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*
Ludwig Institute for Cancer Research, Lausanne Branch, and
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production were lost but Fas (APO1 or CD95)-dependent
cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted
and specific for a photoreactive derivative of the Plasmodium
berghei circumsporozoite peptide PbCS 252–260 (SYIPSAEKI). The
presence of a photoactivatable group in the epitope permitted
assessment of TCR-ligand binding by TCR photoaffinity labeling.
Selective activation of Fas-dependent killing was observed for a
peptide-derivative variant containing a modified photoreactive group. A
similar functional response was obtained after binding of the wild-type
peptide derivative upon blocking of CD8 participation in TCR-ligand
binding. The epitope modification or blocking of CD8 resulted in an
8-fold decrease in TCR-ligand binding. In both cases, phosphorylation
of 
-chain and ZAP-70, as well as calcium mobilization were reduced
close to background levels, indicating that activation of Fas-dependent
cytotoxicity required weaker TCR signaling than activation of
perforin-dependent killing or IFN- production. Consistent with this,
we observed that depletion of the protein tyrosine kinase
p56lck by preincubation of S15 CTL with
herbimycin A severely impaired perforin- but not Fas-dependent
cytotoxicity. Together with the observation that S15 CTL constitutively
express Fas ligand, these results indicate that TCR signaling too weak
to elicit perforin-dependent cytotoxicity or cytokine production can
induce Fas-dependent cytotoxicity, possibly by translocation of
preformed Fas ligand to the cell surface. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(PLC-),3
phosphatidylinositol 3-kinase, and Ras (3, 4, 5). CTL activation generally
elicits three different effector functions. The first and most
prominent function is perforin-dependent cytotoxicity. This lytic
principle involves exocytosis of preformed CTL granules containing
pore-forming perforin and various esterases called granzymes, which in
a synergistic manner induce cell death (1, 2). Second, CTL, upon
activation, express Fas ligand (FasL), which binds to Fas, the receptor
for FasL, present on most cells, and thus induce apoptosis via a
well-characterized signaling mechanism (1, 2, 6, 7, 8). FasL, to exert its
activity, can be cell associated, or secreted in soluble form (9).
While the cellular mechanisms leading to apoptosis are universal, the
induction of FasL expression is not, and in different systems different
signaling pathways seem to exist (10, 11, 12, 13, 14). Third, activation of CTL
usually results in production and secretion of cytokines, such as
TNF-
, IFN-, and various ILs (1, 2, 7, 15, 16). While in general CTL activation by TCR engagement elicits all three of these effector functions, modification of antigenic peptides can result in the activation of only some of them (7, 17, 18). For example, it has been shown that altered self peptide ligands can activate Fas-dependent killing in the absence of perforin-dependent cytotoxicity (7, 17, 18). This suggested that selective activation of Fas-dependent apoptosis may play a role in eliminating cells expressing altered autologous proteins (7, 17). There is, however, no evidence that altered peptide ligands preferentially induce this form of partial agonism in CTL (19). It also seems surprising that cytokine production was maintained in these studies, i.e., that perforin-dependent killing was lost selectively (7, 8). This cytotoxic pathway is very rapid and can be elicited by TCR signaling too weak to induce cytokine production, cell proliferation, or TCR down-modulation (20). It also has been shown that only very few MHC-peptide complexes on target cells, perhaps only one, are sufficient to induce perforin-dependent killing, which clearly is not the case for cytokine responses (20, 21). On the other hand, there exist several reports indicating that induction of Fas-dependent killing requires FasL transcription (1, 2, 7, 10, 22). At least for cytokine production, induction of gene transcription requires sustained TCR signaling for extended periods of time (20).
To address these apparent divergences, we now investigated the activation requirements for selective activation of Fas-dependent cytotoxicity. By screening 12 altered peptide ligands on seven CD8+CTL clones, we have previously observed one case in which perforin- but not Fas-dependent cytotoxicity was lost (18). The CTL clone concerned, S15, is H-2Kd restricted and specific for the Plasmodium berghei circumsporozoite peptide PbCS 252–260 (SYIPSAEKI), modified by replacing PbCS S252 with photoreactive iodo-4-azidosalicylic acid (IASA) and by conjugating PbCS K259 with 4-azidobenzoic acid (ABA) (23). The epitope recognized by this and related CTL clones was the ABA-modified PbCS peptide (23). Selective photoactivation of the IASA group allowed cross-linking to Kd and photoactivation of ABA to TCR (23, 24). This system thus permitted assessment of TCR-ligand binding on living CTL by TCR photoaffinity labeling with covalent soluble Kd-peptide complexes (18, 23, 24). Remarkably, S15 CTL replacement of the ABA group with 4-azidosalicylic acid (ASA, i.e., introduction of a hydroxy substituent in position 2 of ABA) resulted in a variant (K259(ASA)), which was only inefficiently recognized, possibly only via Fas-dependent cytotoxicity (18).
