Abstract
CTL and NK cells use two distinct cytocidal pathways: 1) perforin and granzyme based and 2) CD95L/CD95 mediated. The former requires perforin expression by the effectors (CTL or NK), whereas the latter requires CD95 (Fas/APO-1) expression by the target. We have investigated how these two factors contribute to tumor immune surveillance by studying the immunity of perforin-deficient mice against the progressor C57BL/6 Lewis lung carcinoma 3LL, which expresses no CD95 when cultured in vitro. Unexpectedly, the results indicated that the perforin-independent CD95L/CD95 pathway of CTL/NK plays a role in acting against D122 and Kb39.5 (39.5) high and low metastatic sublines, respectively, derived from the 3LL tumor. Although no membrane-bound CD95 was detected on cultured D122 and 39.5 cells, surface CD95 expression on both D122 and 39.5 was considerably up-regulated when the tumors were grown in vivo. A similarly enhanced expression of CD95 was observed with three additional tumors; LF−, BW, and P815, injected into syngeneic and allogeneic mice. The finding of up-regulated CD95 expression on tumor cells placed in vivo suggests that a CD95-based mechanism plays a role in tumor immunity at early stages of tumor growth. Consequently, the progressive down-regulation of CD95 expression during tumor progression may indeed be an escape mechanism as previously reported. Together, these results suggest a role for CD95-dependent, perforin-independent immunity against certain tumors.
Various cytocidal lymphocytes (CTL, NK), and lymphokine-activated killer cells have been reported to be potential effectors of tumor immunity (1, 2). Under optimal conditions, at least in vitro, they recognize, bind to, and kill tumor cells by inducing apoptosis within minutes after contact. Here apoptosis is mediated by at least two molecularly distinct, fast-acting mechanisms (3, 4, 5). Briefly, in the granule exocytosis lytic pathway, the secreted lytic protein perforin is believed to produce pores in the target cell’s membrane. Granzymes, cosecreted with perforin, are thought to penetrate the target cell through these pores and consequently induce apoptosis (6, 7). In contrast, in the second pathway, a surface membrane ligand of the killer cell (CD95L, Fas/APO-1L) cross-links with the target cell’s surface death receptor CD95 (Fas/APO-1) to induce apoptosis (5). With both mechanisms, apoptotic lysis is brought about by triggering a complex cascade of intracellular protein-protein interactions and proteolytic events (8).
How these two mechanisms of lymphocytotoxicity contribute to tumor immunosurveillance is not well understood, and this problem has been complicated by recent evidence that some CD95L-expressing tumors may become immune privileged (9, 10). Moreover, CD95 expression and function in malignancy is unclear, as is its role in tumor immunity, although Bradley et al. (11) have recently shown that CD95 makes tumor cells more immunogenic as well as susceptible to CTLs. However, the role of CTL or NK cells that express perforin in tumor immunity is better understood. Indeed, Kagi et al. (12) reported that perforin-deficient (PO)3 mice were considerably more susceptible than perforin-expressing mice for the growth of one million fibrosarcoma MC57G cells, suggesting a central role for perforin-based lymphocytotoxicity in tumor immune surveillance. The MHC nonexpressing tumor cell, RMA-S, derived from the T cell lymphoma RBL-5, was used to show that perforin was also involved in controlling tumor growth mediated by NK cells (12, 13). In addition, Kagi and coworkers showed that perforin plays a crucial role in viral and chemical carcinogenesis, as well as with both naive and primed mice responding to a series of syngeneic tumor cell lines of different histological origin, including the lymphoma EL4, MBL-2 melamona B16, and fibrosarcoma MC57G. These findings were consistent with the failure to find a significant role for the death receptor CD95 in tumor immunosurveillance (13). In contrast, Mori et al. (14) showed that the CD95-based pathway of lymphocyte action was tumor cell dependent. Therefore, we decided to examine the role of perforin and CD95 in immunity against a series of tumors, concentrating on two sublines (D122 and 39.5) of the metastatic Lewis lung carcinoma 3LL of C57BL/6 mice.
