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*
Max Planck Institut für Immunbiologie, Freiburg, Germany; and
Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FasL is predominantly expressed on NK and T effector cells (14, 15). Upon T cell activation, FasL is newly synthesized and delivered to the cell surface either directly (16) or via Ca2+-dependent polarized degranulation of cytoplasmic vesicles, serving as a storage compartment for previously synthesized protein (17). Fas, in contrast, is constitutively expressed, although to greatly varying degrees, on a variety of cells, in particular cells of hemopoietic origin (18). Less is known about the regulation of Fas expression with many cells functionally regarded as being Fas negative under normal physiological conditions. However, it may become induced or be up-regulated on individual cell populations by a number of endogenous and exogenous stimuli, including stress and infectious agents (13, 19). In this context, the level of Fas expression is thought to be a determining factor in the induction of apoptosis in various organs during physiological and pathophysiologic processes (19, 20). Here we show that Fas is up-regulated on target cells as a result of recognition by Tc cells and discuss this phenomenon in the context of recruitment of the Fas pathway in immune attack during infections and antigenic feedback on the regulation of cellular immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (KbDb; B6), CBA/H (KkDk; CBA), BALB/c (KdDd; B/c), AKR/N (KkDk; AKR), B10.HTG (KdDb; HTG), B10.A(2R) (KkDb; 2R), the perforin-deficient mutant (perf-/-) (21), and the triple knockout mouse, perf- plus gzmA- and/or gzmB-deficient mutant (perfxgzmA-/-, perfxgzmB-/-, perfxgzmAxB-/-) 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 Immunobiology. The perfxgzmAxB-/- mice were obtained by crossing the perf-/- mice with the gzmAxB double knockout mice (22). Backcrosses were analyzed by PCR for homozygosity of the mutations using primers as previously described (22). The Fas ligand mutant mice (gld) bred on B6 background were obtained from the Centenary Institute (Sydney, Australia). Mice homozygous for both the gld mutation and perf deficiency (perf -/-xgld), were generated by crossing perforin-/- mice with gld mice and by subsequent intercrossing of heterozygous F1 animals.
For detection of the respective mutations, DNA was analyzed by PCR, using the following primers: perforin-/-, 5'-CCA CTC CAC CTT GAC TTC AAA AAG GCG-3' and 5'-TGG GCA GCA GTC CTG GTT GGT GAC CTT-3'; and neo-primer, 5'-CGG AGA ACC TGC GTG CAA TC-3'.
For detection of the gld point mutation in FasL (23), the PCR approach was: forward primer, 5'-AGG AAC TCT AAG TAT CCT GAG-3'; reverse primers specific for the gld, 5'-AGA TCA TTT TAA AAT GCT TTT GAT TTT AAA GCT TAT ACA AGC CGA GAA G-3'; wild-type primer, 5'-TCT TTT AAA GCT TAT ACA AGC CGA AGA A-3.
Genomic DNA was subjected to amplification by PCR and analyzed as previously described for perforin-/- (22)and for gld (23). All mutant mice were analyzed for their perf and gld genotypes before experimentation. Perf-/-xgld mice were used at 5–7 wk of age. The Fas receptor mutant mice (H-2k-lpr), a backcross of B6 lpr with B10.BR (H-2k) and selected for H-2k plus lpr mutation, were generously provided by C. Goodnow (John Curtin School of Medical Research, Australian National University, Canberra, Australia). Only female animals >12 wk of age were used.
Viruses
The cowpoxviruses (CPV) wild-type and the mutants with a defect in serpin-1 (dlSPI-1) or serpin-2 (dlSPI-2) (24) were grown on CV-1 cell monolayers. The ectromelia virus Moscow strain (Ect) was grown in mouse spleen. All poxviruses were titrated as previously described (25).
Cell lines
The mouse cell lines L929(H-2k), L929-Fas (transfected with human Fas; provided by P. Krammer, Heidelberg, Germany), L1210 (H-2d), L1210.Fas (transfected with mouse Fas; provided by P. Golstein, Marseilles, France), P815 (H-2d), and P815.Fas (transfected with human Fas; used with permission of W. Nishioka) were grown in Eagle’s MEM (EMEM) supplemented with 10% FCS. Mouse embryo fibroblasts (MEF) were obtained from 14- to 16-day-old embryos as previously described (26) and used after one or two in vitro passages.
