Antigen-Dependent Release of IFN-γ by Cytotoxic T Cells Up-Regulates Fas on Target Cells and Facilitates Exocytosis-Independent Specific Target Cell Lysis This information is current as of September 27, 2021. Arno Müllbacher, Mario Lobigs, Ron Tha Hla, Thao Tran, Thomas Stehle and Markus M. Simon J Immunol 2002; 169:145-150; ; doi: 10.4049/jimmunol.169.1.145

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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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Antigen-Dependent Release of IFN-␥ by Cytotoxic T Cells Up-Regulates Fas on Target Cells and Facilitates Exocytosis-Independent Specific Target Cell Lysis

Arno Mu¬llbacher,1* Mario Lobigs,* Ron Tha Hla,* Thao Tran,† Thomas Stehle,† and Markus M. Simon†

Effector cytolytic T (Tc) lymphocytes, deficient in the exocytosis-mediated pathway of target cell lysis, induce Fas on target cells and, in turn, delayed cell death and apoptosis via the Fas ligand-Fas interaction. The induction of Fas can be blocked by anti- IFN-␥ Abs. This Fas up-regulation on initially Fas-negative target cells is not mediated by TCR-MHC/peptide signaling per se, but by secreted IFN-␥ from Tc cells after Ag engagement. The Fas up-regulation by Tc cells can be mimicked by treatment of target cells with rIFN-␥. Tc cells from IFN-␥ knockout mice do not induce Fas expression on target cells. Tc cell-mediated Fas expression Downloaded from on third party, bystander, target cells does not enhance their susceptibility to lysis by these nominal effector cells. The results are discussed as to the possible relevance of the phenomenon in efficiency and regulation of the Tc cell response to infections by viruses. The Journal of Immunology, 2002, 169: 145Ð150.

D8ϩ effector T cells exert their biological activity via We have shown previously (17) that delayed target cell lysis and

two quite distinct means upon specific encounter with apoptosis by alloreactive cytolytic T cells from perf-deficient http://www.jimmunol.org/ C target cells. One is the synthesis and release of cytokines (perfϪ/Ϫ) mice proceed via the Fas pathway. This conclusion was such as IFN-␥ (1) and TNF-␣ (2), which may act proximal and reached for the following reasons: 1) Fas expression, on originally distal to the effector cell. The other is direct cytolysis and apoptosis Fas-negative (Fasneg) targets, after incubation with Tc cells from Ϫ Ϫ of the bound target cell. These two distinct effector mechanisms perf / mice, was increased in a time-dependent manner; 2) target are not necessarily executed by the same CD8ϩ T cell. Evidence cells had to be biosynthetically active, i.e., RNA and protein syn- for such is the vastly different numbers of Ag-specific CD8ϩ T thesis was required as well as protein transport for lysis to occur; cells identified as IFN-␥ producers vs estimates of cytolytic T 3) Abs to Fas or soluble Fas-Fc inhibited cytolysis and nucleolysis; (Tc)2 cells using limiting dilution techniques (3), and our recent 4) poxvirus-encoded serpins, in particular SPI-2, a strong inhibitor finding that in response to flavivirus infection, Tc cells with lytic of the Fas pathway (18Ð20), completely inhibited lysis of target by guest on September 27, 2021 Ϫ Ϫ activity are not always producers of IFN-␥ (4). cells by perf / Tc cells; 5) brefeldin A treatment of Tc cells Apoptosis and lysis of target cells by cytolytic lymphocytes (NK severely reduced lysis, which interferes with polypeptide transport, and Tc cells) can be executed via at least two distinct pathways: but only marginally with concanamycin A, an inhibitor of exocy- one, the exocytosis pathway mediated by perforin (perf) and gran- tosis (21); 6) target cells derived from the Fas-defective mutant zymes (gzm); the other, by the Fas pathway involving the Fas mouse (lpr) were refractory