Materials and Methods
Mice and tumor cells
Two- to 4-mo-old C57BL/6 (B/6) (H-2b), PO (H-2b), DBA/2 (H-2d), and BALB/c (H-2d) mice were supplied by the Animal Breeding Center of this Institute. The PO mice were previously described (15). D122, a highly metastatic and low immunogenic subclone of the Lewis lung carcinoma (3LL) and its H-2Kb transfectant, 39.5, a highly immunogenic and nonmetastatic form of the tumor, were previously described (16). The 3LL tumors were cultured in D122 medium (DMEM with 10% heat-inactivated FCS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, and 1% nonessential amino acids). Single-cell suspensions were obtained by trypsinization followed by two washes. The B/6 (H-2b) T cell leukemia EL4, DBA/2 (H2d) mastocytoma P815, and A/J leukemia YAC-1 were maintained in ascitic form in syngeneic mice or maintained for short periods in RHFM medium (RPMI 1640 containing 10% heat-inactivated FCS, 10 mM HEPES, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5 × 10−5 M 2-ME). Several lines of leukemia L1210 of DBA/2 were used: L1210wt (wild type), L1210–3, selected for low CD95 expression; LF+ transfected with mouse CD95 overexpression construct (17), kindly provided by Dr. Pierre Golstein; and LF−, which expresses low levels of the CD95 Ag as a result of transfection with a CD95 antisense construct (18), kindly provided by Dr. William Clark. Transfected LF+ and LF− cells were resistant to G418, as expected. Cells were maintained in CD95 medium (RPMI 1640 containing 5% heat-inactivated FCS, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5 × 10−5 M 2-ME). RMA-S is a clone of the Rauscher virus-induced lymphoma RBL-5, selected for low MHC (H-2b) expression (19). Cultures were maintained in a RMA-S medium (RPMI with 10% heat-inactivated FCS and 1% combined antibiotics).
Preparing and culturing CTL and NK cells
Alloreactive peritoneal exudate CTLs (PELs) were generated, prepared, and purified as previously described (20). Briefly, mice were injected i.p. with tumor cells (25 × 106/mouse). Eight to 11 days after a primary alloimmunization, or 4 to 5 days after a secondary injection (given 6–12 wk after priming), the mice were killed and their peritoneal cavities rinsed with PBS supplemented with 5% heat-inactivated newborn calf serum (PBS-NCS). The resulting crude peritoneal cells were incubated on nylon wool columns for 60 min at 37°C to deplete adherent cells such as B cells and macrophages. Nonadherent cells were eluted by rinsing the columns with cold PBS-NCS. The eluted cells (PEL) contained >95% T cells, 80–90% of which were CD8+, about half of which formed specific conjugates (21). Syngeneic CTLs were prepared by injecting mice i.p. three times at 7-day intervals (25–160 × 106 for EL4; 2–4 × 106 for 39.5 or B16) with irradiated (10,000 rad) tumor cells. Four to 5 days after the last immunization, PELs were prepared as above. The NK cells used were from freshly isolated spleen cells of naive mice. Splenocytes were suspended in ACK to remove RBC and washed in PBS.
Lymphocytotoxicity
Target cells were labeled with Na251CrO4 (1 h at 37°C) and washed twice with PBS-NCS before use. Lytic assays were conducted in U-shaped 96-well microtiter plates with 3–10 × 104 labeled target cells per well and effector cells at the indicated ratios. The plates were centrifuged at room temperature to promote conjugate formation, incubated for 2–5 h at 37°C, and then recentrifuged to terminate the assay. A 100-μl supernatant from each well was harvested, and its radioactivity was determined using a gamma counter. The percentage lysis was calculated as previously described.
Tumor growth in naive and immunized mice
For immunization, PO and B/6 mice were inoculated s.c. or i.p. with 2 or 4 × 106 irradiated (10,000 rad) D122, Kb39.5, or 120 × 106 EL4 tumor cells. D122, 39.5, EL4, or RMA-S tumor cells (in 100 μl) were injected into the right flank of naive or immunized mice. Tumor size was measured by calipers every second or third day at the greatest right-angle diameters. Life span was recorded up to 70 days after inoculation or until the tumor’s largest diameter reached 2.5 cm.