Infection of target cells with poxvirus
Cell lines were infected with poxviruses using 10–20 PFU/cell for 16 h before being labeled with 51Cr for 1 h or were left uninfected and used for analysis as previously described (25).
FACS analysis
Cells were stained for Fas expression using the FITC-conjugated mAb specific either for mouse Fas (Jo-2) or human Fas (PharMingen, Hamburg, Germany) or for H-2k expression with the PE-conjugated mAb specific for H-2Kk (AF3-12.1, PharMingen, Hamburg, Germany). Cells were examined with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).
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 of EMEM, 10% FCS, and 10-5 M 2-ME.
51Cr release cytotoxicity assay
The methods used for infection and 51Cr labeling of target cell lines have been described previously (25). The duration of the assays varied from 4–20 h as indicated in Results. The percentage of specific lysis was calculated by the formula: % 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 of [methyl-3H]thymidine (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 triplicate at the indicated E:T cell ratio in 200 µl of EMEM supplemented with 2 mg/ml BSA. In some experiments, mAb to Fas (Jo-2) was added to cell cultures before incubation. After the indicated time periods cells were lysed with 25 µl of Triton X-100 (2%)/Tris-HCl (pH 8.0)/EDTA (0.5 M) for 10 min at 37°C. After centrifugation (1200 rpm, 10 min) 25 µl of supernatant was harvested into a solid scintillator plate (LumPlate, Packard, Dreieich, Germany), dried, and counted with TopCount (Packard). For maximum release 25 µl of EMEM was added to the wells, and 25 µl of resuspended cell suspension was used. The percentage of specific lysis was calculated by the formula: % specific lysis = [(experimental release - medium release)/(maximum release - medium release)] x 100. Data are the means of triplicate determinations. SEM values were always <5%.
Inhibition of cytotoxicity
Target cell treatment. L929 target cells were labeled with 51Cr for 1 h and treated with inhibitors at 3 x 106 cells/ml for 1 h. Actinomycin D (Act. D; Sigma, St. Louis, MO) was used at 2.5 µg/ml, and cycloheximide (Sigma) was used at 30 µM. Cells were treated with brefeldin A (BFA; Sigma) at 5 µg/ml. Cells were washed thoroughly before incubation with effector cells for cytotoxicity assays. In the case of inhibition with BFA the assay medium contained BFA at 0.5 µg/ml for the duration of assay.
Effector cell treatment. In vitro generated effector cells (3 x 106 cells/ml) were treated with BFA (5 µg/ml) or 200 nM concanamycin A (CCA; Sigma) for 2 h. Cells were washed thoroughly before incubation with target cells for cytotoxicity assays. In the case of inhibition with BFA the assay medium contained BFA at 0.5 µg/ml for the duration of assay; for inhibition with CCA, a concentration of 100 nM CCA was kept throughout the assay period.
Additions to cytotoxicity culture.
Anti- Fas mAb (Jo2; 30, 10, and 1 µg/ml; PharMingen), soluble mouse
Fas-Fc (10 and 1 µg/ml; R&D Systems, Wiesbaden-Nordenstadt, Germany),
anti-IFN-
mAb (AN18; 100, 30, and 1 µg/ml) (27),
or respective control IgG preparations (hamster IgG, rat IgG) were
added at the indicated concentrations to cultures containing Tc cells
and target cells for the duration of assay.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We, like others (2, 28), have observed that effector
Tc cells derived from wild-type mice or mice defective in granule
exocytosis due to a gene deletion of perf have comparable lytic
activity on Fas-positive target cells. A surprising observation was
that target cells lacking detectable amounts of cell surface expressed
Fas (Fasneg), become susceptible to lysis by
perf-/- alloreactive Tc cells after
lengthy incubation periods. Splenocytes of B6 wild-type (B6) or
perf-/- mutant mice were stimulated in
vitro with either Kk- or
Kd-bearing stimulator cells and tested for lytic
activity on H-2k (L929) or
H-2d (L1210 and P815)
Fasneg target cells or those previously
transfected with either mouse (L1210.Fas) or human (L929.Fas, P815.Fas)
Fas. FACS analysis revealed that Fas is readily detectable on
transfected, but not or only marginally (P815) on untransfected,
targets (Fig. 1
A). B6 Tc cells
lysed Fasneg and Fas-transfected targets at any
of the three time points (4, 8, and 12 h) tested (Fig. 1C) to a similar extent. In contrast,
perf-/- Tc cells lysed
Fasneg targets, if at all, only marginally in a
4-h assay. However, lysis of Fasneg targets
increased substantially over the next 8 h, reaching levels
comparable to those seen with Fas-transfected targets in the case of
L929 and L1210, but not P815 cells. As expected, the cytolytic activity
of perf-/- Tc cells on Fas-transfected
targets was detectable from 4 h on, but was always lower than that
of B6 Tc cells.
