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The Journal of Immunology, 2001, 166: 3240-3247.
Copyright © 2001 by The American Association of Immunologists

CD95 Ligand-Expressing Tumors Are Rejected in Anti-Tumor TCR Transgenic Perforin Knockout Mice

Christian K. Behrens2,*, Frederik H. Igney*, Bernd Arnold*, Peter Möller{dagger} and Peter H. Krammer3,*

* Tumor Immunology Program, German Cancer Research Center, Heidelberg, Germany; and {dagger} Institute for Pathology, University of Ulm, Ulm, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD95 (APO-/Fas) ligand (CD95L) is a member of the TNF family predominantly expressed by activated T and NK cells but also by tumors of diverse cellular origin. CD95L trimerizes surface CD95 expressed by target cells that subsequently undergo apoptosis. The role of the CD95/CD95L system in the down-regulation of an immune response (activation-induced cell death) is established. However, it is so far unclear why tumors express CD95L. To investigate whether tumors use the CD95L to down-regulate an anti-tumor immune response, we established a transgenic (tg) mouse model consisting of 1) apoptosis-resistant tumor cells, designated LKC-CD95L, which express functional CD95L and the model tumor Ag Kb; and 2) perforin knockout (PKO) anti-Kb TCR tg mice. L1210-Fas antisense expressing Kb, crmA, and CD95L (LKC-CD95L) killed CD95+ unrelated tumor targets and Con A-activated splenocytes from anti-Kb TCR tg PKO mice by a CD95L-dependent mechanism in vitro. However, we could not detect any cytotoxic activity against anti-tumor (anti-Kb) T cells in vivo. We also observed reduced growth of LKC-CD95L in nude mice and rapid rejection in anti-Kb TCR tg PKO mice. Because the tumor cells are resistant to CD95L-, TNF-{alpha}-, and TNF-related apoptosis-inducing ligand-induced apoptosis and the mice used are perforin-deficient, the involvement of these four cytotoxicity mechanisms in tumor rejection can be excluded. The histological examination of tumors grown in nude mice showed infiltration of LKC-CD95L tumors by neutrophils, whereas L1210-Fas antisense expressing Kb and crmA (LKC) tumor tissue was neutrophil-free. Chemotaxis experiments revealed that CD95L has no direct neutrophil-attractive activity. Therefore, we conclude that LKC-CD95L cells used an indirect mechanism to attract neutrophils that may cause tumor rejection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD95 and CD95 ligand (CD95L)4 are members of the TNF receptor and the TNF family, respectively. Whereas CD95 is expressed in many tissues, expression of CD95L is mainly found in activated T and NK cells (1, 2, 3, 4). Trimerization of CD95 either by agonistic Abs or by CD95L induces target cell apoptosis (5). The CD95/CD95L system is involved in the apoptotic elimination of T cells upon termination of an immune response, a phenomenon called activation-induced cell death or AICD (6, 7, 8).

However, the functional relevance of CD95L expression by tumors of diverse cellular origin discovered recently in our own and other laboratories is so far unclear. This finding suggested a novel role for CD95L in immune escape of tumors. T cells at defined stages of activation are sensitive to CD95-mediated apoptosis (9) and, hypothetically, may be killed by CD95L+ tumor cells. This may lead to depletion of anti-tumor T cells resulting in immunosuppression and outgrowth of the tumor. To date many "passive" immune escape mechanisms have been described, e.g., MHC down-regulation or absence of costimulatory molecules (10, 11). Elimination of tumor-reactive T cells by cytotoxic CD95L+ tumors may represent an "active" way to circumvent tumor rejection by the host and has been named "tumor counterattack" (12, 13). Tumor counterattack has been described, e.g., for melanoma, hepatocellular carcinoma, and esophagus cancer (14, 15, 16). However, several contradicting results have been published. Mice injected with syngeneic CD95L+ tumor cells did not develop tumors but showed a strong neutrophil-mediated response against the grafted cells (17). Moreover, an s.c. growing syngeneic CD95-negative tumor was rejected immediately when the tumor cells were infected with CD95L-coding adenovirus (18). Immunosuppression by CD95L is also discussed in allotransplantation. Thus, according to Bellgrau et al. (19), testis tissue from wild-type mice (CD95L+) survived longer than testis tissue from gld mice (CD95L mutated) upon transplantation under the kidney capsule of allogeneic mice. Lau et al. (20) prevented the rejection of allogeneic {beta} cells by cotransplantation of CD95L-transfected myoblasts. However, these data have not been confirmed. Under similar experimental settings, infiltration of neutrophilic granulocytes followed by vigorous rejection of the allografts was observed. Transgenic (tg) expression of CD95L on {beta} cells also led to neutrophil infiltration and {beta} islet destruction (21, 22, 23).