Cell staining and flow cytometry
Cells (0.25 × 106) were washed in staining medium (0.5–1% BSA in PBS plus 0.02% azide), pelleted, suspended in 30 μl (0.25 μg) hamster anti-mouse CD95 Ab (Jo2; PharMingen, San Diego, CA), and incubated on ice for 30 min with occasional shaking. After the cells were washed and pelleted, they were suspended in 30 μl of FITC-goat anti-hamster F(ab′)2 Ab (1:100 dilution) (Jackson ImmunoResearch, West Grove, PA) and incubated as above. To analyze surface Fas expression on the 5 [and 6]-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR)-labeled cells (see below), Jo2 Ab was added as above, followed by biotinylated goat anti-hamster (30 μl, 1:250 dilution, incubation on ice and washed as above) and PE-streptavidin (30 μl, 1:150 dilution, 30 min on ice). After a final wash, the cells were resuspended in PBS plus 0.02% azide and analyzed by FACS.
For staining tumor cells with CFSE (22, 23), cells were washed twice and resuspended at 5–10 × 107/ml in PBS. CFSE was added to make a final concentration of 5 μM, and the suspension was incubated for 10 min at 37°C. After having been washed twice in PBS, cells were resuspended in PBS to the desired concentration and naive animals were injected i.p. Injected cells were analyzed by FACS.
RT-PCR for Fas
Total RNA was isolated from 1 × 106 cells, accurately counted, and lysed in 200 μl TriReagent (Molecular Research Center, Cincinnati, OH). Chloroform (50 μl) was added at room temperature, and the mixture was vortexed and incubated for 15 min on ice. After centrifuging in the cold at the maximal Eppendorf speed for 15 min, we mixed 120 μl of the upper layer containing total RNA with 120 μl of isopropanol, and the mixture was vortexed, incubated on ice for 30 min, and centrifuged as before. The pellet was then suspended in 1 ml cold 75% ethanol and vortexed and centrifuged as previously described. After removing the ethanol, we dissolved the air-dried pellet in 11 μl diethyl pyrocarbonate (DEPC) H2O. The Life Technologies method was used for cDNA preparation as follows. The total RNA was incubated at 65°C for 5 min and, after fast chilling, 9 μl of the following mixture was added: 4 μl of 5 × 1st standard buffer, 1 μl of 10 mM dNTPs, 2 μl of 100 mM DTT, 0.5 μl of 1 mg/ml oligo(dT), 0.5 μl RNasin, and 1 μl Moloney murine leukemia virus reverse transcriptase. After incubating the mixture at 37°C for 1.5 h, 180 μl DEPC H2O was added.
cDNAs were analyzed by RT-PCR for Fas expression and compared with GAPDH expression. Each 20-μl portion of the reaction mixture contained 3 μl cDNA, 8.7 μl DEPC H2O, 2 μl of 10× buffer, 3.2 μl of 1.25 nM dNTPs, 1.5 μl downstream primer (5′-346ATG CAC ACT CTG CGA TGAAG365-3′), 1.5 μl upstream primer (5′-670TTG GTA TGG TTT CAC GAC TG689-3′); both primers (derived from mouse cDNA) were from 10 μM and 0.1 μl Taq DNA polymerase (for GAPDH, the same mixture was used with the corresponding primers at 10 μM). Each sample was mixed, briefly centrifuged, overlaid with 30 μl mineral oil, and placed in the thermocycler (Programmable Thermal Controller, MJ Research, Cambridge, MA) set as follows: 35 cycles of 1 min denaturation at 94°C, 1 min annealing at 55°C, 1 min elongation at 72°C, and a final elongation of 5 min at 72°C. The size of the RT-PCR product was 344 bp. The samples were then resolved on a 1% agarose gel containing ethidium bromide (in TBE running buffer, 100 V, 50 min) and observed by UV light. That band intensity reflected the amount of RNA analyzed was established by showing linearity of band intensity as a function of the amount of RNA subjected to RT-PCR.