In this work, we report that this variant also failed to induce
cytokine production by S15 CTL. Surprisingly, the same partial agonism
was also observed for the wild-type peptide derivative in the presence
of anti-CD8ß mAb H35-17 or Fab' fragments of
anti-Kd3 mAb SF1-1.1.1 (SF1-1.1.1 Fab'). These
reagents prevent CD8 from binding MHC molecules that interact with TCR,
which weakens TCR-ligand binding and accelerates TCR-ligand complex
dissociation (25). As CD8 (and CD4) is associated with the T
cell-specific tyrosine kinase p56lck, which
plays a key role in TCR signaling, this blocking of CD8 (and CD4) not
only reduces the avidity of TCR-ligand binding but also prevents
coreceptor-associated p56lck from being
brought to the TCR/CD3 complex (25, 26, 27). Consistent with this, we
observed that blocking of CD8 substantially reduced phosphorylation of
-chain as well as ZAP-70 and mobilization of intracellular calcium
[Ca2+]i, all important early events in T cell
activation (5). Essentially the same findings were obtained for variant
K259(ASA), indicating that in this system Fas-dependent cytotoxicity
was induced by TCR signaling too weak to elicit perforin-dependent
killing or lymphokine production. Since S15 CTL constitutively
expressed FasL, it appears that very limited TCR signaling can elicit
Fas-dependent cytotoxicity, conceivably by inducing translocation of
preformed, intracellular FasL to the cell surface, to become
biologically active.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Amino acids and chemicals for peptide and conjugate synthesis were obtained from Sigma (Buchs, Switzerland) and Bachem Finechemicals AG (Bubendorf, Switzerland). Concanamycin A (CMA) was from Sigma. The synthesis and characterization of photoreactive PbCS peptide derivatives have been described previously (18, 23, 24). In brief, all peptide derivatives were synthesized on an ABI 431 peptide synthesizer (Applied Biosystems, Foster City, CA). K(ABA) and K(ASA) were incorporated as Fmoc-K(ABA)-OH and Fmoc-K(ASA)-OH, respectively. The peptide derivatives were deprotected and cleft from the resin and purified by HPLC on a C-18 column (1 x 25 cm, 5 µm particle size; Marcherey & Nagel, Oensingen, Switzerland). After reacting the purified peptide derivatives with 125I or nonradioactive IASA-N-hydroxysuccinimide ester, they were HPLC purified. 125I iodine was from New England Nuclear (Boston, MA) and had a specific radioactivity of approximately 2000 Ci/mMol. The molecular weight (Mr) of all compounds was verified by mass spectrometry (LDI 7000 mass spectrometer; Linear Scientific, Reno, CA).
Cells and Abs
The P815 mastocytoma cells and P815 transfected with Fas
(P815Fas+) (28) were maintained in DMEM supplemented with
FCS (5%) and HEPES (10 mM). A20 B lymphoma cells and a
Fas- variant, which lacks functional Fas
(A20Fas-) (29), were cultured in the same medium
supplemented with 1 mM 2-ME. Cloned S15 CTL were propagated by weekly
restimulation with IASA-YIPSAEK(ABA)I-pulsed, irradiated P815 cells in
the presence of irradiated BALB/c splenocytes and IL-2, as previously
described (23). Hybridomas producing the following mAb were obtained
from American Type Culture Collection (Manassas, VA): H35-17
(anti-CD8ß), SF1-1.1.1 (anti-Kd3), 20-8-4S
(anti-Kd1), and H57-597 (anti-TCR Cß).
Fab' fragments were prepared following published procedures
(30).
Cytotoxicity assays
Cytolytic activities were assessed by a chromium release assay, as previously described (18, 23). In brief, 51Cr-labeled target cells (5 x 103/well) were preincubated in 96-well plates for 15 min with 3- or 10-fold dilutions of peptide derivative in DMEM supplemented with 5% FCS and 10 mM HEPES. Cloned S15 CTL were added at the indicated E:T ratios, and after 4–6 h of incubation at 37°C, the 51Cr content of supernatants was determined. The specific lysis was calculated as 100 x [(experimental - spontaneous release)/(total - spontaneous release)]. In some experiments, mAb H35-17 (10 µg/ml) or SF1-1.1.1 Fab' (20 µg/ml) was added with the peptide.
