Human Melanoma-Reactive CD4+ and CD8+ CTL Clones Resist Fas -Induced and Use Fas/Fas Ligand-Independent Mechanisms for Tumor Killing This information is current as of September 29, 2021. Licia Rivoltini, Marina Radrizzani, Paola Accornero, Paola Squarcina, Claudia Chiodoni, Arabella Mazzocchi, Chiara Castelli, Paolo Tarsini, Vincenzo Viggiano, Filiberto Belli, Mario P. Colombo and Giorgio Parmiani

J Immunol 1998; 161:1220-1230; ; Downloaded from http://www.jimmunol.org/content/161/3/1220

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Human Melanoma-Reactive CD4؉ and CD8؉ CTL Clones Resist Fas Ligand-Induced Apoptosis and Use Fas/Fas Ligand-Independent Mechanisms for Tumor Killing1

Licia Rivoltini,2* Marina Radrizzani,* Paola Accornero,* Paola Squarcina,* Claudia Chiodoni,* Arabella Mazzocchi,* Chiara Castelli,* Paolo Tarsini,* Vincenzo Viggiano,† Filiberto Belli,‡ Mario P. Colombo,* and Giorgio Parmiani*

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؉ . In this study

we report that tumor-specific T lymphocytes isolated from tumor lesions by repeated in vitro TCR stimulation with relevant Ags Downloaded from (mostly represented by normal self , 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 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 metallo- http://www.jimmunol.org/ protease 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. The Journal of Immunology, 1998, 161: 1220–1230.

umor cells can express antigenic determinants that are Particularly, one of the enigmas of tumor immunology is why detected by specific CD4ϩ and CD8ϩ T lymphocytes in the generally fails to eliminate antigenic tumor T an MHC-restricted fashion, thus stimulating cytotoxic ac- cells or to slow down disease progression. This phenomenon, iden- by guest on September 29, 2021 tivity and release (1). Based on these observations, the tified as tumor immune escape, has been associated with the ability identification of the human tumor Ags recognized by these T cells of cells to take advantage of a variety of strategies for is progressing at an accelerating pace (1). The genes reported to evading immune detection and/or killing. Deficiencies in quantity, encode tumor epitopes belong to different groups, including tumor- processing, presentation, or affinity may reduce the immunogenic- specific Ags, mutated proteins, and normal self molecules (2). In ity of certain tumor Ags (7), and loss of MHC expression, fre- the latter, T cell reactivity can be viewed as a sort of autoreactive quently observed in cancer cells, can make them completely un- phenomenon that escaped peripheral tolerance (3). The large detectable by T cells (8). Lack of expression of costimulatory amount of information collected in the last few years on the nature molecules on the neoplastic cells can lead to anergy of tumor- of Ags and epitopes recognized by T lymphocytes on cancer cells reactive CTL (9), and a similar effect can be mediated by immu- is presently supporting worldwide efforts aimed at identifying ef- nosuppressive factors released by cancer cells at the tumor site fective and widely applicable protocols of immune therapy of (10). Altogether, these factors may contribute in making a tumor cancer (4–6). lesion an immune privileged-like site that Ag-specific T cells can However, the functional interactions occurring in vivo between hardly access. the immune system and the cancer process are just starting to be Recently, a further mechanism potentially used by tumor cells to understood, and little information is still available on the rules neutralize host has been identified as involving governing host immune responses directed against tumor cells. Fas-FasL3 interaction (11, 12). These proteins are key molecules in normal immune development, , and functions (13). Li- gation of FasL to its Fas induces (or apoptosis) and plays important immunologic roles, such as im- Divisions of *Experimental Oncology D, †Medical Oncology B, and ‡Surgical On- mune response termination, T cell activation-induced cell death, cology B, Istituto Nazionale Tumori, Milan, Italy clonal downsizing, and control of peripheral tolerance to self Ags Received for publication December 12, 1997. Accepted for publication April 2, 1998. (14). Based on these features, a central role of Fas-FasL has also The costs of publication of this article were defrayed in part by the payment of page been hypothesized recently in the maintenance of immune privi- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. lege in certain vital organs, such as eye, central nervous system, 1 This work was supported in part by grants from Italian Association for Cancer and testis (15), and dysfunctions in Fas-mediated apoptosis have Research (Milan, Italy) and Istituto Superiore di Sanita`(Rome, Italy). 2 Address correspondence and reprint requests to Dr. L. Rivoltini, Division of Ex- perimental Oncology D, Istituto Nazionale Tumori, Via Venezian 1, 20133 Milan, 3 Abbreviations used in this paper: FasL, Fas ligand; CsA, cyclosporin A; LCL, lym- Italy. phoblastoid cell line; TBS, Tris-buffered saline; TIL, tumor-infiltrating lymphocytes.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 The Journal of Immunology 1221 been associated with autoimmunity in both animal models and and BS239-CD4 recognized an unknown Ag specifically expressed on au- humans (13, 14). Additionally, Fas-FasL interaction is also utilized tologous melanoma cells and presented in the context of HLA-DR1 or 10 by cytotoxic T cells as a death pathway for target killing, second- (our unpublished observations). The Th1 clone 103 has been described previously (27). ary to granule exocytosis in CD8ϩ effectors, but essential for cy- ϩ As for the tumor lines, MeBS, Me1340, Me23682, Me97620, totoxic CD4 cells (16, 17). Me1402/R, and Me37041 were established in our laboratory from mela- Although Fas has been reported to be broadly expressed in dif- noma lesions (either s.c. or visceral metastases, or invaded lymph nodes). ferent tissues, FasL was conventionally thought to be a selective 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 marker of cells belonging to the immunologic compartment (13). provided by Dr. F. M. Marincola (Surgery Branch, National Institutes of However, it has been observed recently that nonimmunologic Health, Bethesda, MD) (28). 256 mel and 272 mel were a kind gift from cells, such as tumor cells, can indeed acquire FasL expression and Dr. D. Rimoldi (Ludwig Institute of Cancer Research, Lausanne, Switzer- use it for delivering death signals to activated, Fasϩ T cells (12, 18, land). A375 mel, HS695 mel, and RPMI 7951 mel were purchased from 19). At the same time, tumor cells seem to be spared from their American Type Culture Collection (Rockville, MD). All of these cell lines, including Jurkat and K562, were maintained in 10% FCS/RPMI 1640. own weapon, due to their resistance to Fas-induced cell death that Normal melanocytes were kindly provided by Dr. M. Herlyn (Wistar In- additionally preserves them from undergoing apoptosis-mediated stitute, Philadelphia, PA). killing by cytotoxic cells (19, 20). Based on these findings, resistance to apoptosis and concomitant mAbs and immunofluorescence analysis ability to neutralize approaching T cells are presently considered as a potential mechanism used by tumor cells to evade immuno- The following mAbs were used: CH-11 (anti-Fas IgM, apoptosis in- Downloaded from logic recognition, despite the expression of antigenic determinants. ducing), ZB4 (anti-Fas IgG, neutralizing), phycoerythrin-conjugated UB2 (anti-Fas, for cell surface staining) (all from Medical and Biolog- Although intriguing, this hypothesis has some limitations due to ical Laboratories, Nagoya, Japan), C-20 (anti-FasL, for cell surface the lack of data regarding the significance of in vivo FasL expres- staining) (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bcl-2, anti- sion by tumor cells and its role in affecting Ag-specific T cell Bcl-x, and anti-Bax (all from PharMingen, San Diego, CA). Fluorescein ϩ responses. In fact, most of the functional data on the ability of anti-rabbit IgG (H L) (Vector Laboratories, Burlingame, CA) was used as a secondary reagent. Suitable negative controls were from Cym- cancer cells to mediate Fas-dependent apoptosis have