The above-mentioned controversial results that described immunosuppression via counterattack, on the one hand, and vigorous inflammatory responses including neutrophil attraction, on the other hand, by CD95L+ tumors suggested the need for a tumor model with well defined components, which allows a more detailed study of the counterattack phenomenon. Therefore, in this study, tumor cells resistant to CD95L-, TNF-{alpha}-, and TNF-related apoptosis-inducing ligand (TRAIL)-mediated killing were transfected with CD95L and the model tumor Ag MHC class I H-2Kb. The use of mice that express an anti-Kb tg TCR enabled specific allogeneic recognition between tumor cells and T cells. The haplotypes of mice and tumor cells have been chosen in a way that only the alloantigen H-2Kb acts as a tumor Ag. Upon injection of CD95L+ tumor cells, we followed the fate of the anti-tumor T cells with the mAb Désiré-1 (anti-clonotype) directed against the anti-Kb TCR (24). Perforin was described to be involved in rejection of tumors (25). To exclude rapid elimination of CD95L+ tumor cells by a perforin/granzyme B-mediated mechanism we performed the in vivo experiments in anti-Kb TCR tg mice backcrossed to a perforin knockout (PKO) background (26, 27).

The L1210-Fas antisense expressing Kb, crmA, and CD95L (LKC-CD95L) tumor cell line we generated had cytotoxic activity against CD95+ tumor targets and Con A-activated splenocytes. However, it showed slow growth kinetics in nude mice and was rapidly rejected in anti-Kb TCR tg PKO mice. The tumor tissue was infiltrated by neutrophils, indicating an important role of these cells in rejection of CD95L+ tumors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

CD1 Swiss nude mice (8–12 wk old) were purchased from Iffa Credo (L’Arbresle Cedex, France). PKO mice were provided by Jürg Tschopp (BIL Biomedical Research Center, Lausanne, Switzerland). Anti-Kb TCR tg PKO (haplotype: H-2dxk) mice were obtained by breeding H-2dxk anti-Kb TCR mice (26) with H-2b PKO mice. TCR tg PKO mice used in the following experiments were in their fourth generation and were 8–12 wk old. The perforin genotype was determined as described (28).

Cell lines

L1210 is a lymphatic leukemia cell line from the DBA/2 mouse (haplotype: H-2d). L1210 wild type, L1210-Fas, and L1210-Fas antisense (29, 30) were provided by Gideon Berke (Weizmann Institute of Science, Rehovot, Israel). PC60 hybridoma cells (31) were used as mediators of membrane-CD95L killing (3). All cell lines were cultured in RPMI 1640 except PC60, which were cultured in DMEM. Cell culture media were supplemented with 10% FCS, 10 mM HEPES, 50 µg/ml gentamicin, 1 mM sodium pyruvate, and 50 µM 2-ME.

Reagents

Mouse recombinant TNF-{alpha} was obtained from Bernd Echtenacher (University of Würzburg, Würzburg, Germany). TNFR1-Fc was generated by transient transfection of COS-7 cells with the pCMV4-TNFR1-Fc plasmid (32). Leucine zipper (LZ)-TRAIL was obtained from Henning Walczak (German Cancer Research Center, Heidelberg, Germany).

Generation of LKC-CD95L and L1210-Fas antisense expressing Kb and crmA (LKC) cells

L1210-Fas antisense cells were transfected with pUC19 vector containing the Kb cDNA under the control of its endogenous promoter (33). Transfectants were stained with the Kb-specific mAb B8-24.3 (34), sorted in a Becton Dickinson (Mountain View, CA) FACSort, and subcloned to yield a clonal population of L1210-Fas antisense-Kb cells. Subsequently, these cells were transfected either with pEF-PGK puro crmA-flag (35) alone (designated LKC) or with pEF-PGK puro crmA-flag plus pFM92-mCD95L (designated LKC-CD95L). Transfectants were selected with 10 µg/ml puromycin (Sigma, Deisenhofen, Germany) and tested for CD95L and crmA-flag expression in a cytotoxicity assay or by immunoblotting, respectively. Each clone was subcloned and retested twice.

Tumor injection

A given number of tumor cells were resuspended in 200 µl PBS and injected s.c. into the right flank of nude or of anti-Kb TCR tg PKO mice. To determine the tumor size, two perpendicular tumor diameters, d1 and d2, were measured with a caliper, and the tumor area A was determined according to the simplified formula A = (d1 x d2)/2.

