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The Journal of Immunology, 1998, 161: 1220-1230.
Copyright © 1998 by The American Association of Immunologists

Human Melanoma-Reactive CD4+ and CD8+ CTL Clones Resist Fas Ligand-Induced Apoptosis and Use Fas/Fas Ligand-Independent Mechanisms for Tumor Killing1

Licia Rivoltini2,*, Marina Radrizzani*, Paola Accornero*, Paola Squarcina*, Claudia Chiodoni*, Arabella Mazzocchi*, Chiara Castelli*, Paolo Tarsini*, Vincenzo Viggiano{dagger}, Filiberto Belli{ddagger}, Mario P. Colombo* and Giorgio Parmiani*

Divisions of * Experimental Oncology D, {dagger} Medical Oncology B, and {ddagger} Surgical Oncology B, Istituto Nazionale Tumori, Milan, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Tumor cells have been shown recently to escape immune recognition by developing resistance to Fas-mediated apoptosis and acquiring expression of Fas ligand (FasL) molecule that they may use for eliminating activated Fas+ lymphocytes. In this study, we report that tumor-specific T lymphocytes isolated from tumor lesions by repeated in vitro TCR stimulation with relevant Ags (mostly represented by normal self proteins, such as MART-1/Melan A and gp100) can develop strategies for overcoming these escape mechanisms. Melanoma cells (and normal melanocytes) express heterogeneous levels of Fas molecule, but they result homogeneously resistant to Fas-induced apoptosis. However, CD4+ and CD8+ CTL clones kill melanoma cells through Fas/FasL-independent, granule-dependent lytic pathway. In these lymphocytes, Ag/MHC complex interaction with TCR does not lead to functional involvement of FasL, triggered, on the contrary, by T cell activation with nonspecific stimuli such as PMA/ionomycin. Additionally, melanoma cells express significant levels of FasL (detectable on the cell surface only after treatment with metalloprotease inhibitors), although to a lesser extent than professional immune cells such as Th1 clones. Nevertheless, antimelanoma CTL clones resist apoptosis mediated by FasL either in soluble form or expressed by Th1 lymphocytes or FasL+ melanoma cells. These results demonstrate that CD4+ and CD8+ antimelanoma T cell clones can be protected against Fas-dependent apoptosis, and thus be useful reagents of immunotherapeutic strategies aimed to potentiate tumor-specific T cell responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tumor cells can express antigenic determinants that are detected by specific CD4+ and CD8+ T lymphocytes in an MHC-restricted fashion, thus stimulating cytotoxic activity and cytokine release (1). Based on these observations, the identification of the human tumor Ags recognized by these T cells is progressing at an accelerating pace (1). The genes reported to encode tumor epitopes belong to different groups, including tumor-specific Ags, mutated proteins, and normal self molecules (2). In the latter, T cell reactivity can be viewed as a sort of autoreactive phenomenon that escaped peripheral tolerance (3). The large amount of information collected in the last few years on the nature of Ags and epitopes recognized by T lymphocytes on cancer cells is presently supporting worldwide efforts aimed at identifying effective and widely applicable protocols of immune therapy of cancer (4, 5, 6).

However, the functional interactions occurring in vivo between the immune system and the cancer process are just starting to be understood, and little information is still available on the rules governing host immune responses directed against tumor cells.

Particularly, one of the enigmas of tumor immunology is why the immune system generally fails to eliminate antigenic tumor cells or to slow down disease progression. This phenomenon, identified as tumor immune escape, has been associated with the ability of cancer cells to take advantage of a variety of strategies for evading immune detection and/or killing. Deficiencies in quantity, processing, presentation, or affinity may reduce the immunogenicity of certain tumor Ags (7), and loss of MHC expression, frequently observed in cancer cells, can make them completely undetectable by T cells (8). Lack of expression of costimulatory molecules on the neoplastic cells can lead to anergy of tumor-reactive CTL (9), and a similar effect can be mediated by immunosuppressive factors released by cancer cells at the tumor site (10). Altogether, these factors may contribute in making a tumor lesion an immune privileged-like site that Ag-specific T cells can hardly access.

Recently, a further mechanism potentially used by tumor cells to neutralize host immune response has been identified as involving Fas-FasL3 interaction (11, 12). These proteins are key molecules in normal immune development, homeostasis, and functions (13). Ligation of FasL to its Fas receptor induces programmed cell death (or apoptosis) and plays important immunologic roles, such as immune response termination, T cell activation-induced cell death, clonal downsizing, and control of peripheral tolerance to self Ags (14). Based on these features, a central role of Fas-FasL has also been hypothesized recently in the maintenance of immune privilege in certain vital organs, such as eye, central nervous system, and testis (15), and dysfunctions in Fas-mediated apoptosis have been associated with autoimmunity in both animal models and humans (13, 14). Additionally, Fas-FasL interaction is also utilized by cytotoxic T cells as a death pathway for target killing, secondary to granule exocytosis in CD8+ effectors, but essential for cytotoxic CD4+ cells (16, 17).

Although Fas has been reported to be broadly expressed in different tissues, FasL was conventionally thought to be a selective marker of cells belonging to the immunologic compartment (13). However, it has been observed recently that nonimmunologic cells, such as tumor cells, can indeed acquire FasL expression and use it for delivering death signals to activated, Fas+ T cells (12, 18, 19). At the same time, tumor cells seem to be spared from their own weapon, due to their resistance to Fas-induced cell death that additionally preserves them from undergoing apoptosis-mediated killing by cytotoxic cells (19, 20).

Based on these findings, resistance to apoptosis and concomitant ability to neutralize approaching T cells are presently considered as a potential mechanism used by tumor cells to evade immunologic recognition, despite the expression of antigenic determinants.

Although intriguing, this hypothesis has some limitations due to the lack of data regarding the significance of in vivo FasL expression by tumor cells and its role in affecting Ag-specific T cell responses. In fact, most of the functional data on the ability of cancer cells to mediate Fas-dependent lymphocyte apoptosis have been obtained using Fas-sensitive conventional targets (such as Jurkat cells or even murine cells, like A20 lymphoma) (18, 19), or lymphocyte bulk cultures whose Ag specificity has not been assessed (12). Moreover, if FasL-expressing tumor cells were able to kill the totality of activated (i.e., Fas+) lymphocytes recognizing them, it would be hard to explain the presence of tumor-specific T cell-infiltrating cancer lesions (2, 21), and the ability to produce clinical regressions once tumor-infiltrating lymphocytes (TIL) are activated in vitro and reinjected into patients (4). These observations suggest that at least some T cells in tumor infiltrates are not subjected to Fas-mediated cell death via interaction with tumor cells.

