HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Müllbacher, A. Articles by Simon, M. M. Search for Related Content PubMed PubMed Citation Articles by Müllbacher, A. Articles by Simon, M. M. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH

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

Arno Müllbacher^1,*, Mario Lobigs^*, Ron Tha Hla^*, Thao Tran, Thomas Stehle and Markus M. Simon

^* Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia; and Max-Planck-Institut für Immunbiologie, Stübeweg, Freiburg, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 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.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD8⁺ effector T cells exert their biological activity via two quite distinct means upon specific encounter with target cells. One is the synthesis and release of cytokines such as IFN- (1) and TNF- (2), which may act proximal and distal to the effector cell. The other is direct cytolysis and apoptosis of the bound target cell. These two distinct effector mechanisms are not necessarily executed by the same CD8⁺ T cell. Evidence for such is the vastly different numbers of Ag-specific CD8⁺ T cells identified as IFN- producers vs estimates of cytolytic T (Tc)² cells using limiting dilution techniques (3), and our recent finding that in response to flavivirus infection, Tc cells with lytic activity are not always producers of IFN- (4).

Apoptosis and lysis of target cells by cytolytic lymphocytes (NK and Tc cells) can be executed via at least two distinct pathways: one, the exocytosis pathway mediated by perforin (perf) and granzymes (gzm); the other, by the Fas pathway involving the Fas ligand (FasL) on the effector cell engaging the Fas receptor (Fas or CD95) on the target cell (5, 6, 7, 8, 9, 10, 11, 12). It is generally believed that the exocytosis pathway is primarily involved in the elimination and/or control of intracellular pathogens such as viruses. The Fas pathway of cytotoxicity, in contrast, was thought to be in essence immunoregulatory (11, 13, 14). However, some recent studies (15), including our own on flavivirus-induced cytotoxicity (16), suggest that in certain virus infections both pathways are operative.

We have shown previously (17) that delayed target cell lysis and apoptosis by alloreactive cytolytic T cells from perf-deficient (perf^-/-) mice proceed via the Fas pathway. This conclusion was reached for the following reasons: 1) Fas expression, on originally Fas-negative (Fas^neg) targets, after incubation with Tc cells from perf^-/- mice, was increased in a time-dependent manner; 2) target cells had to be biosynthetically active, i.e., RNA and protein synthesis was required as well as protein transport for lysis to occur; 3) Abs to Fas or soluble Fas-Fc inhibited cytolysis and nucleolysis; 4) poxvirus-encoded serpins, in particular SPI-2, a strong inhibitor of the Fas pathway (18, 19, 20), completely inhibited lysis of target cells by perf^-/- Tc cells; 5) brefeldin A treatment of Tc cells severely reduced lysis, which interferes with polypeptide transport, but only marginally with concanamycin A, an inhibitor of exocytosis (21); 6) target cells derived from the Fas-defective mutant mouse (lpr) were refractory to lysis; and finally 7) Tc effector cells from perf^-/- x gld mice, defective in both cytolytic effector pathways, did not exhibit this lytic phenotype on Fas^neg target cells.

In this study, we present evidence as to the mechanism by which Tc cells induce Fas expression on Fas^neg target cells, investigate its consequence on bystander killing, and speculate on the significance of this process in recovery from viral infections.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

C57BL/6 (K^bD^b) (B6), AKR/N (K^kD^k) (AKR), B10.HTG (K^dD^b) (HTG), the perf^-/- mutant (8), the triple knockout mouse, deficient in perf plus gzm A and B (perfxgzmAxB^-/-) (17), and the IFN--deficient mouse (IFN-^-/-) (22) were bred under pathogen-free conditions at the Animal Breeding Facility of the John Curtin School of Medical Research or the animal facilities of the Max-Planck-Institute for Immunbiology.

Cell lines

The mouse cell lines L929 (H-2^k), L1210 (H-2^d), and L1210.Fas (transfected with mouse Fas; kindly provided by P. Golstein, Marseilles, France) were grown in Eagle’s MEM (EMEM) supplemented with 10% FCS.

FACS analysis

Cells were stained for Fas expression using the FITC-conjugated mAb specific for mouse Fas (Jo-2; PharMingen, Hamburg, Germany). Cells were examined with a FACScan flow cytometer (BD Biosciences, San Jose, CA).

Treatment of L929 cells with rIFN-

L929 were plated in 24-well Costar (Cambridge, MA) plates and treated with 300 U rIFN- (Genzyme, Cambridge, MA; MG-IFN; 200 U/µl) for 4, 6, and 22 h. Cells were trypsinized and aliquots were stained for cell surface Fas expression and Fas transcript quantitation by PCR.