RT-PCR

Total RNA from cell lines was isolated using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed from 1 µg of total RNA with the GeneAmp RT-PCR kit (Perkin-Elmer, Weiterstadt, Germany) using random hexamers. Amplification of mCD95L, CD95, and {beta}-actin with gene-specific primers was performed under standard conditions. The primer sequences and annealing temperatures (Ta) were as follows: mCD95L CTT GGG CTC CTC CAG GGT CAG T and TCT CCT CCA TTA GCA CCA GAT CC, Ta = 56°C; {beta}-actin ATT GTT ACC AAC TGG GAC GAC ATG and CTT CAT GAG GTA GTC TGT CAG GTC, Ta = 56°C; mCD95 CGC GGA TCC ACC ATG CTG TGG ATC TGG GCT and CGC GAA TTC TCA CTC CAG ACA TTG TCC, Ta = 54°C.

51Cr release assay

When cell lines were used as targets, a 51Cr release assay was performed as follows. Target cells (3 x 106) were labeled in 100 µl (100 µCi) Na251CrO4 solution (NEN, Neu-Isenburg, Germany) for 45 min and washed three times in RPMI 1640. Aliquots (100 µl) of the cell suspension (105 cells/ml) were incubated in triplicate for 4 or 16 h with effector cells at the indicated E:T ratios and analyzed using a Packard Autogamma counter. Specific lysis (L) was calculated according to the formula L = (E - S)/(T - S), with E = count rate of the unknown sample, S = spontaneous release, and T = total release. In all assays, the ratio S/T was smaller than 25%. When PC60 were used as effectors, exponentially growing cells were activated for 4 h with 3.5 ng/ml PMA and 2.1 µg/ml ionomycin.

JAM test

When spleen cells were used as targets, the JAM test was performed essentially as described (36) except that Con A-activated cells were labeled for 8 h in RPMI 1640 containing 25 µCi/ml [3H]thymidine. The plates were harvested in a micro cell harvester and counted in a Wallac 1205 Betaplate Counter. Specific death (S) was calculated with the formula S = (T - E)/T, with T = radioactivity in the absence of effector cells and E = radioactivity of unknown sample.

MTT assay

Target cells (104) were seeded in triplicate in flat-bottom 96-well microtiter plates and incubated for 16 h with the reagents to be tested. MTT solution (25 µl; 5 mg/ml) was added and incubated for 4 h. By the addition of 100 µl 2-propanol containing 5% (v/v) formic acid the formazan crystals were dissolved under vigorous shaking and the absorption at {lambda} = 550 nm (A550 nm)was determined in an ELISA reader. Specific cell death (S) was calculated using S = 1 - (E/T) with T = A550 nm in the absence of cytotoxic stimulus and E = A550 nm of unknown sample.

Immunoblot

Immunoblots were performed as previously described (37). Briefly, postnuclear supernatants were separated under reducing conditions in a 10% polyacrylamide gel and subsequently electroblotted onto a Hybond C nitrocellulose membrane. The membrane was incubated for 1 h with PBS/5% low-fat milk powder followed by three washes in PBS/0.05% Tween 20. It was then incubated with 1 µg/ml M2 anti-flag Ab in PBS/Tween 20 for 16 h at 4°C, washed as above, and further incubated for 1 h with a HRP-coupled goat anti-mouse-IgG Ab (1:20,000 in PBS-Tween 20). Detection was performed using the ECL system according to the manufacturer’s instructions (NEN, Neu-Isenburg, Germany).

Histological analysis

Tumor tissue samples were formalin-fixed and subjected to routine hematoxylin and eosin histology. To specifically stain granulocytes, naphthol-AS-D-chloroacetate esterase staining was performed according to the standard protocol of Leder (38).

Phenotype analysis of mouse spleen and lymph node (LN) cells

Spleen cells and cells from the mesenteric and suprafacial inguinal LN of tumor-injected mice were stained using Des-FITC Abs specific for the anti-Kb tg TCR (24), PE-labeled anti-B220 (clone RA3-6B2; PharMingen, Hamburg, Germany), and biotinylated TCR{alpha}{beta} (clone H57-597; PharMingen) and were analyzed in a Becton Dickinson FACScan. Streptavidin-Red670 (Life Technologies, Eggenstein, Germany) was used as secondary reagent.

Chemotaxis

Chemotaxis assays were performed as described by Brenneis et al. (39). Briefly, granulocytes were enriched from human peripheral blood. Erythrocytes were removed by repeated, short incubation, 20 s each, in hypotonic NaCl (0.2%) followed by the addition of the same volume of 1.6% NaCl solution. The remaining cells (>80% granulocytes by FACScan) were taken up in HBSS buffer at a density of 106 cells/ml. Duplicates of the reagents to be tested were entered into Boyden chambers, which were then closed by a membrane (pore size 3 µm; Schleicher und Schüll, Dassel, Germany) and overlaid with 900 µl of granulocyte suspension. After 90 min at 37°C the membranes were stained as follows: 30 s in 2-propanol, 3 min in hematoxylin solution, 3 min in 70% acidic 2-propanol, 3 min in "blueing agent" (166 mM MgSO4, 24 mM NaHCO3), 3 min in 70% 2-propanol, 2x 3 min in 95% 2-propanol, 2x 3 min in 100% 2-propanol, and 2x 3 min in m-xylene. The filters were then embedded in Canada balsam (Roth, Karlsruhe, Germany) and analyzed using an Omnicon Alpha image analyzer (Bausch & Lomb, Dornach, Germany) connected to a motor-driven microscope table counting cells electronically in 10-µm steps. The integral of the distribution curve corresponds to the chemotactic index.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation and characterization of LKC-CD95L transfectants

A prerequisite for our tumor model was a cell line that is resistant to CD95-mediated killing to prevent suicide or killing of neighboring cells in the transplanted CD95L+ tumor. Moreover, resistance toward TNF-{alpha} and TRAIL-mediated cell death was desirable. A cytotoxicity assay using PC60 effector cells that show CD95L-mediated killing revealed a very low sensitivity of the L1210-Fas antisense cell line (data not shown and Ref. 30). These cells were transfected with cDNAs coding for the model tumor Ag Kb and the CD95L. In addition, we cotransfected a cDNA coding for the caspase inhibitor crmA to further increase the resistance toward death receptor-mediated killing (40, 41). The sequential modification steps and their purposes are shown in Fig. 1Go. L1210-Fas antisense cells expressing Kb and crmA-flag were designated LKC, and L1210-Fas antisense cells expressing Kb, crmA, and CD95L were designated LKC-CD95L.



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  		FIGURE 1. Generation of LKC-CD95L and LKC tumor cells. LKC-CD95L and LKC cells were generated from L1210-Fas antisense cells in two subsequent steps. Each newly generated cell line was subcloned and tested as described in Materials and Methods.

 
LKC and LKC-CD95L were characterized with respect to their expression of Kb, crmA-flag, and CD95L, their sensitivity toward CD95L-mediated cell death, and their cytotoxic activity. Expression of the model tumor Ag Kb was demonstrated by staining with B8-24.3 followed by FACS analysis (Fig. 2GoA). Fig. 2GoB shows crmA expression of LKC and LKC-CD95L detected by immunoblotting. Using the M2 anti-flag Ab we found similar crmA expression levels in LKC and LKC-CD95L. The expression of CD95L mRNA by LKC and LKC-CD95L was analyzed by RT-PCR. Although we detected a PCR product corresponding to CD95L in the LKC-CD95L sample, LKC did not express any endogenous CD95L (Fig. 2GoC).



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  		FIGURE 2. Characterization of LKC-CD95L and LKC cells. A, LKC-CD95L and LKC express MHC Kb. L1210-Fas antisense, LKC-CD95L, and LKC were stained with the anti-Kb Ab B8-24.3. PE-labeled goat-anti-mouse Ig served as secondary Ab. B, Detection of crmA expression in LKC-CD95L and LKC by immunoblotting. C, RT-PCR analysis of CD95L expression in LKC-CD95L and LKC cells.

 
We next performed a series of cytotoxicity assays to investigate the sensitivity of LKC and LKC-CD95L for receptor-mediated cell death. First, we showed that LKC and LKC-CD95L were resistant to CD95L-mediated apoptosis using PC60 effector cells. PC60 cells mediate membrane CD95L killing (Refs. 3 and 31 and our experiments). L1210-Fas (29) served as a positive control (Fig. 3GoA). Second, LKC and LKC-CD95L were treated with 500 U/ml of soluble murine TNF-{alpha} (42) in the presence or absence of 10 µg/ml soluble TNF receptor 1 (TNFR1-Fc) using the Kym-1 cell line as a positive control for TNF activity. Kym-1, but not LKC and LKC-CD95L, was found to be sensitive to TNF-{alpha}-mediated cell death (Fig. 3GoB). Third, LKC and LKC-CD95L were tested for their sensitivity to TRAIL-mediated cell death. Although 60% of the positive control cells J16 underwent apoptosis after treatment with 3 µg/ml LZ-TRAIL (43), LKC and LKC-CD95L were not killed by LZ-TRAIL (Fig. 3GoC).



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  		FIGURE 3. Resistance of LKC-CD95L and LKC against death receptor-mediated cell death and cytotoxicity against tumor and T cell targets. A, Resistance of LKC-CD95L and LKC against CD95-mediated apoptosis. PC60 cells were activated for 4 h with 3.5 ng/ml PMA and 2.1 µg/ml ionomycin. The target cells LKC-CD95L ({blacksquare}), LKC ({square}), and LF+ ({diamond}) as a positive control were 51Cr-labeled and coincubated in triplicate with activated PC60 effector cells at the indicated E:T ratios for 14 h. The mean ± SD of specific lysis at a certain E:T ratio is shown. B, LKC, LKC-CD95L, and Kym-1 cells were incubated in triplicate with mouse TNF-{alpha} and TNFR1-Fc as indicated. After 16 h, MTT solution was added and incubated for another 4 h. After the dissolution of formazan crystals, the absorption at {lambda} = 550 nm was measured in an ELISA reader. C, Jurkat T cells ({lozenge}), LKC-CD95L ({blacksquare}), and LKC ({square}) were 51Cr-labeled and coincubated in triplicate with the indicated concentrations of LZ-TRAIL for 14 h. The mean ± SD of specific lysis at a certain E:T ratio is shown. D, Cytotoxic activity of LKC-CD95L against CD95+ tumor target cells. LKC-CD95L ({blacksquare}), LKC ({square}), LKC-CD95L in the presence of human CD95-Fc ({triangleup}) or of human IgG1 ({blacktriangleup}) were coincubated in triplicate with 51Cr-labeled P815-Kb for 14 h at the indicated E:T ratios. The mean ± SD of the specific lysis is shown. E, LKC-CD95L shows cytotoxic activity against Con A blasts. LKC-CD95L ({blacksquare}) and LKC ({square}) were coincubated in triplicate for 14 h with [3H]thymidine-labeled Con A blasts from anti-Kb TCR tg PKO mice at the given E:T ratios (JAM test). 2 mM EGTA/3 mM MgCl2 ({blacktriangleup}) or 20 µg/ml CD95-Fc ({triangleup}) were present where indicated. Shown are the mean ± SD of triplicates.

 
Additionally, LKC and LKC-CD95L were shown to be sensitive to killing via the perforin/granzyme pathway (data not shown). To exclude a rejection of the tumor cells by this mechanism in vivo, PKO mice were used.

To test the cytotoxic activity of LKC and LKC-CD95L against tumor and T cell targets, we performed the killing experiments shown in Fig. 3Go, D and E. The CD95-expressing tumor target cells P815-Kb were lysed by LKC-CD95L, whereas LKC cells did not possess any cytotoxic activity. Cell death induced by LKC-CD95L was mediated by CD95L as it could be blocked by the addition of CD95-Fc fusion protein, a CD95L blocker (Fig. 3GoD). To simulate the in vivo encounter between tumor and T cells we tested whether LKC-CD95L could also kill T cells. Using Con A-activated spleen cells from TCR tg PKO mice as target cells we confirmed that the killing activity of LKC-CD95L was not restricted to tumor targets. By the addition of EGTA/Mg2+, a blocker of perforin-mediated cell death, or CD95-Fc, it was demonstrated that cell death was mediated by CD95L (Fig. 3GoE).

In summary, LKC-CD95L cells express membrane CD95L, which mediates cytotoxic activity against CD95+ tumor and activated T cell targets. LKC-CD95L and LKC express similar levels of Kb and crmA and are completely resistant to killing mediated via death receptors for CD95L, TNF-{alpha}, and TRAIL. Thus, the important difference between LKC and LKC-CD95L is the expression of functional CD95L. In all other aspects tested, particularly with respect to resistance to cytotoxicity, LKC and LKC-CD95L are similar.

Different growth rates of LKC-CD95L and LKC in nude mice

LKC-CD95L and LKC showed equal growth rates in vitro (data not shown). To confirm that the same was true in vivo in the absence of a specific cellular immune response, we s.c. injected 2 x 106 cells into CD1 Swiss nude mice. On days 10, 12, and 16 tumor growth was monitored. On day 16 the animals were killed, and the tumors were tested for Kb expression and resistance to CD95L-mediated killing. No differences were detected between the original tumor cell lines and tumor material excised from nude mice. Furthermore, we confirmed constitutive CD95L expression of ex vivo LKC-CD95L by RT-PCR. In chromium release assays we found that these tumors had the same cytotoxic activity as the original tumor cell line (data not shown). We infer from these experiments that CD95L was also constitutively expressed in vivo.

Unexpectedly and in contrast to the in vitro growth behavior, LKC-CD95L showed significantly slower growth kinetics than the CD95L- control cell line LKC (Fig. 4GoA). Chloroacetate esterase staining of paraffin-embedded tumors showed leukocyte margination (Fig. 4GoB) and a massive neutrophil infiltration in all CD95L-expressing tumors especially in areas of massive tumor cell apoptosis (Fig. 4GoC), whereas none of the control tumors was infiltrated and intratumoral apoptotic rate was low (data not shown). Because the neutrophils were detected within the tumor and not in the border zones, it is conceivable that these cells have a direct cytotoxic effect on the tumor.



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  		FIGURE 4. Growth kinetics and histological analysis of LKC-CD95L and LKC in nude mice. A, Cells (2 x 106) of either LKC-CD95L ({blacksquare}) or LKC ({square}) were s.c. injected into the right flank of CD1 Swiss nude mice (n = 4). On days 10, 12, and 16, tumor growth was measured. B and C, On day 16 the mice were sacrificed, and the tumor tissue was investigated histologically. Chloroacetate esterase staining (red) showed extensive neutrophil margination (B, original magnification x70) and massive infiltration (representative cell clusters are marked by arrows) into the LKC-CD95L tumor tissue, which was paralleled with a high intratumoral apoptotic rate (C, original magnification x280). All animals within a group showed identical results.

 
Rapid rejection of LKC-CD95L in anti-Kb TCR tg PKO mice

Next we examined the growth behavior and the potential immunosuppressive effect of CD95L+ tumor cells in the presence of tumor-specific tg T cells. Thus we s.c. injected different numbers of LKC and LKC-CD95L cells into anti-Kb TCR tg PKO mice (26, 27). The tumor incidence of LKC and LKC-CD95L is shown in Fig. 5Go. Although 105 cells of the CD95L- control cell line LKC formed tumors in anti-Kb TCR tg PKO mice, LKC-CD95L cells were not tumorigenic even when a 10-fold higher number of tumor cells was injected. Although no tumor growth of LKC-CD95L was observed, we examined whether LKC-CD95L had induced deletion of anti-Kb T cells. Thus, we analyzed the spleen and LN composition by triple staining with Abs against the tg TCR, B220, and TCR{alpha}{beta} (data not shown). The percentages of B and T cells in spleens and LN of mice that had been injected with LKC-CD95L or LKC, respectively, did not differ significantly. Moreover, the percentage of clonotype+ T cells within the whole T cell population did not vary between mice injected with CD95L+ and CD95L- tumors. The histological analysis of spleen, LN, thymus, liver, kidney, small intestine, heart, and lung of anti-Kb TCR tg PKO mice injected with either LKC-CD95L or LKC did not reveal any abnormalities (data not shown). Furthermore, to investigate whether anti-tumor T cells were generated we have performed the following experiment: anti-Kb TCR tg PKO mice were injected with LKC-CD95L (no tumor growth). After 4 wk, the mice were rechallenged with LKC tumor cells. No tumor growth was observed, indicating the generation of anti-tumor T cells.



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  		FIGURE 5. Tumor incidence of LKC-CD95L and LKC in anti-Kb TCR tg PKO mice. Five mice per experimental group were injected s.c. into the right flank with the indicated numbers of LKC-CD95L ({blacksquare}) or LKC (), respectively. After 28 days the mice were killed and examined for the presence or absence of tumor cells. The tumor incidence is the number of mice carrying a tumor divided by the number of mice per group.

 
Soluble CD95L is not chemotactic

It is not entirely clear which factors are responsible for the recruitment of neutrophils to the tumor site in CD95L+ tumors. To detect a direct chemotactic activity of CD95L on neutrophils, chemotaxis assays in Boyden chambers were performed (39, 44). Supernatants from COS-7 or CV1-EBNA cells transfected transiently with pcDNA3-hCD95L or LZ-hCD95L cDNA, respectively, were used as potentially chemotactic agents. Yeast-activated normal human serum was used as positive control, and HBSS buffer and the supernatant of COS cells transfected transiently with empty pcDNA3 vector were used as negative controls. The cytotoxic activity of the human CD95L protein preparations on CD95+ Jurkat cells was confirmed (data not shown). Fig. 6Go shows that the chemotactic indices of hCD95L- and LZ-CD95L-containing supernatants correspond to the negative controls. In addition, we did not detect migration of granulocytes upon incubation with wild-type or gld-mutated mouse CD95L (data not shown). These results suggest that the recruitment of neutrophils to the tumor site is due to an indirect rather than a direct effect of the CD95L.



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  		FIGURE 6. Absence of chemotactic activity of CD95L on granulocytes. Granulocytes were isolated from human peripheral blood and resuspended in HBSS. In Boyden chambers the undiluted ({blacksquare}), 1:4 diluted (), or 1:16 diluted ({square}) stimulants as indicated were separated from the granulocytes by a membrane with a pore size of 3 µm. Following the 90-min incubation at 37°C the membranes were stained with hematoxylin, and the chemotactic index was determined.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The detection of CD95L expression on tumors suggested a novel role for this molecule in tumor immune escape. It was hypothesized that tumor counterattack leads to severe depletion of CD95-sensitive anti-tumor T cells. However, the data published so far are contradictory. One set of publications, mainly those describing patient material, supports the existence of counterattack. The other set, mainly those showing data from experimental mouse tumors, speaks against the existence of tumor counterattack. The issue is further complicated by the fact that in some cases different authors obtained different results in similar experimental settings (13). Thus, we generated a tumor model with well defined components, in which the two tumor cell clones to be compared differed exclusively by their CD95L status. Both tumor cells were resistant to death receptor-mediated apoptosis. This is essential because, otherwise, autocrine or fratricidal killing may influence tumor growth. Besides the above features, our cells expressed a defined model Ag, the MHC I alloantigen Kb, which served as the only tumor Ag within the given haplotype combination of mice and tumor cells. Many naturally occurring tumors are known to carry one or several tumor Ags, e.g., the melanoma Ags (MAGE) expressed by melanomas (45). Thus, our tumor cell line may resemble more closely an in situ formed tumor than tumors used in other experimental systems. In addition, we used mice tg for an anti-Kb TCR (26). Thus, a high percentage of the T cells of these mice were tumor-specific and could be identified by surface staining with the mAb Désiré-1 directed against the tg TCR clonotype. In most other tumor models the tumor-specific T cell population is not known and its fate, death or survival, cannot be followed. With the generation of LKC-CD95L we created a model in which in vitro tumor cells could kill T cells (Fig. 3GoE) and T cells from anti-Kb TCR tg perforin wild-type mice could kill tumor cells (data not shown). Depending on the activation pathway, T cells can use both CD95L and perforin/granzyme to kill target cells (46). However, the perforin/granzyme pathway was excluded in the PKO mice we used in our study.

Infiltration of neutrophils into CD95L+ tumoral and nontumoral tissues, e.g., {beta} islets of Langerhans, followed by rejection of tumors or transplants, respectively, has been reported (18, 23). Moreover, it was shown that in vivo depletion of neutrophils with anti-Gr1 allowed subsequent growth of CD95L+ tumors (17). We also detected neutrophils in CD95L+ tumors but never in CD95L- tumors. This finding raised two important questions. First, how are these inflammatory cells recruited to the tumor site and, second, do they play a role in tumor destruction?

Concerning the first question, chemotactic activity of soluble CD95L on neutrophils has been described previously (47, 48). However, we were unable to detect migration of granulocytes upon incubation with either human CD95L and LZ-cross-linked human CD95L (LZ-CD95L) or with wild-type and gld-mutated mouse CD95L (Fig. 6Go and data not shown). In agreement with published data, our human CD95L preparations were cytotoxic, whereas the mouse proteins were not (49). The different observations by Seino et al. and our group may be explained by different preparations and concentrations of CD95L used. Another argument supports the concept that a direct chemotactic activity of soluble CD95L is not responsible for the attraction of neutrophils. It was shown before that, in contrast to the human protein, murine CD95L is not shed or is not active after cleavage from the cell surface (37). Because our tumor cells were transfected with murine CD95L we assume that neutrophil recruitment was mediated by one or more additional factors and/or cell types. For instance, IL-8, a potent neutrophil attractor and activator, was shown to be secreted by endothelial cells after stimulation by CD95L (50, 51). Furthermore, IL-1{beta} has been made responsible for the induction of neutrophil infiltration after i.p. injection of CD95L-transfected tumor cells (52). It remains to be shown whether one of these cytokines causes neutrophil immigration in our in vivo experiments. A recent paper demonstrated a striking difference between the effects induced by soluble and membrane-bound CD95L: tumor cells expressing only membrane-bound CD95L (cleavage of the molecule was prevented by deletion of the metalloproteinase cleavage site) attracted neutrophils to a higher extent than tumor cells expressing wild-type CD95L (soluble and membrane forms) or the CD95L extracellular domain only. Neutrophil chemotaxis correlated highly with the cytotoxic activity of the different CD95L transfectants (53).

Concerning the second question, the role of neutrophils in the destruction of CD95L+ tumors has not been fully defined yet. It was demonstrated that neutrophils were present at the tumor site (our data and Refs. 17, 18) and that upon in vivo depletion of neutrophils CD95L+ tumors were not rejected by the host (17). Thus, it is conceivable that neutrophils kill tumor cells directly. Lysis of microorganisms by neutrophils has been studied extensively (54). A "bystander" killing of host cells by neutrophils as well as the killing of Ab-coated tumor cells has been described (55, 56). Two of the essential effector molecules of neutrophils for the control of microbial infection are inducible NO and different reactive oxygen intermediates (57, 58). In our experimental setup the effector molecules CD95L, TNF, TRAIL, and perforin were excluded as mediators of tumor destruction. One might speculate that NO and reactive oxygen intermediates may be responsible for tumor cell lysis. However, this has to be tested in future experiments.

Complete rejection of CD95L+ tumor cells in nude mice has previously been described (17, 18). However, in our experiments we observed growth of LKC-CD95L, albeit retarded, in these mice. This discrepancy may be explained by different CD95L expression levels. Thus, it remains unclear which level of CD95L expression is required for maximal neutrophil attraction to the tumor site.

We also investigated whether injection of LKC-CD95L caused changes in the T cell compartment of the recipient anti-Kb TCR tg PKO mice. Taken together, no differences were detected between LKC- and LKC-CD95L-injected animals. Two lines of reasoning may explain this finding. First, because we used euthymic animals that form new T cells continuously, the release of naive T cells into the periphery may mask a putative T cell deletion by the tumor. More probably, the strength of interaction between CD95L+ tumor cells and T cells may not have been sufficient to see T cell deletion. In our experimental system as well as the one described by Seino et al. (17) tumors were rejected within 2 days upon transfer (data not shown). This time may be too short to allow for sufficient interaction between T cells and tumor cells. In addition, sensitivity and resistance of T cells to apoptosis is tightly regulated and is dependent on the activation state of the T cells. Naive T cells are resistant to apoptosis. Some time after activation they acquire an apoptosis-sensitive phenotype, finally leading to the down-regulation of the immune response. In vitro, T cells need to be activated in the presence of IL-2 for 6 days to become sensitive toward CD95-mediated apoptosis (9). In anti-Kb TCR tg PKO mice the rejection of CD95L- tumor cells 4 wk after a first challenge with CD95L+ tumor cells indicated the development of an anti-tumor immune response (data not shown). In contrast, no activation of anti-Kb T cells was detectable by immunostaining for the activation markers CD25, CD44, and CD69. Future experiments will show whether anti-Kb TCR tg T cells acquire an apoptosis-sensitive phenotype after Kb+ tumor cell injection.

Taken together, we show that despite the impairment of four major T cell killing systems, CD95L+ tumors are eliminated in TCR tg PKO mice. The presence of neutrophils at the tumor site suggests a causative role of these cells. However, it still remains to be elucidated which mechanism of tumor destruction is used. Finally, we did not observe any effect of CD95L+ tumors on T cells in the sense of counterattack.


   Acknowledgments
 
We thank R. Kuner for critical reading of the manuscript and G. M. Hänsch for help with the chemotaxis assays. PKO mice were provided by Jürg Tschopp (Lausanne, Switzerland).


   Footnotes
 
1 This work was supported by European Union Grant BI04-972151 (to B.A.). Back

2 Current address: BASF-LYNX Bioscience AG, Im Neuenheimer Feld 515, 69120 Heidelberg, Germany. Back

3 Address correspondence and reprint requests to Prof. Peter H. Krammer, German Cancer Research Center, Tumor Immunology Program, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Back

4 Abbreviations used in this paper: CD95L, CD95 ligand; LKC, L1210-Fas antisense expressing Kb and crmA; LKC-CD95L, L1210-Fas antisense expressing Kb, crmA, and CD95L; LN, lymph node; LZ, leucine zipper; TRAIL, TNF-related apoptosis-inducing ligand; PKO, perforin knockout; Ta, annealing temperature; tg, transgenic. Back

Received for publication May 8, 2000. Accepted for publication December 14, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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