to lysis; and finally 7) Tc effector cells Ϫ/Ϫ ligand (FasL) on the effector cell engaging the Fas receptor (Fas or from perf ϫ gld mice, defective in both cytolytic effector path- neg CD95) on the target cell (5Ð12). It is generally believed that the ways, did not exhibit this lytic phenotype on Fas target cells. exocytosis pathway is primarily involved in the elimination and/or In this study, we present evidence as to the mechanism by which neg control of intracellular pathogens such as viruses. The Fas pathway Tc cells induce Fas expression on Fas target cells, investigate its of cytotoxicity, in contrast, was thought to be in essence immu- consequence on bystander killing, and speculate on the signifi- noregulatory (11, 13, 14). However, some recent studies (15), in- cance of this process in recovery from viral infections. cluding our own on flavivirus-induced cytotoxicity (16), suggest that in certain virus infections both pathways are operative. Materials and Methods Animals C57BL/6 (KbDb) (B6), AKR/N (KkDk) (AKR), B10.HTG (KdDb) (HTG), the perfϪ/Ϫ mutant (8), the triple knockout mouse, deficient in perf plus Ϫ Ϫ *Division of Immunology and Cell Biology, John Curtin School of Medical Research, gzm A and B (perfϫgzmAϫB / ) (17), and the IFN-␥-deficient mouse Australian National University, Canberra, Australia; and †Max-Planck-Institut fu¬r Im- (IFN-␥Ϫ/Ϫ) (22) were bred under pathogen-free conditions at the Animal munbiologie, Stu¬beweg, Freiburg, Germany Breeding Facility of the John Curtin School of Medical Research or the Received for publication February 28, 2002. Accepted for publication May 1, 2002. animal facilities of the Max-Planck-Institute for Immunbiology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Cell lines with 18 U.S.C. Section 1734 solely to indicate this fact. The mouse cell lines L929 (H-2k), L1210 (H-2d), and L1210.Fas (trans- 1 Address correspondence and reprint requests to Dr. Arno Mu¬llbacher, Division of fected with mouse Fas; kindly provided by P. Golstein, Marseilles, France) Immunology and Cell Biology, John Curtin School of Medical Research, Australian were grown in Eagle’s MEM (EMEM) supplemented with 10% FCS. National University, P.O. Box 334, Canberra, ACT 2601, Australia. E-mail address: [email protected] FACS analysis 2 Abbreviations used in this paper: Tc, cytolytic T; DC, dendritic cell; Ect, Ectrome- lia; EMEM, Eagle’s MEM; FasL, Fas ligand; gzm, granzyme; neg, negative; perf, Cells were stained for Fas expression using the FITC-conjugated mAb perforin. specific for mouse Fas (Jo-2; PharMingen, Hamburg, Germany). Cells

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 146 Tc CELLS UP-REGULATE Fas ON TARGETS BY SECRETED IFN-␥ were examined with a FACScan flow cytometer (BD Biosciences, San lation. Initially, we explored whether signaling by the Tc cell via Jose, CA). MHC class I on the target cell was responsible for inducing Fas neg Treatment of L929 cells with rIFN-␥ expression on Fas targets. We failed to mimic Fas up-regulation on Fasneg L929 (H-2k) targets by a panel of anti-Kk or anti-Dk Abs. L929 were plated in 24-well Costar (Cambridge, MA) plates and treated neg ␥ ␮ In addition, Fas L929 target cells transfected with MHC class I with 300 U rIFN- (Genzyme, Cambridge, MA; MG-IFN; 200 U/ l) for 4, d 6, and 22 h. Cells were trypsinized and aliquots were stained for cell sur- K cDNA, lacking the cytoplasmic domain, still induced Fas after d face Fas expression and Fas transcript quantitation by PCR. recognition by K -reactive Tc cells (data not shown). Thus, it was unlikely that Fas expression was induced by signaling via MHC Quantitation of Fas transcripts class I directly. Therefore, we considered the second option, i.e., mRNA was isolated from cell lines using TriReagent, following the in- cytokine-induced up-regulation