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IFN- Promotes Fas Ligand- and Perforin-Mediated Liver Cell Destruction by Cytotoxic CD8 T Cells¹

Institute for Medical Microbiology and Hygiene, Department of Immunology, University of Freiburg, Freiburg, Germany

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
To study liver cell damage by CTL, CD8 T cells from P14 TCR transgenic (tg) mice specific for the gp33 epitope of lymphocytic choriomeningitis virus with either deficiency in IFN- (P14.IFN-°), functional Fas ligand (P14.gld), or perforin (P14.PKO) were transferred into H8 tg mice ubiquitously expressing gp33 Ag. Treatment of H8 recipient mice with agonistic anti-CD40 Abs induced vigorous expansion of the transferred P14 T cells and led to liver cell destruction determined by increase of glutamate dehydrogenase serum levels and induction of caspase-3 in hepatocytes. Liver injury was mediated by the Fas/Fas ligand (FasL) pathway and by perforin, because P14.gld and P14.PKO T cells failed to induce increased glutamate dehydrogenase levels despite strong in vivo proliferation. In addition, H8 tg mice lacking Fas were resistant to the pathogenic effect of P14 T cells. Besides FasL and perforin, IFN- was also required for liver cell damage, because P14.IFN-° T cells adoptively transferred into H8 mice failed to induce disease. Moreover, Fas expression on hepatocytes from H8 recipient mice was increased after transfer of wild-type compared with P14.IFN-° T cells, and wild-type P14 T cells expressed higher levels of FasL than P14 T cells lacking IFN-. Thus, our data suggest that IFN- released by activated CD8 T cells upon Ag contact facilitates liver cell destruction.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is now well established that the activation state of APC determines whether interaction of T cells with Ag leads to tolerance induction or to proliferation and acquisition of effector cell function (1, 2, 3, 4, 5, 6). In an autoimmune setting, stimulation of self-reactive CD8 T cells by activated APC could result in CTL-mediated tissue destruction (7, 8). To study these processes in an in vivo model, we have established a transfer system with CD8 T cells from P14 TCR transgenic (tg)³ mice specific for the gp33 epitope of lymphocytic choriomeningitis virus (LCMV) and H8 tg recipient mice ubiquitously expressing gp33 (9). After adoptive transfer into H8 mice, P14 T cells were tolerized rapidly by peripheral deletion and induction of anergy. However, bacterial and viral infections interfered with tolerance induction and led to CTL-mediated immunopathology. Moreover, CD40 ligation of APC by agonistic mAb could mimic these inflammatory processes and induced vigorous expansion of adoptively transferred P14 T cells in H8 tg mice, which caused CTL-mediated liver cell destruction (10). CTL are thought to destroy their target cells primarily through granule exocytosis, which requires perforin to facilitate entry of granzymes into the cytosol (11, 12), and through the Fas ligand (FasL)/Fas pathway (13, 14, 15). The inflammatory cytokine IFN- represents an additional effector molecule released by activated CTL. In this study, we have used the P14/H8 transfer model described above to analyze the role of FasL, perforin, and IFN- in liver cell destruction by self-reactive CTL in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

C57BL/6 (B6) mice were obtained from our breeding colony and from Harlan Winkelmann (Borchen, Germany). P14 TCR-tg mice (line 318) specific for aa 33–41 (= gp33 epitope) of the LCMV glycoprotein (16) and H8 tg mice ubiquitously expressing the LCMV gp33 epitope as a transgene (9) have been described previously. P14 mice deficient in Fas, FasL, perforin, or IFN- were generated by breeding P14 mice with B6.Fas° (17) (M. Simon, MPI, Freiburg, Germany), B6.gld/gld (H. Eibel, University of Freiburg), B6.PKO (18) (M. Simon, MPI, Freiburg, Germany), or B6.IFN-° (19) (M. Kopf, ETH, Zürch, Switzerland) mice, respectively. Fas-deficient H8 tg mice were generated by breeding H8 tg mice with B6.Fas° mice (17). P14 mice used in this study had been backcrossed eight times to B6, B6.Fas°, and B6.PKO mice were originally made on a B6 background using B6 embryonal stem cells. B6.IFN-° mice, originally made on a mixed 126/B6 background, had been backcrossed six times to B6.P14.Fas°, and P14.gld mice were used at 8–10 wk of age. At this age, the mice had not yet developed lymphadenopathy; the frequency of double-negative T cells in lymph node and spleen was below 5%; and all P14 T cells exhibited a naive phenotype (CD62L^high, CD25^low, CD44^low). All other mice, female or male, were used at 8–16 wk of age. Mice were bred and kept in a conventional animal house facility.

