, 18 hours at 371C. After 18 hours incubation supernatants respectively. These cells were maintained in RPMI 1640 (50 ml) were mixed with scintillation fluid and measured in supplemented by 10% heat-inactivated fetal bovine serum a b-counter. The percentage of specific lysis was and gentamicin (50 mg/ml). calculated as follows: % Lysis ¼ {(experimental {35S} release – spontaneous Antibodies and reagents release)/(maximal release – spontaneous release)} Â 100. Spontaneous and maximum release were determined by Recombinant human soluble TRAIL (sTRAIL) kit was incubating target cells with medium alone or with 0.1 N purchased from Alex Corporation. These reagents were NaOH, respectively. used for induction of apoptosis in in vitro assays. FITC- labeled anti-mouse Fas mAbs (Jo2) were purchased from BD PharMingen (San Diego, CA). Anti-mouse H-2Kb Detection of apoptosis (Caltech. Lot-04010404), Db (Lot No. G127-M4787) and Cells were incubated with sTRAIL (100 ng/ml) overnight. CD 80 mAbs (Clone 1G10) labeled FITC (SouthernBio- After 24 hours, apoptotic cells were detected by flow tech, BA) were all used in FACS analyses. A broad- cytometry after staining with FITC-conjugated Annexin spectrum caspsae inhibitor (z-VAD-fmk: BIOMOL Re- V and propidium iodine (PI) using a commercial kit search Laboratories), caspase-8 inhibitor II (z-IETD-fmk) (Annexin V-FITC Kit: Bender MedSystems, Vienna, and caspase-9 inhibitor I (z-LEHD-fmk):Calbiochem, Austria).

Cancer Gene Therapy Tumor rejection by CTL independent of perforin/FasL S-H Lee et al 239 In vivo tumor growth and spontaneous metastasis Depletion of immunocytes and neutralization of gIFN or a Mice (eight mice in each experimental group) were TNF- in vivo inoculated intrafootpad (i.f.p.) with 2 Â 105–1 Â 106 tu- Monoclonal antibodies against CD8 (YTS-169.1:rat mor cells in 50 ml PBS in the right hind leg. Local tumor mAb, IgG2b) were injected to deplete subsets of immune growth was determined by measuring the diameter of the cells. R4-6A2 (rat mAb) against mouse gIFN, and 1F3F3 footpads with calipers. To quantitate spontaneous lung (rat IgM) against mouse TNF-a22 were used to neutralize metastasis, tumor-bearing feet were amputated below the gIFN and TNF-a in vivo, respectively. Mice were injected knee when the tumors reached 8–9 mm in diameter and intravenously (i.v.) with 0.5 ml hybridoma ascites and the the mice were killed 60 days following amputation or following day, tumor cells (2 Â 105 cells/mouse) were monitored daily and killed when mice were moribund. inoculated into the footpads of the mice. Starting on day 6 Survival is defined as the number of days until moribund after inoculation, mice were injected i.p. with 250 mlof mice were sacrificed. Metastatic load was assayed by diluted hybridoma ascites weekly until tumor-bearing feet weighing the lungs. were amputated. Purified rat IgM was injected into parallel groups as control.

NK assays Statistical analysis Mice were injected intraperitoneally (i.p.) with 200 mg The significance between the experimental and control Poly I:C (polyinosinic–polycytidylic acid : Sigma) per groups in all experiments of this study was determined by mouse, in 0.5 ml PBS. After 18 hours, spleens were Student’s t-test. Two-tailed P-values of o.05 were removed, splenocytes teased and RBC lysed. The spleno- considered significant. cytes were washed 2–3 times with PBS and admixed at different ratios with [35S]-methionine target cells. The plates were incubated for 5 hours at 371C, are 5% CO 2 Results and 50 ml supernatant were collected for counting as a above. Fas- and TRAIL-mediated apoptotic pathways in paretnal D122 and its transfectants We tested Fas expression on the high metastatic and low In vitro cytotoxicity assays immunogenic Lewis lung carcinoma clone D122 cells, and Mice were immunized, intraperitoneally with 2 Â 106 on the H-2Kb and B7-1 (CD80) transfectant (39.5-B7) as irradiated (5000 rad) parental D122 cells or transfectants, well as various cytokine transfectants. Fas expression on three times at 7-day intervals. Spleens were taken from RNA levels was examined by RT–PCR. Levels of Fas immunized mice 10 days after the third booster and expression were markedly upregulated in NAT.A (gIFN restimulated in vitro for 4 days on a monolayer of nonsecretor), DIF-2 (gIFN secretor) and DIL6-7 (IL-6 irradiated (5000 rad) and mitomycin C-treated (80 mg/107 secretor) cells compared to D122 parental cells (Fig 1a). cells, for 45 minutes at 371C) immunizing cells, in RPMI gIFN secretor, DIF-2 cells showed high Fas expression on medium supplemented with 10% FCS, 2 mM glutamine the cell surface while D122 cells stained negative. In and 2 Â 10À5 M b-mercaptoethanol. Viable lymphocytes addition, we observed that Fas expression on 39.5-B7 cells were separated by Lympholyte-M (Cenderlane, Ontario, was slightly increased. Enhancement of Fas receptor Canada) gradient centrifugation, admixed at different expression on DIF-2 cells was proportional to the density ratios with [35S]methionine target cells in U-shaped of the culture and increased with the incubation times (48, microtiter wells and incubated at 371C for 5 hours or 18 72 and 96 hours) in vitro; however, expression levels of hours. Cultures were terminated by centrifugation at Fas on 39.5-B7 cells did not change with the time of 250 g for 10 minutes at 4 1C, and then 50 ml supernatants incubation (data not shown). This observation is con- were harvested and monitored in a mix with scintillation sistent with reports that gIFN can effectively enhance fluid. Percent-specific lysis was calculated as in the cell expression of Fas receptor on the cell surface.23 Although killing assay. For the inhibition of perforin-based we could not detect apoptotic death mediated by soluble cytotoxicity, effectors were pretreated with Concanamy- FasL (sFasL) (1 mg/ml) under various experimental cin A (CMA:Sigma-Aldrich) for 1 hour at 100 nM, and conditions, coincubation with FasL-expressing cells then CMA-treated effectors were incubated with their (39.5-B7-FasL) for 18 hours did induce apoptosis of target cells for indicated times at 371C, 5% CO2. Fas-expressing cells, including L1210-fas as a positive For the inhibition experiments using various caspase control. FasL nonexpressor (39.5-B7) did not induce inhibitors, various caspase inhibitors (50 mM) were apoptosis (Fig 2a). These results indicate that membrane- distributed into 96-well plates containing [35S]-labeled bound FasL can more effectively induce apoptosis in Fas- target cells. After pre-incubation for 30 minutes, the plates expressing cells than soluble FasL.24 Recently, it has been were added with effector cells. Diluted hybridoma ascites reported that the TRAIL-mediated killing pathway on containing specific mAbs (anti-CD8, anti-TNF-a and immune cells also plays an important role in elimination anti-gIFN) were filtered by a 0.45 mm filter and added into of tumor cells.25 We tested whether soluble TRAIL would 96-well plates that were distributed with effector cells. induce apoptotic cell death of parental D122 and cytokine Purified rat IgG was used as a negative control for mAb. transfectants. RT–PCR results show that the TRAIL