bus Bioscience (Southampton, U.K.). Cell surface Fas and FasL ex- http://www.jimmunol.org/ been obtained using Fas-sensitive conventional targets (such as pression was detected as previously described (29). Fas expression was Jurkat cells or even murine cells, like A20 lymphoma) (18, 19), or increased by culturing melanoma cells or melanocytes for 24 to 48 h lymphocyte bulk cultures whose Ag specificity has not been as- with 1000 U/ml IFN-␥, as reported by others (20). sessed (12). Moreover, if FasL-expressing tumor cells were able to FasL expression on melanoma cells was evaluated after 24-h treatment ϩ with 250 ␮M 1,10-phenanthroline (Sigma, Poole, U.K.), a metalloprotease kill the totality of activated (i.e., Fas ) lymphocytes recognizing inhibitor known to block soluble FasL release, and thus able to increase them, it would be hard to explain the presence of tumor-specific T FasL surface expression. For intracellular staining (Bcl-2, Bcl-x, and Bax), cell-infiltrating cancer lesions (2, 21), and the ability to produce cells (5 ϫ 105/sample) were fixed with 3% paraformaldehyde in PBS (10 clinical regressions once tumor-infiltrating lymphocytes (TIL) are 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); activated in vitro and reinjected into patients (4). These observa- 0.1% /Triton X-100 in TBS was used as buffer during immunofluorescence. by guest on September 29, 2021 tions suggest that at least some T cells in tumor infiltrates are not subjected to Fas-mediated cell death via interaction with tumor cells. Susceptibility to Fas-mediated killing by CH-11 mAb, rFas-L, In the present study, we identified CD4ϩ and CD8ϩ T cell and Th1 clone 103 clones obtained from melanoma lesions that resist FasL-mediated Different concentrations of agonistic CH-11 mAb or rFasL (kindly pro- apoptosis, induced by FasL either in soluble form or expressed on vided by Dr. J. Tschopp, Institute of Biochemistry, University of Lausanne, ϩ ␮ FasL cells, including melanoma. At the same time, these lym- Switzerland) were both diluted in final 150 l/well 10% FCS/RPMI and incubated for 16 h at 37°C with 51Cr-labeled 104/well target cells in 96- phocytes exclusively rely on Fas/FasL-independent mechanisms of well V-bottom plates. To prove that cell killing was Fas mediated, the tumor killing, and hence are not influenced by melanoma resis- assay was also performed in the presence of 50 ng/ml neutralizing anti-Fas tance to FasL. These data indicate that, like other pathologic sit- mAb ZB4, as previously described (27). At the end of incubation, 50 ␮l/ uations such as autoimmune diseases, a proportion of Ag-specific well of supernatant was harvested and counted, and killing was calculated T cells involved in tumor-directed immune responses can survive 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 Fas/FasL-mediated tumor counterattack. 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 Materials and Methods as positive and negative control of Fas-mediated killing, respectively. Lymphocyte clones, melanoma lines, and normal melanocytes Lymphocyte clones 501-CD8 and GDN-CD8 were obtained from TIL of Cytotoxic activity by lymphocytes and its inhibition by melanoma patient 501 and GDN, respectively (22). Clone BS-CD8, BS57- MgCl /EGTA, CsA, or anti-Fas mAb CD4, and BS239-CD4 were derived from a tumor-invaded lymph node of 2 patient BS (23). Clones were generated by limiting dilution in the presence Cytotoxic activity of T cell clones was measured by 51Cr release assay of autologous tumor cells and 20 to 50 Cetus U/ml IL-2 (23) and selected (25). Labeled targets (1000 cells/well) were incubated for 6 or 16 h with after 3 to 4 wk of culture by screening cytotoxic, CD3-mediated activity different concentrations of effector cells. Peptide pulsing was performed by against autologous melanoma cells. GDN-CD8, deriving from a TIL line incubating T2 cells with 1 ␮M peptide for 1 to2hat37°C. Ag-specific generated from tumor lesion by in vitro culture with IL-2, was cloned using presentation to CD4ϩ clones was obtained by culturing for 24 h at 37°C soluble OKT3 (30 ng/ml), as described elsewhere (24). Clones were main- autologous EBV-B-transformed cells (106 cells/well, in 24-well plates) tained by weekly restimulation with autologous tumor cells, autologous with tumor lysate from BS-Mel (generated from 106 cell, by quick freezing 2ϩ ϩ lymphoblastoid cell line (LCL), allogeneic feeder (PBMC), and 20 to 50 and thawing). For inhibiting Ca -dependent killing, EGTA MgCl2 (at U/ml IL-2. The clonality of these effectors (all bearing different V␤- and 6 and 3 mM, respectively) were added to the assay. For blocking FasL- V␣-chains) was proved by TCR analysis (data not shown), as described mediated lysis, effector cells were incubated with CsA (5 ␮g/106/ml) for elsewhere (23). 501-CD8 and GDN-CD8 recognize the nonapeptide 27–35 3 h at 37°C, washed three times in PBS before adding to the assay. Oth- from the melanoma Ag MART-1/Melan A in the context of HLA-A2.1 erwise, the assay was performed in the presence of neutralizing anti-Fas (25). Clone BS-CD8 reacts with the nonapeptide 17–25 from the differ- mAb ZB4, as described above. Activation with PMA-ionomycin was in- entiation Ag gp100, presented in the context of HLA-A3 (26). BS57-CD4 duced by incubating cells with as described above for Th1 clone 103 (27). 1222 ANTIMELANOMA CTLs OVERCOME Fas/FasL TUMOR IMMUNE ESCAPE