IFN- assay
P815 target cells (5 x 103/well) were
sensitized with peptide derivatives in presence or absence of Abs, as
described for the chromium release assay. After incubation with cloned
S15 CTL (1 x 104/well) at 37°C for 24 h, the
content of IFN- in supernatants was determined by ELISA, using
anti-IFN- mAb R4-6A2 as the first and biotinylated
anti-IFN- mAb AN18 as the second Ab. Horseradish
peroxidase-conjugated streptavidin (Amersham, Arlington Heights, IL)
was used to detect the biotinylated Ab, followed by incubation with
o-phenylenediamine hydrochloride (Sigma). The color was
measured at 490 nm using an ELISA reader (MR7000; Dynatech
Laboratories, Chantilly, VA).
Calcium flux
Calcium flux was measured as previously described (16). In brief, S15 CTL were labeled with Indo-1-AM (Sigma) (2 mM, 1 x 106 cells/ml) at 37°C for 45 min. After one wash, CTL were mixed at an E:T ratio of 1:3 with P815 cells, previously pulsed with the indicated concentrations of IASA-YIPSAEK(ABA)I or IASA-YIPSAEK(ASA)I. After centrifugation for 1 min at 1500 x g and incubation at 37°C for 1 min, calcium-dependent fluorescence of Indo-1 was assessed by flow cytometry on a FACStar cytofluorometer (Becton Dickinson), by gating on a forward light scattering corresponding to conjugates. The total time of recording was 8 min. For experiments with Abs, calcium flux was first acquired for 2 min, the sample tube was removed, and H35-17 mAb and SF1-1.1.1 Fab' were added at 20 µg/ml. Then the calcium flux was reacquired for the remaining time.
FasL expression
To assess FasL message by reverse-transcriptase PCR, poly(A) mRNA was extracted from 107 S15 cells previously incubated for 4 h either without or with 1 µM of IASA-YIPSAEK(ABA)I or IASA-YIPSAEK(ASA)I, using the mini message maker kit from Ingenius (R&D Systems, Wiesbaden, Germany), according to the protocol of the manufacturer. cDNA was generated from poly(A) mRNA and the respective downstream primers using the first-strand cDNA synthesis kit from Pharmacia (Uppsala, Sweden). For the subsequent PCR, the following primers were used: for FasL, 5'-TAG CTG ACC TGT TGG ACC TTG C and 5'-CAC TCA AGG TCC ATC CCT CTG; and for actin, 5'-ATC AAG ATC CTG ACC GAG CG and 5'-TAC TTG CGC TCA GGA GGA GC. Thirty-five PCR cycles were performed at 94°C for 1 min, at 55°C for 1 min, and at 72°C for 2 min in a volume of 25 µl. PCR samples (10 µl) were analyzed on a 2% agarose gel. Alternatively, FasL protein expression was assessed by Western blot analysis. S15 CTL, pretreated the same way, were lysed in 2% Triton X-114, and the proteins of the lower phase were recovered by precipitation with CHCl3/MeOH (1:4 v/v). After separation by SDS-PAGE (10%, reducing conditions) and transfer onto nitrocellulose membrane, FasL was detected with PE62 anti-FasL Ab (28) and horseradish peroxidase-conjugated goat anti-rabbit Igs and revealed by chemoluminescence (ECL; Amersham).
TCR photoaffinity labeling
Photoaffinity labeling of soluble Kd molecules and
TCR with 125IASA-YIPSAEK(ABA)I or derivatives was performed
as described previously (24). In brief,
125IASA-YIPSAEK(ABA)I or 125IASA-YIPSAEK(ASA)I
(108 cpm/ml) was incubated with purified soluble
Kd (50 µg) in the presence of
ß2-microglobulin (2, 5 µg) at room temperature for
2 h. After UV irradiation at 350 nm for 20 s with 3000 W
UVA irradiator (Mutzhaas, Munich, Germany), the covalent
Kd-peptide derivative complexes were purified by
gel-filtration fast protein liquid chromatography. For TCR
photoaffinity labeling, S15 CTL (3 x 106 cells) were
incubated with soluble covalent monomeric Kd-peptide
derivative complexes (5–10 x 106 cpm) for 2 h
at 0°C in presence or absence of H35-17 mAb (10 µg/ml) or SF1-1.1.1
Fab' (20 µg/ml), followed by UV irradiation at 312 ± 40 nm for
25 s with a 90 W mercury fluorescence lamp (BioBlock Scientific,
Illkirch, France). TCR immunoprecipitation with mAb H57-597 and
SDS-PAGE analysis (10%, reducing conditions) were performed as
described (18, 23, 24). For quantification of covalent TCR-ligand
complexes, the dried gels were evaluated by phosphor imaging using a
PhosphorImager and Image Quant software (Molecular Dynamics, Sunnyvale,
CA). Mean values and SDs were calculated from at least three
independent experiments.