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One possible explanation for the observed cytolytic potential of perf-/- effectors on Fasneg targets was that lysis is mediated by gzm in a perf-independent way. This possibility was tested by employing mice deficient in perf plus gzmA and gzmB (perfxgzmAxB-/-).
H-2d-alloreactive Tc cells from B6 or
perf-/- mutant mice, including those with
additional deficiencies in gzmA
(perfxgzmA-/-), gzmB
(perfxgzmB-/-), or both
(perfxgzmAxB-/-), were tested for both
cytolytic (51Cr release) and nucleolytic
([3H]DNA release) activities in short (4-h) and
long term (6- and 20-h) cultures (Fig. 2). Only Tc cells from B6 but not from
any of the perf-/- mice were able to
induce 51Cr release or DNA fragmentation in L1210
targets when tested at 4 h of incubation. However, in long term
cultures (6 and 20 h), Tc cells of all
perf-/- mutant mice expressed similar cytolytic
and nucleolytic activities on L1210 targets, which were most pronounced
after 20-h incubation. As expected, Tc cells from both B6 and each of
the perf-mutant mice were cytolytic and nucleolytic for L1210.Fas
targets in 4-, 6-, and 20-h assays, where the higher lytic potential
was always seen with B6-derived splenocytes. Similar results were
obtained using H-2Kk-alloreactive Tc cells from
perfxgzmAxB-/- mice and L929 target cells
(data not shown). These data demonstrate that in the absence of perf,
neither of the gzms contributed to cytolysis/nucleolysis of either
Fas-transfected or Fasneg targets.
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The Ag specificity of Fasneg target cell
lysis by perf-deficient Tc cells was proven by comparing the lytic
potential of in vitro-derived alloreactive
(H-2Kk- or H-2Kd-specific)
splenocytes from B6 and
perfxgzmAxB-/- mice on
H-2Kk-expressing L929, L929.Fas, and
H-2Kd-expressing L1210 and L1210.Fas target
cells (Fig. 3). When assayed between
4 and 12 h, H-2Kd-reactive
perfxgzmAxB-/- Tc cells lysed L1210.Fas and
L1210 targets, although with differing kinetics, but gave only
background lysis on L929 and L929.Fas targets.
H-2Kk-reactive
perfxgzmAxB-/- Tc cells responded in a
similar Ag-specific pattern. A similar specificity pattern was observed
with the respective alloreactive B6 Tc cells.
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We next investigated the effects of specific inhibitors of
transcription, translation, or protein transport on Tc cell-mediated
lysis of Fasneg target cell. L929 target cells
were pretreated with inhibitors of RNA synthesis (Act. D), protein
synthesis (cycloheximide), or vesicular transport (BFA) before the
addition of H-2k alloreactive Tc cells from B6 or
perf-/- mice.
51Cr release was measured after 4, 8, and 12
h of assay (Fig. 4A).
B6-derived effectors lysed target cells regardless of treatment with
inhibitors equally well at the 4 and 8 h points. At 12 h of
assay, mock-treated targets gave slightly higher
51Cr release at low E:T cell ratios than any of
the treated targets. Perf-/- and
perfxgzmAxB-/- Tc cells did not lyse any
target at 4 h. Significant lysis at high E:T cell ratios was
observed on mock-treated targets at 8 h of incubation, which
increased with assay time (12 h). No lysis was induced by either
perf-deficient Tc cell population on any of the pretreated targets at 8
or 12 h of assay. This provides strong evidence that for
perf-independent lysis of Fasneg target cells, de
novo transcription and translation and cell surface transport of newly
synthesized proteins are required.