In the present study, we identified CD4+ and CD8+ T cell clones obtained from melanoma lesions that resist FasL-mediated apoptosis, induced by FasL either in soluble form or expressed on FasL+ cells, including melanoma. At the same time, these lymphocytes exclusively rely on Fas/FasL-independent mechanisms of tumor killing, and hence are not influenced by melanoma resistance to FasL. These data indicate that, like other pathologic situations such as autoimmune diseases, a proportion of Ag-specific T cells involved in tumor-directed immune responses can survive Fas/FasL-mediated tumor counterattack.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Lymphocyte clones, melanoma lines, and normal melanocytes

Lymphocyte clones 501-CD8 and GDN-CD8 were obtained from TIL of melanoma patient 501 and GDN, respectively (22). Clone BS-CD8, BS57-CD4, and BS239-CD4 were derived from a tumor-invaded lymph node of patient BS (23). Clones were generated by limiting dilution in the presence of autologous tumor cells and 20 to 50 Cetus U/ml IL-2 (23) and selected after 3 to 4 wk of culture by screening cytotoxic, CD3-mediated activity against autologous melanoma cells. GDN-CD8, deriving from a TIL line generated from tumor lesion by in vitro culture with IL-2, was cloned using soluble OKT3 (30 ng/ml), as described elsewhere (24). Clones were maintained by weekly restimulation with autologous tumor cells, autologous lymphoblastoid cell line (LCL), allogeneic feeder (PBMC), and 20 to 50 U/ml IL-2. The clonality of these effectors (all bearing different Vß- and V{alpha}-chains) was proved by TCR analysis (data not shown), as described elsewhere (23). 501-CD8 and GDN-CD8 recognize the nonapeptide 27–35 from the melanoma Ag MART-1/Melan A in the context of HLA-A2.1 (25). Clone BS-CD8 reacts with the nonapeptide 17–25 from the differentiation Ag gp100, presented in the context of HLA-A3 (26). BS57-CD4 and BS239-CD4 recognized an unknown Ag specifically expressed on autologous melanoma cells and presented in the context of HLA-DR1 or 10 (our unpublished observations). The Th1 clone 103 has been described previously (27).

As for the tumor lines, MeBS, Me1340, Me23682, Me97620, Me1402/R, and Me37041 were established in our laboratory from melanoma lesions (either s.c. or visceral metastases, or invaded lymph nodes). BS-LCL, a B lymphoblastoid cell line, was obtained by infecting B cells from patient BS with EBV. 501 mel, GDNmel, and 624.28 mel were kindly provided by Dr. F. M. Marincola (Surgery Branch, National Institutes of Health, Bethesda, MD) (28). 256 mel and 272 mel were a kind gift from Dr. D. Rimoldi (Ludwig Institute of Cancer Research, Lausanne, Switzerland). A375 mel, HS695 mel, and RPMI 7951 mel were purchased from American Type Culture Collection (Rockville, MD). All of these cell lines, including Jurkat and K562, were maintained in 10% FCS/RPMI 1640. Normal melanocytes were kindly provided by Dr. M. Herlyn (Wistar Institute, Philadelphia, PA).

mAbs and immunofluorescence analysis

The following mAbs were used: CH-11 (anti-Fas IgM, apoptosis inducing), ZB4 (anti-Fas IgG, neutralizing), phycoerythrin-conjugated UB2 (anti-Fas, for cell surface staining) (all from Medical and Biological Laboratories, Nagoya, Japan), C-20 (anti-FasL, for cell surface staining) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bcl-2, anti-Bcl-x, and anti-Bax (all from PharMingen, San Diego, CA). Fluorescein anti-rabbit IgG (H+L) (Vector Laboratories, Burlingame, CA) was used as a secondary reagent. Suitable negative controls were from Cymbus Bioscience (Southampton, U.K.). Cell surface Fas and FasL expression was detected as previously described (29). Fas expression was increased by culturing melanoma cells or melanocytes for 24 to 48 h with 1000 U/ml IFN-{gamma}, as reported by others (20).

FasL expression on melanoma cells was evaluated after 24-h treatment with 250 µM 1,10-phenanthroline (Sigma, Poole, U.K.), a metalloprotease inhibitor known to block soluble FasL release, and thus able to increase FasL surface expression. For intracellular staining (Bcl-2, Bcl-x, and Bax), cells (5 x 105/sample) were fixed with 3% paraformaldehyde in PBS (10 min on ice), washed in TBS (50 mM Tris-HCl, pH 7.5, in saline solution), then permeabilized with 0.25% Triton X-100 in TBS (5 min on ice); 0.1% /Triton X-100 in TBS was used as buffer during immunofluorescence.

Susceptibility to Fas-mediated killing by CH-11 mAb, rFas-L, and Th1 clone 103

Different concentrations of agonistic CH-11 mAb or rFasL (kindly provided by Dr. J. Tschopp, Institute of Biochemistry, University of Lausanne, Switzerland) were both diluted in final 150 µl/well 10% FCS/RPMI and incubated for 16 h at 37°C with 51Cr-labeled 104/well target cells in 96-well V-bottom plates. To prove that cell killing was Fas mediated, the assay was also performed in the presence of 50 ng/ml neutralizing anti-Fas mAb ZB4, as previously described (27). At the end of incubation, 50 µl/well of supernatant was harvested and counted, and killing was calculated as percentage of lysis (25). Comparable results were obtained using targets labeled with [3H]TdR or 125I-labeled UdR (data not shown). The Th1 clone 103 was pretreated with ZB4 mAb, then activated with PMA-ionomycin, as previously described (27), and used at different ratios as effector in a 16-h 51Cr release assay. The lines Jurkat and K562 were always included as positive and negative control of Fas-mediated killing, respectively.

Cytotoxic activity by lymphocytes and its inhibition by MgCl2/EGTA, CsA, or anti-Fas mAb

Cytotoxic activity of T cell clones was measured by 51Cr release assay (25). Labeled targets (1000 cells/well) were incubated for 6 or 16 h with different concentrations of effector cells. Peptide pulsing was performed by incubating T2 cells with 1 µM peptide for 1 to 2 h at 37°C. Ag-specific presentation to CD4+ clones was obtained by culturing for 24 h at 37°C autologous EBV-B-transformed cells (106 cells/well, in 24-well plates) with tumor lysate from BS-Mel (generated from 106 cell, by quick freezing and thawing). For inhibiting Ca2+-dependent killing, EGTA + MgCl2 (at 6 and 3 mM, respectively) were added to the assay. For blocking FasL-mediated lysis, effector cells were incubated with CsA (5 µg/106/ml) for 3 h at 37°C, washed three times in PBS before adding to the assay. Otherwise, the assay was performed in the presence of neutralizing anti-Fas mAb ZB4, as described above. Activation with PMA-ionomycin was induced by incubating cells with as described above for Th1 clone 103 (27).

Fas-mediated killing of lymphocyte by melanoma cells

Melanoma cells (at different concentrations, starting from 105/well, in 96-well U-bottom plates) (Costar, Cambridge, MA) were cultured in 10% FBS/RPMI for 48 h at 37°C, to allow cell adhesion. No supernatant was removed during these 48 h, to avoid any elimination of FasL in soluble form. 51Cr-labeled T cell clones or Jurkat cells (at 104 cells/well), in the presence or absence of neutralizing anti-Fas mAb ZB4 (50 ng/ml final concentration), were then added as target cells, and the plates were incubated for 16 h. For coculturing assay, 106/well adherent nonirradiated melanoma cells (in 24-well plates) cultured with an equivalent number of lymphocytes, in 10% FBS/RPMI supplemented with 20 U/ml IL-2. After 1 wk, lymphocytes were harvested, counted, and tested for cytotoxic activity in a 4-h 51Cr release assay.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fas expression and susceptibility to Fas-mediated apoptosis of melanoma cells and normal melanocytes

Alterations in Fas expression or function have been described recently to occur in tumor cells. Mutations have been indicated to be involved in tumorigenesis of these neoplastic cells and in their proliferative advantage as compared with the normal counterpart (30). Specifically, melanoma cell lines have been reported by Hahne et al. to lack significant expression of Fas molecules, and consequently to resist Fas-mediated killing (19). However, despite this potential resistance, melanoma cells are usually efficiently recognized, at least in vitro, by Ag-specific T cells either from the CD8+ or the CD4+ lineage (22, 23, 25, 31).

To assess the role of Fas-FasL system in the lymphocyte/melanoma interaction, we first analyzed a large panel of melanoma lines for Fas expression, evaluated by fluorescence staining with the anti-Fas mAb UB2. Normal melanocytes were also included to see whether any observed phenomenon was a feature of cells belonging to melanocytic lineage, or was restricted to the neoplastic phenotype.