-chain and ZAP-70 phosphorylation
Phosphorylation of -chain was assessed as described
previously (16). Briefly, P815 cells (1 x 106/ml),
preincubated with peptide derivatives (1 µM) in presence or absence
of mAb H35-17 (10 µg/ml), or SF1-1.1.1 Fab' (20 µg/ml) or mAb
20-8-4S (10 µg/ml) for 15 min at 37°C, were added to S15 CTL
(2 x 107/ml). After centrifugation for 1 min at
1000 x g and incubation for 8 min at 37°C, cells
were lysed in 0.5 ml ice-cold lysis buffer (1% Triton X-100, 50 mM
Tris, pH 7.5, 150 mM NaCl, 10 mM EDTA, 1 mM PMSF, 10 µg/ml aprotinin,
and 10 mM Na3VO4). Detergent-insoluble material
was removed, and the supernatants were incubated with anti- mAb
H146 absorbed on protein A-Sepharose for 4 h at 4°C. The
immunoprecipitates were washed three times and subjected to SDS-PAGE
(15%, reducing conditions). After transfer on poly(vinylidene
difluoride) membranes (New England Nuclear) and immunoblotting
with antiphosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid,
NY), the blots were developed using the enhanced chemoluminescence
technique (ECL; Amersham). Phosphorylation of ZAP-70 was determined
likewise, except that for immunoprecipitation, polyclonal
anti-ZAP-70 Abs (Upstate Biotechnology) were used and for SDS-PAGE,
8% gels.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have observed previously that cloned S15 CTL only
inefficiently lysed target cells sensitized with IASA-YIPSAEK(ASA)I
(18). Further analysis now showed that this was also true for the
derivative that either had an additional iodine substituent
(IASA-YIPSAEK(IASA)I) or lacked the N-terminal IASA group
(YIPSAEK(ASA)I) (data not shown). To verify whether this
inefficient lysis was accounted for by loss of perforin-dependent
cytotoxicity, the 51Cr release assay shown in Fig. 1
was performed. S15 CTL efficiently
killed A20 target cells pulsed with IASA-YIPSAEK(ABA)I but
inefficiently lysed A20 cells pulsed with IASA-YIPSAEK(ASA)I (Fig. 1A). The same inefficient lysis of A20 cells was observed
for the wild-type peptide derivative in the presence of CMA, a drug
that specifically inhibits perforin-dependent cytolysis (31). Together
with the finding that CMA barely affected lysis of A20 cells pulsed
with K259(ASA), this indicates that the low lysis observed for the
variant epitope was not accounted for by perforin-dependent lysis.
Consistent with this, we observed that A20Fas- cells,
which can only be lysed by perforin-mediated lysis (29), were
efficiently lysed when pulsed with IASA-YIPSAEK(ABA)I but not when
pulsed with IASA-YIPSAEK(ASA)I (Fig. 1B). Again this lysis
was inhibited by CMA to near background levels. The same findings were
obtained when esterase release by S15 CTL was assessed, which is in
agreement with the fact that perforin-dependent lysis involves
exocytosis of CTL granules, which contain perforin and a mixture of
serine/threonine esterases (granzymes) (data not shown and 2 .
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C). Because P815 cells were more
efficiently lysed than A20 cells, a shorter incubation period (4
instead of 6 h) and a lower E:T ratio (1:4 instead of 1:20) were
used in this experiment. Since P815 cells were efficiently killed via
the perforin but not the Fas pathway, we repeated this experiment using
P815Fas+ cells that express high levels of Fas (28). These
cells were efficiently killed in the presence of either the wild-type
or the variant peptide derivative (Fig. 1D). Moreover, CMA
barely affected either lysis, indicating that P815Fas+,
unlike normal P815, were killed efficiently via the Fas pathway. These
results demonstrate that S15 CTL lysed IASA-YIPSAEK(ASA)I- pulsed A20
or P815 cells only via Fas-dependent cytotoxicity. This is consistent
with reports showing that certain altered peptide ligands can activate
Fas- but not perforin-dependent cytotoxicity (7, 17). Blocking of CD8 impairs perforin- but not Fas-dependent cytotoxicity by S15 CTL
In the presence of anti-CD8ß mAb H35-17 S15, CTL
inefficiently recognized IASA-YIPSAEK(ABA)I (18). To find out whether
this was accounted for by selective Fas-dependent killing, we performed
a chromium release assay using A20 and A20Fas- cells as
targets (Fig. 2). In the presence of mAb
H35-17, S15 CTL lysed A20 target cells sensitized with
IASA-YIPSAEK(ABA)I as inefficiently as they lysed A20 cells sensitized
with IASA-YIPSAEK(ASA)I (Fig. 2, A and B).