Quantitation of Fas transcripts

mRNA was isolated from cell lines using TriReagent, following the instruction of the manufacturer (T9424; Sigma-Aldrich, St. Louis, MO). After treatment with 2 µl (2 µg) DNase (Roche Molecular Biochemicals, Mannheim, Germany), mRNA was incubated with 1 µl oligo(dT)_12–18 primer (500 ng; Pharmacia, Freiburg, Germany) and Omniscript RT (4 U; Qiagen, Hilden, Germany). RT-PCR was done as described by Qiagen. Aliquots of 50–100 ng cDNA were used as template for PCR amplification of mouse Fas using the primer pairs, as described (17, 23). PCR products were amplified with 35 cycles, separated by 1.5% agarose gel electrophoresis, and visualized by ethidium bromide staining. Quantitation of PCR products was done using the NIH Image software (version 1.62; freeware from National Institutes of Health download page) by analyzing the density of the scanned bands.

Generation of cytotoxic T cells

For the generation of alloreactive Tc cells, 8 x 10⁷ responder splenocytes were cocultured with 4 x 10⁷ irradiated (2000 rad) allogeneic stimulator cells for 5–6 days in 40 ml EMEM, 10% FCS, plus 10^-5 M 2-ME.

⁵¹Cr release cytotoxicity assay

The methods used for ⁵¹Cr labeling of target cell lines have been described (24). Duration of the assays varied from 4 to 22 h, as indicated in Results. Percentage of specific lysis was calculated by the formula: percentage of specific lysis = ((experimental release - medium release)/(maximum release - medium release)) x 100. Data given are the means of triplicate determinations. SEM values were always <5%.

[³H]DNA release assay

To assay DNA fragmentation, target cells (2 x 10⁵/ml) were prelabeled with 5 mCi/ml [³H]thymidine (thymidine, methyl[³H]; aqueous solution; 1 mCi/ml; DuPont NEN, Bad Homburg, Germany) in complete EMEM overnight, washed, and used as targets in cytotoxicity assays. Effector cells were mixed with 1–2 x 10⁴ labeled target cells in triplicates at the indicated E:T ratio in 200 µl EMEM supplemented with 2 mg/ml BSA. In some experiments, mAb to Fas (Jo-2) was added to cell cultures before incubation. After indicated time periods, cells were lysed with 25 µl 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 µl supernatant was harvested into a solid scintillator plate (LumPlate; Packard, Dreieich, Germany), dried, and counted with TopCount (Packard). For maximum release, 25 µl EMEM was added to the wells, and 25 µl resuspended cell suspension was used. Percentage of specific lysis was calculated by the formula: percentage of specific lysis = ((experimental release - medium release)/(maximum release - medium release)] x 100. Data given are the means of triplicate determinations. SEM values were always <5%.

Inhibition of cytotoxicity

Anti-IFN- mAb (AN18) (25) or control IgG (hamster IgG, rat IgG) was added at the indicated concentrations to cultures containing Tc cells and target cells for the duration of assay.

Double-chamber experiments

Experiments were performed in six-well Transwell (Costar catalogue 3412) tissue culture plates. A total of 1 x 10⁶ Fas^neg L929 indicator cells was cultured in the bottom chamber separated by a 0.4-µm-pore-size polycarb membrane. Upper chamber contained 1 x 10⁶ L1210 target cells and effector alloreactive Tc cells, as indicated. After 20-h assay time, L929 cells were trypsinized and Fas expression was analyzed by FACS.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Two possibilities as to the mechanism by which alloreactive Tc cells induce Fas on Fas^neg target cells were considered: first, direct TCR/MHC class I signaling; second, cytokine-mediated up-regulation. Initially, we explored whether signaling by the Tc cell via MHC class I on the target cell was responsible for inducing Fas expression on Fas^neg targets. We failed to mimic Fas up-regulation on Fas^neg L929 (H-2^k) targets by a panel of anti-K^k or anti-D^k Abs. In addition, Fas^neg L929 target cells transfected with MHC class I K^d cDNA, lacking the cytoplasmic domain, still induced Fas after recognition by K^d-reactive Tc cells (data not shown). Thus, it was unlikely that Fas expression was induced by signaling via MHC class I directly. Therefore, we considered the second option, i.e., cytokine-induced up-regulation of Fas on target cells.