As listed in Table IGo, the two melanoma lines, 256 mel and 272 mel, previously described as Fas negative by Hahne et al. (19), expressed no significant level of Fas molecules. However, in contrast to this previous report, the additional melanoma lines tested in this study were characterized by heterogeneous surface expression of Fas, ranging from levels comparable (in percentage of positive cells and mean fluorescence intensity) with the positive control Jurkat, to intermediate or low levels. A similar variable distribution was observed in normal melanocytes, although such cells could be analyzed on a limited number of cases. In accordance with results reported for other tumors (20), the level of Fas expression could be increased significantly by in vitro culture with IFN-{gamma} in both melanoma and melanocytes (data not shown).


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  		Table I. Phenotypic expression of Fas molecules on melanoma cells and normal melanocytesa

 
The functional role of Fas was then analyzed by assessing sensitivity to Fas-mediated apoptosis induced by agonistic anti-Fas mAb or rFasL molecules. Melanoma cells and normal melanocytes were cultured in the presence of different concentrations of CH-11 mAb or rFasL, and apoptosis was measured in a 16-h 51Cr release assay. As reported in Table IIGo, regardless of the amount of Fas expression, all of the lines tested showed a complete resistance to Fas-mediated killing by CH-11 and rFasL, which were, however, effective in inducing significant apoptosis of the Fas-sensitive line Jurkat. The same resistance was observed when killing through Fas was mediated by the Th1 clone 103, known to up-regulate FasL and induce apoptosis of target cells after activation with PMA-ionomycin (27). No change in sensitivity was detected after treatment of melanoma and melanocyte lines with IFN-{gamma} (data not shown), which was previously reported to reverse the Fas-resistant phenotype in breast cancer cell lines (20). Interestingly, the lack of sensitivity to Fas-induced apoptosis (either by agonist Abs or by the natural ligand) was not limited to melanoma cells, and thus appeared to be a feature of cells deriving from the melanocytic lineage. Altogether, these data indicate that cells from melanocytic lineage (either normal or neoplastic) can express Fas on their surface, but this expression does not correlate with the ability of this molecule to transmit apoptotic signals when adequately ligated by FasL. We cannot completely rule out the possibility that the apparent defect of Fas signaling in melanocytic cell lines could be related to their in vitro selection as part of cell line establishment. However, it should be underlined that resistance to Fas-mediated killing could be observed starting from very early passages (such as second or third), as tested with Me23682, Me97620, and Me37041 (Table IGo).


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  		Table II. Susceptibility to Fas-mediated lysis (via CH-11 or rFasL) of human melanoma cells and normal melanocytesa

 
Lytic mechanisms used by melanoma-specific CD4+ and CD8+ T cells to kill autologous tumor cells

The observed resistance of melanoma cells to Fas-mediated apoptosis could have relevant implications for the susceptibility of these cells to killing activity by T lymphocytes. CD4+ or CD8+ Ag-specific CTL recognizing tumor cells in MHC-restricted, TCR-dependent fashion have been described extensively in melanoma patients (1, 2). CD8+CTL are known to use mainly Ca2+-dependent perforin-mediated lysis for killing their targets, although Fas-mediated cytotoxicity can also be used as a lytic tool (32, 33). On the contrary, CD4+ T lymphocytes have been described to rely for killing mostly on Fas-mediated apoptosis (33, 34). Therefore, resistance to the apoptotic effect of Fas molecule could contribute significantly to impair susceptibility of tumor cells to the cytotoxic activity of Ag-specific CD4+ and CD8+ CTL.

To evaluate this hypothesis, we characterized the lytic mechanisms used by melanoma-specific CD4+ and CD8+ CTL clones generated from the infiltrate of metastatic tumor lesions. CD4+ T cell clones were derived from a tumor-invaded lymph node of a melanoma patient and were shown to recognize an HLA-DR-restricted Ag expressed on autologous melanoma cells (our unpublished observations). We dissected the lytic mechanisms utilized by these clones to kill autologous tumor, by inhibiting Ca2+-dependent and perforin-mediated cytotoxicity or by interfering with Fas-mediated lysis. This was done, for granule-dependent cytotoxicity, by incubating in Ca2+-chelating conditions in the presence of MgCl2/EGTA (35), and by blocking FasL synthesis in T cells through treatment with CsA (36) or inhibiting Fas-FasL interaction with the anti-Fas antagonist mAb ZB4 (27), for testing Fas-mediated killing.

As clearly reported in Figure 1Go (A and B), both of the CD4+ clones tested, which significantly lysed autologous tumor cells, were blocked completely in their lytic activity when Ca2+-chelating agents were added to the assay, while no significant effect was observed after treatment with CsA or ZB4. These data indicate that our antimelanoma CD4+ T cell clones, in contrast with most of the CD4+ clones and cultures described to date (32, 33), lysed tumor cells through a Ca2+-dependent Fas-independent mechanism. This independence was not due to a general inability to up-regulate functional FasL. In fact, after treatment with PMA-ionomycin (stimuli known to potently induce FasL production in T cells) (27), both CD4+ clones were able to lyse Fas-sensitive cells such as Jurkat in a Fas-dependent fashion, since this lysis was blocked efficiently by treatment with CsA or ZB4 mAb (Fig. 2Go, A and B). The predominant role of perforin and other granule-related enzymes in the killing activity of melanoma cells by antimelanoma CD4+ clones was also supported by the detection of BLT esterase (N-{alpha}-benzyloxycarbonyl-L-lysine-thiobenzyl esterase) release in response to autologous tumor cells or solid-phase OKT3 (data not shown).



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  		FIGURE 1. Melanoma-specific CD4+ and CD8+ T cell clones kill autologous tumor cells in a Ca2+-dependent, FasL-independent fashion. Effector cells were tested in a 6-h 51Cr release assay against autologous melanoma cells in the presence of MgCl2/EGTA (3 and 6 mM, respectively), or after treatment with CsA (5 µg/106/ml) for 3 h, followed by three washings in 10% HS RPMI. Lysis block with 50 ng/ml anti-Fas ZB4 mAb was perfomed as previously described (29). Comparable data were obtained in a 16-h 51Cr release assay. A, Clone BS57-CD4; B, clone BS239-CD4; C, clone 501-CD8; D, GDN-CD8.

 


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  		FIGURE 2. Antimelanoma CD4+ and CD8+ T cell clones can utilize Fas/FasL mechanism after activation with PMA-ionomycin. Lymphocytes, activated with PMA-ionomycin for 3 h, as previously described (27), were tested in a 16-h 51Cr release assay against Jurkat cells. For blocking FasL synthesis, T cells were additionally treated with CsA (5 µg/106/ml). Blocking with anti-Fas mAb ZB4 was performed as previously described (29). A, Clone BS57-CD4; B, clone BS239-CD4; C, clone 501-CD8; D, clone GDN-CD8.

 
These data show that specific CD4+ clones displaying significant Fas-independent cytotoxic activity against autologous tumor cells can be found as a component of antitumor T cell response; these results also confirm that perforin/granzyme expression is not necessarily confined to CD8+ T cells.

Although CD8+CTL are conventionally known to use the perforin-dependent granule exocytosis as the main cytotoxic pathway, reports have shown recently that these cells can also take advantage of the Fas-FasL interaction as a concomitant mechanism of target killing (32, 33, 37). To assess this hypothesis in our system, we analyzed lytic mechanisms used by melanoma-specific CD8+ clones to kill Ag-positive targets. Both of the CD8+ clones used recognized the immunodominant peptide 27–35 from the melanoma Ag MART-1/Melan A (clone 501-CD8 and GDN-CD8) (25), presented in the context of HLA-A2.1. It should be underlined that this Ag is a self differentiation-related protein, expressed on both melanoma and normal melanocytes (2).