Table I. Phenotypic expression of Fas molecules on melanoma cells anoma cells (in 24-well plates) cultured with an equivalent number of and normal melanocytes a 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. % Mean Positive Fluorescence Cell Line Cell Type Cells Intensity

Jurkat Positive control 99 43 Results K562 Negative control 1 17 Fas expression and susceptibility to Fas-mediated apoptosis of 501mel Melanoma 94 15 melanoma cells and normal melanocytes MeBS Melanoma 84 95 A375mel Melanoma 74 21 Alterations in Fas expression or function have been described re- GDNmel Melanoma 62 21 cently to occur in tumor cells. Mutations have been indicated to be Me1340 Melanoma 50 16 involved in tumorigenesis of these neoplastic cells and in their HS695mel Melanoma 24 36 proliferative advantage as compared with the normal counterpart Me23682b Melanoma 15 64 Me97620b Melanoma 14 75 (30). Specifically, melanoma cell lines have been reported by Me37041b Melanoma 2 46 Hahne et al. to lack significant expression of Fas molecules, and RPMI7951mel Melanoma 2 29 consequently to resist Fas-mediated killing (19). However, despite Mel402/R Melanoma 1 16 this potential resistance, melanoma cells are usually efficiently rec- 624.28mel Melanoma 0 11 ognized, at least in vitro, by Ag-specific T cells either from the

256mel Melanoma 1 10 ϩ ϩ Downloaded from 272mel Melanoma 2 51 CD8 or the CD4 lineage (22, 23, 25, 31). FM2030 Melanocytes 82 54 To assess the role of Fas-FasL system in the lymphocyte/mel- FM2081 Melanocytes 63 27 anoma interaction, we first analyzed a large panel of melanoma FM2093 Melanocytes 51 50 lines for Fas expression, evaluated by fluorescence staining with FM1094 Melanocytes 24 19 the anti-Fas mAb UB2. Normal melanocytes were also included to a Melanoma cells and normal melanocytes were mechanically removed from cul- see whether any observed phenomenon was a feature of cells be- ture flasks and stained for Fas expression using the anti-Fas mAb UB2-PE (see Ma- http://www.jimmunol.org/ terials and Methods). Positivity was then evaluated by flow cytometry. longing to melanocytic lineage, or was restricted to the neoplastic b Tested at early in vitro passage (p2 to p3). phenotype. As listed in Table I, the two melanoma lines, 256 mel and 272 mel, previously described as Fas negative by Hahne et al. (19), Fas-mediated killing of lymphocyte by melanoma cells expressed no significant level of Fas molecules. However, in con- Melanoma cells (at different concentrations, starting from 105/well, in 96- trast to this previous report, the additional melanoma lines tested in well U-bottom plates) (Costar, Cambridge, MA) were cultured in 10% this study were characterized by heterogeneous surface expression FBS/RPMI for 48 h at 37°C, to allow cell adhesion. No supernatant was of Fas, ranging from levels comparable (in percentage of positive removed during these 48 h, to avoid any elimination of FasL in soluble 51 4 cells and mean fluorescence intensity) with the positive control form. Cr-labeled T cell clones or Jurkat cells (at 10 cells/well), in the by guest on September 29, 2021 presence or absence of neutralizing anti-Fas mAb ZB4 (50 ng/ml final Jurkat, to intermediate or low levels. A similar variable distribu- concentration), were then added as target cells, and the plates were incu- tion was observed in normal melanocytes, although such cells bated for 16 h. For coculturing assay, 106/well adherent nonirradiated mel- could be analyzed on a limited number of cases. In accordance

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

% Lysis by CH-11b % Lysis by recFasLc

Cell Line Cell Type 1 ␮g 50 ng 2.5 ng 50 ng 5 ng 0.5 ng

Jurkat Positive control 75 (30) 56 (20) 34 (0) 68 (11) 47 (13) 28 (1) K562 Positive control 80 (22) 66 (29) 46 (7) 71 (18) 55 (14) 36 (6) T2 Negative control 2 (1) 0 (0) 0 (0) 2 (2) 1 (0) 0 (0) A375 Melanoma 5 (4) 0 (0) 0 (0) 3 (2) 0 (0) 0 (0) HS695 Melanoma 0 (0) 0 (0) 0 (0) 2 (0) 0 (0) 0 (0) RPMI7951 Melanoma 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) BSmel Melanoma 3 (3) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) GDNmel Melanoma 5 (2) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 501mel Melanoma 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 23582mel Melanoma 3 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 97620mel Melanoma 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 37041mel Melanoma 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 256mel Melanoma 3 (2) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 272mel Melanoma 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) FM1094 Melanocytes 5 (4) 0 (0) 0 (0) NDd ND ND FM2488 Melanocytes 7 (5) 0 (0) 0 (0) ND ND ND FM2030 Melanocytes 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) FM2081 Melanocytes 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) FM2093 Melanocytes 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)

a Melanoma cells and normal melanocytes were mechanically removed from culture flasks and evaluated for susceptibility to Fas-mediated apoptosis by incubating 51Cr- labeled cells (104/well) for 16 h with different concentration of the agonist anti-Fas mAb CH-11 or the human rFasL. b Percentage of lysis obtained in the presence of the indicated doses of CH-11. Lysis data concerning the addition of 50 ng/ml of the neutralizing anti-Fas mAb ZB4 are reported in parentheses. c Percentage of lysis obtained in the presence of the indicated doses of rFasL. Lysis data concerning the addition of 50 ng/ml of the neutralizing anti-Fas mAb ZB4 are reported in parentheses. d ND, not done The Journal of Immunology 1223