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B). As shown previously (30), cytolysis and nucleolysis of L929 and L929.Fas cells by B6 Tc cells was seen at all time point and was greatly inhibited by CPV, dlSPI-1, and Ect and only marginally inhibited by dlSPI-2. As expected, perf-/- Tc cells also induced 51Cr release and DNA fragmentation in mock-infected L929.Fas target cells at both time points, and in L929 targets only after prolonged (12/24-h) assay times. Infection of either targets with Ect or CPV completely suppressed target cell lysis under those conditions. Infection with dlSPI-2, but not dISPI-1, partially restored cytolysis. The same pattern was observed for DNA release.
This inhibition of 51Cr release and nucleolysis is not due to interference with Fas expression on target cells. Infection with Ect, CPV, and dlSPI-2 increased rather than decreased Fas expression on L929 and L929.Fas target cells, as determined by Jo-2 mAb staining over a 20-h period (data not shown).
In vitro-derived H-2k alloreactive Tc cells
from perfxgzmAxB-/- mice were also tested for
cytolysis and nucleolysis on L929 target cells in the absence or the
presence of increasing amounts of anti-Fas (Jo-2) mAbs in a
6-h assay. Both, cytolysis and nucleolysis induced by the Tc cell
population on L929 cells were blocked by Jo-2 mAb in a dose-dependent
manner, but not by isotype-matched control Ab (Fig. 4C).
Similar inhibition of cytolysis and nucleolysis was obtained using a
soluble mouse Fas-Fc preparation (data not shown).
Induction of Fas expression on target cells during coincubation with Tc cells
The previous experiment indicated that functionally active
Fas becomes available on the cell surface of normal Fas-negative L929
targets during their encounter with Tc cells. In fact, mRNA analysis
revealed that L929 cells contain small amounts of mouse Fas-specific
transcripts, but are negative when tested for surface expression of the
protein (Fig. 1, A and B). Surprisingly, when the
same targets were incubated with Tc cells from either
perf-/- or
perfxgzmAxB-/- mice for 6–20 h, cell
surface expression of Fas increased to the same level with both
effector populations (Fig. 5).
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Tc cells from B6, perf-/-, and
perfxgzmAxB-/- mice were preincubated before
assays with either BFA, an inhibitor of protein transport, or CCA,
which has been shown to selectively inhibit exocytosis
(31). As shown in Fig. 6A, BFA severely inhibited
lysis of L929 and L929.Fas targets by any of the three Tc cell
populations at 4, 8, and 12 h of assay. In contrast, CCA inhibited
lysis of L929 and L929.Fas targets and L1210 and L1210.Fas targets
(Fig. 6B) to varying degrees depending on the source of Tc
effectors. Lysis of the Fasneg targets by
perf-/- effectors at 8 and 12 h
assay times was insignificantly reduced. Lysis of the same targets by
gld Tc cells, which predominantly exerted their function via
the exocytosis pathway, was totally inhibited by CCA at the 4 h
point and was significantly inhibited also at later time points (8 and
12 h). The effect of CCA observed on target cell lysis by B6 Tc
cells was partially inhibited, confirming previous work
(31). This further provides evidence that the lysis seen
on Fasneg targets is mediated by the Fas
pathway.
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To assess whether the cytolytic activity seen on the
Fasneg targets by perf-/-
effectors at 8 and 12 h assay times is solely due to lysis via the
Fas pathway, we bred homozygous mice from
F1(gldxperf-/-)
hybrids. Unknown to us and not reported previously (32)
and in only one of two recent studies (33, 34) in which
such mice were used, most homozygous mice died between 3–5 wk of age.
A pool of splenocytes from two homozygous surviving offspring was
stimulated in vitro with either 2R
(anti-H-2Kk) or HTG
(anti-H-2Kd) stimulator cells. Phenotypic
analysis of their splenocytes revealed similar proportions of
CD4+, CD8+, and B cells as
those observed in B6 control splenocytes (data not shown). No lysis of
either the Fasneg targets after 8–12 h of assay
or their Fas-transfected variants was observed (Fig. 6B).