Ag-mediated release of soluble mediators induces Fas expression

Double-chamber experiments were performed to investigate whether soluble mediators were responsible for the up-regulation of Fas on Fas^neg L929 targets. Splenocytes from perfxgzmAxB^-/- mice (H-2^b) deficient in exocytosis-mediated cytotoxicity were cocultured in vitro with B10.HTG (K^dD^b) stimulator cells and tested for their ability to lyse Fas^neg L1210 targets after 6- and 20-h assay time (Fig. 1A). As shown previously (17), lysis of Fas^neg L1210 targets was negligible at 6 h, but became highly significant at 20 h of incubation. The same effectors and targets were used in the double-chamber experiment (Fig. 1B), with Fas^neg L929 indicator cells in the bottom chamber. Induction of Fas expression on L929 cells was analyzed by FACS using FITC anti-Fas mAb (Jo-2). Background fluorescence of L929 cells is indicated by the left two bars (0:1, E:T), and refers to L929 cultivated with either no cells (filled) or only L1210 target cells (hatched) in the upper chamber. A substantial increase in Fas expression on L929 cells was observed when the upper chamber contained both effector and target cells (hatched bars), but not effector cells alone (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). This suggests that Ag was not limiting at conditions used, and at high effector numbers the putative mediator(s) may have also been utilized by the Tc cell population.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. A and B, Induction of Fas on Fasneg bystander cells by soluble mediators released by Tc cells. A, 51Cr release from L1210 targets. Splenocytes from perfxgzmAxB-/- mice were cocultured with HTG stimulator cells in vitro. Effector cells were tested for lysis of L1210 target cells. Cytotoxic assay time was 6 and 20 h. Each point constitutes the mean of percentage of specific 51Cr release of three separate wells. Spontaneous 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 a membrane. A total of 1 x 106 L929 cells was cultured in the bottom of Costar Transwell cell culture chamber. The upper chambers contained no (left histograms) or 1, 3, and 9 x 106 Tc effector cells assayed for lytic activity, as shown in A, together with 1 x 106 specific L1210 target cells (shaded histograms), or left without target cells (filled histograms). After 18-h culture, L929 cells were trypsinized labeled with FITC anti-Fas (Jo-2) mAb, and fluorescence was determined by FACScan.

rIFN- induces Fas expression on Fas^neg L929 targets

IFN- and TNF- are two dominant cytokines known to be released from Tc cells following activation (1, 2). We have argued previously that it is unlikely that TNF- is involved in Fas up-regulation, as conditions optimal for determining TNF activity (treatment of effector cells with actinomycin D) actually inhibited Fas expression on target cells (17, 26). We thus investigated whether L929 cells, used in our study, can be induced to express Fas upon treatment with rIFN- in vitro, as has been shown previously (27). L929 cells were incubated with rIFN- for 4, 6, and 22 h, and were analyzed for cell surface Fas expression by FACS, and the presence of Fas-specific transcripts by RT-PCR (Fig. 2, A and B). Cell surface Fas expression was evident at the first (4-h) time point measured, declined slightly by 6 h, but stayed elevated 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 double the intensity, and decreased to 1.5 times the intensity as compared with untreated control by 22 h (Fig. 2B). The low levels of Fas message observed in mock-treated L929 target cells suggest that Fas cell surface expression is also regulated at the translational level and may facilitate a faster response after an appropriate signal.

View larger version (47K): [in this window] [in a new window]   FIGURE 2. A and B, IFN--induced Fas expression 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-specific transcripts. Isolated mRNA from L929 cells incubated 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.

Inhibition of DNA fragmentation and ⁵¹Cr release by Tc cells deficient in exocytosis-mediated cytotoxicity using anti-IFN- Ab

H-2^k-reactive Tc cells from perfxgzmAxB^-/- mice were tested for their ability to induce ⁵¹Cr and [³H]DNA release on Fas^neg L929 targets in the presence of anti-IFN- and control Abs (Fig. 3). T cell-mediated lysis and apoptosis of targets were partially inhibited, in a dose-dependent manner, by anti-IFN- mAb, compared with isotype control Abs over a 6-h assay period.

View larger version (21K): [in this window] [in a new window]   FIGURE 3. Effect of anti-IFN- mAb on cytolysis and nucleolysis of L929 cells by anti-H-2k alloreactive Tc cells from perfxgzmAxB-/- mice. Anti-AKR/N (H-2k) alloreactive Tc cells from perfxgzmAxB-/- mice were incubated with L929 cells for 6 h in the presence of anti-IFN- mAb (10, 100, 300 µg/ml; AN-18) or similar amounts of control rat IgG. Each point constitutes the mean percentage of specific 51Cr or [3H]DNA release of three separate wells. Spontaneous release was always <20%. SEM values were always <5%.