Results
Tumor growth in perforin-containing (B/6) and PO mice
To determine how perforin and the CD95L/CD95 pathways contribute to tumor immunity, we tested the growth of the EL4, D122, and 39.5 tumor cell lines, all of B/6 origin, in both B/6 and perforin-deficient PO mice (both H-2b). The results (Table I⇓) show that while both the B/6 and PO mice were susceptible to the progressively growing D122 and EL4, the PO mice exhibited greater innate resistance to the 39.5 line, which had been derived from the H-2Kb-transfected D122 line (16). We next tested tumor growth in PO and B/6 mice that had been vaccinated with x-irradiated tumor cells. PO mice vaccinated against either 39.5, D122, or EL4 were more resistant to 39.5 or D122 tumor cells. Vaccinated with EL4, both strains were resistant to the homologous tumor, but B/6 mice were resistant to neither D122 nor 39.5 (Table I⇓). Hence PO mice showed superior acquired immunity against the otherwise three progressor tumors studied.
Tumor growth in naive and immunized PO and B/6 micea
NK and CTL activity of perforin-containing and PO mice
Resistance to the growth of at least some tumors, observed in both naive and vaccinated animals, could be mediated by NK cells or by CTL or both. Confirming a previous report (13), we found that spleen cells procured from PO mice exhibited only minimal NK-like activity, compared with perforin-expressing mice (Table II⇓). Hence, the adaptive resistance of PO mice to tumor growth (Table I⇑) was probably not due to NK cell activity. In support of this conclusion, we found (data not shown) that mutant, MHC class I-nonexpressing RMA-S tumor cells grew faster in PO mice than in perforin-expressing mice, as was previously reported (24). CTLs appeared more likely to mediate the tumor immunoprotective activity seen in PO mice, because CTLs had been observed in both PO and C57BL/6 mice immunized against the syngeneic leukemia EL4; in some cases, PO showed higher specific and nonspecific CTL activity compared with B/6 mice (Table III⇓). However, the nonspecific activity against the allogeneic L1210 leukemia of DBA/2 (H-2d) mice was CD95-dependent, because only the CD95-transfected L1210 cells (LF+) were lysed.
The lytic activity of NK from PO and B/6 micea
The lytic activity of CTL from PO and B/6 micea
CD95 is up-regulated in vivo
The induction of adaptive tumor immunity in PO mice combined with the nonspecific lysis of CD95-expressing targets (Tables I⇑ and III⇑, respectively) implied a CD95L/CD95-based mechanism against the tumor. Surprisingly, using a CD95 mAb (Jo2; Phar-Mingen), we detected virtually no cell-surface CD95 on cultured D122 and 39.5 cells. To resolve this enigma, we tested the hypothesis that cell-surface CD95 expression was up-regulated on tumor cells upon their injection in vivo. Indeed, we found that D122 and 39.5 tumors grown in vitro and expressing little CD95 underwent massive CD95 up-regulation when injected into PO mice (Table IV⇓). This unexpected finding prompted us to test CD95 expression on a series of other tumors that were injected into both syngeneic and allogeneic recipients. As with D122 and 39.5, a moderate to marked enhancement of CD95 expression in a series of otherwise low CD95-expressing cells (L1210, LF-, BW, P815) was found (Table IV⇓). Hence, CD95 up-regulation upon in vivo inoculation appears to be a more general phenomenon, a finding relevant to our understanding of CD95 expression and function in tumor immunity.