of Fas on target cells. struction of the manufacturer (T9424; Sigma-Aldrich, St. Louis, MO). Af- ter treatment with 2 ␮l(2␮g) DNase (Roche Molecular Biochemicals, Ag-mediated release of soluble mediators induces Fas Mannheim, Germany), mRNA was incubated with 1 ␮l oligo(dT) 12Ð18 expression primer (500 ng; Pharmacia, Freiburg, Germany) and Omniscript RT (4 U; Qiagen, Hilden, Germany). RT-PCR was done as described by Qiagen. Double-chamber experiments were performed to investigate Aliquots of 50Ð100 ng cDNA were used as template for PCR amplification whether soluble mediators were responsible for the up-regulation of mouse Fas using the primer pairs, as described (17, 23). PCR products Ϫ Ϫ of Fas on Fasneg L929 targets. Splenocytes from perfϫgzmAϫB / were amplified with 35 cycles, separated by 1.5% agarose gel electrophore- b sis, and visualized by ethidium bromide staining. Quantitation of PCR mice (H-2 )deficient in exocytosis-mediated cytotoxicity were cocul- products was done using the NIH Image software (version 1.62; freeware tured in vitro with B10.HTG (KdDb) stimulator cells and tested for from National Institutes of Health download page) by analyzing the density their ability to lyse Fasneg L1210 targets after 6- and 20-h assay time Downloaded from of the scanned bands. (Fig. 1A). As shown previously (17), lysis of Fasneg L1210 targets was Generation of cytotoxic T cells negligible at 6 h, but became highly significant at 20 h of incubation. ϫ 7 The same effectors and targets were used in the double-chamber ex- For the generation of alloreactive Tc cells, 8 10 responder splenocytes neg were cocultured with 4 ϫ 107 irradiated (2000 rad) allogeneic stimulator periment (Fig. 1B), with Fas L929 indicator cells in the bottom cells for 5Ð6 days in 40 ml EMEM, 10% FCS, plus 10Ϫ5 M 2-ME. chamber. Induction of Fas expression on L929 cells was analyzed by FACS using FITC anti-Fas mAb (Jo-2). Background fluorescence of http://www.jimmunol.org/ 51Cr release cytotoxicity assay L929 cells is indicated by the left two bars (0:1, E:T), and refers to The methods used for 51Cr labeling of target cell lines have been described L929 cultivated with either no cells (filled) or only L1210 target cells (24). Duration of the assays varied from 4 to 22 h, as indicated in Results. (hatched) in the upper chamber. A substantial increase in Fas expres- Percentage of specific lysis was calculated by the formula: percentage of specific lysis ϭ ((experimental release Ϫ medium release)/(maximum re- sion on L929 cells was observed when the upper chamber contained lease Ϫ medium release)) ϫ 100. Data given are the means of triplicate both effector and target cells (hatched bars), but not effector cells alone determinations. SEM values were always Ͻ5%. (filled bars), with similar levels obtained at E:T ratios of 1:1 and 3:1, and a slight decrease at 9:1 (target cell numbers were held constant). [3H]DNA release assay This suggests that Ag was not limiting at conditions used, and at high by guest on September 27, 2021 To assay DNA fragmentation, target cells (2 ϫ 105/ml) were prelabeled effector numbers the putative mediator(s) may have also been utilized 3 3 with 5 mCi/ml [ H]thymidine (thymidine, methyl[ H]; aqueous solution; 1 by the Tc cell population. mCi/ml; DuPont NEN, Bad Homburg, Germany) in complete EMEM over- night, washed, and used as targets in cytotoxicity assays. Effector cells ␥ neg were mixed with 1Ð2 ϫ 104 labeled target cells in triplicates at the indi- rIFN- induces Fas expression on Fas L929 targets cated E:T ratio in 200 ␮l EMEM supplemented with 2 mg/ml BSA. In IFN-␥ and TNF-␣ are two dominant cytokines known to be re- some experiments, mAb to Fas (Jo-2) was added to cell cultures before leased from Tc cells following activation (1, 2). We have argued incubation. After indicated time