Alloreactive Tc cells from IFN--deficient mice do not up-regulate Fas on Fas^neg target cells

To test whether IFN- is the sole mediator responsible for the up-regulation of Fas on Fas^neg targets, we tested whether alloreactive Tc cells from IFN--deficient mice facilitate Fas expression in a double-chamber experiment. Splenocytes from B6, perf^-/-, and IFN-^-/- mice were stimulated in in vitro culture with HTG stimulator splenocytes. K^d-reactive effectors were tested for lysis of L1210 and L1210.Fas target cells after 6- and 20-h assay times (Fig. 4A). Effectors from perf^-/- mice lysed L1210 targets only marginally at 6 h, but significantly at 20-h assay times, as expected. Lysis of L1210.Fas targets was above that of L1210 targets with all effectors. IFN-^-/--derived effectors lysed both target cells slightly more efficiently than B6-derived effectors, an observation made previously (22). The same effector cells used in the cytotoxic assay were used in a double-chamber experiment. Indicator Fas^neg L929 cells were placed in the bottom chamber and assayed for Fas cell surface expression using anti-Fas mAb (Jo2) after a 20-h incubation (Fig. 4B). The presence of B6 and IFN-^-/- effectors, in the absence of Ag, resulted only in a slight increase of fluorescence above the control (dotted line) and was similar for both cell populations. When effectors were cocultured with L1210 target cells, indicator Fas^neg L929 cells had significantly increased fluorescence from wells with B6 and perf^-/- effectors, but not with IFN-^-/- effectors. This indicates that IFN- is the sole mediator released by Tc cells, which induces the expression of Fas on Fas^neg bystander cells.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. A and B, Induction of Fas on Fasneg bystander cells by Tc cells from B6 wild-type, perf-/-, and IFN--/- mice. A, 51Cr release from L1210 targets. Splenocytes from B6, perf-/-, and IFN--/- mice were cocultured with HTG stimulator cells in vitro. Effector cells were tested for 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 percentage of specific 51Cr release of three separate wells. Spontaneous 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 a membrane. A total of 1 x 106 L929 cells was cultured in the bottom of Costar Transwell cell culture chamber. The upper chambers contained no (dotted line) or 1, 3, and 9 x 106 Tc effector cells assayed for lytic activity, as shown in A, together with 1 x 106 specific L1210 target cells (open symbols), or left without target cells (filled symbols). After 18-h culture, L929 cells were trypsinized and labeled with FITC anti-Fas (Jo-2) mAb, and fluorescence was determined by FACScan.

Role of IFN--induced Fas expression on bystander killing

The availability of Tc cells lacking in IFN- gene and thus unable to induce Fas on Fas^neg L929 cells enabled us to investigate whether up-regulation of Fas on bystander cells leads to their lysis by Tc cells. H-2K^d alloreactive Tc cells from B6, perf^-/-, and IFN-^-/- mice were tested for lysis of target cell mixtures of equal proportions of L1210 (H-2^d, the specific target) and L929 (H-2^k, bystanders) with only one of the cell type labeled with ⁵¹Cr. 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-^-/- mice lysed L1210 targets to a similar extent at all time points tested, whereas perf^-/- effectors did not lyse L1210 targets at this low E:T at the early time points and only marginally after 20-h assay time. Lysis of L929 cells was low and insignificant up to the 8-h point. At 20-h assay time, however, significant cross-reactivity by K^d-reactive Tc cells on H-2^k targets can be observed, predominantly with effectors from B6 and IFN-^-/- mice (Fig. 5B). However, most importantly, effectors from B6 mice did not lyse L929 target cells, which by this time express Fas, to a greater extent than those cultured with effectors from IFN-^-/- mice. The latter effector population was actually more cross-reactive than B6 effectors. This clearly indicates that Tc cell-mediated up-regulation of Fas on bystander cells does not render them more susceptible to lysis by irrelevant (third party) effectors, even in the presence of functioning FasL/Fas and exocytosis pathways.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. A and B, A 51Cr release of L1210-specific and L929 bystander target cells. Splenocytes from B6, perf-/-, and IFN--/- mice were cocultured with HTG stimulator cells in vitro. Effector cells were tested for lysis of L1210 and L929 target cell mixtures after 4-, 8-, and 20-h assay time. A, L1210 were labeled with 51Cr; L929 were left unlabeled. B, L929 were labeled with 51Cr; L1210 were left unlabeled. Each point constitutes the mean of percentage of specific lysis at E:T ratio of 0.7:1 derived from regression analysis from a 4-fold E:T titration.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The evidence that a secretable mediator of Tc cells, rather than direct cell contact-mediated signaling, is responsible for induced Fas expression, thus able to exert its biological effect distal to the effector cell, may have additional biological implications. We speculated previously (17) that the induction of Fas expression on target cells may constitute a back-up cytolytic mechanism for granule exocytosis-mediated cytotoxicity in the event of a defective perf/gzm machinery (this is mimicked in the perf^-/- mouse) or exhaustion in effector molecules of the exocytosis-dependent killing pathway during an extended Tc cell response. Furthermore, the option of Tc cells to switch from the perf/gzm pathway to the FasL/Fas pathway may facilitate survival from infections with pathogens that have evolved means to evade the granule exocytosis-mediated mechanisms of apoptosis and cell death. One such evasion strategy has recently been described for adenoviruses, in which the adenovirus late assembly protein, L4-100K, inhibits gzmB-mediated cell death (28). This would indicate that one of the cytolytic mechanisms, exocytosis-mediated cell death or Fas-mediated cell death, is redundant. Such complementary cytolytic mechanisms operating in the recovery from primary viral infections or immunopathology would obscure clear-cut phenotypes in granule exocytosis-mediated cytotoxicity-deficient mice. This is actually observed in most studies on viral infections using perf- or gzm-deficient mice (13, 15, 16, 29, 30, 31). The one prominent exception is Ectromelia (Ect), mouse pox (32, 33, 34). In this case, the observed absolute requirement for perf- and gzm-mediated mechanisms for recovery from Ect infection is most likely the consequence of the inhibition of the Fas-mediated pathway by Ect-encoded serpin SPI-2 (32, 35) and has implications for the use of poxviruses in bioterrorism (32).