Cancer Gene Therapy Tumor rejection by CTL independent of perforin/FasL S-H Lee et al 240 a 80 a 39.5-B7 70 D122 39.5-B7 NAT.A DIF--2 DIL2-102 DIL6-7 BD9 DG4 A-20 39.5-B7-FasL 60

Specific Lysis (%) Specific Lysis 20

b 150 150 10 D122 39.5-B7 120 120 0 D12239.5-B7 DIF-2 L1210-fas 90 90 M1 Target cells Counts 60 Counts 60 b 30 30 NAT.A D122 DIF-2 DIL-6-7 0 0 100 101 102 103 104 100 101 102 103 104 FL1-H FL1-H 150 200 DIF-2 L1210-fas 480 bp 120 160 Figure 2 Sensitivity of the cells to FasL and TRAIL. (a)[35S]methio- 90 120 nine target cells (5 Â 105 cells/ml) were coincubated with FasL- M1 M1 60 80 expressing cells (39.5-B7-FasL) or with nonexpressor (39.5-B7) for Counts Counts 18 h and release of radioactivity was monitored. L1210-fas cells were 30 40 used as a positive control. The data represent average 7SD of triplicate in one of three independent experiments. (b) Expression of 0 0 mTRAIL receptor (DR5) on parental D122 and cytokine transfectants 100 101 102 103 104 100 101 102 103 104 (NAT.A, DIF-2 and DIL6-7) was tested according to Materials and FL1-H FL1-H methods. (c) Cells (1 Â 106 cells) were incubated with sTRAIL (100 ng/ml) overnight, and cells were analyzed by flow cytomery Figure 1 Murine Fas expression on parental D122 and its gene- after staining with Annexin and PI. The data shows one of two modified transfectants. (a) cDNAs were prepared according to independent experiments. Materials and methods. Murine Fas mRNA expression was detected by PCR amplification of cDNA from the cells. GAPDH mRNA expression was used as control for PCR amplification. (b) Cells were stained with anti-Fas Ab (Jo2: solid line histograms) or control DIF-2, were sensitive to FasL-expressing tumor cells, but hamster IgG (filled histograms) and monitor by FACS analyses. 39.5- not to sTRAIL. B7-D122 transfected by H-2Kb and B7-1; NAT.A-D122 transfected by mgIFN gene, nonsecretor; DIF-2-D122 transfected by a gIFN Tumorigenicity and metastatic phenotype of the gene, high secretor; DIL2-102-D122 transfected by a human IL-2 parental D122, the Kb transfectant (39.5. 1.11) and the gene; DIL6-7-D122 transfected by a human IL-6 gene; BD9-D122 Kb and B7-1 (CD80) transfectant (39.5-B7) in C57BL/6 transfected by a mIL-2 gene; DG4-D122 transfected by a GM-CSF and mutant mice gene; A-20-B cell lymphoma as positive controls. The data shown are representative of three independent experiments. We next tested how abrogation of perforin, Fas and FasL expression in the host affects tumorigenicity and metastatic phenotype of the parental D122, the H-2Kb transfectant (39.5.1.11) and the H-2Kb and receptor, DR5, is expressed in the transfectants, NAT.A, B7-1 transfectant (39.5-B7). Taking advantage of perforin DIF-2 and IL6-7, and is equivalent to that of D122 knockout (B6-PKO) Fas mutant B6-MRL and FasL parental cells, suggesting that cytokines (gIFN, IL-6) do mutant B6-Smn mice, the tumorigenicity and metastatic not modulate transcription of TRAIL receptor expression phenotype of the various cells was tested. FACS analyses (Fig 2b). Whereas Jurkat T cells, which are sensitive to were performed to confirm expression of H-2Kb,Db sTRAIL, were markedly induced to undergo apoptotic and B7-1 (CD80) in D122 and the two transfectants cell death, D122 and the transfectants were not killed (Fig 3). The results show that, in D122 cells, H-2Kb during coincubated with sTRAIL (100 ng/ml) for 24 expression is very low, but H-2Db is expressed at hours (Fig 2c). Therefore, D122 derivatives, 39.5-B7 and relatively high levels. The expression levels of H-2Kb in