periods, cells were lysed with 25 ␮l TTE, except maximum release (2% T-X-100/80 mM Tris-HCl, pH 8.0, 5 M EDTA, pH 8.0), for 10 min at 37¡C. After centrifugation (1200 rpm; 10 min), 25 ␮l supernatant was harvested into a solid scintillator plate (LumPlate; Packard, Dreieich, Germany), dried, and counted with Top- Count (Packard). For maximum release, 25 ␮l EMEM was added to the wells, and 25 ␮l resuspended cell suspension was used. Percentage of specific lysis was calculated by the formula: percentage of specific lysis ϭ ((experimental release Ϫ medium release)/(maximum release Ϫ medium release)] ϫ 100. Data given are the means of triplicate determinations. SEM values were always Ͻ5%. Inhibition of cytotoxicity Anti-IFN-␥ mAb (AN18) (25) or control IgG (hamster IgG, rat IgG) was neg added at the indicated concentrations to cultures containing Tc cells and FIGURE 1. A and B, Induction of Fas on Fas bystander cells by sol- target cells for the duration of assay. uble mediators released by Tc cells. A, 51Cr release from L1210 targets. Splenocytes from perfϫgzmAϫBϪ/Ϫ mice were cocultured with HTG Double-chamber experiments stimulator cells in vitro. Effector cells were tested for lysis of L1210 target Experiments were performed in six-well Transwell (Costar catalogue cells. Cytotoxic assay time was 6 and 20 h. Each point constitutes the mean 3412) tissue culture plates. A total of 1 ϫ 106 Fasneg L929 indicator cells of percentage of specific 51Cr release of three separate wells. Spontaneous was cultured in the bottom chamber separated by a 0.4-␮m-pore-size poly- release was always less than 20%. B, Cell surface expression of Fas on carb membrane. Upper chamber contained 1 ϫ 106 L1210 target cells and L929 cells after culture in presence of Tc effectors and targets separated by effector alloreactive Tc cells, as indicated. After 20-h assay time, L929 a membrane. A total of 1 ϫ 106 L929 cells was cultured in the bottom of cells were trypsinized and Fas expression was analyzed by FACS. Costar Transwell cell culture chamber. The upper chambers contained no (left histograms)or1,3,and9ϫ 106 Tc effector cells assayed for lytic Results activity, as shown in A, together with 1 ϫ 106 specific L1210 target cells Two possibilities as to the mechanism by which alloreactive Tc (shaded histograms), or left without target cells (filled histograms). After neg cells induce Fas on Fas target cells were considered: first, direct 18-h culture, L929 cells were trypsinized labeled with FITC anti-Fas (Jo-2) TCR/MHC class I signaling; second, cytokine-mediated up-regu- mAb, and fluorescence was determined by FACScan. The Journal of Immunology 147 previously that it is unlikely that TNF-␣ is involved in Fas up- Alloreactive Tc cells from IFN-␥-deficient mice do not up- regulation, as conditions optimal for determining TNF activity regulate Fas on Fasneg target cells (treatment of effector cells with actinomycin D) actually inhibited To test whether IFN-␥ is the sole mediator responsible for the Fas expression on target cells (17, 26). We thus investigated up-regulation of Fas on Fasneg targets, we tested whether allo- whether L929 cells, used in our study, can be induced to express reactive Tc cells from IFN-␥-deficient mice facilitate Fas ex- Fas upon treatment with rIFN-␥ in vitro, as has been shown pre- pression in a double-chamber experiment. Splenocytes from viously (27). L929 cells were incubated with rIFN-␥ for 4, 6, and Ϫ Ϫ Ϫ Ϫ B6, perf / , and IFN-␥ / mice were stimulated in in vitro 22 h, and were analyzed for cell surface Fas expression by FACS, culture with HTG stimulator splenocytes. Kd-reactive effectors and the presence of Fas-specific transcripts by RT-PCR (Fig. 2, A were tested for lysis of L1210 and L1210.Fas target cells after and B). Cell surface Fas expression was evident at the first (4-h) 6- and 20-h assay times (Fig. 4A). Effectors from perfϪ/Ϫ mice time point measured, declined slightly by 6 h, but stayed elevated lysed L1210 targets only marginally at 6 h, but significantly at till at least 22 h (Fig. 2A). Low levels of Fas message were present in mock-treated cells, increased at 4 h after rIFN-␥ treatment to 20-h assay times, as expected. Lysis of L1210.Fas targets was ␥Ϫ/Ϫ double the intensity, and decreased to 1.5 times the intensity as above that of L1210 targets with all effectors. IFN- -de- compared with untreated control by 22 h (Fig. 2B). The low levels rived effectors lysed both target cells slightly more efficiently of Fas message observed in mock-treated L929 target cells suggest than B6-derived effectors, an observation made previously (22). that Fas cell surface expression is also regulated at the translational The same effector cells used in the cytotoxic assay were used in neg level and may facilitate a faster response after an appropriate a double-chamber experiment. Indicator Fas L929 cells were signal. placed in the bottom chamber and assayed for Fas cell surface Downloaded from expression using anti-Fas mAb (Jo2) after a 20-h incubation (Fig. 4B). The presence of B6 and IFN-␥Ϫ/Ϫ effectors, in the 51 Inhibition of DNA fragmentation and Cr release by Tc cells absence of Ag, resulted only in a slight increase of fluorescence ␥ deficient in exocytosis-mediated cytotoxicity using anti-IFN- Ab above the control (dotted line) and was similar for both cell H-2k-reactive Tc cells from perfϫgzmAϫBϪ/Ϫ mice were tested populations. When effectors were cocultured with L1210 target 51 3 neg neg for their ability to induce Cr and [ H]DNA release on Fas cells, indicator Fas L929 cells had significantly increased http://www.jimmunol.org/ Ϫ Ϫ L929 targets in the presence of anti-IFN-␥ and control Abs (Fig. fluorescence from wells with B6 and perf / effectors, but not 3). T cell-mediated lysis and apoptosis of targets were partially with IFN-␥Ϫ/Ϫ effectors. This indicates that IFN-␥ is the sole inhibited, in a dose-dependent manner, by anti-IFN-␥ mAb, com- mediator released by Tc cells, which induces the expression of pared with isotype control Abs over a 6-h assay period. Fas on Fasneg bystander cells. by guest on September 27, 2021

FIGURE 2. A and B, IFN-␥-induced Fas expres- sion in L929 cells. A, Cell surface expression of Fas on L929 cells. L929 cells were incubated without or with rIFN-␥ for the indicated time and analyzed for surface staining by FACS analysis, as described in Materials and Methods. B, Expression of Fas-spe- cific transcripts. Isolated mRNA from L929 cells in- cubated without or with rIFN-␥ for the indicated time or from control cell lines, L1210.3 and L1210. Fas were analyzed by RT-PCR amplification using the mouse Fas-specific primer pairs, as described in Materials and Methods. 148 Tc CELLS UP-REGULATE Fas ON TARGETS BY SECRETED IFN-␥ Downloaded from http://www.jimmunol.org/ FIGURE 3. Effect of anti-IFN-␥ mAb on cytolysis and nucleolysis of L929 cells by anti-H-2k alloreactive Tc cells from perfϫgzmAϫBϪ/Ϫ mice. Anti-AKR/N (H-2k) alloreactive Tc cells from perfϫgzmAϫBϪ/Ϫ mice were incubated with L929 cells for6hinthepresence of anti-IFN-␥ FIGURE 4. A and B, Induction of Fas on Fasneg bystander cells by Tc mAb (10, 100, 300 ␮g/ml; AN-18) or similar amounts of control rat IgG. cells from B6 wild-type, perfϪ/Ϫ, and IFN-␥Ϫ/Ϫ mice. A, 51Cr release from Each point constitutes the mean percentage of specific 51Cr or [3H]DNA L1210 targets. Splenocytes from B6, perfϪ/Ϫ, and IFN-␥Ϫ/Ϫ mice were release of three separate wells. Spontaneous release was always Ͻ20%. cocultured with HTG stimulator cells in vitro. Effector cells were tested for SEM values were always Ͻ5%. lysis of L1210 (filled symbols) and L1210.Fas (open symbols) target cells.

Cytotoxic assay time was 6 and 20 h. Each point constitutes the mean of by guest on September 27, 2021 percentage of specific 51Cr release of three separate wells. Spontaneous Role of IFN-␥-induced Fas expression on bystander killing release was always less than 20%. B, Cell surface expression of Fas on L929 cells after culture in presence of Tc effectors and targets separated by The availability of Tc cells lacking in IFN-␥ gene and thus unable a membrane. A total of 1 ϫ 106 L929 cells was cultured in the bottom of to induce Fas on Fasneg L929 cells enabled us to investigate Costar Transwell cell culture chamber. The upper chambers contained no whether up-regulation of Fas on bystander cells leads to their lysis (dotted line) or 1, 3, and 9 ϫ 106 Tc effector cells assayed for lytic activity, by Tc cells. H-2Kd alloreactive Tc cells from B6, perfϪ/Ϫ, and as shown in A, together with 1 ϫ 106 specific L1210 target cells (open IFN-␥Ϫ/Ϫ mice were tested for lysis of target cell mixtures of symbols), or left without target cells (filled symbols). After 18-h culture, equal proportions of L1210 (H-2d, the specific target) and L929 L929 cells were trypsinized and labeled with FITC anti-Fas (Jo-2) mAb, (H-2k, bystanders) with only one of the cell type labeled with 51Cr. and fluorescence was determined by FACScan. Fig. 5A shows 51Cr release from L1210 targets at three time points at an E:T of 0.7:1. Effector cells from B6 and IFN-␥Ϫ/Ϫ mice lysed L1210 targets to a similar extent at all time points tested, whereas diated up-regulation of Fas on target cells. In particular, the find- Ϫ Ϫ perf / effectors did not lyse L1210 targets at this low E:T at the ings that treatment of L929neg targets with rIFN-␥ up-regulates Fas early time points and only marginally after 20-h assay time. Lysis and the lack of induction of Fas expression on L929neg cells, when of L929 cells was low and insignificant up to the 8-h point. At 20-h Tc effector cells from IFN-␥ Ϫ/Ϫ mice were used, underpin this assay time, however, significant cross-reactivity by Kd-reactive Tc conclusion. The discovery of this phenomenon was made possible cells on H-2k targets can be observed, predominantly with effectors by the fortuitous use of Tc effector cells deficient in exocytosis- Ϫ Ϫ from B6 and IFN-␥ / mice (Fig. 5B). However, most impor- mediated cytotoxicity, in cytotoxic assays over extended assay pe- tantly, effectors from B6 mice did not lyse L929 target cells, which riods (17), and reveals another possible facet of Tc cell-mediated by this time express Fas, to a greater extent than those cultured apoptosis. Ϫ Ϫ with effectors from IFN-␥ / mice. The latter effector population The evidence that a secretable mediator of Tc cells, rather than was actually more cross-reactive than B6 effectors. This clearly direct cell contact-mediated signaling, is responsible for induced indicates that Tc cell-mediated up-regulation of Fas on bystander Fas expression, thus able to exert its biological effect distal to the cells does not render them more susceptible to lysis by irrelevant effector cell, may have additional biological implications. We (third party) effectors, even in the presence of functioning FasL/ speculated previously (17) that the induction of Fas expression on Fas and exocytosis pathways. target cells may constitute a back-up cytolytic mechanism for granule exocytosis-mediated cytotoxicity in the event of a defec- Discussion tive perf/gzm machinery (this is mimicked in the perfϪ/Ϫ mouse) The data presented in this study provide clear evidence that IFN-␥ or exhaustion in effector molecules of the exocytosis-dependent is the principal mediator responsible for the Tc effector cell-me- killing pathway during an extended Tc cell response. Furthermore, The Journal of Immunology 149

the relevant MHC class I/peptide complex or allogeneic MHC class I (17). However, Fas-mediated cell death in the absence of cognate Ag has been reported to be operative in peripheral T cell deletion. The mechanism by which this is controlled is not clear (36), but may be dependent on cell type (37). In addition, by- stander killing mediated via the Fas pathway has been reported by other groups under conditions of simultaneous recognition of cog- nate Ag on one APC and Fas on the bystander cell (38, 39). How- ever, in these experiments, lysis was only seen in cells expressing high Fas levels from the beginning of the cytotoxic assay. IFN-␥-mediated up-regulation of Fas may also play a role in processes of feedback regulation of an immune response by acting on APCs, including dendritic cells (DC), rendering them more sen- sitive to FasL/Fas-mediated apoptosis by Tc cells at the early FIGURE 5. A and B,A51Cr release of L1210-specific and L929 by- Ϫ Ϫ Ϫ Ϫ stages of the Tc cell response when cytolytic granule formation has stander target cells. Splenocytes from B6, perf / , and IFN-␥ / mice were cocultured with HTG stimulator cells in vitro. Effector cells were not yet matured. However, reports that DC cells are refractory to tested for lysis of L1210 and L929 target cell mixtures after 4-, 8-, and 20-h Fas-mediated apoptosis (37) and evidence that the perf rather than assay time. A, L1210 were labeled with 51Cr; L929 were left unlabeled. B, the Fas pathway is responsible for elimination of Ag-presenting L929 were labeled with 51Cr; L1210 were left unlabeled. Each point con- DC (40) suggest otherwise. Downloaded from stitutes the mean of percentage of specific lysis at E:T ratio of 0.7:1 derived Finally, Fas up-regulation by IFN-␥ may only concern regula- from regression analysis from a 4-fold E:T titration. tion of Tc cells themselves by self-regulating clonal expansion. The evidence of greatly reduced apoptosis and lymphoproliferative disease in the Fas mutant mouse lpr/lpr (36), an elevated Tc cell the option of Tc cells to switch from the perf/gzm pathway to the response in IFN-␥Ϫ/Ϫ mice (Ref. 22 and our own unpublished

FasL/Fas pathway may facilitate survival from infections with results), and the essential requirement for IFN-␥ in activation-in- http://www.jimmunol.org/ pathogens that have evolved means to evade the granule exocyto- duced cell death of T effector cells in vitro (unpublished results) sis-mediated mechanisms of apoptosis and cell death. One such are in agreement with this hypothesis. evasion strategy has recently been described for adenoviruses, in which the adenovirus late assembly protein, L4-100K, inhibits References gzmB-mediated cell death (28). This would indicate that one of the 1. Boehm, U., T. Klamp, M. Groot, and J. C. Howard. 1997. Cellular responses to cytolytic mechanisms, exocytosis-mediated cell death or Fas-me- interferon-␥. Annu. Rev. Immunol. 15:749. 2. Vassalli, P. 1992. The pathophysiology of tumor necrosis factor. Annu. Rev. diated cell death, is redundant. 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