Lysis of autologous melanoma cells by these clones was inhibited completely by MgCl2/EGTA, with no effect of CsA or mAb ZB4 (Fig. 1Go, C and D), suggesting that perforin-dependent (and not Fas-dependent) cytotoxicity was the crucial mechanism involved. Again, the absence of any role of Fas-mediated killing was not due to the inability of these T cells to synthesize FasL, since treatment with PMA-ionomycin promoted Fas-mediated cytotoxic activity against Jurkat cells (although to a lesser extent than that observed in CD4+) that could be blocked by anti-Fas mAb or CsA (Fig. 2Go, C and D).

Antimelanoma CD4+ or CD8+ clones do not trigger FasL in response to interaction between TCR and the relevant MHC/Ag complex

From the above data, it was not clear whether the lack of any role of Fas in tumor killing by T cells was due to Fas resistance of melanoma cells or to the lack of FasL up-regulation in T cells encountering the relevant Ag. In fact, T cells have been described to up-regulate FasL in response to TCR-activating stimuli (33), but little is known about the functional pathways triggered in tumor-specific T cells by the interaction with tumor-expressed epitopes.

To see whether the resistance of melanoma cells to Fas-mediated apoptosis may contribute to significantly reduce the lytic potentials of tumor-specific T cell clones, we analyzed the mechanisms used by our CD4+ and CD8+ T cell clones to kill Fas-sensitive targets expressing the relevant Ag. For CD8+ clones, the recognized peptide MART-127–35 was pulsed at the concentration of 1 µM, on HLA-A2.1+ T2 cells, a TAP-deficient cell line efficient in presenting exogenous peptides and highly sensitive to Fas-mediated killing (Table IIGo). As reported in Figure 3Go (A and B), no significant part of the total cytotoxic activity on peptide-pulsed T2 cells appeared to be mediated by Fas-FasL (as detected by the inhibition of lysis caused by CsA or ZB4), while most of the killing was Ca2+ dependent and probably perforin mediated. These data clearly indicate that antimelanoma CD8+ T cells did not significantly use FasL when their TCR is triggered by the interaction with the relevant Ag.



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  		FIGURE 3. CD4+ and CD8+ T cell clones kill Fas-sensitive APC, presenting relevant tumor Ag in a FasL-independent fashion. CD8+ T cell clones were tested in a 6-h 51Cr release assay against T2 cells prepulsed with MART-127–35 peptide (1 µM) (A and B). Comparable data were obtained in a 16-h 51Cr release assay. CD4+ T cell clones were tested against autologous BS-LCL incubated overnight with cell lysate derived from MeBS (16-h 51Cr release assay) (C and D). A, Clone 501-CD8; B, GDN-CD8; C, BS57-CD4; D, clone BS239-CD4.

 
Comparable results were observed with CD4+ clones. Since the melanoma Ag recognized by CD4+ clones was unknown in this case, the autologous LCL was incubated overnight with lysate from autologous melanoma cells, a procedure that has been shown to induce processing and presentation of exogenous tumor Ags by B cells (31). The totality of the cytotoxic activity exerted by CD4+ clones on pulsed autologous LCL cells with tumor lysate was inhibited by Ca2+-chelating agents, with no effect of treatments impairing Fas-mediated lysis (Fig. 3Go, C and D). As observed in the CD8+ system, TCR triggering by tumor Ag did not lead to significant FasL involvement in melanoma-specific CD4+ clones as well.

FasL expression on melanoma cells

In the previous studies, FasL expression on melanoma level was, however, analyzed by techniques that detect the total pool of intracellular and membrane-bound FasL present in a sample (such as Western blot and immunohistochemistry) (19). However, no quantitative evaluation at the single cell level, with particular attention to the surface fraction, was reported, especially in comparison with the level of expression detectable in immune cells that professionally use this molecule for their immune functions (such as Th1 lymphocytes). With the aim of quantifying the amounts of FasL expressed on melanoma cells and its functional relevance in immune modulation, phenotypic analysis by flow cytometry was performed using the anti-FasL mAb C-20, which detects the membrane-bound form of this protein. It should be underlined that, in contrast to what has been reported by others (19), no significant and reproducible level of FasL could be observed in our melanoma cell lines without previous treatment with inhibitors of metalloproteases that are known to block FasL shedding from cell membrane (13) (data not shown). On the contrary, a significant percentage of positive cells expressing high levels of FasL could be found in the Th1 clone 103 after activation with OKT3 (Fig. 4Go). Thus, heterogeneous but clear expression of FasL in the majority of the melanoma lines analyzed could be demonstrated after an overnight treatment with 250 µM phenanthroline, a metalloprotease inhibitor. A comparison between the profiles obtained with melanoma lines such as A375 mel, HS695 mel, and Me23682 (expressing the highest FasL levels) and the profile obtained with Th1 clone 103 clearly showed that T cells that are professionally committed to use FasL for their killing activity express significantly higher amounts of this molecule on their surface (evaluated as mean fluorescence intensity) (Fig. 4Go). This quantitative difference was confirmed by RT-PCR, which showed a 10- to 100-fold stronger FasL message in activated clone 103 as compared with positive melanoma lines (data not shown). The reduced amounts of FasL expression by melanoma lines in comparison with T cells could also explain the functional data reported in Table IIIGo, showing that melanoma cells induced apoptosis of Jurkat cells at level significantly lower than that observed in the presence of the Th1 clone.



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  		FIGURE 4. Surface expression of FasL by melanoma cell lines in comparison with the Th1 clone 103. FasL expression was evaluated by flow cytometry using the anti-FasL mAb C-20. Melanoma cells were tested after 24-h treatment with 250 µM 1,10-phenanthroline. The Th1 clone 103 was treated overnight with 20 µg/ml solid-phase OKT3 for inducing FasL expression. Closed areas, staining with C-20 mAb; open areas, staining with negative control. Percentage of FasL+ cells and mean fluorescence intensity (in parenthesis) are indicated.

 

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  		Table III. Susceptibility to Fas-mediated lysis of human anti-melanoma CD4+ and CD8+ T cell clonesa

 
Resistance of antitumor CTL clones to Fas-FasL-mediated apoptosis

To conclusively analyze the role of FasL in down-regulating Ag-specific T cell responses in the system, we tested the susceptibility of our CD4+ and CD8+ T cell clones to FasL-mediated apoptosis, induced by different means, i.e., by an agonist anti-Fas Ab (CH-11), the human rFasL molecule, the Th1 clone 103 (known to mediate FasL-dependent killing after activation with PMA-ionomycin) (27), and by autologous melanoma cells.

As shown in Table IIIGo, all of the CD8+ and CD4+ clones tested displayed resistance to rFasL or to Th1-mediated apoptosis (comparable with what was observed with the Fas-negative line K562). As expected, Jurkat cells underwent massive apoptosis when incubated in the same conditions. This resistance was not due to lack of Fas expression, since all T cell clones were found to be highly positive for this molecule when analyzed by flow cytometry (data not shown).

In agreement with what we observed with rFasL and Th1 clone 103 (Table IIIGo), the antitumor CD4+ and CD8+ T cell clones tested did not show any susceptibility to apoptosis induced by FasL+ melanoma cells, either autologous or allogeneic. Although different from what has been described in other T cell models (38, 39), this resistance appears to be an intrinsic feature of these T cells, since it was independent from TCR triggering. However, Jurkat cells underwent significant apoptosis after exposure to rFasL, Th1 clone 103, and some melanoma cells (Table IIIGo). As discussed above, the level of killing mediated by tumor cells was lower than expected on the basis of the data previously published by Hahne (19). However, this could be due to an overall lower susceptibility of human Jurkat T cells to FasL-dependent apoptosis in comparison with murine A20 cells used by these authors. The level of killing observed with melanoma cells on Jurkat was also compatible with the amount of FasL expressed by these tumor cells, which was significantly lower than that observed on the Th1 clone 103 (Fig. 4Go).