IFN- release by Tc cells will induce Fas on bystander cells proximal and distal to the effector T cell. In the event that these cells express the cognate Ag, i.e., are infected with the relevant parasite, this should facilitate a more efficient control of the infection. The possibility that this may lead to killing of innocent (noninfected) bystander cells has been investigated by using Tc effector cells from IFN-^-/- mice. If innocent bystander lysis is occurring due to Fas expression, the following prediction should be fulfilled. Tc cells from B6 mice upon Ag encounter induce Fas on bystanders as a result of IFN- release in a time-dependent manner. Thus, at early time points, only Fas-independent (cross-reactive) lysis takes place. With increasing assay times and Fas expression, the rate of bystander killing is predicted to increase and should result in an increase in the magnitude and kinetics of killing when compared with Tc cells from IFN-^-/- mice in which no Fas expression takes place. Experiments shown in Fig. 5 fail to provide evidence that IFN--mediated up-regulation of Fas on target cells makes them increasingly susceptible to lysis by Tc cells without Ag specificity. In accordance with this observation are our findings that Fas-positive targets are not lysed by activated Tc cells expressing FasL in the absence of TCR ligation, i.e., expression of 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, bystander killing mediated via the Fas pathway has been reported by other groups under conditions of simultaneous recognition of cognate Ag on one APC and Fas on the bystander cell (38, 39). However, 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 sensitive to FasL/Fas-mediated apoptosis by Tc cells at the early stages of the Tc cell response when cytolytic granule formation has not yet matured. However, reports that DC cells are refractory to Fas-mediated apoptosis (37) and evidence that the perf rather than the Fas pathway is responsible for elimination of Ag-presenting DC (40) suggest otherwise.

Finally, Fas up-regulation by IFN- may only concern regulation 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 response in IFN-^-/- mice (Ref. 22 and our own unpublished results), and the essential requirement for IFN- in activation-induced cell death of T effector cells in vitro (unpublished results) are in agreement with this hypothesis.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Arno Müllbacher, Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia. E-mail address: arno.mullbacher{at}anu.edu.au

² Abbreviations used in this paper: Tc, cytolytic T; DC, dendritic cell; Ect, Ectromelia; EMEM, Eagle’s MEM; FasL, Fas ligand; gzm, granzyme; neg, negative; perf, perforin.