Alloreactive Tc cells from perforin-deficient mice do not lyse Fas-defective target cells
To obtain additional evidence that lysis of
Fasneg target cells by
perf-/- Tc cells is mediated via the Fas
pathway, we made use of target cells derived from Fas- mutant
(lpr, H-2k) and Fas-expressing
(CBA, H-2k) mice. Primary MEF obtained from
embryos of both mouse strains were stained with Fas-specific mAb Jo-2
and analyzed by FACS. In contrast to CBA MEFs, which expressed high
levels of Fas, lpr MEFs did not stain with the mAb (Fig. 7A). The same cell populations
were used as targets for B6 and perf-/-
H-2Kk alloreactive Tc cells in a 4- to 12-h
cytotoxic assay (Fig. 7B). L929 and L929.Fas targets served
as controls. B6 effectors lysed all H-2k targets
at any time point tested, however with different efficiency where MEF
targets were lysed to a lesser extent. This was probably due to
their low expression of cell surface MHC class I (26). Tc
cells from perf-/- mice
(perf-/-,
perfxgzmAxB-/-) lysed L929.Fas targets
at all time points. Moreover, the same effectors lysed L929 and CBA/MEF
with increasing efficiency in assays from 8 h onward. Again, MEF
cells were lysed less efficiently than L929. In contrast, no lysis was
observed on MEF target cells derived from the Fas receptor mutant mouse
lpr.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The finding that perf-/- and
perfxgzmAxB-/- T cells exert similar delayed
cytolytic and nucleolytic activities on Fasneg
targets corroborate published data regarding the independence of the
Fas pathway from perforin (35). In addition, it
demonstrates that Fas-mediated cytolysis is also not influenced by
either of the two granzymes. A possible contribution of TNF, a
cytolytic protein produced by mouse Tc cells (36), to this
late target cell lysis cannot be formally excluded. However, this would
require a number of ad hoc assumptions for the following reasons.
treatment of L929 target cells with Act. D at concentrations used for
TNF assays (37) prevented, rather than augmented, killing
(Fig. 4A). The lpr MEFs, defective in Fas, but
not TNF, receptors, were refractory to killing by
perf-/- and
perfxgzmAxB-/- Tc cells, and Tc effectors
from perf-/-xgld mice, not
known to be defective in TNF production, did not lyse untransfected or
Fas-transfected L929 and L1210 targets, even after incubation for up to
12 h. Furthermore, preliminary data using double-chamber
experiments showed that bystander cells separated from Tc cell assay
cultures (targets plus Tc effectors) by permeable membranes showed
increased Fas expression but no cell death (our unpublished
observations).
At present, the mechanism by which Fas is induced by Tc cells on
Fasneg cells is elusive, but two possibilities
come to mind. Fas biosynthesis and expression may be induced either
directly by TCR engagement with MHC and signaling via the cytoplasmic
tail of MHC class I, including any of the accessory molecules, by
soluble mediators released by Tc cell effectors such as IFN-, or by
both. The former hypothesis is unlikely, because to date attempts to
induce Fas expression on the same target cells as those used for Tc
cell assays with mAb to the relevant MHC class I have failed (data not
shown). The fact that Tc cells produce IFN- (38), which
has been shown to induce Fas-specific mRNA in various target cells,
including L929 (18) (data not shown), the increase in Fas
expression in bystander experiments mentioned above, as well as the
finding that cytolysis of targets by
perfxgzmAxB-/- Tc cells is inhibited at least
partially by anti-IFN- Abs (data not shown) support this
assumption.
The more intriguing question concerns the biological relevance of such an Ag-specific Tc cell up-regulation of Fas on target cells. One possibility is the involvement in activation-induced cell death of APCs by Tc cells as a means of down-modulating the immune response. This has at least been shown to occur with FasL-expressing CD4+ T cells, which are able to kill B cells (8, 39) and macrophages (40) via the Fas pathway, and probably also works with CD8+ T cells. Whether Fas-mediated induction of cell death by Tc cells also affects the regulation of dendritic cells, the most potent of the known APCs (41), is unclear in light of the reported resistance of dendritic cells to Fas-mediated apoptosis (50). However, such modulation of Tc cell activation would differ fundamentally from the Fas- or perf-mediated control of expansion and persistence of T effector cells, as described recently (34, 42, 43). In accordance with this hypothesis one could suggest that poxvirus-associated serpins have evolved to delay or circumvent this process. The severe splenomegalies and liver and lympho-hyperplasia seen after poxvirus infection in mice are consistent with this hypothesis (44).