Cancer Gene Therapy Tumor rejection by CTL independent of perforin/FasL S-H Lee et al 241 H-2Kb H-2Db B7-1(CD80) 200 200 200 81.2(%) 160 160 M1 160 120 120 120 D122 80 80 80 Counts Counts 40 40 40

0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FL1-H FL1-H FL1-H

200 200 200 71.6(%) 76(%) 160 160 M1 M1 160 120 120 120 39.5.1.11 80 Counts 80 80 Counts Counts Counts 40 40 40

0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FL1-H FL1-H FL1-H

200 200 200 86.8(%) 84.4(%) 160 83.3(%) 160 160 M1 M1 M1 120 120 120 39.5-B7 Counts

Counts 80 80 80 Counts

40 40 40

0 0 0 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 FL1-H FL1-H FL1-H Figure 3 Expression of H-2Kb, H-2Db and B7-1 (CD80) on transfectants and parental D122 tumor cells. Cells were stained with anti-Kb, anti-Db and anti-CD80 mAb conjugated with FITC for 30 min and analyzed by flow cytomery. Filled histograms show background staining with isotype- matched control mAb. The numbers indicate the percentage of positive cells. The data shows a representative experiment of three independent experiments. both transfectants, 39.5.1.11 and 39.5-B7, is remarkably 24 days postinoculation. These observations suggest higher than in parental D122 cells. Levels of H-2Db that rejection of 39.5-B7 tumors is not strictly dependent expression of the transfectants are similar to those on perforin and Fas/FasL expression by the host (Fig 4). of parental D122, showing that transfection does not Furthermore, Table 1 shows that, despite the fact that affect H-2Db expression levels of parental D122 cells. the tumor growth pattern of 39.5.1.11 cells is similar We could only observe B7-1 expression in 39.5-B7 cells to that of parental D122 cells, mice inoculated with (Fig 3). C57BL/6 mice, B6-PKO and Fas/FasL 39.5.1.11 tumor do not succumb to metastatses. All (B6-MRL/Smn) mutant mice were inoculated with D122 B6-PKO, 5/8 B6-MRL and 5/8 B6-Smn mice survived and the two transfectants, 39.5.1.11 and 39.5-B7, until all the D122-bearing mice died of metastases within at various doses (2 Â 105,5Â 105 and 1 Â 106 cells/mouse) 30 days after amputation. At 60 days post amputation, and tumor growth followed. Figure 4 shows the results; lung weights from the surviving mice showed that at the highest inoculum (106 cells/mouse), local tumor metastases did not occur in the mice. These results growth of 39.5.1.11 cells in the mice was not statistically indicate that, although H-2Kb expression on the parental different from that of parental D122 cells. Local tumor D122 cells does not induce reduction of tumorigenicity, progression of parental D122 and 39.5.1.11 cells in their metastatic phenotype is abolished due to expression syngenic C57BL/6 mice was delayed relative to all mutant of H-2Kb, in vivo. In addition, both perforin and mice, indicating that perforin and functional Fas/FasL Fas/FasL expression in the host did not affect their low expression, by the host can influence tumorigenicity metastatic phenotype. Survival in B6-PKO mice inocu- in vivo. Notably, 39.5-B7 local tumors were rejected in lated with D122 cells was significantly shorter than that of the mice, including B6-PKO and B6-MRL/Smn other mouse strains (p value o.0001), indicating that mice. Local 39.5-B7 tumors in B6-PKO and B6-MRL/ perforin expression by the host plays a major role in the Smn mice seemed to develop at early stages, but these kinetics of spontaneous metastases from D122 local palpable tumors regressed and were finally rejected tumors.

Cancer Gene Therapy Tumor rejection by CTL independent of perforin/FasL S-H Lee et al 242

Figure 4 Tumorigenicity of D122, 39.5.1.11 and 39.5-B7 cells in syngeneic C57BL/6, B6-PKO and Fas and FasL mutant B6-MRL and B6-Smn mice. Tumor cells, 106/mouse, (8 mice/group) were inoculated into the footpads of C57BL/6, B6-KPO and B6-MRL/Smn mice. Tumor diameters were measured by calipers. The numbers in parenthesis indicate the fraction of tumor-free mice in each group. C57BL/6 versus B6-PKO, Po.01; C57BL/6 versus B6-MRL, Po.05; C57BL/6 versus B6-Smn, P4.05 at 21 days after D122 tumor cells inoculation. The data shown are representative of three independent experiments.