Received for publication February 28, 2002. Accepted for publication May 1, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Boehm, U., T. Klamp, M. Groot, J. C. Howard. 1997. Cellular responses to interferon-. Annu. Rev. Immunol. 15:749.[Medline]
2. Vassalli, P.. 1992. The pathophysiology of tumor necrosis factor. Annu. Rev. Immunol. 10:411.[Medline]
3. Lefkovits, I., H. Waldmann. 1984. Limiting dilution analysis of the cells of immune system. I. The clonal basis of the immune response. Immunol. Today 5:265.
4. Regner, M., M. Lobigs, R. V. Blanden, A. Müllbacher. 2001. Effector cytolytic function but not IFN- production in cytotoxic T cells triggered by virus-infected target cells in vitro. [Published erratum appears in 2001 Scand. J. Immunol. 54:640.]. Scand. J. Immunol. 54:366.[Medline]
5. Griffiths, G. M.. 1995. The cell biology of CTL killing. Curr. Opin. Immunol. 7:343.[Medline]
6. Henkart, P. A.. 1994. Lymphocyte-mediated cytotoxicity: two pathways and multiple effector molecules. Immunity 1:343.[Medline]
7. Heusel, J. W., R. L. Wesselschmidt, S. Shresta, J. H. Russell, T. J. Ley. 1994. Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells. Cell 76:977.[Medline]
8. Kägi, D., B. Ledermann, K. Burki, P. Seiler, B. Odermatt, K. J. Olsen, E. R. Podack, R. M. Zinkernagel, H. Hengartner. 1994. Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369:31.[Medline]
9. Kägi, D., F. Vignaux, B. E. A. Ledermann. 1994. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 265:528.[Abstract/Free Full Text]
10. Lowin, B., M. Hahne, C. Mattmann, J. Tschopp. 1994. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature 370:650.[Medline]
11. Rouvier, R., M.-F. Luciani, P. Golstein. 1993. Fas involvement in Ca²⁺-independent T-cell-mediated cytotoxicity. J. Exp. Med. 177:195.[Abstract/Free Full Text]
12. Simon, M. M., M. Hausmann, T. Tran, K. Ebnet, J. Tschopp, R. ThaHla, A. Müllbacher. 1997. In vitro and ex vivo-derived cytolytic leukocytes from granzyme AxB double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells. J. Exp. Med. 186:1781.[Abstract/Free Full Text]
13. Kägi, D., B. Ledermann, K. Bürki, R. M. Zinkernagel, H. Hengartner. 1995. Lymphocyte-mediated cytotoxicity in vitro and in vivo: mechanisms and significance. Immunol. Rev. 146:95.[Medline]
14. Nagata, S.. 1997. Apoptosis by death factor. Cell 88:355.[Medline]
15. Doherty, P. C., D. J. Topham, R. A. Tripp, R. D. Cardin, J. W. Brooks, P. G. Stevenson. 1997. Effector CD4⁺ and CD8⁺ T-cell mechanisms in the control of respiratory virus infections. Immunol. Rev. 159:105.[Medline]
16. Licon Luna, R. M., E. Lee, A. Müllbacher, R. V. Blanden, R. Langman, M. Lobigs. 2002. Lack of both Fas ligand and perforin protects from flavivirus-mediated encephalitis in mice. J. Virol. 76:3202.[Abstract/Free Full Text]
17. Simon, M. M., P. Waring, M. Lobigs, A. Nil, T. Tran, R. T. Hla, S. Chin, A. Müllbacher. 2000. Cytotoxic T cells specifically induce Fas on target cells, thereby facilitating exocytosis-independent induction of apoptosis. J. Immunol. 165:3663.[Abstract/Free Full Text]
18. Macen, J. L., R. S. Garner, P. Y. Musy, M. A. Brooks, P. C. Turner, R. W. Moyer, G. McFadden, R. C. Bleackley. 1996. Differential inhibition of Fas- and granule-mediated cytolysis pathways by the orthopoxvirus cytokine response modifier A/SPI-2 and SPI-1 protein. Proc. Natl. Acad. Sci. USA 93:9108.[Abstract/Free Full Text]
19. Müllbacher, A., R. Wallich, R. W. Moyer, M. M. Simon. 1999. Poxvirus encoded serpins do not prevent cytolytic T cell mediated recovery from primary infections. J. Immunol. 162:7315.[Abstract/Free Full Text]
20. Tewari, M., W. G. Telford, R. A. Miller, V. M. Dixit. 1995. CrmA, a poxvirus-encoded serpin, inhibits cytotoxic T-lymphocyte-mediated apoptosis. J. Biol. Chem. 270:22705.[Abstract/Free Full Text]
21. Kataoka, T., N. Shinohara, K. Takayama, S. Takaku, S. Yonehara Kondo, K. Nagai. 1996. Concanamycin A, a powerful tool for characterization and estimation of contribution of perforin- and Fas-based lytic pathways in cell-mediated cytotoxicity. J. Immunol. 156:3678.[Abstract]
22. Dalton, D. K., S. Pitts-Meek, S. Keshav, I. S. Figari, A. Bradley, T. A. Stewart. 1993. Multiple defects of immune cell function in mice with disrupted interferon- genes. Science 259:1739.[Abstract/Free Full Text]