FIGURE 1. Melanoma-specific CD4ϩ and CD8ϩ T cell clones kill autologous tumor cells in aCa2ϩ-dependent, FasL-independent fashion. Ef- fector cells were tested in a 6-h 51Cr release assay against autologous melanoma cells in the pres- ence of MgCl2/EGTA (3 and 6 mM, respective- ly), or after treatment with CsA (5 ␮g/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). Downloaded from 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. http://www.jimmunol.org/ by guest on September 29, 2021 with results reported for other tumors (20), the level of Fas ex- as second or third), as tested with Me23682, Me97620, and pression could be increased significantly by in vitro culture with Me37041 (Table I). ␥ IFN- in both melanoma and melanocytes (data not shown). ϩ ϩ The functional role of Fas was then analyzed by assessing sen- Lytic mechanisms used by melanoma-specific CD4 and CD8 sitivity to Fas-mediated apoptosis induced by agonistic anti-Fas T cells to kill autologous tumor cells mAb or rFasL molecules. Melanoma cells and normal melanocytes The observed resistance of melanoma cells to Fas-mediated apo- were cultured in the presence of different concentrations of CH-11 ptosis could have relevant implications for the susceptibility of mAb or rFasL, and apoptosis was measured in a 16-h 51Cr release these cells to killing activity by T lymphocytes. CD4ϩ or CD8ϩ assay. As reported in Table II, regardless of the amount of Fas Ag-specific CTL recognizing tumor cells in MHC-restricted, TCR- expression, all of the lines tested showed a complete resistance to dependent fashion have been described extensively in melanoma Fas-mediated killing by CH-11 and rFasL, which were, however, patients (1, 2). CD8ϩCTL are known to use mainly Ca2ϩ-depen- effective in inducing significant apoptosis of the Fas-sensitive line dent -mediated lysis for killing their targets, although Fas- Jurkat. The same resistance was observed when killing through Fas mediated cytotoxicity can also be used as a lytic tool (32, 33). On was mediated by the Th1 clone 103, known to up-regulate FasL the contrary, CD4ϩ T lymphocytes have been described to rely for and induce apoptosis of target cells after activation with PMA- killing mostly on Fas-mediated apoptosis (33, 34). Therefore, re- ionomycin (27). No change in sensitivity was detected after treat- sistance to the apoptotic effect of Fas molecule could contribute ment of melanoma and melanocyte lines with IFN-␥ (data not significantly to impair susceptibility of tumor cells to the cytotoxic shown), which was previously reported to reverse the Fas-resistant activity of Ag-specific CD4ϩ and CD8ϩ CTL. phenotype in breast cancer cell lines (20). Interestingly, the lack of To evaluate this hypothesis, we characterized the lytic mecha- sensitivity to Fas-induced apoptosis (either by agonist Abs or by nisms used by melanoma-specific CD4ϩ and CD8ϩ CTL clones the natural ligand) was not limited to melanoma cells, and thus generated from the infiltrate of metastatic tumor lesions. CD4ϩ T appeared to be a feature of cells deriving from the melanocytic cell clones were derived from a tumor-invaded lymph node of a lineage. Altogether, these data indicate that cells from melanocytic melanoma patient and were shown to recognize an HLA-DR-re- lineage (either normal or neoplastic) can express Fas on their sur- stricted Ag expressed on autologous melanoma cells (our unpub- face, but this expression does not correlate with the ability of this lished observations). We dissected the lytic mechanisms utilized molecule to transmit apoptotic signals when adequately ligated by by these clones to kill autologous tumor, by inhibiting Ca2ϩ-de- FasL. We cannot completely rule out the possibility that the ap- pendent and perforin-mediated cytotoxicity or by interfering with parent defect of Fas signaling in melanocytic cell lines could be Fas-mediated lysis. This was done, for granule-dependent cytotox- related to their in vitro selection as part of cell line establishment. icity, by incubating in Ca2ϩ-chelating conditions in the presence of

However, it should be underlined that resistance to Fas-mediated MgCl2/EGTA (35), and by blocking FasL synthesis in T cells killing could be observed starting from very early passages (such through treatment with CsA (36) or inhibiting Fas-FasL interaction 1224 ANTIMELANOMA CTLs OVERCOME Fas/FasL TUMOR IMMUNE ESCAPE

FIGURE 2. Antimelanoma CD4ϩ and CD8ϩ T cell clones can utilize Fas/FasL mechanism after activation with PMA-ionomycin. Lymphocytes, activated with PMA-ionomycin for 3 h, as previ- ously 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 ␮g/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. Downloaded from

with the anti-Fas antagonist mAb ZB4 (27), for testing Fas-medi- Again, the absence of any role of Fas-mediated killing was not due ated killing. to the inability of these T cells to synthesize FasL, since treatment As clearly reported in Figure 1 (A and B), both of the CD4ϩ with PMA-ionomycin promoted Fas-mediated cytotoxic activity clones tested, which significantly lysed autologous tumor cells, against Jurkat cells (although to a lesser extent than that observed http://www.jimmunol.org/ were blocked completely in their lytic activity when Ca2ϩ-chelat- in CD4ϩ) that could be blocked by anti-Fas mAb or CsA (Fig. 2, ing agents were added to the assay, while no significant effect was C and D). observed after treatment with CsA or ZB4. These data indicate that ϩ ϩ our antimelanoma CD4ϩ T cell clones, in contrast with most of the Antimelanoma CD4 or CD8 clones do not trigger FasL in CD4ϩ clones and cultures described to date (32, 33), lysed tumor response to interaction between TCR and the relevant MHC/Ag cells through a Ca2ϩ-dependent Fas-independent mechanism. This complex independence was not due to a general inability to up-regulate From the above data, it was not clear whether the lack of any role