Up regulation of CD95 expression on tumor cellsa
To ascertain the authentic origin of the Fas-up-regulated tumor cells in vivo, and to exclude Fas-expressing lymphoid cells infiltrating the peritoneal cavity in response to the tumor, we labeled low Fas-expressing tumor cells (LF− and BW) with CFSE before injection. CFSE penetrates cells and subsequently covalently and stably binds to cellular proteins, which allows direct measurement and sorting by flow cytometry over several cell generations before the dye is diluted out (22, 23). Sorting the CFSE-labeled LF− and BW (injected at 25/50 × 106 and 80 × 106 cells per mouse, respectively) was done on cells withdrawn from the peritoneal cavity 4 days after injection. Large, strongly CFSE-labeled tumor cells were separated from the small, nonlabeled peritoneal cells to exclude Fas-expressing lymphocytes. An aliquot of the sorted CFSE-labeled cells was seeded in petri dishes to examine Fas expression upon returning to in vitro conditions. Fas expression on the CFSE-labeled and sorted cells was determined after double staining by the Fas Ab Jo2, before and 4 days after injection. Four independent experiments were performed, unequivocally proving that Fas up-regulation took place in most original CFSE-labeled cell populations injected into PO and B/6 mice (Table V⇓). The reversibility of Fas-expression was rigorously proven by showing that the CFSE-labeled, high (up-regulated) Fas-expressing cells down-regulated their surface Fas upon culturing (and dividing) in vitro (Table V⇓). RT-PCR performed with RNA from CFSE-labeled, sorted cells indicated that the up- and down-regulated cell-surface Fas expression upon in vivo and in vitro growth, respectively, correlated well with the amount of Fas mRNA detected (Fig. 1⇓).
Fas mRNA RT-PCR. Sorted LF− cells were processed by RT-PCR, and the Fas and GAPDH products were analyzed by agarose gel electrophoresis as described in Materials and Methods. Lane 1, Water control; lane 2, cultured LF+; lane 3, cultured LF−; lanes 4 and 5, in vivo grown LF− cells and recultured LF− cells, respectively; lanes 6–11, three repeats of lanes 4 and 5. DNA sequencing of the PCR products showed >99% identity in nucleotide and 100% in amino acid sequence compared with the consensus Fas sequence.
Up- and down-regulation of CD95 expression on CFSE-labeled tumor cellsa
The role of CD95 in tumor growth and immunity
To test the hypothesis that CD95 up-regulation plays a role in tumor immune surveillance, we monitored the growth of LF+ and LF− in syngeneic (perforin-expressing) DBA/2 mice. The LF+ and LF− cells were derived from leukemia L1210 of DBA/2 mice and were then transfected with CD95-overexpression (LF+) and antisense constructs (LF−), respectively (17, 18). Fig. 2⇓ shows surface CD95 expression on in vitro-cultured LF+ and LF− cells; whereas 77% (76–96% in eight experiments) of LF+ expressed CD95, only 2.2% of LF− expressed it, consistent with their differential apoptotic response to the CD95 Ab Jo2 and to PO CTL (Table VI⇓). Note that immunization of PO mice with either LF+ or LF− resulted in CTLs that lysed LF+ but not LF− (Table VI⇓). The induction of PO CTL by LF−, and its eventual rejection, supports the notion of CD95 up-regulation. This has been confirmed by FACS (Tables IV⇑ and V⇑) and by lymphocytotoxicity of in vivo-grown D122 (Table III⇑) and LF− (Table VI⇓) found to be more sensitive to apoptotic lysis than cultured cells.
FACS analysis of Fas expression on cultured LF+ and LF− tumor cells.
Lysis of LF+ and LF− induced by Fas-Ab (Jo2) and by PO-CTL
After syngeneic (DBA/2) mice were inoculated (s.c. or i.p.), we observed enhanced growth of the CD95 nonexpressing tumors (LF−) compared with the expressing tumors (LF+, Fig. 3⇓, A and B), despite the fact that CD95 expression per se had no effect on the tumor doubling time measured in vitro (Fig. 3⇓C). Interestingly, enhanced CD95 expression on LF− in vivo was detected before the onset of tumor growth and an inverse expression pattern of CD95 was found with the slower growing CD95-expressing (LF+) tumors (data not shown). Hence, CD95 up-regulation upon in vivo inoculation of LF- cells appears to have profound consequences on their susceptibility to CD95-based apoptosis as well as in vivo growth.
LF+ and LF− growth in syngenic DBA/2 mice. A, Cells (1 × 105) were injected i.p. into DBA/2 mice. The results of one of three experiments with similar results are presented. B, Cells (1 × 105) were injected s.c. into the flank of DBA/2 mice. Tumor size was measured by calipers at the indicated times; the averages of two experiments are presented. C, In vitro growth of LF+ and LF− cells. Cells (0.5 × 106) were cultured and viable cells were counted at the indicated times.