Table 1 Spontaneous metastasis of D122, 39.5.1.11 and 39.5-B7 transfectants in syngeneic C57BL/6, B6-PKO and B6-MRL and B6-Smn mice Spontaneous metastasis lung weight (mg) Survival days post amputation

% tumor-free % of surviving Mice Cells Mean7SD Med mice Mean7SD Med Pa mice

C57BL/6 D122 795.37226 783 — 2871.52 28 — — 39.5.1.11 163.878.60 164 1/8 460b — 7/8 39.5-B7 — — 8/8 — — — —

B6-PKO D122 712.87215.8 773.5 — 1773.3 17 o0.0001 — 39.5.1.11 158.8712.30 153.5 — 460 — 8/8 39.5-B7 — — 8/8 — — — — B6-MRL D122 934.37404.3 1093 — 2977.2 29 0.57 — 39.5.1.11 182723.68 189 — 42710.4 48c — 5/8 39.5-B7 — — 8/8 — — — —

B6-Smm D122 641.87356 712 — 2374 23 0.007 — 39.5.1.11 192.4746.9 191 — 38718.7 35c — 5/8 39.5-B7 — — 8/8 — — — — D122, 39.5.1.11 and 39.5-B7 transfectants were implanted i.f.p (8 mice/group) into syngeneic C57BL/6, B6-PKO B6-MRL and B6-Smn mice. Local tumors were amputated when they reached a diameter 8–9 mm and survival postamputation were monitored, when mice died, lungs were weighed to determine metastatic load. aComparsion of survival days between syngeneic C57BL/6 mice and other mouse strains injected with D122 cells (two-tailed P-value). bMice incoculated with 39.5.1.11 tumor cells were killed 30 days after D122 control mice died of spontaneous metastasis. Lungs from the mice were weighed to evaluate metastasis. cSurvival days were calculated for 3/8 mice that died. Other mice were killed 60 days after amputation.

NK and CTL responses against 39.5-B7 cells in B6-PKO, examined. Poly I:C (200 mg/0.5 ml per mouse) was injected B6-MRl and B6-Smn mice into mice (C57BL/6, B6-PKO, B6-MRL/Smn) and 18 hours later, splenocytes were coincubated with 35S-labeled To test which immunocytes mediated the rejection of target cells (D122, 39.5.1.11, 39.5-B7) including Yac cells 39.5-B7 local tumors, NK and CTL activity was (NK-sensitive target cells) as a positive control. The

Cancer Gene Therapy Tumor rejection by CTL independent of perforin/FasL S-H Lee et al 243 results (Fig 5a) show that target cell lysis by NK cells Generation of CTL in different mouse strains obtained from B6-PKO mice was completely abolished, indicating that NK cytotoxicity against the targets is Although CTL and NK act through peforin-and Fas/ dominantly induced by the perforin-mediated killing FasL-mediated pathways in acute target cells lysis, it was pathway. Although perforin-dependent killing seems to also shown that other killing mechanisms, such as TNF-a be dominant in NK, reduced NK activity was also and gIFN, play a critical role in controlling cytotoxicity in monitored in splenocytes from B6-Smm mice, especially vitro and in vivo.28,29 We tested the possibility that other against D122 and its derivatives. Moreover, 39.5.1.11 killing mechanisms independent of either perforin or Fas/ target cell lysis by NK cells from C57BL/6 and B6-MRL FasL would contribute to rejection of 39.5-B7 tumors in mice was significantly reduced compared to parental vivo. Since 39.5-B7 cells are sensitive to exogenous FasL- D122 and 39.5-B7 cells. However, D122 target sensitivity expressing cells, we could not exclude that 39.5-B7 local to NK cells is statistically similar to 39.5-B7 cells. In tumors in the mice were possibly eliminated through the agreement with previous reports, expression of MHC FasL-mediated pathway of immune cells. CTL cytotoxi- class I (Kb) inhibits NK activity through inhibitory city from the mice (C57BL/6, B6-PKO and B6-Smn) receptors like the Ly49 family members,26 while expres- implanted with 39.5-B7 cells were compared in 5 and 18 sion of B7-1 triggers NK activation.27 hours in vitro assays. Splenocytes were extracted 10 days after inoculation of 39.5-B7 tumor cells (1 Â 106 cells/ mouse), and effectors were resensitized and coincubated a 35 Ya c with [ S]-labeled 39.5-B7 target cells in the presence of 30 39.5-B7 CMA, an inhibitor of perforin-based cytotoxicity. B6-S mm 39.5.1.11 D122 The results (Fig 5b) show that 39.5-B7 cell lysis by CTLs obtained from B6-PKO mice is abrogated in a

B6-MRL short-term (5 hour) assay. Consistently, when CMA- treated effectors from syngenic C57BL/6 or B6-Smn mice were incubated with the cells under the same conditions, cells lysis was completely abrogated compared with no- B6-PKO treated and DMSO-treated CTL as controls. These results convincingly demonstrate that the perforin-mediated cytotoxicity by CTL is a dominant killing pathway in C57BL/6 elimination of the 39.5-B7 cells for a short-term (5 hours) incubation (Fig 5b). However, in an 18 hour assay, target 0 20406080100 lysis by effectors was markedly increased even when 39.5- Specific Lysis (%) B7 cells were coincubated with effectors in the presence of CMA. In addition, effectors that are treated with CMA b 80 from C57BL/6 and B6-Smn mice effectively induce in None 70 their target, 39.5-B7 cells, lysis with similar levels CMA 60 DMSO compared to B6-PKO mice. Thus, whereas the perforin- mediated killing pathway by CTL is essentially required 50 to induce target lysis for 5-hour incubation, other killing 40 mechanisms that are not dependent on FasL or perforin 30 play a major role in eliminating targets at a later time.