functional FasL. In fact, after treatment with PMA-ionomycin of Fas in tumor killing by T cells was due to Fas resistance of by guest on September 29, 2021 (stimuli known to potently induce FasL production in T cells) (27), melanoma cells or to the lack of FasL up-regulation in T cells both CD4ϩ clones were able to lyse Fas-sensitive cells such as encountering the relevant Ag. In fact, T cells have been described Jurkat in a Fas-dependent fashion, since this lysis was blocked to up-regulate FasL in response to TCR-activating stimuli (33), but efficiently by treatment with CsA or ZB4 mAb (Fig. 2, A and B). little is known about the functional pathways triggered in tumor- The predominant role of perforin and other granule-related en- specific T cells by the interaction with tumor-expressed epitopes. zymes in the killing activity of melanoma cells by antimelanoma To see whether the resistance of melanoma cells to Fas-mediated CD4ϩ clones was also supported by the detection of BLT esterase apoptosis may contribute to significantly reduce the lytic potentials of (N-␣-benzyloxycarbonyl-L-lysine-thiobenzyl esterase) release in tumor-specific T cell clones, we analyzed the mechanisms used by our response to autologous tumor cells or solid-phase OKT3 (data not CD4ϩ and CD8ϩ T cell clones to kill Fas-sensitive targets expressing shown). the relevant Ag. For CD8ϩ clones, the recognized peptide MART- ϩ ␮ ϩ These data show that specific CD4 clones displaying signifi- 127–35 was pulsed at the concentration of 1 M, on HLA-A2.1 T2 cant Fas-independent cytotoxic activity against autologous tumor cells, a TAP-deficient cell line efficient in presenting exogenous pep- cells can be found as a component of antitumor T cell response; tides and highly sensitive to Fas-mediated killing (Table II). As re- these results also confirm that perforin/granzyme expression is not ported in Figure 3 (A and B), no significant part of the total cytotoxic necessarily confined to CD8ϩ T cells. activity on peptide-pulsed T2 cells appeared to be mediated by Fas- Although CD8ϩCTL are conventionally known to use the per- FasL (as detected by the inhibition of lysis caused by CsA or ZB4), forin-dependent granule exocytosis as the main cytotoxic pathway, while most of the killing was Ca2ϩ dependent and probably perforin reports have shown recently that these cells can also take advan- mediated. These data clearly indicate that antimelanoma CD8ϩ T tage of the Fas-FasL interaction as a concomitant mechanism of cells did not significantly use FasL when their TCR is triggered by the target killing (32, 33, 37). To assess this hypothesis in our system, interaction with the relevant Ag. we analyzed lytic mechanisms used by melanoma-specific CD8ϩ Comparable results were observed with CD4ϩ clones. Since the clones to kill Ag-positive targets. Both of the CD8ϩ clones used melanoma Ag recognized by CD4ϩ clones was unknown in this case, recognized the immunodominant peptide 27–35 from the mela- the autologous LCL was incubated overnight with lysate from autol- noma Ag MART-1/Melan A (clone 501-CD8 and GDN-CD8) ogous melanoma cells, a procedure that has been shown to induce (25), presented in the context of HLA-A2.1. It should be under- processing and presentation of exogenous tumor Ags by B cells (31). lined that this Ag is a self differentiation-related , expressed The totality of the cytotoxic activity exerted by CD4ϩ clones on on both melanoma and normal melanocytes (2). pulsed autologous LCL cells with tumor lysate was inhibited by Lysis of autologous melanoma cells by these clones was inhib- Ca2ϩ-chelating agents, with no effect of treatments impairing Fas- ϩ ited completely by MgCl2/EGTA, with no effect of CsA or mAb mediated lysis (Fig. 3, C and D). As observed in the CD8 system, ZB4 (Fig. 1, C and D), suggesting that perforin-dependent (and not TCR triggering by tumor Ag did not lead to significant FasL involve- Fas-dependent) cytotoxicity was the crucial mechanism involved. ment in melanoma-specific CD4ϩ clones as well. The Journal of Immunology 1225

FIGURE 3. CD4ϩ and CD8ϩ T cell clones kill Fas-sensitive APC, presenting relevant tumor Ag in a FasL-independent fashion. CD8ϩ T cell clones were tested in a 6-h 51Cr release assay against T2 cells prepulsed with MART-127–35 peptide (1 ␮M) (A and B). Comparable data were obtained in a 16-h 51Cr release assay. CD4ϩ 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; Downloaded from D, clone BS239-CD4. http://www.jimmunol.org/

FasL expression on melanoma cells positive melanoma lines (data not shown). The reduced amounts of FasL expression by melanoma lines in comparison with T cells

In the previous studies, FasL expression on melanoma level was, by guest on September 29, 2021 however, analyzed by techniques that detect the total pool of in- could also explain the functional data reported in Table III, show- tracellular and membrane-bound FasL present in a sample (such as ing that melanoma cells induced apoptosis of Jurkat cells at level Western blot and immunohistochemistry) (19). However, no quan- significantly lower than that observed in the presence of the Th1 titative evaluation at the single cell level, with particular attention clone. to the surface fraction, was reported, especially in comparison with the level of expression detectable in immune cells that profession- Resistance of antitumor CTL clones to Fas-FasL-mediated ally use this molecule for their immune functions (such as Th1 apoptosis lymphocytes). With the aim of quantifying the amounts of FasL To conclusively analyze the role of FasL in down-regulating Ag- expressed on melanoma cells and its functional relevance in im- specific T cell responses in the system, we tested the susceptibility mune modulation, phenotypic analysis by flow cytometry was per- of our CD4ϩ and CD8ϩ T cell clones to FasL-mediated apoptosis, formed using the anti-FasL mAb C-20, which detects the mem- induced by different means, i.e., by an agonist anti-Fas Ab (CH- brane-bound form of this protein. It should be underlined that, in 11), the human rFasL molecule, the Th1 clone 103 (known to contrast to what has been reported by others (19), no significant mediate FasL-dependent killing after activation with PMA-iono- and reproducible level of FasL could be observed in our melanoma mycin) (27), and by autologous melanoma cells. cell lines without previous treatment with inhibitors of metallo- As shown in Table III, all of the CD8ϩ and CD4ϩ clones tested proteases that are known to block FasL shedding from cell mem- displayed resistance to rFasL or to Th1-mediated apoptosis (com- brane (13) (data not shown). On the contrary, a significant per- parable with what was observed with the Fas-negative line K562). centage of positive cells expressing high levels of FasL could be As expected, Jurkat cells underwent massive apoptosis when in- found in the Th1 clone 103 after activation with OKT3 (Fig. 4). cubated in the same conditions. This resistance was not due to lack Thus, heterogeneous but clear expression of FasL in the majority of Fas expression, since all T cell clones were found to be highly of the melanoma lines analyzed could be demonstrated after an positive for this molecule when analyzed by flow cytometry (data overnight treatment with 250 ␮M phenanthroline, a metallopro- not shown). tease inhibitor. A comparison between the profiles obtained with In agreement with what we observed with rFasL and Th1 clone melanoma lines such as A375 mel, HS695 mel, and Me23682 103 (Table III), the antitumor CD4ϩ and CD8ϩ T cell clones (expressing the highest FasL levels) and the profile obtained with tested did not show any susceptibility to apoptosis induced by Th1 clone 103 clearly showed that T cells that are professionally FasLϩ melanoma cells, either autologous or allogeneic. Although committed to use FasL for their killing activity express signifi- different from what has been described in other T cell models (38, cantly higher amounts of this molecule on their surface (evaluated 39), this resistance appears to be an intrinsic feature of these T as mean fluorescence intensity) (Fig. 4). This quantitative differ- cells, since it was independent from TCR triggering. However, ence was confirmed by RT-PCR, which showed a 10- to 100-fold Jurkat cells underwent significant apoptosis after exposure to stronger FasL message in activated clone 103 as compared with rFasL, Th1 clone 103, and some melanoma cells (Table III). As 1226 ANTIMELANOMA CTLs OVERCOME Fas/FasL TUMOR IMMUNE ESCAPE Downloaded from http://www.jimmunol.org/