A detectable level of apoptosis could be observed when melanoma-specific T cell clones were exposed to the agonist anti-Fas mAb CH-11 (see Table IIIGo). This killing was, however, significantly lower than that observed with Jurkat cells. A discrepancy between data obtained with the natural FasL (either recombinant or displayed on cell surface) and the effect mediated by agonist Ab has been described already by others (40, 41). In fact, this phenomenon has been related to the triggering of different signaling pathways that could be limited in terms of physiologic implications when agonist Abs, and not natural ligands, are used. T cell death could also be observed in these clones after overnight treatment with high (10 µg/ml) concentrations of immobilized OKT3 mAb (data not shown). Such data suggest that the resistance of antimelanoma T cell clones to apoptosis was not an absolute phenomenon, and that these cells were still susceptible to certain forms of immunologic control.

To confirm the absence of any functional effect of melanoma-expressed FasL on tumor-specific T cells, we performed a coculture assay for different times, in which CD4+ or CD8+ clones were incubated with a high number (at ratio 1:1) of FasL-positive nonirradiated melanoma cells. At the end of this culture, lymphocytes were harvested, evaluated for viability and number, and tested for antimelanoma cytotoxic activity, to detect possible long-term effects of FasL on T cell functions. 256 mel was included in this analysis, since previously reported by Hanhe et al. to express significant levels of FasL (19). The presence of either autologous or allogeneic melanoma cells did not negatively affect lymphocyte short-term or long-term (24 h vs 7 days) growth as compared with culture with IL-2 alone. As expected, when an HLA-matched target was present (i.e., autologous melanoma or the HLA-A2+ melanoma line 256 mel, both expressing MART-1, the target Ag of clone 501-CD8), significant killing of tumor cells with a proliferative boost was observed (data not shown).

The culture in the presence of FasL+ melanoma cells failed also to functionally affect either CD4+ or CD8+ T cell clones, which maintained their potent antitumor cytotoxic activity and Ag specificity. Figure 5Go reports data concerning cytotoxic activity of T cell clones after 7 days of culture in the presence of melanoma cells. Comparable results were obtained when lymphocyte clones were cocultured with melanoma cells for a shorter time (24 h) and cytotoxic activity was evaluated using the original cell count in the calculation of E:T ratios (data not shown). These results further support the conclusion that these antimelanoma clones are resistant to FasL-mediated apoptosis, and explain why these cells can survive in vivo in the tumor lesion and receive proliferative signals from repeated in vitro stimulation with autologous tumor cells.



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  		FIGURE 5. Cytotoxic activity of antimelanoma CD4+ and CD8+ T cell clones after coculture with or without autologous or allogeneic FasL-positive melanoma cells. Lymphocytes were cultured in the presence of IL-2 or IL-2 + autologous, nonirradiated melanoma cells (ratio 1:1). The allogeneic FasL+ melanoma line 256 mel was also used as positive control. After 1 wk, lymphocytes were recovered, counted, and tested against relevant targets in a 51Cr release assay. A, Clone 501-CD8; B, GDN-CD8. Both CD8+ T cell clones were tested against T2 pulsed with MART-127–35 peptide (open bars), T2 alone (dashed bars), and autologous melanoma cells (closed bars); C, BS57-CD4, tested against autologous LCL, as negative control (dashed bars) or autologous melanoma cells (closed bars).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Great interest has been raised recently by the possibility that the Fas-FasL system may represent one of the tools used by tumor cells to evade immunologic control. In view of the central role of Fas-FasL in the regulation of immune responses, this mechanism has also been indicated as responsible for immune depletion and esczape in other pathologic situations such as HIV infection (29, 43).

In this study, we provide the first evidence that the immune system can develop strategies for overcoming this escape mechanism developed by the tumor cells. In fact, by repeated Ag stimulations, we could isolate from melanoma lesions tumor-specific CD4+ and CD8+ T cell clones that apparently rely on Fas-independent mechanisms for mediating tumor cell killing and that show resistance to FasL-induced apoptosis, either to soluble FasL or to FasL expressed on Th1 lymphocytes and tumor cells.

Although recent data indicate that Fas-mediated apoptosis is a crucial guardian in the immune regulation, including maintenance of peripheral self tolerance, the role of Fas and FasL in the homeostatic regulation of immune responses appears to be much more complex than initially thought. Qualitative differences in how cells perceive signals through Fas exist in distinct T cell subpopulations, probably depending on the differentiation state and Ag specificity (42). T cells engaged either by APCs presenting antigenic peptides or by Ab to TCR/CD3 can even undergo a series of activation events, instead of apoptotic death, following Fas-FasL interaction (42, 44). Moreover, recent reports have shown that the immune privilege cannot be ascribed to a single protective Fas-FasL-dependent mechanism, but rather involves an intricate orchestration of different processes (45). The identification of a proinflammatory role of FasL in transplanted grafts or in murine tumor models, which can even lead to a faster rejection of FasL-positive cells (46, 47), confirms the complexity of Fas-FasL immunologic functions. Moreover, the possibility that a proportion of T cells involved in a given immune reaction is programmed to develop intrinsic features, enabling them to survive FasL-triggered death signals, is emerging from many recent reports (42, 48). In other words, not all T cells follow the conventional rule of undergoing death by apoptosis after ligation of FasL to cell surface expressed Fas molecules.

In the present work, we described antitumor cytotoxic T cell clones that are characterized by immunologic features different from most of the conventional Ag-specific T cells described in the literature. It should be first underlined that our effectors were derived from tumor lesions or tumor-invaded lymph nodes (i.e., from sites in which they most likely had the chance of encountering and possibly being primed by the relevant Ag). Additionally, they have been generated by multiple in vitro restimulations, which likely select for cells resistant to Ag-induced cell death.

In terms of cytotoxic mechanisms, granule-dependent cytolysis is clearly the predominant killing pathway used by our antimelanoma CD8+ and CD4+ T cell clones. CD8+ T cells are known to use mainly perforin to lyse their targets, although they can potentially up-regulate FasL once activated and induce apoptosis of Fas-positive cells, as a secondary lytic pathway (16). On the contrary, CD4+ cytotoxic T cells are thought to lack perforin and other enzyme-containing granules, and thus to exert target cell killing through alternative mechanisms, such as Fas-FasL interaction (32, 33). However, several instances of cytotoxic CD4+ T cells using granule-depending pathway have been described recently, mostly involving autoreactive cells (49, 50). Our findings additionally show that cytotoxic CD4+ T cells can be found as a significant component of the antitumor T cell response in melanoma, as powerful mediators of autologous tumor recognition, exerted through significant lysis and cytokine release (our unpublished data). This observation is in contrast with another immunologic dogma considering lytic CD4 as exclusive components of an immunoregulatory population devoted to negatively control detrimental immune responses such as autoreactivity (51).

The lack of a functional role of Fas-FasL interaction in Ag-specific killing was not due to defects in FasL-up-regulating mechanisms, since both CD4+ and CD8+ CTL clones do utilize FasL and kill Jurkat cells when nonspecifically activated with PMA-ionomycin. At odds from other systems, TCR interaction with the relevant MHC/peptide complex, either expressed endogenously by tumor cells or exogenously as peptide presented by APCs, does not appear to trigger FasL involvement in our T cells. This phenomenon could be related to a particular feature of antitumor effectors that are characterized by the expression of TCRs with low affinity for their specific ligand (52), as reported for other self-reactive T cells (53). A limited TCR affinity could explain why antimelanoma T cells do require strong stimuli, such as PMA-ionomycin, for a full activation of killing pathways involving Fas-FasL interaction. Alternatively, two distinct TCR transduction signals could be required for separate activation of Fas- and perforin-mediated killing, as recently shown by Esser et al. (54), thus accounting for the different lytic pathways observed in our T cell clones in response to the relevant Ag as compared with PMA-ionomycin.

The possible lack of FasL functional involvement in response to TCR engagement by MHC/peptide complex may also play a role in protecting these effector cells from Fas-FasL-mediated activation-induced cell death, and allow their survival to repeated Ag stimulations, a situation occurring not only in vitro, during cloning, but also in vivo, in which a high tumor burden is often present.

Another interesting feature of our antimelanoma CTL clones is their resistance to apoptotic death mediated by either soluble or cell membrane-bound FasL. Tumor cells have been hypothesized recently to impair lymphocyte function by expressing functionally active FasL, and thus inducing apoptosis of Fas+-activated lymphocytes (11, 12, 55).

However, the possibility of recovering TIL with tumor-specific reactivity (2, 4, 21) suggests that at least a certain component of the antitumor immune response has developed strategies for surviving tumor counterattack. This resistance is constitutive (and does not require TCR triggering, as described in other systems) (38, 39) and it is not due to a reduced Fas expression, as our clones express levels of this molecule comparable with those present on Jurkat cells. Moreover, our clones do not show an intrinsic resistance to apoptotic death, since they undergo apoptosis in response to high dose OKT3 and after treatment with agonist anti-Fas Abs such as CH-11. The latter result confirms the already described phenomenon that Ab agonists and natural ligand can stimulate different signaling pathways, and further emphasizes the limitations of defining the physiologic role of Fas-FasL solely by Ab agonists (40, 41).

Antimelanoma T cell resistance to FasL-induced death signal is not an unexpected finding, and several lines of evidence could support it. One first explanation could be related to the observation that antitumor T cells present in vivo in a tumor lesion may be anergized by different mechanisms (including immune suppressive factors) (11, 21). Due to this anergy, T cells could thus survive to tumor-mediated apoptosis, as described in other systems (56). Additionally, it has been reported recently that certain components of the TCR/CD3 complex, such as the {zeta}-chain, are required for T cells to undergo Fas-mediated apoptosis (57), and this specific chain has been found to lack in fresh TIL obtained from tumor lesions (58).

An optimistic interpretation of our data could suggest that these antimelanoma T cell clones can resist FasL-induced apoptosis because of their memory phenotype. Although this hypothesis is hard to prove given the absence of phenotypic markers for memory cells (with the exception of CD45RO, which is usually highly expressed in long-term cultured T cells, including our clones) (42, 59), functional characterizations of the T cell responses to some melanoma Ags such as MART-1 have shown that sensitization to these Ags has occurred in vivo in melanoma patients (60).

CD4+ cells with Th2 or Th0 phenotype have been shown in some systems to be resistant to FasL-induced death (38, 39), although our previous data do not fully agree with this observation (29). It is hence possible that our CD4 T cell clones, which make significant amounts of IFN-{gamma} and IL-4 in response to autologous tumor (data not shown), could be resistant due to their Th0 phenotype.

T cells primed in the presence of TGF-ß (61), cytokine that is usually produced in situ by melanoma cells, or costimulated via CD28 (62) (that could be provided by local APCs) have been shown to acquire resistance to apoptosis. Finally, resistance to apoptotic death has been hypothesized recently to be responsible for the escape from peripheral tolerance of autoreactive T cells (13, 41), thus providing a further explanation for FasL resistance of our T cell clones that mostly recognize normal self Ags (2).

In some of the systems described above, the molecular mechanisms responsible for T cell resistance to FasL-induced cell death have been identified as involving apoptosis regulators such as Bcl-x, Bcl-2, Bax, and others (13). However, preliminary data showed that none of these seem to play a role in providing our T cells with a reduced susceptibility to apoptotic death (our unpublished observations). Investigations are presently ongoing on additional factors, including the potential involvement of molecules such as FLIP, which has been shown recently to inhibit apoptosis by blocking FLICE/procaspase-8 activation (63).

The apparent failure of the immune system to control in vivo tumor growth remains an unsolved paradox, and immunologists still have to deal with tumor immune escape in designing new protocols for effective immunotherapy. However, our data showing that antitumor CD4+ and CD8+ T cell subpopulations resistant to FasL-induced apoptosis do exist, suggest that this mechanism of immune escape may not play a central role in melanoma.

We cannot rule out the possibility that FasL-mediated apoptosis may play a role in eliminating the most effective component of the antitumor T cell response, which perhaps is already lost when TIL come to our observation. However, our data support the emerging idea that complex and subtle mechanisms regulate T cell susceptibility to FasL-induced apoptosis; such mechanisms should be investigated extensively in Ag-specific settings. The identification of the different pathways of T cell immune reactivities will help exploit these resistance mechanisms to provide new tools of immunotherapeutic intervention in cancer patients.


   Acknowledgments
 
We thank Drs. P. Romero, D. Rimoldi (Ludwig Institute of Cancer Research, Lausanne, Switzerland), P. Golstein (Centre d’Immunologie, Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique (INSERM-CNRS), Marseille, France), J. Tschopp (Institute of Biochemistry, University of Lausanne, Switzerland), V. Bronte (University of Padova, Italy), T. Z. Zaks (Surgery Branch, National Cancer Institute/National Institutes of Health, Bethesda, MD), and P. Perego (Division of Experimental Oncology B, Istituto Nazionale Tumori, Milan, Italy) for providing useful reagents and helpful suggestions to the project. We are grateful to Dr. D. J. Loftus (Laboratory of Cell Biology, National Cancer Institute/National Institutes of Health) for reviewing the manuscript. We are also grateful to Ms. Francesca Rini for technical support, and Ms. Grazia Barp for the excellent secretarial assistance.


   Footnotes
 
1 This work was supported in part by grants from Italian Association for Cancer Research (Milan, Italy) and Istituto Superiore di Sanità (Rome, Italy). Back

2 Address correspondence and reprint requests to Dr. L. Rivoltini, Division of Experimental Oncology D, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, Italy. Back

3 Abbreviations used in this paper: FasL, Fas ligand; CsA, cyclosporin A; LCL, lymphoblastoid cell line; TBS, Tris-buffered saline; TIL, tumor-infiltrating lymphocytes. Back