Specific Lysis (%)Specific Lysis 20 Mice To examine the ability of 39.5-B7 transfectants to 10 induce CTL generations, as well as the target sensitivity to 0 CTL, CTL were prepared from mice immunized with C57BL/6 B6-PKO B6-Smn C57BL/6 B6-PKO B6-Smn irradiated 39.5-B7 cells. The cytotoxicity against parental Mice D122 and the two transfectants was compared with that Figure 5 Susceptibility of tumor cells (D122, 39.5.11 and 39.5-B7) to of CTL prepared from tumor-bearing mice as described in NK and CTL in various mouse strains. (a) Mice (C57BL/6, B6-PKO previous experiments. Effectors were prepared by resti- B6-MRL and B6-Smn) were injected with Poly I:C (200 mg/0.5 ml mulation with irradiated parental D122 or 39.5-B7 cells 35 mouse) 18 h before assays. NK assays were performed as described for 4 days. The effectors were co-incubated with [ S]- in Materials and methods. Radioactivity was measured after 5 h labeled different target cells for 18 hours. The results (Fig coincubation (E:T ¼ 200:1). 39.5.1.11 versus 39.5-B7 in C57BL/6, 6) indicate that inoculation as well as immunization with Po.01; D122 versus 39.5.B7 in C57BL/6, P4.05. (b) Mice (C57BL/6, 39.5-B7 cells strongly stimulate CTL responses in all B6-PKO and B6-Smn) were inoculated with 39.5-B7 cells 6 mouse strains. Moreover, 39.5-B7 target lysis was (1 Â 10 cells/mouse). At 10 days after inoculation, effector cells were effectively induced by all effectors. Abrogation of perforin prepared according to Materials and methods. A 5 h cytotoxic assay and Fas/FasL expression in the host did not remarkably or 18 h assay (E:T ¼ 50:1) were performed in the presence or absence of CMA (100 nM) or DMSO. Data represent average 7SD of affect 39.5-B7 target lysis (Fig 6b, d). In contrast, as we triplicate samples. The same results were obtained in two indepen- expected, CTL responses in mice both inoculated or dent experiments. In the 18 h assay, CMA versus DMSO in C57BL/6 immunized with parental D122 were lower. Anti-D122 and B6-Smn. Po.05. One of two independent experiments. CTL lysed D122 and 39.5.1.11 cells at low levels, but

Cancer Gene Therapy Tumor rejection by CTL independent of perforin/FasL S-H Lee et al 244 ab 90 90 C57BL/6 80 80 C57BL/6 B6-PKO B6-PKO 70 70 B6-MRL B6-MRL 60 B6-Smn 60 B6-Smn 50 50 40 40 30 30

Specific Lysis (%) Specific Lysis 20 20 10 10 0 0 D122 39.5.1.11 39.5-B7 D122 39.5.1.11 39.5-B7 cd 90 90 80 80 70 70 60 60 50 50 40 40 30 30

Specific Lysis (%) Specific Lysis 20 20 10 10 0 0 D122 39.5.1.11 39.5-B7 D122 39.5.1.11 39.5-B7 Target cells Target cells Figure 6 Comparison of CTL responses from tumor-bearing mice with responses of mice immunized with irradiated cells in 18 h assays. Effectors were prepared from (a) D122, (b) 39.5-B7 tumor-bearing syngeneic C57BL/6, B6-PKO and B6-MRL/Smn mice as in Figure 5. (c) Irradiated D122, (d) irradiated 39.5-B7 cells were used to immunize the mice three times weekly. At 10 days after last immunization, effectors were prepared according to Materials and methods. Effectors were coincubated with [35S]-labeled target cells for 18 h (E:T ¼ 20:1). Upper panels (a) CTL from D122, (b) CTL from 39.5-B7 tumor-bearing mice, and lower panels are CTL derived from mice immunized with (c) D122, (d) 39.5- B7 cells. Data represent average 7SD of triplicate samples. Similar results were obtained in two independent experiments.