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 ␮M 1,10-phenanthroline. The Th1 clone 103 was treated overnight with 20 ␮g/ml solid-phase OKT3 for inducing FasL expression. Closed areas, staining with C-20 mAb; open areas, staining with negative control. Percentage of FasLϩ cells and mean fluorescence intensity (in parenthesis) are indicated. by guest on September 29, 2021 discussed above, the level of killing mediated by tumor cells was cytes were harvested, evaluated for viability and number, and lower than expected on the basis of the data previously published tested for antimelanoma cytotoxic activity, to detect possible long- by Hahne (19). However, this could be due to an overall lower term effects of FasL on T cell functions. 256 mel was included in susceptibility of human Jurkat T cells to FasL-dependent apoptosis this analysis, since previously reported by Hanhe et al. to express in comparison with murine A20 cells used by these authors. The significant levels of FasL (19). The presence of either autologous level of killing observed with melanoma cells on Jurkat was also or allogeneic melanoma cells did not negatively affect lymphocyte compatible with the amount of FasL expressed by these tumor short-term or long-term (24 h vs 7 days) growth as compared with cells, which was significantly lower than that observed on the Th1 culture with IL-2 alone. As expected, when an HLA-matched tar- clone 103 (Fig. 4). get was present (i.e., autologous melanoma or the HLA-A2ϩ mel- A detectable level of apoptosis could be observed when mela- anoma line 256 mel, both expressing MART-1, the target Ag of noma-specific T cell clones were exposed to the agonist anti-Fas clone 501-CD8), significant killing of tumor cells with a prolifer- mAb CH-11 (see Table III). This killing was, however, signifi- ative boost was observed (data not shown). cantly lower than that observed with Jurkat cells. A discrepancy The culture in the presence of FasLϩ melanoma cells failed also between data obtained with the natural FasL (either recombinant or to functionally affect either CD4ϩ or CD8ϩ T cell clones, which displayed on cell surface) and the effect mediated by agonist Ab maintained their potent antitumor cytotoxic activity and Ag spec- has been described already by others (40, 41). In fact, this phe- ificity. Figure 5 reports data concerning cytotoxic activity of T cell nomenon has been related to the triggering of different signaling clones after 7 days of culture in the presence of melanoma cells. pathways that could be limited in terms of physiologic implica- Comparable results were obtained when lymphocyte clones were tions when agonist Abs, and not natural ligands, are used. T cell cocultured with melanoma cells for a shorter time (24 h) and cy- death could also be observed in these clones after overnight treat- totoxic activity was evaluated using the original cell count in the ment with high (10 ␮g/ml) concentrations of immobilized OKT3 calculation of E:T ratios (data not shown). These results further mAb (data not shown). Such data suggest that the resistance of support the conclusion that these antimelanoma clones are resistant antimelanoma T cell clones to apoptosis was not an absolute phe- to FasL-mediated apoptosis, and explain why these cells can sur- nomenon, and that these cells were still susceptible to certain vive in vivo in the tumor lesion and receive proliferative signals forms of immunologic control. from repeated in vitro stimulation with autologous tumor cells. To confirm the absence of any functional effect of melanoma- expressed FasL on tumor-specific T cells, we performed a cocul- ture assay for different times, in which CD4ϩ or CD8ϩ clones Discussion were incubated with a high number (at ratio 1:1) of FasL-positive Great interest has been raised recently by the possibility that the Fas- nonirradiated melanoma cells. At the end of this culture, lympho- FasL system may represent one of the tools used by tumor cells to The Journal of Immunology 1227

Table III. Susceptibility to Fas-mediated lysis of human anti-melanoma CD4ϩ and CD8ϩ T cell clones a

% Lysis of Target Cells

Jurkat K562 501-CD8 GDN-CD8 BS-CD8 BS57-CD4 BS239-CD4 BS-LAKb

Effectors Med ZB4 Med ZB4 Med ZB4 Med ZB4 Med ZB4 Med ZB4 Med ZB4 Med ZB4

CH-11 1 ␮g 68 21 0 0 20 8 18 3 33 20 36 14 21 10 45 21 50 ng 56 18 0 0 15 0 5 0 23 10 16 12 10 3 32 9 2.5ng4480040 001804 0 0 0261 rFasL 50 ng 51 11 2 0 5 3 4 1 8 0 6 0 1 0 49 18 5ng47130000 20 00 0 0 0 0387 0.5ng3940000 00 00 0 0 0 0221 cl. 103 10:1 64 40 11 10 10 8 5 7 6 8 9 11 5 0 55 18 5:1 64 20 11 9 0 0 8 8 0 0 0 0 0 0 49 8 2:1 58 18 9 9 0 0 0 0 0 0 0 0 0 0 37 5 501mel 700000 00 00 0 0 0 0 6 0 MeBS 402200 00 00 0 0 0 0 3 0 Downloaded from A375mel 000000 00 00 0 0 0 0 0 0 GDNmel 701402 00 00 0 0 0 0 5 0 HS695mel 840000 00 00 0 0 0 0 7 0 Me23682 000000 00 00 0 0 0 0 0 0 Me97620 830000 00 00 0 0 0 0 5 0 Me37041 000000 00 00 0 0 0 0 0 0 RPMI7951mel 000000 00 00 0 0 0 0 0 0