Since variant K259(ASA) was recognized by S15 CTL only in a
Fas-dependent manner (Fig. 1), this suggested that H35-17 mAb abolished
perforin-dependent cytotoxicity by S15 CTL. Consistent with this, we
observed that mAb H35-17 abolished perforin-dependent lysis of
IASA-YIPSAEK(ABA)I-sensitized A20Fas- targets (Fig. 2C). Conversely, this Ab had no significant effect on
Fas-dependent killing of A20 cells sensitized with K259(ASA) (Fig. 1B). The same results were obtained when H35-17 Fab' were
used (data not shown).
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3 mAb SF1-1.1.1
(SF1-1.1.1 Fab'), which block only participation of CD8 in TCR-ligand
binding but not CD8-mediated adhesion (25). This reagent inhibited
partially lysis of A20 cells sensitized with IASA-YIPSAEK(ABA) but
abrogated lysis of A20Fas- cells (Fig. 2, A and
C). The reason for this differential effect is not clear but
may be related to the more efficient killing of A20 as compared with
A20Fas- cells (half-maximal lysis of A20 cells required
about 100-fold lower concentrations of IASA-YIPSAEK(ABA)I) (Fig. 2, A and C). As mAb H35-17, SF1-1.1.1 Fab' had no
effect on the Fas-dependent killing of A20 cells sensitized with
IASA-YIPSAEK(ASA)I (Fig. 2A–C). Together these results
indicate that anti-CD8ß mAb H35-17 and less efficiently SF1-1.1.1
Fab' inhibited perforin-dependent cytotoxicity of S15 CTL but had no
effect on Fas-dependent mediated killing, i.e., had the same effect as
the epitope modification under study. The finding that SF1-1.1.1 Fab'
had a similar effect as mAb H35-17 suggests that for perforin- but not
Fas-dependent cytotoxicity, participation of CD8 in TCR-ligand binding
was important.
Epitope modification K259(ASA) or blocking of CD8 abolishes IFN-
production
We next examined what effect epitope modification and blocking of
CD8 have on IFN- production by S15 CTL. As shown in Fig. 3A, S15 CTL incubated with A20
in the presence of graded concentrations of IASA-YIPSAEK(ABA)I produced
IFN- in a dose-dependent manner. In contrast, no IFN- production
was detectable when variant K259(ASA) was used (Fig. 3B).
The IFN- response elicited by IASA-YIPSAEK(ABA)I was abolished by
mAb H35-17 and substantially diminished by SF1-1.1.1 Fab' (Fig. 3A). The same findings were obtained when P815 or
P815Fas+ cells were used as targets (data not shown). Thus,
in contrast to previous studies, in which either altered peptide
ligands or mutant CTL exhibited Fas-dependent killing and IFN-
response, but no perforin-dependent cytotoxicity (7, 8), only
Fas-dependent killing was preserved.
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A unique feature of our system is that TCR-ligand binding can be
assessed by TCR photoaffinity labeling (18, 23, 24). To this end,
soluble monovalent Kd was photo-cross-linked with
IASA-YIPSAEK(ABA)I and IASA-YIPSAEK(ASA)I, respectively, and
incubated with S15 CTL in the absence or presence of mAb H35-17
SF1-1.1.1 Fab'. As shown in Fig. 4, S15
TCR photoaffinity labeling by the variant ligand was approximately
ninefold less efficient as compared with the wild-type ligand. In the
presence of mAb H35-17 or SF1-1.1.1 Fab', S15 TCR photoaffinity
labeling by either ligand was reduced to background levels, observed in
the presence of anti-Kd1 mAb 20-8-4S, which blocks
specific TCR-ligand binding (Fig. 4 and 24 . Thus, either the
epitope modification or blocking of CD8 reduced the avidity of
TCR-ligand binding by 8-fold.
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-chain and ZAP-70
We next examined what effect the epitope modification and blocking
of CD8 had on phosphorylation of -chain and ZAP-70. To this end, S15
CTL were incubated in the absence or presence of mAb H35-17 or
SF1-1.1.1 Fab' with P815 cells, previously pulsed with
IASA-YIPSAEK(ABA)I and IASA-YIPSAEK(ASA)I, respectively. P815 rather
than A20 cells were used as targets, because they express higher levels
of Kd, and hence induce more extensive phosphorylations. As
shown in Fig. 5A, the
wild-type epitope elicited considerably more extensive -chain
phosphorylation than variant K259(ASA) (lanes 1 and
6), and the ratio between the pp23 and pp21 phospho- was
reduced by about 40% (Fig. 5B). The pp23 phospho- is
typically elicited by peptide agonists, and the pp21 -phospho by
partial agonists or antagonists (26, 32). While -chain
phosphorylation was barely detectable in the presence of
anti-Kd1 mAb 20-8-4S (lane 5),
weak -chain phosphorylation, mainly of the pp21 form, was observed
in the presence of mAb H35-17 or SF1-1.1.1 Fab'
(lanes 3 and 4). Remarkably, this
-chain phosphorylation was little increased as compared with the one
observed in the absence of antigenic peptide (lane
1). Similarly, significant phosphorylation of ZAP-70 on S15 CTL
was only observed following incubation with
IASA-YIPSAEK(ABA)I-pulsed P815 cells but not for the K(ASA) variant
or upon blocking of CD8 (Fig. 5C, lanes 3–5).
The phosphorylated ZAP-70 observed in case of the wild-type peptide was
associated with TCR/CD3, as seen by the coprecipitation of
phosphorylated CD3
and -chains (Fig. 5C, lane
3); thus, in this case, ZAP-70 was significantly recruited to
TCR/CD3 and activated by phosphorylation.
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-chain,
mainly of the pp23 phospho-form and recruitment, as well as activation
of ZAP-70. Epitope modification or blocking of CD8 impairs mobilization of [Ca2+]i
T cell activation typically provokes a rapid rise in
[Ca2+]i, which can be detected by
calcium-dependent fluorescence of Indo-1 and flow cytometry (16, 20).
As shown in Fig. 6B,
incubation of S15 CTL with P815 cells, previously pulsed with 1 nM
IASA-YIPSAEK(ABA)I, caused a rapid increase of
[Ca2+]i. This calcium level was stable during
the assayed 5 min, and was the same whether APC were pulsed with 1 or
100 nM IASA-YIPSAEK(ABA)I (data not shown). In contrast, S15 CTL
incubated with P815 cells pulsed with 100 nM IASA-YIPSAEK(ASA)I
exhibited [Ca2+]i levels just slightly above
background levels (Fig. 6C). A similar marginal increase in
[Ca2+]i was observed when S15 CTL were
incubated with IASA-YIPSAEK(ABA)I-pulsed P815 cells in the presence
of mAb H35-17, 20-8-4S, or SF1-1.1.1 Fab' (Fig. 6, D–F).
These results indicate that the epitope modification or blocking of CD8
resulted in a very small, yet significant, increase in
[Ca2+]i in S15 CTL.
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Overnight incubation of CTL with herbimycin A results in depletion
of p56lck in a dose-dependent manner (33).
As shown in Fig. 7A, gradual
depletion of p56lck severely impaired
perforin-dependent killing of P815 cells sensitized with
IASA-YIPSAEK(ABA)I by S15 CTL. Fifty percent inhibition was observed
when using 1 µM of herbimycin A. In contrast, S15 CTL preincubated
with 3 µM of herbimycin still efficiently killed P815Fas+
cells pulsed with IASA-YIPSAEK(ASA)I (Fig. 7B). This
Fas-dependent lysis was reduced by less than 60% when S15 CTL were
preincubated with 10 µM of herbimycin A, a treatment that reduced the
mainly perforin-dependent lysis of P815 cells by 85%. These findings
suggest that the perforin/granzyme-mediated cytotoxicity in this system
is more p56lck-dependent than the
Fas-dependent one. Since p56lck in part is
CD8 associated, this is consistent with the finding that blocking of
CD8 impaired perforin- but not Fas-dependent cytotoxicity (Fig. 2). It
must be noted, however, that herbimycin as well as other tyrosine
kinase inhibitors have limited specificity.
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To find out whether the Fas-dependent killing observed in this
study requires induction of FasL, we assessed FasL mRNA by
reverse-transcriptase PCR. As shown in Fig. 8A, nonactivated S15 CTL
constitutively expressed substantial levels of FasL message. Upon
incubation of S15 CTL with P815 cells pulsed with IASA-YIPSAEK(ABA)I,
FasL message slightly increased, and even less when IASA-YIPSAEK(ASA)I
was used. The messages for actin or Fas were similar for S15 CTL that
were either untreated or incubated with P815 cells in the presence of
either peptide. Essentially, the same findings were obtained when FasL
protein was assessed by Western blotting (Fig. 8B,
lanes 2, 4, and 5). This analysis also
showed that S15 CTL express significantly more FasL as compared with
P815 cells, and that this expression was not significantly increased
upon mixing the cells in either the absence or presence of either
peptide derivative. Similar findings were obtained when the incubation
period was increased from 4 to 12 h, or when S15 CTL were used 1
or 2 wk after restimulation (data not shown). These findings suggest
that S15 CTL have a pool of intracellular FasL, which upon activation,
is either translocated to the cell surface or released in soluble form.
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To find out whether the Fas-dependent killing observed in
this system was mediated by soluble or cell-associated FasL, we
performed a bystander cytolytic assay, in which IASA-YIPSAEK(ASA)I
was photo-cross-linked with Kd molecules on
P815Fas+ cells. In the experiment shown in Fig. 9A, 51Cr-labeled
sensitized targets were mixed with equal numbers of untreated
P815Fas+ cells and incubated with S15 CTL for 4 h. In
contrast to the high specific lysis observed in this experiment, no
lysis was detectable when the peptide was on the cold targets (Fig. 9B). The lack of bystander killing indicates that the
Fas-dependent killing of P815Fas+ cells required CTL/target
cell contact, i.e., did not involve soluble FasL, nor other soluble
cytotoxic molecules, such as TNF-.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF-, and various ILs, we described in this work a case in
which the only detectable CTL function was Fas-dependent cytotoxicity.
Selective activation of Fas-dependent killing has been observed
previously, either for altered peptide ligands on CD8+CTL
and CD4+Th clones or for mutant CTL clones (7, 8, 17, 18, 34). However, in contrast to these studies, we observed no cytokine
production, i.e., no IFN- response and, as deduced from the lack of
bystander killing, also no TNF- production (Figs. 3 and 9).
The lack of cytokine production and perforin-dependent killing observed
in our study is consistent with the barely detectable increase in
[Ca2+]i and the minimal -chain
phosphorylation, mainly of the pp21 form, as well as the lack of
significant phosphorylation of ZAP-70 (Figs. 5 and 6). While activation
of perforin-dependent cytotoxicity is very rapid and induced by brief
TCR engagement, induction of cytokine production requires sustained TCR
signaling; yet activation of both CTL functions relies on similar,
often overlapping, signaling pathways, which involve mobilization of
[Ca2+]i, significant -chain
phosphorylation, namely of the pp23 form, and recruitment and
phosphorylation/activation of ZAP-70 (3, 4, 5, 32). Consistent with this,
we observed that on closely related CTL clones, peptide modifications
generally affected perforin-mediated cytotoxicity and IFN-
production in good correlation, although occasional divergences can
occur (15, 16).
A main finding of the present study was that upon blocking of CD8, the
wild-type peptide exhibited the same selective Fas-dependent
cytotoxicity as observed for variant K259(ASA) (Fig. 2). Similar
results were obtained when anti-CD8ß mAb H35-17, its Fab'
fragments, or Fab' fragments of anti-Kd3 mAb
SF1-1.1.1 were used (Figs. 2 and 3, and unpublished results), which
have in common that they block coordinate binding of CD8 to
Kd molecules that interact with TCR (25). This blocking of
CD8 substantially decreased the avidity of TCR-ligand binding (Fig. 4).
A similar, although less dramatic reduction of TCR-ligand binding was
observed for the epitope modification (Fig. 4). Based on the
observation that blocking of CD8 or weakening of TCR-ligand binding by
peptide modification typically accelerates TCR-ligand complex
dissociation, one would expect this also to be the case in these
situations (16, 25, 26, 27). Since the coreceptor is associated with
p56lck, its coordinate binding to
TCR-associated MHC brings this src kinase to the CD3/
complex, which is hampered by acceleration of TCR-ligand complex
dissociation or blocking of the coreceptor (16, 25, 26). As this
tyrosine kinase plays an essential role in phosphorylation of CD3/
and ZAP-70, this may explain the faint -chain phosphorylation
observed upon blocking of CD8 or for the low affinity variant K259(ASA)
and the lack of significant recruitment and phosphorylation of ZAP-70
(Fig. 5). Together with the observation that depletion of
p56lck by herbimycin A little affected
Fas-dependent cytotoxicity (Fig. 7), these findings suggest that
p56lck, especially CD8-associated one, is
not important for activating S15 CTL for this cytotoxicity. This is in
accordance with reports showing that p56lck
and ZAP-70 are not required for induction of Fas-dependent apoptosis
and that minimal -chain phosphorylation can be sufficient for its
activation (14, 34).
Different immunoreceptor tyrosine-based activation motifs of CD3/
are clearly not identical in terms of initiating downstream signaling
cascades (35, 36). Importantly, it has been shown that induction of
Fas-dependent killing requires phosphorylation of the first but not the
second or third immunoreceptor tyrosine-based activation motif of
-chain (37). This is consistent with the finding that selective
activation of S15 CTL for Fas-dependent killing involved limited
-chain phosphorylation, mainly of the pp21 form, and that this
phosphorylation was different from the one elicited by peptide
antagonists, because variant K259(ASA) is not an antagonist for S15 CTL
(Fig. 5 and 18 . Conceivably, this form of -chain
phosphorylation is mediated either by
p56lck, which is not associated with CD8,
or by another tyrosine kinase, possibly
p59fyn.
Moreover, the finding that blocking of CD8 or the peptide variant
resulted in barely detectable increases in
[Ca2+]i suggests also that phosphorylation of
PLC-[ was impaired (Fig. 6). This lipase, upon activation by
phosphorylation, releases from membrane lipids inositol triphosphate
and diacylglycerol (5). While these secondary messengers have various
effects, inositol triphosphate stimulates mobilization of calcium from
intracellular stores (5). This is consistent with the observation that
activation of Fas-dependent killing by CTL requires limited influx of
extracellular but not mobilization of intracellular Ca2+
(38). The indifference of the selective Fas-dependent cytotoxicity
observed in this study to depletion of
p56lck correlates with the lack of ZAP-70
phosphorylation, which in turn correlates with the very marginal
increase in [Ca2+]i, i.e., activation of
PLC-, as both kinases are needed for its activation (
Figs. 5–7 and
Refs. 3–5).
It is interesting to note that in several systems there is a clear divergence between the activation requirements for induction of FasL expression and Fas-dependent cytotoxicity. While several studies have clearly demonstrated that p56lck and ZAP-70 are required for FasL induction (13, 39, 40, 42), other studies show that induction of Fas-dependent apoptosis does not, or minimally, require p56lck (14, 41). In agreement with our findings, Chung et al. observed that induction of TCR-induced cell death, in contrast to lymphokine production, is relatively lck independent and in poor correlation with FasL expression (42).
It is important to realize that Fas-dependent killing can be mediated
either by soluble or cell-associated FasL, and that for some cell
types, Fas-dependent killing requires FasL transcription, whereas other
cells, including macrophages, CTL, or melanocytes, have significant
amounts of intracellular FasL, which upon activation, can be
translocated to the plasma membrane (29, 43, 44). This latter pathway
seems to be realized in our system. On one hand, cloned S15 CTL clearly
killed via cell-associated and not soluble FasL (Fig. 9). On the other
hand, S15 CTL expressed constitutively a high level of FasL message and
protein that was little increased upon TCR triggering, especially in
the case of variant K259(ASA) (Fig. 8). The observation that S15 CTL,
which for propagation need to be stimulated periodically by Ag,
exhibited significant -chain phosphorylation, even after 2 wk of
stimulation, suggests that these cells are sufficiently activated to
express significant levels of FasL (Figs. 5 and 8).
Even though S15 CTL are very CD8 dependent, i.e., their
perforin-dependent cytotoxicity and IFN- production were effectively
blocked by anti-CD8 mAb, their ability to induce Fas-dependent
killing was not (Figs. 2 and 3, and 18 . Our study shows that
induction of Fas-dependent killing by CTL can occur even when CD8 is
blocked or by low affinity altered peptide ligands. The TCR signaling
required for this activation is extremely weak, and is unable to induce
significant IFN- or FasL production, and not even perforin-dependent
cytotoxicity or esterase release. This mechanism may allow eradication
of cells in the absence of apparently any other CTL function and may
play a role in eliminating cells expressing altered self proteins,
e.g., proteins that were modified in vivo by reactive chemicals but
also of cells expressing MHC class I molecules with defect CD8 binding.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. I. F. Luescher, Ludwig Institute for Cancer Research, Ch. des Boveresses 155, 1066 Epalinges, Switzerland. E-mail address:
3 Abbreviations used in this paper: PLC, phospholipase C; ABA, 4-azidobenzoic; ASA, 4-azidosalicylic acid; [Ca2+]i, intracellular calcium; CMA, concanamycin A; FasL, Fas ligand; IASA, iodo-4-azidosalicylic acid.
Received for publication May 15, 1998. Accepted for publication August 31, 1998.
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