39.5-B7 cells were also effectively lysed by them (Fig 6a, weekly. At 10 days after last immunization, effectors were c). Importantly, parental D122 cells were not eliminated prepared according to Materials and methods. Effectors by their effectors prepared from B6-PKO mice. Thus, were coincubated with [35S]-labeled target cells for 18 h unlike parental D122 cells, the high immunogenic 39.5-B7 (E:T ¼ 20:1). Upper panels (a) CTL from D122, (b) CTL cells can strongly elicit CTL responses. In addition, 39.5- from 39.5-B7 tumor-bearing mice, and lower panels are B7 cells are lysed by CTL, in long-term assays, through a CTL derived from mice immunized with (c) D122, (d) mechanism other than the mechanisms mediated by 39.5-B7 cells. Data represent average 7SD of triplicate perforin and FasL. samples. Similar results were obtained in tow independent experiments.. Effectors were coincubated with [35S]- abeled 39.5-B7, in the presence of the 10 m hybridoma In vivo depletion of CD8 T-cells and neutralization of ascites containing mAb, as above for 18 h(E:T ¼ 20:1). In TNF-a and gIFN (b), non treated versus anti-CD8 in both C57BL/6 and In order to verify which cell population is responsible for B6-PKO, Po0.05. In (c) black and white bar indicate rejection of 39.5-B7 local tumors in vivo, in vivo depletion C57BL/6 B6-PKO-derived CTL, respectively. The data experiments were performed. Since perforin, gIFN and shown average 7SD of triplicate samples. Similar results TNF-a were identified as critical pathways of effector were obtained in three independent experiments. show mechanisms that characterize therapeutic anti-tumor T that local tumors of C57BL/6 and B6-PKO mice cells,31 Hybridoma ascites containing specific antibodies significantly progressed when the mice were depleted against CD8, gIFN and TNF-a were intravenously from the CD8 T-cell subpopulation. But, depletion of injected into C57BL/6 and B6-PKO mice 1 day prior to other immune cells, such as NK cells and CD4 T cells had inoculation of 39.5-B7 cells. Starting on day 6 after tumor no effect on abrogation of local tumors (data not shown). cell inoculation, mice were further injected with hybrido- The mice died of spontaneous metastasis as a result of ma ascites weekly until local tumors were amputated. The depletion of CD8 T cells (data not shown). Therefore, results in Figure 7a. (c) Irradiated D122, (d) irradiated CD8 T cells, in C57BL/6 and B6-PKO mice inoculated 39.5-B7 cells were used to immunize the mice three times with 39.5-B7 cells, mainly contribute to tumor rejection,

Cancer Gene Therapy Tumor rejection by CTL independent of perforin/FasL S-H Lee et al 245 as well as to the prevention of metastasis. However, Initially, local tumors of B6-PKO mice injected with neutralization of gIFN and TNF-a activity using specific antibodies, including control IgM, developed, but the antibodies in vivo did not prevent rejection of the local palpable tumors regressed 26 days after injection and were tumors in the C57BL/6 and B6-PKO mice (Fig 7a). finally rejected. In contrast, local tumors in the mice depleted from CD8 T cells continuously developed. In parallel, we also tested whether the antibodies can inhibit a CTL cytotoxicity against 39.5-B7 target during in vitro 100 Control assays. The results show that 39.5-B7 target lysis by C57BL/6 and B6-PKO derived effectors is significantly 80 YTS R4-6A2 reduced compared to only effector cells and rat IgG TNF-alpha groups in the presence of anti-CD8 antibodies (Fig 7b). 60 Rat lgM Moreover, neutralization of cytokines during CTL assays 40 using the same anti-gIFN, and anti-TNF-a antibodies did not inhibit CTL activity (Fig 7c). These results indicate 20 that CD8 T-cells are directly responsible for rejection of 39.5-B7 tumors in the C57BL/6 and B6-PKO mice in vivo. 0 Interestingly, elimination of 39.5-B7 cells by CD8 T-cells 1419 24 29 34 is not dependent on both TNF-a-and gIFN-mediated 100 mechanisms in vivo.

80 Caspase-dependent apoptosis in tumor cells

Local tumour diameter (X0.1mm) Since it was reported that Fas/FasL-and perforin/ 60 granzyme B-mediated pathways induce caspase-depen- 40 dent as well as caspase-independent apoptosis in the cells, we studied whether caspase activation is required for lysis 13,14,32 20 of 39.5-B7 cells by CTL. Moreover, we wanted to examine which caspases have essential functions to induce 0 apoptosis by CD8 T cells using caspase inhibitors. 1419 24 29 34 CTL from C57BL/6 and B6-PKO mice immunized with Days after injection irradiated 39.5-B7 cells were tested in 18 hour assays, in the presence and absence of the broad caspase inhibitors, b 80 z-VAD-fmk (50 mM) and inhibitors of caspase-8 (z-IETD- None 70 fmk, 50 mM) or caspase-9 (z-LEHD-fmk, 50 mM)(Fig 8). Anti-CD8 The results show partial inhibition of apoptosis mediated 60 Rat IgG by C57BL/6 derived CTL and complete inhibition of 50 apoptosis mediated by B6-PKO derived CTL in the presence of z-VAD-fmk. These results strongly suggest 40 that caspase activation is required to induce 39.5-B7 cells 30 lysis through the perforin-independent killing mechanism. Specific Lysis (%) Specific Lysis However, target cell lysis involving a perforin-mediated 20 killing pathway can also be induced without caspases 10 activations. Besides in vitro assays, when DNA fragmen-