256mel 1202100 00 00 0 0 0 0 8 0 http://www.jimmunol.org/

a Anti-melanoma CD4ϩ and CD8ϩ T cell clones were evaluated for susceptibility to Fas-mediated apoptosis by incubating 51Cr-labeled cells (104/well) for 16 h with different concentration of the agonist anti-Fas mAb CH-11 or the human rFasL. For detecting apoptosis mediated by FasL expressed on melanoma, tumor cells (105/well) were plated in 96-well plates and allowed to adhere and grow for 48 h, targets were then added without removing any culture medium (to avoid dilution soluble FasL possibly released by tumor cells). Jurkat and K562 were used as positive and negative control, respectively. Each cell line was incubated with medium (Med) or with 50 ng/ml of the neutralizing anti-Fas mAb ZB4. b BS-LAK cells (obtained from BS-PBMC after 3-day culture with 1000 Cetus U/ml IL-2) were also used as positive control for sensitivity to apoptosis.

evade immunologic control. In view of the central role of Fas-FasL in death signals, is emerging from many recent reports (42, 48). In by guest on September 29, 2021 the regulation of immune responses, this mechanism has also been other words, not all T cells follow the conventional rule of under- indicated as responsible for immune depletion and escape in other going death by apoptosis after ligation of FasL to cell surface pathologic situations such as HIV infection (29, 43). expressed Fas molecules. In this study, we provide the first evidence that the immune In the present work, we described antitumor system can develop strategies for overcoming this escape mecha- clones that are characterized by immunologic features different nism developed by the tumor cells. In fact, by repeated Ag stim- from most of the conventional Ag-specific T cells described in the ulations, we could isolate from melanoma lesions tumor-specific literature. It should be first underlined that our effectors were de- CD4ϩ and CD8ϩ T cell clones that apparently rely on Fas-inde- rived from tumor lesions or tumor-invaded lymph nodes (i.e., from pendent mechanisms for mediating tumor cell killing and that sites in which they most likely had the chance of encountering and show resistance to FasL-induced apoptosis, either to soluble FasL possibly being primed by the relevant Ag). Additionally, they have or to FasL expressed on Th1 lymphocytes and tumor cells. been generated by multiple in vitro restimulations, which likely Although recent data indicate that Fas-mediated apoptosis is a select for cells resistant to Ag-induced cell death. crucial guardian in the immune regulation, including maintenance In terms of cytotoxic mechanisms, granule-dependent cytolysis is of peripheral self tolerance, the role of Fas and FasL in the ho- clearly the predominant killing pathway used by our antimelanoma meostatic regulation of immune responses appears to be much CD8ϩ and CD4ϩ T cell clones. CD8ϩ T cells are known to use more complex than initially thought. Qualitative differences in mainly perforin to lyse their targets, although they can potentially how cells perceive signals through Fas exist in distinct T cell sub- up-regulate FasL once activated and induce apoptosis of Fas-positive populations, probably depending on the differentiation state and cells, as a secondary lytic pathway (16). On the contrary, CD4ϩ cy- Ag specificity (42). T cells engaged either by APCs presenting totoxic T cells are thought to lack perforin and other enzyme-contain- antigenic peptides or by Ab to TCR/CD3 can even undergo a series ing granules, and thus to exert target cell killing through alternative of activation events, instead of apoptotic death, following Fas- mechanisms, such as Fas-FasL interaction (32, 33). However, several FasL interaction (42, 44). Moreover, recent reports have shown instances of cytotoxic CD4ϩ T cells using granule-depending path- that the immune privilege cannot be ascribed to a single protective way have been described recently, mostly involving autoreactive cells Fas-FasL-dependent mechanism, but rather involves an intricate (49, 50). Our findings additionally show that cytotoxic CD4ϩ T cells orchestration of different processes (45). The identification of a can be found as a significant component of the antitumor T cell re- proinflammatory role of FasL in transplanted grafts or in murine sponse in melanoma, as powerful mediators of autologous tumor rec- tumor models, which can even lead to a faster rejection of FasL- ognition, exerted through significant lysis and cytokine release (our positive cells (46, 47), confirms the complexity of Fas-FasL im- unpublished data). This observation is in contrast with another im- munologic functions. Moreover, the possibility that a proportion of munologic dogma considering lytic CD4 as exclusive components of T cells involved in a given immune reaction is programmed to an immunoregulatory population devoted to negatively control detri- develop intrinsic features, enabling them to survive FasL-triggered mental immune responses such as autoreactivity (51). 1228 ANTIMELANOMA CTLs OVERCOME Fas/FasL TUMOR IMMUNE ESCAPE

FIGURE 5. Cytotoxic activity of antimelanoma CD4ϩ and CD8ϩ 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 ϩ autologous, nonirradi- ated melanoma cells (ratio 1:1). The allogeneic FasLϩ melanoma line 256 mel was also used as pos- itive control. After 1 wk, lymphocytes were recov- ered, counted, and tested against relevant targets in a 51Cr release assay. A, Clone 501-CD8; B, GDN- CD8. Both CD8ϩ 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 au- tologous LCL, as negative control (dashed bars) or autologous melanoma cells (closed bars). Downloaded from http://www.jimmunol.org/

The lack of a functional role of Fas-FasL interaction in Ag- do not show an intrinsic resistance to apoptotic death, since they specific killing was not due to defects in FasL-up-regulating mech- undergo apoptosis in response to high dose OKT3 and after ϩ ϩ anisms, since both CD4 and CD8 CTL clones do utilize FasL treatment with agonist anti-Fas Abs such as CH-11. The latter and kill Jurkat cells when nonspecifically activated with PMA- result confirms the already described phenomenon that Ab ago- ionomycin. At odds from other systems, TCR interaction with the nists and natural ligand can stimulate different signaling path- relevant MHC/peptide complex, either expressed endogenously by ways, and further emphasizes the limitations of defining the tumor cells or exogenously as peptide presented by APCs, does not physiologic role of Fas-FasL solely by Ab agonists (40, 41).