Another possibility is that the observed phenomenon contributes to the
overall efficiency of Tc cells to eliminate intracellular pathogens.
Thus, killing of target cells in affected tissues and simultaneous
destruction of pathogens may proceed via Fas in an Ag-specific manner
in addition to exocytosis-mediated lysis or even in the absence or
during suppression of exocytosis or production of cytolytic cytokines
such as TNF-
. The fact that perf-/- mice
recover from a variety of viral diseases (45), but not
lymphocytic choriomeningitis virus (46) and ectromelia
(47), is in line with this concept. The fact that
poxviruses totally inhibit Fas-mediated kill and that
perfxgzmAxB-/- mice are unable to control
mouse pox ectromelia (6) is further indirect evidence for
the possible contribution of Fas to the recovery from pathogens other
than ectromelia.
Finally, Ag-specific Tc cell up-regulation of Fas on target cells may contribute to the pathogenesis of diseases. Recent studies indicated that Fas expression on hepatocytes is up-regulated by viral components (48) and that FasL expressed on pathogen-specific Tc cells is responsible for apoptosis of hepatocytes expressing hepatitis B virus surface Ag, thus leading to hepatitis (49). Together with the present report this suggests that Fas-mediated development of tissue failures, including hepatitis, can be differentially regulated by both pathogens and Tc cells and depends on the densities of Fas and FasL generated in situ. In any event, the data indicate that the T effector cell-mediated up-regulation of Fas on target cells has an important role in T cell activation and Tc cell-mediated cytotoxicity and pathology.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Arno Mullbacher, Division of Immunology and Cell Biology, John Curtain School of Medical Research, Australian National University, P.O. Box 334, Canberra, ACT 2601, Australia.
3 Abbreviations used in this paper: Tc cells, cytotoxic T cells; perf, perforin; gzm, granzyme; FasL, Fas ligand; EMEM, Eagle’s MEM; MEF, mouse embryo fibroblast; Act. D, actinomycin D; BFA, brefeldin A; CCA, concanamycin A; SPI, serpin; Ect, ectromelia virus Moscow strain; CPV, cowpoxvirus.
Received for publication May 1, 2000. Accepted for publication July 14, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 J. H. Stewart IV, D. M. Nguyen, G. A. Chen, D. S. Schrump, and S. V. Rusch Induction of apoptosis in malignant pleural mesothelioma cells by activation of the Fas (Apo-1/CD95) death-signal pathway J. Thorac. Cardiovasc. Surg., February 1, 2002; 123(2): 295 - 302. [Abstract] [Full Text] [PDF]
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A. Mullbacher and M. Lobigs Creation of Killer Poxvirus Could Have Been Predicted J. Virol., September 15, 2001; 75(18): 8353 - 8355. [Full Text] [PDF]
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S. Balkow, A. Kersten, T. T. T. Tran, T. Stehle, P. Grosse, C. Museteanu, O. Utermohlen, H. Pircher, F. von Weizsacker, R. Wallich, et al. 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., September 15, 2001; 75(18): 8781 - 8791. [Abstract] [Full Text] [PDF]
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V. Screpanti, R. P. A. Wallin, H.-G. Ljunggren, and A. Grandien A Central Role for Death Receptor-Mediated Apoptosis in the Rejection of Tumors by NK Cells J. Immunol., August 15, 2001; 167(4): 2068 - 2073. [Abstract] [Full Text] [PDF]
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), L929.Fas (
), L1210 (•), L1210.Fas
(
), P815 (
), and P815.Fas (
) target cells were tested for
lysis by B6 and perf-/- anti-2R
(anti-Kk) and B6 and perf-/-
anti-HTG (anti-Kd) alloreactive Tc cells,
respectively. The cytotoxic assay times were 4, 8, and 12 h. Each
point constitutes the mean percent specific lysis of three separate
wells. Spontaneous release was always <20%.