Adoptive cell transfers and anti-CD40 Ab treatment

Spleen cells of TCR tg mice containing 10⁴, 10⁵, or 10⁶ P14 TCR⁺ (V2⁺/V8⁺) cells were injected (i.v.) into nonirradiated H8 or H8.Fas° mice. Anti-CD40 treatment was performed by i.p. injection of 100 µg of anti-CD40 mAb, clone FGK45 (20). Abs were given on the day of cell transfer and 2 days afterward.

Disease score

Illness of the mice was scored using the following scale: 0 = healthy; 1 = slightly ruffled fur; 2 = ruffled fur, but active; 3 = ruffled fur and inactive; 4 = ruffled fur, inactive, and hunched; 5 = moribund. Mice illness scores were monitored 1 wk after cell transfer.

Flow cytometry

Lymphocytes were resuspended in PBS containing 2% FCS and 0.1% NaN₃ at a concentration of 10⁶-10⁷ cells/ml, followed by incubation at 4°C for 20 min with 100 µl of appropriately diluted mAb. For PBL staining, 2 U/ml heparin was added to the staining buffer. The following mAbs were used: anti-CD8 (clone 53-6.7), anti-CD44 (clone IM7), anti-CD62L (clone MEL-14), anti-TCR V2 (clone B20.1), anti-TCR V8 (clone MR5-2), anti-Fas (clone Jo2), and anti-FasL (clone MFL3). FasL staining of hepatic lymphocytes was performed after 4-h stimulation with 10 µg/ml plate-bound anti-CD3 mAb (clone 17A2) and 20 µM metalloproteinase inhibitor TAPI-1 (Calbiochem-Merck, Darmstadt, Germany) to avoid the cleavage of FasL from the cell surface. All Abs were purchased from BD PharMingen (San Diego, CA). The mAbs were directly labeled with FITC, PE, or APC, or were biotinylated. For the latter, APC-streptavidin (BD PharMingen) was used as a secondary reagent for detection. Before analysis of PBL, RBC were lysed using FACS lysing solution (BD PharMingen). Cells were analyzed on a FACSort flow cytometer (BD Biosciences, Mountain View, CA) using CellQuestPro software.

Preparation of hepatocytes and hepatic lymphocytes

Hepatocytes were prepared, as described in detail elsewhere (21), with slight modifications. Briefly, mice were anesthetized and the peritoneal cavity was opened. The inferior vena cava was cannulated with a 0.45 x 25-mm needle (Sterican, Braun, Melsungen) in the area of the lower abdomen, and perfusion was started. After a few seconds, the portal vein was sectioned. The liver was perfused sequentially with 5 ml HBSS without Ca²⁺ and Mg²⁺ (Biochrom, Berlin, Germany), containing 1% w/v glucose and 0.5 mM EGTA, followed by 10 ml collagenase solution (HBSS, containing 1% w/v glucose, 5 mM CaCl₂, and 0.1 mg/ml liberase blendzyme 3 (Roche Diagnostics, Mannheim, Germany)). The liver was removed and filtered through a sterile 100-µm nylon cell strainer (BD Biosciences). The cells were washed twice by centrifugation at 50 x g for 2 min and resuspended in IMDM containing 10% FCS. Flow cytometry analysis of hepatocytes was performed, as described by Kaulek et al. (22). A detector value of E-01 was used, and gating was performed with a logarithmic amplification for forward scatter and a linear amplification for side scatter. With these parameters, all different cell populations were obtained in the same plot.

To isolate hepatic lymphocytes, liver was perfused with 10 ml PBS without Ca²⁺ and Mg²⁺, removed, and cut in small pieces. After digestion with PBS containing 0.1% collagenase, 0.01% hyaluronidase, and 0.002% DNase I (all from Sigma-Aldrich, Steinheim, Germany) for 30 min at 37°C, liver was filtered through a sterile 100-µm nylon cell strainer (BD Biosciences) and washed twice with IMDM, and lymphocytes were isolated by Ficoll gradient centrifugation (Ficoll-Paque^Plus; Amersham Biosciences, Uppsala, Sweden).

Immunohistochemistry

Spleen or liver sections (5–7 µm) were cut on a cryostat microtome, air dried, fixed in acetone, and blocked with TBS containing 5% mouse serum and the DAKO biotin blocking system (DAKO, Hamburg, Germany). Liver sections were treated additionally with 0.3% H₂O₂ in PBS to neutralize endogenous peroxidases. Anti-CD8 biotin and anti-B220 biotin (all BD PharMingen) were used as primary mAb, followed by streptavidin-conjugated alkaline phosphatase (StreptAB Complex/AP; DAKO) and alkaline phosphatase substrate kit I (Vector Laboratories, Burlingame, CA). For double staining, anti-CD4 FITC (BD PharMingen) was used, followed by rabbit anti-FITC (DAKO), peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), and peroxidase substrate kit 3-amino-9-ethylcarbazole (Vector Laboratories). Staining of apoptotic cells was performed using purified polyclonal rabbit anti-active caspase 3 Abs (BD PharMingen), followed by biotinylated goat anti-rabbit Igs (BD PharMingen), AB complex-HRP (DAKO), and 3-amino-9-ethylcarbazole peroxidase substrate kit (Vector Laboratories). Sections were counterstained with Mayer’s hemalum (Calbiochem-Merck).

Serum glutamate dehydrogenase activity

Blood (200 µl) taken from the tail vein was collected in serum separator tubes (MICROTAINER brand serum separator tube; BD Biosciences) and centrifuged for 20 min at 3300 x g, and sera were analyzed for glutamate dehydrogenase (GLDH U/L).

⁵¹Cr release assay

Cytolytic activity of ex vivo isolated spleen cells was tested in a standard 5-h ⁵¹Cr release assay. EL-4 cells coated with the gp33 peptide (KAVYNFATM) or the control adenovirus E1A 234–243 peptide (SGPSNTPPEI) at a concentration of 10^-6 M were used as target cells. The number of P14 T cells in the effector cell population was determined by flow cytometry using TCR V2- and V8-specific mAb.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Essential role of both IFN- and FasL in P14 T cell-mediated immunopathology

CD8 T cells (10⁵) from P14 TCR tg mice specific for aa 33–41 (= gp33 epitope) of the LCMV glycoprotein were adoptively transferred into anti-CD40-treated H8 tg mice, ubiquitously expressing the gp33 epitope. One week after T cell transfer, all H8 mice that received wild-type P14 T cells showed severe clinical symptoms, including hunched posture, cachexia, and ataxia (Fig. 1A). To determine the role of IFN-, FasL, and perforin in this model system, P14.IFN-° T cells deficient in IFN-, P14.gld T cells lacking functional FasL, and perforin-deficient P14.PKO cells were used. The results were strikingly clear-cut. In contrast to wild-type transfers, H8 recipient mice of P14.IFN-° or P14.gld T cells exhibited only mild symptoms (slightly ruffled fur), but no cachexia or ataxia. The disease score induced by P14.PKO T cells was reduced only slightly when compared with wild-type P14 T cells.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Essential role of IFN- and FasL in P14 T cell-mediated immunopathology. A, H8 recipient mice were injected with 105 P14 T cells from the indicated donor mice and were treated with anti-CD40 mAb, as described in Materials and Methods. After 1 wk, the illness scores of individual mice were monitored. Illness was scored using the following standard scale: 0 = healthy; 1 = slightly ruffled fur; 2 = ruffled fur, but active; 3 = ruffled fur and inactive; 4 = ruffled fur, inactive, and hunched; 5 = moribund. B, The percentage of P14 T cells (V2+/V8+) in PBL of the indicated recipient mice was determined by flow cytometry 1 wk after cell transfer and anti-CD40 mAb treatment. Dots indicate values from individual mice.

To exclude the possibility that the differences in disease severity in the various donor/host combinations were due to different proliferation capacities of the transgenic donor T cells, the frequency of P14 T cells in anti-CD40-treated H8 mice was determined using mAb (V2/V8) specific for the P14 TCR. As illustrated in Fig. 1B, similar percentages of P14 T cells in host PBL were observed in all donor/host combinations. In all recipients, 80–95% of total CD8 T cells in spleen and PBL were derived from the small number (10⁵) of P14 donor T cells injected (data not shown). Taken together, these results demonstrate an essential role of IFN- and of the FasL/Fas pathway in the P14 T cell-induced immunopathological processes in H8 mice.

Similar activation status of wild-type P14, P14.IFN-°, P14.gld, and P14.PKO T cells in H8 mice

After transfer into anti-CD40-treated H8 mice, P14 T cells became activated and acquired cytolytic activity (Fig. 2). The absence of severe pathological effects of P14 T cells lacking IFN- or functional FasL was not due to impaired activation, because wild-type, IFN--deficient, and P14.gld T cells transferred into H8 mice exhibited a similar activated phenotype with up-regulated CD44 and down-regulated CD62L expression (Fig. 2A). Furthermore, the cytolytic activity of P14.IFN-° and P14.gld T cells did not differ from wild-type P14 T cells when tested in a standard ⁵¹Cr release assay using gp33 peptide-loaded EL-4 target cells (Fig. 2B). Similar to wild-type P14 T cells, P14.PKO T cells also exhibited a highly activated phenotype. However, EL-4 target cells were not lysed by these activated P14.PKO T cells. This result is compatible with previous findings that EL-4 target cells are resistant to FasL-mediated lysis and can only be lysed by perforin-dependent mechanisms (26, 27). Taken together, these results demonstrated that, upon transfer in anti-CD40-treated H8 mice, wild-type P14 T cells and P14 T cells lacking IFN-, functional FasL, or perforin were activated similarly.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Phenotype and cytolytic activity of wild-type P14, P14.IFN-°, P14.gld, and P14.PKO T cells in anti-CD40-treated H8 mice. H8 recipient mice were injected with 105 P14 T cells from the indicated donor mice and treated with anti-CD40 mAb. After 1 wk, H8 recipient mice were sacrificed and spleen cells were analyzed. A, Expression of CD44 and CD62L gated on CD8 T cells. The P14 TCR (V2/V8) was expressed on 80–95% of the CD8 T cells analyzed (data not shown). Isotype controls are shown as filled histograms. For better comparison, staining of naive P14 T cells is included. B, Ex vivo cytolytic activity analyzed in a 5-h 51Cr release assay. As target cells, EL-4 cells coated with gp33 (filled symbols) or control adenovirus E1A peptide (open symbols) were used. The P14 T cell to target cell ratio was determined by flow cytometry using TCR V2/V8-specific mAb. Data from three individual mice per group are shown.

Liver injury by P14 T cells is dependent on IFN-, FasL, and perforin

Macroscopic evaluation of livers from anti-CD40-treated H8 mice adoptively transferred with wild-type P14, P14 IFN-°, P14.gld T cells, and P14.PKO T cells revealed marked differences. Livers from wild-type P14 T cell transfers were interspersed with necrotic areas, which appeared as white or hemorrhagic spots. In contrast, livers from H8 recipient mice of P14 IFN-°, P14.gld, and P14.PKO T cells exhibited no macroscopically visible alterations (Fig. 3A, and data not shown).

View larger version (22K): [in this window] [in a new window]   FIGURE 3. Liver injury by P14 T cells is dependent on IFN-, FasL, and perforin. A, Macroscopically visible liver damage by wild-type, but not by P14.IFN-° and P14.gld, T cells. One week after transfer of the indicated P14 T cells (105) into anti-CD40-treated H8 mice, mice were sacrificed and livers were photographed. B, GLDH levels in the sera of the indicated groups of mice were determined 1 wk after P14 T cell (105) transfer. C, P14 (104) and P14.gld (106) T cells were transferred into H8 mice. Seven days after transfer and anti-CD40 treatment, the percentage of P14+ T cells in PBL and the GLDH level in the sera of the recipient mice were determined. Dots indicate individual mice.

Caspase-3 has been implicated as a key protease that is activated during the signaling pathway of apoptosis. Immunohistological analysis of liver sections from H8 mice that had received wild-type P14 T cells revealed a high number of caspase 3-expressing cells, localized mainly around the necrotic areas (Fig. 4B). In contrast, only a few positive cells were found in liver sections of H8 mice that had been transferred with P14.IFN-° or P14.gld T cells (Fig. 4, C and D). Similarly, caspase 3-positive cells were almost undetectable in Fas-deficient H8 mice after transfer of P14.Fas° T cells (data not shown) or in H8 control mice treated only with anti-CD40 mAb (Fig. 4A). Quantitative analysis of infiltrating P14 T cells indicated that the reduced hepatocyte damage in the absence of donor T cell-derived IFN-, FasL/Fas interaction, or perforin was not due to impaired liver infiltration of the P14.IFN-°, P14.gld, and P14.PKO T cells (Fig. 4, I and J).

View larger version (55K): [in this window] [in a new window]   FIGURE 4. Differences in induction of caspase 3 in hepatocytes despite comparable liver infiltration by CD8 T cells. H8 recipient mice were injected with 105 P14 T cells from the indicated donor mice and treated with anti-CD40 mAb. After 1 wk, mice were sacrificed. A–H, Frozen sections of liver were stained with anti-caspase 3 Abs (A–D) and anti-CD8 mAb (E–H). The P14 TCR (V2/V8) was expressed on 80–95% of the CD8 T cells from the mice analyzed (data not shown). I–J, Absolute cell numbers of total hepatic (I) and P14+ hepatic (J) lymphocytes were determined by flow cytometry following isolation, as described in Materials and Methods.

IFN- up-regulates Fas expression in hepatocytes and FasL expression in P14 T cells

IFN- is known to increase Fas expression in several tumor cell lines in vitro (28, 29, 30). Thus, IFN- released by activated P14 T cells may up-regulate Fas expression in hepatocytes in this in vivo model also and thereby facilitate FasL-mediated lysis. Indeed, Fas expression analyzed by flow cytometry of isolated hepatocytes was increased 2-fold (mean fluorescence intensity) in H8 mice adoptively transferred with P14 T cells, compared with mice treated with anti-CD40 mAb only. Moreover, Fas up-regulation on hepatocytes was significantly higher after transfer with wild-type compared with P14.IFN-° T cells (Fig. 5A). Besides the difference in Fas up-regulation on hepatocytes, we also observed a 2-fold decrease in FasL expression of P14.IFN-° splenocytes compared with wild-type P14 T cells (Fig. 5B). Similarly, P14 T cells infiltrating into the H8 mouse liver also expressed higher levels of FasL than P14.IFN-° (Fig. 5C). Taken together, these results indicated that IFN- released by activated P14 T cells promotes FasL-mediated destruction of hepatocytes by increasing Fas expression on target cells and FasL expression on attacking CTL.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Fas expression on hepatocytes and FasL expression on CD8+ splenic and hepatic lymphocytes. H8 mice were left untreated (dotted line) or were injected with wild-type (thin line) and IFN--deficient (thick line) P14 T cells. Afterward, mice were treated with anti-CD40 mAb. One week later, hepatocytes (A), CD8+ splenic (B), and CD8+ hepatic lymphocytes (C) were analyzed by flow cytometry using mAb specific for Fas or FasL, as described in Materials and Methods. The P14 TCR (V2/V8) was expressed on 80–95% of the CD8 T cells from the mice analyzed (data not shown). Isotype controls are shown as filled histograms. Numbers indicate mean fluorescence intensities of the histograms.

Destruction of the splenic architecture by P14 T cells is independent of IFN-, FasL, and perforin

Expansion of P14 T cells in anti-CD40-treated H8 mice was accompanied by a severe loss of host lymphocytes and destruction of the splenic architecture. The few remaining CD4 T cells manifested a diffuse localization, and the size of B cell follicles was reduced strongly compared with anti-CD40-treated control mice. In contrast to the important role of IFN-, FasL, and perforin in liver cell damage, destruction of the splenic architecture by P14 T cells occurred in the absence of IFN-, FasL (Fig. 6), or perforin (data not shown). Thus, the effector mechanisms that caused immunopathology in the liver appeared to be redundant for immunopathological destruction and depletion of host lymphocytes in the spleen.

View larger version (164K): [in this window] [in a new window]   FIGURE 6. Destruction of the splenic architecture by P14 T cells is independent of IFN- and FasL. H8 recipient mice were injected with 105 P14 T cells from the indicated donor mice and were treated with anti-CD40 mAb. After 1 wk, mice were sacrificed and frozen sections of spleen were double stained with anti-CD4 (brown) and anti-B220 (blue) mAb.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the model described in this work, monoclonal CD8 T cells with defined Ag specificity were used. The results revealed important roles of IFN-, FasL, and perforin as mediators of CTL-induced liver cell injury in vivo. A nonredundant function of FasL has been reported previously by Kondo et al. (41), using an adoptive transfer system with a hepatitis B virus surface Ag (HBsAg)-specific CTL clone and HBsAg-tg mice. However, this finding could not be confirmed by Chisari and colleagues (42), although the same CTL clone and the same hepatitis B virus-tg mouse line were used. In the model described by Kennedy et al. (43), injection of soluble OVA peptide into OT-1 TCR-tg mice specific for OVA led to hepatocyte damage and increase in transaminase levels in the sera. As in our system, Fas expression on hepatocytes was required for disease, but the role of IFN-, FasL, and perforin was not examined.

How can the dramatic effect of IFN- on CTL-mediated liver damage be rationalized? IFN- is known to up-regulate Fas expression in various tumor cell lines, liver macrophages, and parenchymal cells in vitro (28, 29, 30). In vivo, ectopic expression of IFN- in the liver of tg mice has been shown to increase Fas at the mRNA level (44). Moreover, IFN- produced by activated CD8 T cells in an acute graft-vs-host disease model has been reported to increase Fas expression on host lymphocytes (45). In the liver, up-regulation of Fas mRNA expression by Con A treatment was reduced to 50% in IFN--deficient mice when compared with IFN-^+/- mice (37). At the functional level, Ag-dependent release of IFN- by activated CTL has recently been demonstrated to facilitate target cell lysis in vitro (46). Our results demonstrate that Fas expression on hepatocytes was increased after transfer with wild-type compared with IFN--deficient P14 T cells. This suggests that IFN- released by activated P14 T cells upon Ag contact up-regulates Fas expression on hepatocytes, making them more susceptible for FasL-mediated T cell attack. Besides the difference in Fas expression on hepatocytes, we also observed higher levels of FasL on wild-type compared with P14.IFN-° T cells. This result is in line with the finding of Shustov et al. (45) that anti-IFN- Ab treatment reduced FasL expression on donor CD8 T cells in an acute graft-vs-host disease model in mice. IFN- may regulate FasL expression via IFN regulatory factor-1 and IFN regulatory factor-2 that are induced in T cells after IFN- treatment (47) and that are thought to be involved in the transcriptional control of the FasL gene (48, 49).

GLDH levels in H8 recipient mice of P14.gld and P14.PKO T cells were reduced similarly, indicating that both FasL- and perforin-dependent signals were involved in hepatocyte damage by P14 T cells. This result fits well with previous data in the HBsAg-tg system using CTL clones from gld/gld and PKO mice (42). In this context, IFN- produced by activated P14 T cells may enhance MHC class I expression on hepatocytes that might facilitate perforin-mediated lysis in vivo. In addition, IFN- has been shown to induce genes involved in signaling of cell death (35, 50, 51) or to down-regulate antiapoptotic molecules (52). An indispensable role for TNF and IFN- in liver injury mediated byHBsAg-specific CD4 T cells has been reported recently by Ohta et al. (53). In contrast to our data with CD8 T cells, FasL was not essential in this Th1-mediated model of liver injury. Thus, IFN- may also exert direct cytotoxic effects on hepatocytes in vivo, as has been suggested by in vitro data (54). In addition, inducible NO synthase induced by IFN- and TNF may participate in the liver cytotoxicity of this cytokine (55).

Five to 7 days after T cell transfer, H8 mice that received P14.PKO T cells developed only slightly reduced signs of illness (cachexia, ataxia, hunched posture, moribund) compared with wild-type P14 T cells, although serum GLDH levels were significantly lowered in P14.PKO transfers. This may indicate that P14 T cell-mediated immunopathology in organs other than the liver contributed to the clinical symptoms observed. In this context, it is noteworthy that massive infiltration of donor P14 T cells was also found in lung and bone marrow, but not in brain of the recipient mice. In contrast to classical graft-vs-host reactions induced by transfer of allogeneic T cells into irradiated recipient mice, allopecia or ulceration of the skin was not observed in H8 mice after P14 T cell transfer (data not shown).

In conclusion, we have used a transfer model with TCR-tg and Ag-tg mice to analyze the mechanism of liver cell damage by CTLs in vivo. The results revealed a crucial role of IFN-, FasL, and perforin in liver cell destruction. It is important to stress that these effector molecules were not essential for destruction of the splenic architecture and depletion of host lymphocytes by P14 T cells in the same model. This indicates that the effector molecules required for target cell destruction in vivo could vary with the cell type (i.e., hepatocytes vs lymphocytes) attacked by CTLs.

	Acknowledgments

 
We thank Peter Aichele and Stephen Batsford for comments on the manuscript; Manfred Kopf, Markus Simon, and Hermann Eibel for providing breeding pairs of B6.IFN-°, B6.Fas°, and B6.gld mice, respectively; and Theresa Treuer, Sonja Wagenknecht, Rainer Bronner, and Thomas Imhof for animal husbandry.

	Footnotes

 
¹ This work was supported by the Deutsche Forschungsgemeinschaft (Pi 295/4-1).

² Address correspondence and reprint requests to Dr. Hanspeter Pircher, Institute for Medical Microbiology and Hygiene, Department of Immunology, Hermann-Herder-Strasse 11, University of Freiburg, D-79104 Freiburg, Germany. E-mail address: pircher{at}UKL.uni-freiburg.de

³ Abbreviations used in this paper: tg, transgenic; GLDH, glutamate dehydrogenase; HBsAg, hepatitis B virus surface Ag; LCMV, lymphocytic choriomeningitis virus.

Received for publication March 25, 2003. Accepted for publication November 13, 2003.

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