appear to trigger FasL involvement in our T cells. This phenom- Antimelanoma T cell resistance to FasL-induced death signal is by guest on September 29, 2021 enon could be related to a particular feature of antitumor effectors not an unexpected finding, and several lines of evidence could that are characterized by the expression of TCRs with low affinity support it. One first explanation could be related to the observation for their specific ligand (52), as reported for other self-reactive T that antitumor T cells present in vivo in a tumor lesion may be cells (53). A limited TCR affinity could explain why antimelanoma anergized by different mechanisms (including immune suppressive T cells do require strong stimuli, such as PMA-ionomycin, for a factors) (11, 21). Due to this anergy, T cells could thus survive to full activation of killing pathways involving Fas-FasL interaction. tumor-mediated apoptosis, as described in other systems (56). Ad- Alternatively, two distinct TCR transduction signals could be re- ditionally, it has been reported recently that certain components of quired for separate activation of Fas- and perforin-mediated kill- the TCR/CD3 complex, such as the ␨-chain, are required for T ing, as recently shown by Esser et al. (54), thus accounting for the cells to undergo Fas-mediated apoptosis (57), and this specific different lytic pathways observed in our T cell clones in response chain has been found to lack in fresh TIL obtained from tumor to the relevant Ag as compared with PMA-ionomycin. lesions (58). The possible lack of FasL functional involvement in response to An optimistic interpretation of our data could suggest that these TCR engagement by MHC/peptide complex may also play a role antimelanoma T cell clones can resist FasL-induced apoptosis be- in protecting these effector cells from Fas-FasL-mediated activa- cause of their memory phenotype. Although this hypothesis is hard tion-induced cell death, and allow their survival to repeated Ag stimulations, a situation occurring not only in vitro, during cloning, to prove given the absence of phenotypic markers for memory but also in vivo, in which a high tumor burden is often present. cells (with the exception of CD45RO, which is usually highly ex- Another interesting feature of our antimelanoma CTL clones is pressed in long-term cultured T cells, including our clones) (42, their resistance to apoptotic death mediated by either soluble or 59), functional characterizations of the T cell responses to some cell membrane-bound FasL. Tumor cells have been hypothesized melanoma Ags such as MART-1 have shown that sensitization to these Ags has occurred in vivo in melanoma patients (60). recently to impair lymphocyte function by expressing functionally ϩ active FasL, and thus inducing apoptosis of Fasϩ-activated CD4 cells with Th2 or Th0 phenotype have been shown in lymphocytes (11, 12, 55). some systems to be resistant to FasL-induced death (38, 39), However, the possibility of recovering TIL with tumor-spe- although our previous data do not fully agree with this obser- cific reactivity (2, 4, 21) suggests that at least a certain com- vation (29). It is hence possible that our CD4 T cell clones, ponent of the antitumor immune response has developed strat- which make significant amounts of IFN-␥ and IL-4 in response egies for surviving tumor counterattack. This resistance is to autologous tumor (data not shown), could be resistant due to constitutive (and does not require TCR triggering, as described their Th0 phenotype. in other systems) (38, 39) and it is not due to a reduced Fas T cells primed in the presence of TGF-␤ (61), cytokine that is expression, as our clones express levels of this molecule com- usually produced in situ by melanoma cells, or costimulated via parable with those present on Jurkat cells. Moreover, our clones CD28 (62) (that could be provided by local APCs) have been The Journal of Immunology 1229 shown to acquire resistance to apoptosis. Finally, resistance to ap- 8. Ferrone, S., and F. M. Marincola. 1995. Loss of HLA class I antigens by mela- optotic death has been hypothesized recently to be responsible for noma cells: molecular mechanisms, functional significance and clinical rele- vance. Immunol. Today 16:487. the escape from peripheral tolerance of autoreactive T cells (13, 9. Chen, L., P. McGowan, S. Ashe, J. Johnston, Y. Li, I. Hellstro¨m, and K. E. 41), thus providing a further explanation for FasL resistance of our Hellstro¨m. 1994. Tumor immunogenicity determines the effect of B7 costimula- T cell clones that mostly recognize normal self Ags (2). tion on T-cell-mediated tumor immunity. J. Exp. Med. 179:523. 10. Kiessling, R., K. Kono, M. Petersson, and K. Wasserman. 1996. Immunosup- In some of the systems described above, the molecular mecha- pression in human tumor-host interaction: role of and alterations in nisms responsible for T cell resistance to FasL-induced cell death signal-transducing molecules. Springer Semin. Immunopathol. 18:227. 11. Williams, N. 1996. Tumor cells fight back to beat immune system. Science 274: have been identified as involving apoptosis regulators such as 1302. Bcl-x, Bcl-2, Bax, and others (13). However, preliminary data 12. Walkler, P. R., P. Saas, and P.-Y. Dietrich. 1997. Role of Fas ligand (CD95L) in showed that none of these seem to play a role in providing our T immune escape: the tumor cell strikes back. J. Immunol. 158:4521. cells with a reduced susceptibility to apoptotic death (our unpub- 13. Nagata, S. 1997. Apoptosis by death factor. Cell 88:355. 14. Abbas, A. K. 1996. Die and let live: eliminating dangerous lymphocytes. Cell lished observations). Investigations are presently ongoing on ad- 84:655. ditional factors, including the potential involvement of molecules 15. Griffith, T. S., and T. A. Ferguson. 1997. The role of FasL-induced apoptosis in such as FLIP, which has been shown recently to inhibit apoptosis immune privilege. Immunol. Today 18:240. 16. Henkart, P. A. 1994. Lymphocyte-mediated cytotoxicity: two pathways and mul- by blocking FLICE/procaspase-8 activation (63). tiple effector molecules. Immunity 1:343. The apparent failure of the immune system to control in vivo 17. Lowin, B., M. Hahne, C. Mattmann, and J. Tschopp. 1994. Cytolytic T-cell tumor growth remains an unsolved paradox, and immunologists cytotoxicity is mediated through perforin and Fas lytic pathway. Nature 370:650. 18. O’Connell, J., G. C. O’Sullivan, J. K. Collins, and F. Shanahan. 1996. The Fas still have to deal with tumor immune escape in designing new counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas Downloaded from protocols for effective immunotherapy. However, our data show- ligand. J. Exp. Med. 184:1075. ϩ ϩ 19. Hahne, M., D. Rimoldi, M. Schroter, P. Romero, M. Schreier, L. E. French, ing that antitumor CD4 and CD8 T cell subpopulations resistant P. Schneider, T. Bornand, A. Fontana, D. Lie´nard, J. Cerottini, and J. Tschopp. to FasL-induced apoptosis do exist, suggest that this mechanism of 1996. Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for immune escape may not play a central role in melanoma. tumor immune escape. Science 274:1363. 20. Keane, M. M., S. A. Ettenberg, G. A. 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