Received for publication December 12, 1997. Accepted for publication April 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Boon, T., P. van der Bruggen. 1996. Human tumor antigens recognized by T lymphocytes. J. Exp. Med. 183:725.[Free Full Text]
  2. Rosenberg, S. A., Y. Kawakami, P. F. Robbins, R. Wang. 1996. Identification of the genes encoding cancer antigens: implications for cancer immunotherapy. Adv. Cancer Res. 70:145.[Medline]
  3. Houghton, A. N.. 1994. Cancer antigens: immune recognition of self and altered self. J. Exp. Med. 180:1.[Free Full Text]
  4. Rosenberg, S. A., J. R. Yannelli, J. C. Yang, S. L. Topalian, D. J. Schwartzentruber, J. S. Weber, D. R. Parkinson, C. A. Seipp, J. H. Einhorn, D. E. White. 1994. Treatment of patients with metastatic melanoma with autologous tumor-infiltrating lymphocytes and interleukin 2. J. Natl. Cancer Inst. 86:1159.[Abstract/Free Full Text]
  5. Jr Slingluff, C. L.. 1996. Tumor antigens and tumor vaccines: peptides as immunogens. Semin. Surg. Oncol. 12:446.[Medline]
  6. Houghton, A. N.. 1997. Times are a changin’: the era of peptide immunization begins. Cancer J. Sci. Am. 3:4.[Medline]
  7. Bennink, J. R., R. Anderson, I. Bacik, J. Cox, P. Day, Y. Deng, C. Lapham, H. Link, G. Russ, J. W. Yewdell. 1993. Antigen processing: where tumor-specific T-cell responses begin. J. Immunother. 14:202.
  8. Ferrone, S., F. M. Marincola. 1995. Loss of HLA class I antigens by melanoma cells: molecular mechanisms, functional significance and clinical relevance. Immunol. Today 16:487.[Medline]
  9. Chen, L., P. McGowan, S. Ashe, J. Johnston, Y. Li, I. Hellström, K. E. Hellström. 1994. Tumor immunogenicity determines the effect of B7 costimulation on T-cell-mediated tumor immunity. J. Exp. Med. 179:523.[Abstract/Free Full Text]
  10. Kiessling, R., K. Kono, M. Petersson, K. Wasserman. 1996. Immunosuppression in human tumor-host interaction: role of cytokines and alterations in signal-transducing molecules. Springer Semin. Immunopathol. 18:227.[Medline]
  11. Williams, N.. 1996. Tumor cells fight back to beat immune system. Science 274:1302.[Free Full Text]
  12. Walkler, P. R., P. Saas, P.-Y. Dietrich. 1997. Role of Fas ligand (CD95L) in immune escape: the tumor cell strikes back. J. Immunol. 158:4521.[Abstract]
  13. Nagata, S.. 1997. Apoptosis by death factor. Cell 88:355.[Medline]
  14. Abbas, A. K.. 1996. Die and let live: eliminating dangerous lymphocytes. Cell 84:655.[Medline]
  15. Griffith, T. S., T. A. Ferguson. 1997. The role of FasL-induced apoptosis in immune privilege. Immunol. Today 18:240.[Medline]
  16. Henkart, P. A.. 1994. Lymphocyte-mediated cytotoxicity: two pathways and multiple effector molecules. Immunity 1:343.[Medline]
  17. Lowin, B., M. Hahne, C. Mattmann, J. Tschopp. 1994. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathway. Nature 370:650.[Medline]
  18. O’Connell, J., G. C. O’Sullivan, J. K. Collins, F. Shanahan. 1996. The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J. Exp. Med. 184:1075.[Abstract/Free Full Text]
  19. Hahne, M., D. Rimoldi, M. Schroter, P. Romero, M. Schreier, L. E. French, P. Schneider, T. Bornand, A. Fontana, D. Liénard, J. Cerottini, J. Tschopp. 1996. Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape. Science 274:1363.[Abstract/Free Full Text]
  20. Keane, M. M., S. A. Ettenberg, G. A. Lowrey, E. K. Russell, S. Lipkowitz. 1996. Fas expression and function in normal and malignant breast cell lines. Cancer Res. 56:4791.[Abstract/Free Full Text]
  21. Whiteside, T. L., G. Parmiani. 1994. Tumor-infiltrating lymphocytes: their phenotype, functions and clinical use. Cancer Immunol. Immunother. 39:15.[Medline]
  22. Kawakami, Y., S. Eliyahu, C. H. Delgado, P. F. Robbins, K. Sakaguchi, E. Appella, J. R. Yannelli, G. J. Adema, T. Miki, S. A. Rosenberg. 1994. Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection. Proc. Natl. Acad. Sci. USA 91:6458.[Abstract/Free Full Text]
  23. Mazzocchi, A., W. J. Storkus, C. Traversari, P. Tarsini, M. J. Maeurer, L. Rivoltini, C. Vegetti, F. Belli, A. Anichini, G. Parmiani, C. Castelli. 1996. Multiple melanoma-associated epitopes recognized by HLA-A3-restricted CTLs and shared by melanomas but not melanocytes. J. Immunol. 157:3030.[Abstract]
  24. Walter, E. A., P. D. Greenberg, M. J. Gilbert, R. J. Finch, K. S. Watanabe, E. D. Thomas, S. R. Riddell. 1995. Reconstitution of cellular immunity against cytomegalovirus in recipients of allogeneic bone marrow by transfer of T-cell clones from the donor. N. Engl. J. Med. 333:1038.[Abstract/Free Full Text]
  25. Rivoltini, L., Y. Kawakami, K. Sakaguchi, S. Southwood, A. Sette, P. F. Robbins, F. M. Marincola, M. L. Salgaller, J. R. Yannelli, E. Appella, S. A. Rosenberg. 1995. Induction of tumor-reactive CTL from peripheral blood and tumor-infiltrating lymphocytes of melanoma patients by in vitro stimulation with an immunodominant peptide of the human melanoma antigen MART-1. J. Immunol. 154:2257.[Abstract]
  26. Castelli, C., A. Mazzocchi, F. Rini, P. Tarsini, L. Rivoltini, M. Maio, G. Gallino, F. Belli, G. Parmiani. 1998. Immunogenicity of the ALLAVGATK (gp10017–25) peptide in HLA-A3.1 melanoma patients. Eur. J. Immunol. 28:1543.
  27. Radrizzani, M., P. Accornero, D. Delia, R. Kurrle, M. P. Colombo. 1997. Apoptosis induced by HIV-gp120 in a Th1 clone involves the generation of reactive oxygen intermediate downstream CD95 triggering. FEBS Lett. 411:87.[Medline]
  28. Rivoltini, L., Y. Kawakami, K. Sakaguchi, S. Southwood, A. Sette, P. F. Robbins, F. M. Marincola, M. L. Salgaller, J. R. Yannelli, E. Appella, S. A. Rosenberg. 1995. Quantitative correlation between HLA class I allele expression and recognition of melanoma cells by antigen-specific CTL. Cancer Res. 55:3149.[Abstract/Free Full Text]
  29. Accornero, P., M. Radrizzani, D. Delia, F. Gerosa, R. Kurrle, M. P. Colombo. 1997. Differential susceptibility to HIV-GP120-sensitized apoptosis in CD4+ T-cell clones with different T-helper phenotypes: role of CD95/CD95L interactions. Blood 89:558.[Abstract/Free Full Text]
  30. Nagata, S.. 1996. Apoptosis: telling cells their time is up. Curr. Biol. 6:1241.[Medline]
  31. Topalian, S. L., L. Rivoltini, M. Mancini, N. R. Markus, P. F. Robbins, Y. Kawakami, S. A. Rosenberg. 1994. Human CD4+ T cells specifically recognize a shared melanoma-associated antigen encoded by the tyrosinase gene. Proc. Natl. Acad. Sci. USA 91:9461.[Abstract/Free Full Text]
  32. Berke, G.. 1997. Killing mechanisms of cytotoxic lymphocytes. Curr. Opin. Hematol. 4:32.[Medline]
  33. Kagi, D., B. Ledermann, K. Burki, R. M. Zinkernagel, H. Hengartner. 1996. Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu. Rev. Immunol. 14:207.[Medline]
  34. Stalder, T., S. Hahn, P. Erb. 1994. Fas antigen is the major target molecule for CD4+ T cell-mediated cytotoxicity. J. Immunol. 152:1127.[Abstract]
  35. Young, J. D., Z. A. Cohn. 1987. Cellular and humoral mechanisms of cytotoxicity: structural and functional analogies. Adv. Immunol. 41:269.[Medline]
  36. Anel, A., M. Buferne, C. Boyer, A. M. Schmitt-Verhulst, P. Golstein. 1994. TCR-induced Fas ligand expression in cytotoxic T lymphocyte clones is blocked by protein tyrosine kinase inhibitors and cyclosporin A. Eur. J. Immunol. 24:2469.[Medline]
  37. Kagi, D., F. Vignaux, B. Ledermann, K. Burki, V. Depraetere, S. Nagata, H. Hengartner, P. Golstein. 1994. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 265:528.[Abstract/Free Full Text]
  38. Zhang, X., T. Brunner, L. Carter, R. W. Dutton, P. Rogers, L. Bradley, T. Sato, J. C. Reed, D. Green, S. L. Swain. 1997. Unequal death in T helper cell (Th)1 and Th2 effectors: Th1, but not Th2, effectors undergo rapid Fas/FasL-mediated apoptosis. J. Exp. Med. 185:1837.[Abstract/Free Full Text]
  39. Varadhachary, A. S., S. N. Perdow, C. Hu, M. Ramanarayanan, P. Salgame. 1997. Differential ability of T cell subsets to undergo activation-induced cell death. Proc. Natl. Acad. Sci. USA 94:5778.[Abstract/Free Full Text]
  40. Thilenius, A. R. B., K. Braun, J. H. Russell. 1997. Agonist antibody and Fas ligand mediate different sensitivity to death in the signaling pathways of Fas and cytoplasmic mutants. Eur. J. Immunol. 27:1108.[Medline]
  41. Zipp, F., R. Martin, R. Lichtenfels, W. Oth, J. Dichgans, P. H. Krammer, M. Weller. 1997. Human autoreactive and foreign-specific T cells resist apoptosis induced by soluble recombinant CD95 ligand. J. Immunol. 159:2108.[Abstract/Free Full Text]
  42. Lynch, D. H., F. Ramsdell, M. R. Alderson. 1995. Fas and FasL in the homeostatic regulation of immune response. Immunol. Today 16:569.[Medline]
  43. Xiao-Ning, X., G. R. Screaton, F. Gotch, T. Dong, R. Tan, N. Almond, B. Walker, R. Stebbings, K. Kent, S. Nagata, J. E. Stott, A. McMichael. 1997. Evasion of CTL responses by Nef-dependent induction of Fas ligand (CD95L) expression on simian immunodeficiency virus-infected cells. J. Exp. Med. 186:7.[Abstract/Free Full Text]
  44. Alderson, M. R., R. J. Marmitage, E. Marashakovsky, T. W. Tough, E. Roux, K. Schooley, F. Ramsdell, D. H. Lynch. 1993. Fas transduces activation signals in normal human T lymphocytes. J. Exp. Med. 178:2231.[Abstract/Free Full Text]
  45. Lau, H. T., C. J. Stoeckert. 1997. FasL: too much of a good thing?. Nat. Med. 3:727.[Medline]
  46. Kang, S.-M., D. B. Schneider, Z. Lin, D. Hanahan, D. A. Dichek, P. G. Stock, S. Baekkeskov. 1997. Fas ligand expression in islets of Langerhans does not confer immune privilege and instead targets them for rapid destruction. Nat. Med. 3:738.[Medline]
  47. Seino, K., N. Kayagaki, K. Okumura, H. Yagita. 1997. Antitumor effect of locally produced CD95 ligand. Nat. Med. 3:165.[Medline]
  48. Kishi, S., S. Saijyo, M. Arai, S. Karasawa, S. Ueda, M. Kannagi, Y. Iwakura, M. Fujii, S. Yonehara. 1997. Resistance to Fas-mediated apoptosis of peripheral T cells in human T lymphocyte virus type I (HTLV-I) transgenic mice with autoimmune arthropathy. J. Exp. Med. 186:57.[Abstract/Free Full Text]
  49. Vergelli, M., B. Hemmer, P. A. Muraro, L. Tranquill, W. E. Biddison, A. Sarin, H. F. McFarland, R. Martin. 1997. Human autoreactive CD4+ T cell clones use perforin- or Fas/Fas ligand-mediated pathways for target cell lysis. J. Immunol. 158:2756.[Abstract]
  50. Williams, N. S., V. H. Engelhard. 1996. Identification of a population of CD4+ CTL that utilizes a perforin- rather than a Fas ligand-dependent cytotoxic mechanism. J. Immunol. 156:153.[Abstract]
  51. Piazza, C., M. S. Gilardini Montani, S. Moretti, E. Cundari, E. Piccolella. 1997. CD4+ T cells kill CD8+ T cells via Fas/Fas ligand-mediated apoptosis. J. Immunol. 158:1503.[Abstract]
  52. Gervois, N., Y. Guilloux, E. Diez, F. Jotereau. 1996. Suboptimal activation of melanoma infiltrating lymphocytes (TIL) due to low avidity of TCR/MHC-tumor peptide interactions. J. Exp. Med. 183:2403.[Abstract/Free Full Text]
  53. Theobald, M., J. Biggs, J. Hernández, J. Lustgarten, C. Labadie, L. A. Sherman. 1997. Tolerance to p53 by A2.1-restricted CTL. J. Exp. Med. 185:833.[Abstract/Free Full Text]
  54. Esser, M. T., B. Krishnamurthy, V. L. Braciale. 1996. Distinct TCR signaling requirements for perforin- or FasL-mediated cytotoxicity. J. Exp. Med. 183:1697.[Abstract/Free Full Text]
  55. Strand, S., W. J. Hofmann, H. Hug, M. Muller, G. Otto, D. Strand, S. M. Mariani, W. Stremmel, P. H. Krammer, P. R. Galle. 1996. Lymphocyte apoptosis induced by CD95 (APO-1/Fas) ligand-expressing tumor cells: a mechanism of immune evasion?. Nat. Med. 2:1361.[Medline]
  56. Miethke, T., R. Vabulas, R. Bittlingmaier, K. Heeg, H. Wagner. 1996. Mechanisms of peripheral T cell deletion: anergized T cells are Fas resistant but undergo proliferation-associated apoptosis. Eur. J. Immunol. 26:1459.[Medline]
  57. Combadiere, B., M. Freedman, L. Chen, E. W. Shores, P. Love, M. J. Lenardo. 1997. Qualitative and quantitative contributions of the TCR {zeta} chain to mature T cell apoptosis. J. Exp. Med. 183:2109.[Abstract/Free Full Text]
  58. Ochoa, A. C., and D. L. Longo. 1995. Alteration of signal transduction in T cells from cancer patients. Important Adv. Oncol. p. 43.
  59. Lenardo, M. J.. 1996. Fas and the art of lymphocyte maintenance. J. Exp. Med. 183:721.[Free Full Text]
  60. Cormier, J. N., M. L. Salgaller, T. Prevette, K. C. Barracchini, L. Rivoltini, N. P. Restifo, S. A. Rosenberg, F. M. Marincola. 1997. Enhancement of cellular immunity in melanoma patients immunized with a peptide from MART-1/Melan A. Cancer J. Sci. Am. 3:4.
  61. Cerwenka, A., H. Kovar, O. Majdic, W. Holter. 1996. Fas- and activation-induced apoptosis are reduced in human T cells preactivated in the presence of TGF-ß1. J. Immunol. 156:459.[Abstract]
  62. Radvanyi, L. G., Y. Shi, H. Vaziri, A. Sharma, R. Dhala, G. B. Mills, R. G. Miller. 1996. CD28 costimulation inhibits TCR-induced apoptosis during a primary T cell response. J. Immunol. 156:1788.[Abstract]
  63. Irmler, M., M. Thome, M. Hahne, P. Schneider, K. Hofmann, V. Steiner, J. L. Bodmer, M. Schroter, K. Burns, C. Mattmann, D. Rimoldi, L. E. French, J. Tschopp. 1997. Inhibition of death receptor signals by cellular FLIP. Nature 388:190.[Medline]



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