23. Chu, J. L., J. Drappa, A. Parnassa, K. B. Elkon. 1993. The defect in Fas mRNA expression in MRL/lpr mice is associated with insertion of the retrotransposon, ETn. J. Exp. Med. 178:723.[Abstract/Free Full Text]
24. Müllbacher, A., A. Hill, R. Blanden, W. Cowden, N. King, R. Tha Hla. 1991. Alloreactive cytotoxic T cells recognize MHC class I antigen without peptide specificity. J. Immunol. 147:1765.[Abstract]
25. Simon, M. M., U. Hochgeschwender, U. Brugger, S. Landolfo. 1986. Monoclonal antibodies to interferon- inhibit interleukin 2-dependent induction of growth and maturation in lectin/antigen-reactive cytolytic T lymphocyte precursors. J. Immunol. 136:2755.[Abstract]
26. Hogan, M. M., S. N. Vogel. 1994. Measurement of tumor necrosis factor and . J. E. Coligan, and A. M. Kruisbeek, and D. H. Margulies, and E. M. Shevach, and W. Strober, eds. Current Protocols in Immunology, Vol. 1 6.10.1.-6.10.5. Wiley, New York.
27. Watanabe-Fukunaga, R., C. I. Brannan, N. Itoh, S. Yonehara, N. G. Copeland, N. A. Jenkins, S. Nagata. 1992. The cDNA structure, expression, and chromosomal assignment of the mouse Fas antigen. J. Immunol. 148:1274.[Abstract]
28. Andrade, F., H. G. Bull, N. A. Thornberry, G. W. Ketner, L. A. Casciola-Rosen, A. Rosen. 2001. Adenovirus L4-100K assembly protein is a granzyme B substrate that potently inhibits granzyme B-mediated cell death. Immunity 14:751.[Medline]
29. Balkow, S., A. Kersten, T. T. Tran, T. Stehle, P. Grosse, C. Museteanu, O. Utermohlen, H. Pircher, F. von Weizsacker, R. Wallich, et al 2001. Concerted action of the FasL/Fas and perforin/granzyme A and B pathways is mandatory for the development of early viral hepatitis but not for recovery from viral infection. J. Virol. 75:8781.[Abstract/Free Full Text]
30. Riera, L., M. Gariglio, G. Valente, A. Müllbacher, C. Museteanu, S. Landolfo, M. M. Simon. 2000. Murine cytomegalovirus replication in salivary glands is controlled by both perforin and granzymes during acute infection. Eur. J. Immunol. 30:1350.[Medline]
31. Topham, D. J., R. A. Tripp, P. C. Doherty. 1997. CD8⁺ T cells clear influenza virus by perforin or Fas-dependent processes. J. Immunol. 159:5197.[Abstract]
32. Müllbacher, A., M. Lobigs. 2001. Creation of killer poxvirus could have been predicted. J. Virol. 75:8353.[Free Full Text]
33. Müllbacher, A., R. Tha Hla, C. Museteanu, M. M. Simon. 1999. Perforin is essential for the control of ectromelia virus but not related poxviruses in mice. J. Virol. 73:1665.[Abstract/Free Full Text]
34. Müllbacher, A., P. Waring, R. Tha Hla, T. Tran, S. Chin, T. Stehle, C. Museteanu, M. M. Simon. 1999. Granzymes are the essential downstream effector molecules for the control of primary infections by cytolytic leukocytes. Proc. Natl. Acad. Sci. USA 96:13950.[Abstract/Free Full Text]
35. Simon, M. M., A. Müllbacher. 2000. Role of granzymes in target cell lysis and viral infections. M. V. Sitkovsky, and P. A. Henkart, eds. Cytotoxic Cells: Basic Mechanisms and Medical Applications 197.-211. Lippincott Williams & Wilkins, Philadelphia.
36. Mogil, J. S., P. Flodman, M. A. Spence, W. F. Sternberg, B. Kest, B. Sadowski, J. C. Liebeskind, J. K. Belknap. 1995. Oligogenic determination of morphine analgesic magnitude: a genetic analysis of selectively bred mouse lines. Behav. Genet. 25:397.[Medline]
37. Rescigno, M., V. Piguet, B. Valzasina, S. Lens, R. Zubler, L. French, V. Kindler, J. Tschopp, P. Ricciardi-Castagnoli. 2000. Fas engagement induces the maturation of dendritic cells (DCs), the release of interleukin (IL)-1, and the production of interferon in the absence of IL-12 during DC-T cell cognate interaction: a new role for Fas ligand in inflammatory responses. J. Exp. Med. 192:1661.[Abstract/Free Full Text]
38. Kojima, H., K. Eshima, H. Takayama, M. V. Sitkovsky. 1997. Leukocyte function-associated antigen-1-dependent lysis of Fas⁺ (CD95⁺/Apo-1⁺) innocent bystanders by antigen-specific CD8⁺ CTL. J. Immunol. 159:2728.[Abstract]
39. Thilenius, A. R., K. A. Sabelko-Downes, J. H. Russell. 1999. The role of the antigen-presenting cell in Fas-mediated direct and bystander killing: potential in vivo function of Fas in experimental allergic encephalomyelitis. J. Immunol. 162:643.[Abstract/Free Full Text]
40. Ludewig, B., W. V. Bonilla, T. Dumrese, B. Odermatt, R. M. Zinkernagel, H. Hengartner. 2001. Perforin-independent regulation of dendritic cell homeostasis by CD8⁺ T cells in vivo: implications for adaptive immunotherapy. Eur. J. Immunol. 31:1772.[Medline]