0 C57BL/6 B6-PFP Figure 7 The influence of CD8 þ , gIFN and TNF-a depletion on tumorigenicity and CTL of 39.5-B7. (a) Syngeneic C57BL/6 and B6- c 80 PKO mice were injected i.v. with hybridoma ascites containing antibodies against CD8 (YTS-164), gIFN (R4-6A2), TNF-a or rat IgM 70 as control, The following day, 39.5-B7 tumor cells (2 Â 105 cells/ mouse) were inoculated into the footpads of the mice. Tumor growth 60 was measured at different time intervals. Additional mAb doses were 50 injected i.p. weekly to maintain the depleted state. The data shows a representative experiment of three independent experiments. (b and 40 c) CTL assays were performed using effectors from mice immunized 35 30 like in Figure 6. Effectors were coincubated with [ S]- labeled 39.5- B7, in the presence of the 10 ml hybridoma ascites containing mAb,

Specific Lysis (%) Specific Lysis 20 as above for 18 h (E:T ¼ 20:1). In (b), nontreated versus anti-CD8 in both C57BL/6 and B6-PKO, P .05. In (c) black and white bar 10 o indicate C57BL/6 B6-PKO-derived CTL, respectively. The data 0 shown average 7SD of triplicate samples. Similar results were None Anti-γIFN Anti-TNFα Rat IgG obtained in three independent experiments.

Cancer Gene Therapy Tumor rejection by CTL independent of perforin/FasL S-H Lee et al 246 90 lysis through both perforin-and Fas/FasL-independent mechanisms. In addition, we showed that, whereas 39.5- 80 B7 cell lysis by NK cells was dependent on perforin, 70 coincubation with CTL obtained from C57BL/6, B6-PKO and B6-Smn mice in the presence of CMA could 60 effectively eliminate 39.5-B7 cells in an 18 hour assay. 50 The CTL cytotoxicity against 39.5-B7 cells was responsible for rejection of the tumor in vivo, but did not involve 40 TNF-a-and gIFN-mediated cytotoxicity. Furthermore, 30 the perforin independent CTL lysis of 39.5-B7 cells was

Specific Lysis (%) Specific Lysis blocked by the broad caspase inhibitor, z-VAD-fmk 20 indicating that caspase activation in the cells was required 10 to induce 39.5-B7 cell lysis by CTL through the perforin- independent cytotoxicity. However, caspase-8 and cas- 0 pase-9 inhibitors did not markedly inhibit 39.5-B7 lysis. None z-VAD-fink z-IETD-fink z-LEHD-fink Thus, these results suggest that CD8 T-cells, which are Caspase Inhibitors activated by inoculation with H-2Kb and B7-1 expressing Figure 8 Inhibition assays using different caspase inhibitors to test tumor cells, contribute to rejection of 39.5-B7 local caspases activation in target cells. Effectors were coincubated with tumors in the mice. The CTL can induce apoptosis of [35S]-labeled 39.5-B7 target cells in the presence of following their target through neither perforin-nor Fas/FasL- caspase inhibitors, Caspase inhibitor I (z-VAD-fmk: 50 mM), cas- mediated mechanisms, but caspase activation in the cells pase-8 inhibitor II (z-IETD-fmk: 50 mM) and caspase-9 inhibitor I is needed to eliminate 39.5-B7 cells by the CTL. (z-LEHD-fmk: 50 mM) for 18 h (E:T ¼ 10:1). The black and white bars Parental D122 cells were resistant to FasL-expressing indicate C57BL/6 and B6-PKO derived CTL, respectively. The data 7 cells due to lack of Fas expression on the cell surface. But, shows average SD of triplicate samples. The same results were gIFN secreting D122 cells, DIF-2, can effectively enhance obtained in three independent experiments. Fas expression and increase their sensitivity to FasL- expressing cells (Figs 1,2). Unlike DIF-2 cells, levels of Fas tations of 39.5-B7 target cells were tested after co expression on 39.5-B7 cells are not influenced by cell incubation with CTL under the same conditions, we density, indicating that the slight increase in Fas expression observed typical DNA ladders on gels, indicating that on the cells resulted most probably from a cloning effect of apoptotsis was induced in 39.5-B7 cells (data not shown). the transfectants. Nevertheless, these results show that, in Transmission of apoptotic signals mediated by cell surface contrast to other tumors, the Fas-mediated apoptotic Fas receptor is mainly dependent upon activation of pathway on D122 cells is not blocked by inhibitors or is not caspase-8 or caspase-9, which can be activated through defective (Fig 2). Bergmann-Leitner and Abrams34,35 have molecules released from the mitochondria as an internal compared lytic sensitivity of primary colon adenocarcino- signal. We assumed that if Fas expression on 39.5-B7 cells ma cells with that of metastatic colon tumor cells to an has a crucial role in induced target cell lysis by CD8 T established CTL line after gIFN treatment of the tumor cells, CTL cytotoxicity against 39.5-B7 cells can be cells. In contrast to increased lytic sensitivity of primary markedly inhibited in the presence of these caspase colon tumor cells by Fas-dependent and -independent inhibitors in in vitro assays.10,33 However, incubation pathways, metastatic colon tumor cells were not sensitive with 39.5-B7 cells in the presence of caspase-8 inhibitors to Fas-dependent cytotoxic activity, suggesting that the (z-IETD-fmk) or caspase-9 inhibitors (z-LEHD-fmk) did metastatic phenotype may be associated with resistance to not significantly inhibit CTL cytotoxicity (Fig 8). Thus, Fas-mediated lysis in response to tumor-specific T-cells CTL cytotoxicity mediated by the perforin-independent attack. Whether enhancement of Fas receptor expression pathway does not involved the Fas/FasL-mediated killing on DIF-cells is directly related to increased immunogeni- mechanism, yet caspases activation is a prerequisite for city should be further investigated. the induction of 39.5-B7 cell lysis by CD8 T cells. NK lysis of target cells can be regulated by activatory and inhibitory signals.36 Mouse NK function can be inhibited by Ly49 receptor families that are specific for classical MHC class I molecule on NK cells.26 In this Discussion study, we can confirm previous reports demonstrating that B7-1 expressing target cells are more susceptible to We demonstrated, in the present study, that perforin NK lysis than nonexpressor cells.27,37 Here, our results expression by the host mainly contributes to prevention of show that, although levels of H-2Kb on the 39.5-B7 spontaneous metastasis from the highly metastatic D122 transfectant are equivalent to the levels H-2Kb on local tumors. However, although 39.5-B7 cells were 39.5.1.11 cells, 39.5-B7 target cells were significantly more eliminated by the human FasL transfectants, abrogation sensitive to NK lysis. This result indicates that B7-1 of both perforin and Fas/FasL expression in the host did expression on NK target functions as an additional not affect rejection of 39.5-B7 tumors in vivo. These activation signal that overcomes reduced killing activity results suggest that immune cells can induce 39.5-B7 cell by H-2Kb molecules (Fig 5).

Cancer Gene Therapy Tumor rejection by CTL independent of perforin/FasL S-H Lee et al 247 Despite the fact that 39.5-B7 cells could be eliminated transmit death signals to the cells. Malyguine et al44 have by FasL-expressing cells, effectors that were blocked in reported that the caspase inhibitor zVAD-fmk had no or both peforin-and Fas/FasL-mediatedkilling could still significantly lower effect on B6-Smn T-cells cytotoxicity effectively lyse their target cells in 18-hour assays. This against A20 cells. In contrast, the same concentrations of observation is also supported by the fact that 39.5-B7 zVAD-fmk practically blocked the lytic activity of B6- local tumors are rejected in both B6-PKO and B6-MRL/ PKO mice T cells. This observation strongly suggests that Smn mice in vivo. Besides the two main killing pathways, perforin-independent apoptotic pathways mediated by it has been reported that other killing mechanisms, for CTL are different from the perforin-mediated lytic example the proinflammatory cytokines gIFN and TNF- mechanisms. It might be necessary to further investigate a, have important roles in rejection of pulmonary how perforin-independent lytic activity can be triggered to metastasis of tumor cells.38 In the study of Poehlein induce target lysis by CTL. et al,31 adoptive transfer of effector T cells, which are In summary, we demonstrated, in this study, that taken from perforin and gIFN double knockout mice whereas perforin-mediated cytotoxicity has a dominant (PKO/GKO), mediated complete tumor regression and function in elimination of highly metastatic D122 cells, cured wild-type animals with established pulmonary the H-2Kb and B7-1 transfected D122, 39.5-B7 cells were metastasis of melanoma cells by a TNF-a mediated sensitive to CTL killing through neither perforin nor Fas/ killing mechanism. In addition, Smyth et al39 have FasL-mediated lysis. Caspase activation was required to reported that, using the Renca carcinoma tumor model, elicit cell lysis by CTL. Furthermore, this perforin-and TRAIL expression on NK and NKT cells has effective FasL-independent lytic activity by CD8 T cells played a antimetastatic functions in vivo mediated by a gIFN- major role in rejection of 39.5-B7 cells in vivo. dependent immune mechanism. In contrast to the tumor models used in previous studies, we demonstrated here that the high metastatic Lewis lung carcinoma clone, Acknowledgments D122 cells were not sensitive to Fas or TRAIL-mediated apoptotic pathways. Perforin expression plays a major This study was supported by grants from the Israel role in protection of the host from tumor progression, as Science Foundation, The ISRAEL CANCER ASSOCIA- well as in the kinetics of spontaneous metastasis. But, TION, the Israel Cancer Research Fund, The Lombroso 39.5-B7 cells, which are high immunogenic cells expres- fund and the Ornest family fund (to L. Eisenbach). sing both H-2Kb and B7-1(CD80), could be rejected in C57BL/6 and B6-PKO mice neutralized with anti-TNF-a and anti-gIFN antibodies, but not in mice depleted from References CD8 T cells (Fig 7). These results indicated that TNF-a- or gIFN-mediated cytotoxic mechanisms did not play an 1. Thompson CB, Lindsten T, Ledbetter JA, et al. CD28 important function in rejection of tumors by CD8 T cells activation pathway regulates the production of multiple T- in vivo. In vitro assays, using antibodies that were used for cell-derived lymphokines/cytokines. Proc Natl Acad Sci USA. 1989;86:1333–1337. in vivo experiments, confirmed that cytokine-mediated 2. Boise LH, Minn AJ, Noel PJ, et al. CD28 costimulation can cytotoxicity was not involved in 39.5-B7 cells lysis by CD8 promote T cell survival by enhancing the expression of Bcl- T cells. Except these killing mechanisms, what kind of XL. Immunity. 1995;3:87–98. cytotoxic activity can potentially be responsible for 3. 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