Discussion
While recognizing the significant role of perforin in tumor immunity (13, 24), our study lends support to the theory that the CD95/CD95L mechanism of CTL acts in both innate and acquired immunity against certain tumors. Using perforin-deficient mice (15) and three tumors of C57BL/6 origin (clones D122 and 39.5 of 3LL origin and leukemia EL4), which grow progressively and kill both C57BL/6 and PO mice (both H-2b), we have shown that vaccination with x-irradiated tumor cells protected PO mice against a lethal challenge of nonirradiated tumor cells (Table I⇑). Simultaneously, we have confirmed reduced NK cell activity in PO mice (Table II⇑) and consequently an impaired ability to control the growth of RMA-S cells (MHC nonexpressing (syngeneic) leukemia RBL-5) (data not shown), as previously reported (24). Hence, while perforin-expressing immune effector cells appear to play an obligatory role against some tumors, a CTL-mediated CD95/CD95L-based mechanism appears to be involved in immunity against certain tumors.
CD95 is a membrane-anchored protein that can bring about apoptosis upon cross-linking by certain CD95 Abs or by the cell-bound or soluble natural ligand CD95L (Table VI⇑). The CD95 gene is defective in lymphoproliferative (lpr) mice due to the insertion of a retrotransposome that causes premature termination of CD95 mRNA transcripts or a point mutation in its cytoplasmic death domain. Homozygous mutant mice develop lymphadenopathy and lupus-like autoimmunity (25). Interestingly, a similar mutation in the human CD95 homologue gene results in an autoimmune lymphoproliferative syndrome (26). The emergence of tumor cell resistance to Fas-based apoptosis may be fundamental to tumor progression. Apoptosis-resistant human lymphoma cells have been described; these express splicing variants of CD95, coding for truncated CD95 that lacks the intracellular death domain of 80 aa (27). Reduced CD95 expression or function has been found in melanoma (9), colon cancer (28, 29), and testicular cancer (30). In contrast, expression of CD95L is up-regulated in some normal and malignant tissues (9, 10, 31). Hence, coordinate expression of CD95 and its ligand may be an important factor in tumor growth and progression. Although Nagata (8) extensively studied CD95 and showed that it plays a role in immunoregulation and in some immunopathologies (liver, heart), little has been known about the role of CD95 in tumor immunity and immunosurveillance. Debatin et al. (32) established that an externally administered CD95 Ab can retard the growth of certain tumors, although CD95-Ab therapy of tumors seems unlikely because of its severe side effects (mostly liver). In contrast, CD95-based specific CTL/NK cell action may be an important and powerful immune mechanism in controlling tumor growth metastases (11) .
Down-regulation or lack of the CD95 protein in malignant cells may be a primary mechanism in tumor escape from immune surveillance. Because CD95 expression varies widely among tumors and within a given tumor, studies on the regulation of CD95 expression in a well-defined in vivo setting are important (33). We observed an unexpected sharp up-regulation of CD95 expression on D122, as well as on its H-2Kb-transfected clone, 39.5, upon inoculation of the two tumors into susceptible PO mice (Table IV⇑). We found a similar up-regulation of CD95 expression in vivo with a series of other common laboratory tumors (Tables IV⇑ and V⇑) that express very little cell-surface CD95, including the Fas antisense transfected LF− cells. In vitro, the expression of the normal Fas gene in LF− cells is likely to be retarded by the Fas anti-sense transcripts; this appears to be reversed under the in vivo conditions that favor the expression of the normal gene, as reflected at both the protein and mRNA levels, as well as by the enhanced susceptibility to Fas-based Ab and CTL-mediated lysis. Up-regulated CD95 expression took place in the very same cells injected in either PO or B/6 mice, and expression was down-regulated upon returning to in vitro culture (Table V⇑). Fig. 1⇑ presents evidence correlating the extent of Fas mRNA expression and up- and down-regulation of surface membrane CD95 in LF− cells shown in Tables IV⇑ and V⇑. Whether the difference in Fas mRNA expression are due to changes in transcription or also to an altered half-life of the Fas message is not known at present. Moreover, mRNA-based control of Fas expression may apply to some but not all tumors. For example, in the human lung adenocarcinoma, Fas is sequestered within the cytoplasm (34). It appears that the failure to detect CD95 expression on in vitro-cultured tumor cells may not indicate its absence in the tumor cells when placed in vivo. The up-regulation of CD95 expression on the D122 and 39.5 as well as LF− tumors upon inoculation in vivo, which resulted in increased susceptibility to Fas-based lysis mediated by CTL (Tables III⇑ and VI⇑), supports the hypothesis that, at least in PO mice, immunity against these tumors is CD95 based.
In a separate set of experiments, we found that the CD95-nonexpressing tumor, LF− (CD95 anti-sense transfected), had a clear growth advantage compared with its CD95-overexpressing counterpart, LF+ (CD95 transfected), in vivo, but not in vitro (Fig. 3⇑). Paradoxically, the enhanced growth of LF− (Fig. 3⇑, A and B), grown i.p. as well as s.c., occurred concurrently with the up-regulation of CD95 expression; conversely, with the slower-growing CD95-expressing tumor, LF+, we observed down-regulated CD95 expression (data not shown). Importantly, both tumors grew progressively in syngeneic (DBA/2) recipients (Fig. 3⇑, A and B), eventually killing them. As a minimal working hypothesis, we have postulated that the enhanced growth of LF− in vivo was related to the lack of normal CD95 gene expression. Consequently, tumor growth was not retarded by CD95-based innate immunity (CTL NK cell-mediated). In fact, LF+ cells showed a low susceptibility to natural cytotoxicity (Fig. 3⇑). While we are inclined to attribute the enhanced growth of the LF− tumor to their (initial) low level CD95 expression, which may help escape immune surveillance, it is also possible that the growth advantage of LF− over LF+ is due to a more complex interaction of LF− cells with the in vivo environment. The acquisition of CD95 upon progressive syngeneic growth of LF− in vivo probably reflects the overall effects of the natural and acquired immune responses against the tumor and is likely to be mediated by cytokines (see below). However, with LF+ cells, constitutive CD95 expression presumably retards tumor growth at least initially by the innate immune system (Fig. 3⇑, A and B), resulting in the partial deletion of high CD95-expressing LF+ cells (data not presented). However, this does not alter the fate of the host with either tumor, because tumor growth overrides and progressive growth follows, with the host eventually succumbing to the tumor. Factors generated by innate and adaptive immunity, in response to tumors, such as IFN-γ and TNF-α, can bring about enhanced CD95 expression, and the combination of these cytokines synergistically enhance CD95 expression (35, 36). Further studies on the roles that cytokines play in the regulation of CD95 expression in well-defined tumor settings in vivo are required because CD95 expression varies widely among tumors, even within a given tumor. The present findings stress the need to scrutinize the regulation and function of the CD95 system in tumor immunity in an in vivo setting. This information is relevant to adaptive tumor immunotherapy in conjunction with cytokines, as well as to tumor vaccination programs. The extent to which a given tumor may be subject to CD95 up-regulation may, by itself, predict the success (or failure) of immunotherapy.
Footnotes
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↵1 This work was supported by the Israel Science Foundation and by an anonymous source through the Weizmann Institute of Science, Rehovot, Israel.
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↵2 Address correspondence and reprint requests to Prof. Gideon Berke, Department of Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel. E-mail address: gideon.berke{at}weizmann.weizmann.ac.il
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↵3 Abbreviations used in this paper: PO, perforin-deficient mice; PEL, peritoneal exudate lymphocytes; wt, wild type; CFSE, 5 [and 6]-carboxyfluorescein diacetate succinimidyl ester; NCS, newborn calf serum; DEPC, diethyl pyrocarbonate.
- Received March 19, 1999.
- Accepted January 3, 2000.
- Copyright © 2000 by The American Association of Immunologists
References
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