This article has been cited by other articles:

				S. Charni, J. I. Aguilo, J. Garaude, G. de Bettignies, C. Jacquet, R. A. Hipskind, D. Singer, A. Anel, and M. Villalba ERK5 Knockdown Generates Mouse Leukemia Cells with Low MHC Class I Levels That Activate NK Cells and Block Tumorigenesis J. Immunol., March 15, 2009; 182(6): 3398 - 3405. [Abstract] [Full Text] [PDF]

				J. S. Woodworth, Y. Wu, and S. M. Behar Mycobacterium tuberculosis-Specific CD8+ T Cells Require Perforin to Kill Target Cells and Provide Protection In Vivo J. Immunol., December 15, 2008; 181(12): 8595 - 8603. [Abstract] [Full Text] [PDF]

				B. Shrestha and M. S. Diamond Fas Ligand Interactions Contribute to CD8+ T-Cell-Mediated Control of West Nile Virus Infection in the Central Nervous System J. Virol., November 1, 2007; 81(21): 11749 - 11757. [Abstract] [Full Text] [PDF]

				Y. Wang, M. Lobigs, E. Lee, A. Koskinen, and A. Mullbacher CD8+ T cell-mediated immune responses in West Nile virus (Sarafend strain) encephalitis are independent of gamma interferon J. Gen. Virol., December 1, 2006; 87(12): 3599 - 3609. [Abstract] [Full Text] [PDF]

				J. K. Ahn, H. Chung, D.-s. Lee, Y. S. Yu, and H. G. Yu CD8brightCD56+ T Cells Are Cytotoxic Effectors in Patients with Active Behcet's Uveitis J. Immunol., November 1, 2005; 175(9): 6133 - 6142. [Abstract] [Full Text] [PDF]

				E. C. Morris, A. Tsallios, G. M. Bendle, S.-a. Xue, and H. J. Stauss A critical role of T cell antigen receptor-transduced MHC class I-restricted helper T cells in tumor protection PNAS, May 31, 2005; 102(22): 7934 - 7939. [Abstract] [Full Text] [PDF]

				U. Christen, R. Darwiche, H. E. Thomas, T. Wolfe, E. Rodrigo, A. Chervonsky, R. A. Flavell, and M. G. von Herrath Virally Induced Inflammation Triggers Fratricide of Fas-Ligand-Expressing {beta}-Cells Diabetes, March 1, 2004; 53(3): 591 - 596. [Abstract] [Full Text] [PDF]

				E. Roth and H. Pircher IFN-{gamma} Promotes Fas Ligand- and Perforin-Mediated Liver Cell Destruction by Cytotoxic CD8 T Cells J. Immunol., February 1, 2004; 172(3): 1588 - 1594. [Abstract] [Full Text] [PDF]

				P. Zamiri, Q. Zhang, and J. W. Streilein Vulnerability of Allogeneic Retinal Pigment Epithelium to Immune T-Cell-Mediated Damage In Vivo and In Vitro Invest. Ophthalmol. Vis. Sci., January 1, 2004; 45(1): 177 - 184. [Abstract] [Full Text] [PDF]

				B.-G. Xiao, X.-C. Wu, J.-S. Yang, L.-Y. Xu, X. Liu, Y.-M. Huang, B. Bjelke, and H. Link Therapeutic potential of IFN-{gamma}-modified dendritic cells in acute and chronic experimental allergic encephalomyelitis Int. Immunol., January 1, 2004; 16(1): 13 - 22. [Abstract] [Full Text] [PDF]

				T. Murakami, A. R. Cardones, S. E. Finkelstein, N. P. Restifo, B. A. Klaunberg, F. O. Nestle, S. S. Castillo, P. A. Dennis, and S. T. Hwang Immune Evasion by Murine Melanoma Mediated through CC Chemokine Receptor-10 J. Exp. Med., November 3, 2003; 198(9): 1337 - 1347. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Müllbacher, A. Articles by Simon, M. M. Search for Related Content PubMed PubMed Citation Articles by Müllbacher, A. Articles by Simon, M. M. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH