From the aInstitute for Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland, the dLudwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland, cINSERM U563, CHU Purpan, 31059 Toulouse, France, the eKantonsspital, University of Basel, 4031 Basel, Switzerland, and the fCentre Pluridisciplinaire d'Oncologie, University of Lausanne Medical School, 1066 Epalinges, Switzerland
Received for publication, March 17, 2003 , and in revised form, April 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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Cloned CTLs are propagated by periodic re-stimulation, and hence are
activated T cells, which express high levels of LFA-1 and 1
and
3 integrins. The latter interact with extracellular
matrix (ECM) proteins, like fibronectin, vitronectin, and collagen, as well as
with counter receptors (e.g. vasular cell adhesion molecule) on other
cells
(1317).
Whereas LFA-1-mediated adhesion requires TCR triggering,
1
and
3 integrin-mediated adhesion of activated T cells,
although enhanced upon TCR triggering, also takes place spontaneously
(16,
18,
19). Therefore these integrins
can sense changes in the extracellular environment, e.g. when T cells
leave the vasculature and enter secondary lymphoid organs or inflamed tissues,
where they become strongly exposed to ECM proteins
(16). Integrin-mediated
adhesion to ECM proteins results in activation and recruitment at the contact
sites of the focal adhesion kinases FAK
(20,
21), Itk
(22), and Pyk2
(2330),
which promotes their association with the cytoskeleton linkers paxillin and
talin (20,
23,
30,
31) and the Src kinases Fyn
(29) and Lck
(28,
30). Pyk2 is translocated to
the T cell-target cell contact site after TCR triggering and plays an
important role in degranulation of CTLs and natural killer cells
(4,
32). Although the avidity and
the redistribution of integrins is promoted by TCR signaling, the contribution
of integrin-mediated signals to T cell activation is not well understood.
The availability of soluble recombinant MHC·peptide complexes triggered various studies aimed to elucidate the molecular basis of T cell activation (3337), which often reached diverging conclusions. For example, monomeric MHC·peptide complexes have been reported to activate CD8+ T cells by cross-linking of TCR and CD8 (35) or by transfer of peptide from soluble to cell-associate MHC molecules (38, 39). By contrast, other studies concluded that activation of CD8+ T cells requires multimeric MHC·peptide complexes and co-engagement of CD8 (33, 34, 37). These discrepancies suggest that activation of CD8+ T cells involves additional factors.
To elucidate these divergences and to define the minimal molecular requirements for the activation of perforin-dependent cytotoxicity, we studied the MHC·peptide-driven activation of cloned T1 CTLs and CD8+ T cells from T1 TCR transgenic mice. The T1 TCR recognizes the Plasmodium berghei circumsporozite (PbCS) peptide 252260 (SYIPSAEKI) conjugated with photoreactive 4-azidobenzoic acid on Lys-259 (PbCS(ABA)) in the context of Kd (40, 41). In the absence of cell adhesion, i.e. on CTLs in suspension, MHC·peptide complexes by co-engaging CD8 and TCR·CD3 promote the formation of raft-associated TCR·CD3-CD8/Lck adducts (34). Cross-linking of these adducts results in Lck activation, CD3 phosphorylation, recruitment and activation of ZAP-70, phosphorylation of LAT, and mobilization of intracellular calcium (34, 42).
Here we show that CTLs in suspension do not degranulate in response to
MHC·peptide complexes and that for this cell adhesion is required. The
1 and
3 integrin-mediated adhesion of CTLs
to ECM induces strong tyrosine phosphorylation and association of Pyk2 with
the cytoskeleton linker paxillin and the Src kinases Lck and Fyn. These
adhesion-induced, raft-associated molecular aggregates also contain
TCR·CD3 and are able to integrate and to amplify adhesion- and
TCR-mediated signals, thus promoting MHC·peptide-driven CTL
degranulation.
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EXPERIMENTAL PROCEDURES |
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The following antibodies were from Upstate Biotechnology (New York, NY):
anti-Pyk2 (polyclonal), anti-paxillin (5H11), anti-Lck (3A5),
anti-phosphotyrosine (4G10), and anti-ZAP-70 (polyclonal). Anti-Lck (2102),
anti-CD3 (M-20) were from Santa Cruz Biotechnology (Santa Cruz, CA).
Anti-LFA-1 (FD44.8), anti-
2 integrin (2E6), anti-CD8
mAbs H35.17 and KT112 were from American Type Culture Collection (ATCC)
(Manassas, VA). Anti-CD45 (clone 69), anti-CD29 (clone Ha 2/5), anti-CD3
(1452C11-PE), and anti-CD61 (clone 2C9.G2) were from BD Pharmingen (San
Diego, CA). Anti-fibronectin was from Molecular Probes (Eugene, OR). Anti-Fyn
(polyclonal) was from Dr. M. F. White (Harvard Medical School) linear GRGDS,
GRGES, and cyclic GRGDS peptides were from Bachem (Bubendorf, Switzerland).
Monomeric cholera toxin B subunit-peroxidase and -fluorescein isothiocyanate
conjugates were from Sigma (Buchs, Switzerland). Western blotting,
immunoprecipitations, and immunodetection were performed as described
(34) using the ECL detection
system (Amersham Biosciences, Little Chalfond, UK). Soluble monomeric and
tetrameric Kd·PbCS(ABA) complexes were prepared, as
described previously (34,
44). Cy5-labeled PbCS(ABA) was
obtained by reacting Fmoc
(N-(9-fluorenyl)methoxycarbonyl)-Dap-YIPSAEK(ABA)I in
Me2SO/dimethylformamide/diisopropylethylamine (5/4.5/0.5) with
Cy5-N-hydroxysuccinimide ester (5% molar excess) at room temperature
for 4 h. A 3-fold larger volume of piperidine (10% in dimethylformamide) was
added, and after 9 min of incubation the peptide derivative was precipitated
with diethylether purified by reversed-phase high performance liquid
chromatography and analyzed by mass spectrometry as described previously
(34).
FACS and FRETFor FACS and FRET analysis, CTLs clones were
stained at 4 °C in PBS containing 1% BSA and 0.1% sodium azide.
Cell-associated fluorescence was measured using a FACSCalibur (BD
Biosciences). For FRET, T1 CTLs were stained at 4 °C for 40 min with
Cy5-labeled anti-CD8 mAb KT112-Cy5 and PE-labeled anti-CD3
mAb
1452C11 in the absence or presence of 1 µM
Kd·PbCS(ABA), and FRET was measured by the Cy5 fluorescence
measured upon excitation of PE as described previously
(42).
Calcium Mobilization, Esterase Release, and in Vitro Kinase AssaysCTLs in serum-free DMEM were adhered for 30 min at 37 °C on polystyrene tissue culture plates or glass coverslips previously coated overnight at 4 °C with 2 µg/ml of superfibronectin (Sigma) and incubated at 37 °C for 5 min with 100 nM monomeric or 50 nM tetrameric Kd·PbCS(ABA) complexes or P815 cells pulsed with 10 nM PbCS(ABA) peptide. For calcium mobilization T cells were loaded with 5 µM fura-2/AM (Sigma) and adhered onto glass coverslips coated with fibronectin. After washing off non-adhered cells, calcium-dependent fura-2 fluorescence was measured on a Zeiss Axiovert microscope equipped with a charge-coupled device camera and a light monochromator (Princeton Instruments). The camera output was recorded and analyzed using the Metafluor calcium-imaging software (Universal Imaging Corp.). Alternatively, T cells were loaded with 5 µM indo-1/AM (Sigma) for 45 min at 37 °C and calcium-dependent indo-1 fluorescence was measured on a FACStar (BD Biosciences, Erembodegen, Belgium) as described (34).
For esterase release experiments, T1 CTLs, untreated or preincubated for 30 min at room temperature with the indicated anti-integrin antibodies (10 µg/ml) or the peptides GRGDS or GRGES (1.5 mg/ml) or cyclic GRGDS (60 µg/ml), were washed and incubated in DMEM for 90 min at 37 °C with P815, A20, or L-cells (effector/target of 1/1) previously sensitized for 120 min at 37 °C with the indicated concentrations of PbCS(ABA) peptide. For esterase release in response to soluble Kd·PbCS(ABA) complexes, T1 CTLs in serum-free DMEM (106 cells/ml) were either previously adhered to fibronectin-coated plates or kept in suspension in rotating polycarbonate vials. Released esterases were measured in the supernatants as described (36); 100% of esterase release refers to the value measured upon lysis of the cells in 1% Triton X-100.
For measurements of the kinase activity of CD8-associated Lck, T1 CTLs,
either adhered to fibronectin or in suspension, were incubated at 37 °C
for 2.5 min with monomeric (100 nM) or tetrameric (50
nM) soluble Kd·PbCS(ABA) complexes. After lysis
in n-octylglucoside (80 mM) CD8 was immunoprecipitated
with anti-CD8 mAb 53.6.72. The immunoprecipitates were incubated at 37
°C for 5 min with [32P]ATP and biotinylated peptide
corresponding to the immunoreceptor tyrosine-based activation motif c of
CD3
, and its phosphorylation was assessed as described previously
(34).
Adhesion AssayThe adhesion of T1 CTLs to immobilized
fibronectin was performed as described
(45). In brief, CTLs
pretreated or not for 30 min at 37 °C with 30 µM PP2
(Calbiochem, San Diego, CA), 100 µM cytochalasin D (Calbiochem),
100 µM piceatannol, or 10 mM
methyl--cyclodextrin (Sigma), were incubated in 96-well plates
(Polylabo, Illkirch, France) coated with fibronectin. The peptides GRGDS or
GRGES (1.5 mg/ml) were added at the beginning of the incubations. After
incubation for 30 min at 37 °C, adherent cells were stained with crystal
violet, and the optical density of their lysates was measured at 570 nm.
Isolation of Rafts and Confocal MicroscopyRafts were
isolated as previously described
(34), except that 0.2% Brij 96
(Fluka, Buchs, Switzerland) was used instead of 1% Triton X-100. Rafts were
solubilized in octyl--D-glucoside (50 mM)
containing EDTA (5 mM). The remaining insoluble material contained
none of the molecules under study, as judged by SDS-PAGE and Western blotting.
For confocal microscopy, T1 CTLs were adhered to fibronectin-coated Lab-Tek
chambered coverglass (Nalge Nunc, Naperville, IL), incubated for 10 min with
monomeric Kd·PbCS(ABA), washed twice with pre-warmed DMEM,
and fixed for 10 min at room temperature with 3% paraformaldehyde in PBS.
Alternatively, T1 CTLs were washed and fixed directly. CTLs were permeabilized
for 10 min at room temperature with 0.1% Brij 96, washed twice with PBS, and
blocked for 20 min with PBS containing 1% BSA or 2% gelatin for detection of
phosphotyrosine. Fixed cells were incubated with the different antibodies for
30 min at room temperature in the same buffer. Following three washes with
PBS, the cells were incubated with anti-mouse Alexa 488 or anti-rabbit Cy3
(Molecular Probes, Eugene, OR) and washed twice with PBS, and sections of the
cells parallel to the coverslip were analyzed on an LSM510 Zeiss confocal
microscope (Zeiss, Germany). For analysis of conjugates,
Kd-transfected L cells were pulsed with 0.1 µM
Dap(Cy5)-YIPSAE(ABA)I, washed, adhered to Lab-Tek chambered coverglass, and
conjugated for 15 min at 37 °C with T1 CTLs and then fixed, permeabilized
with 0.02% Triton X-100, and analyzed as described above. For co-localization
images were recorded in multitracking mode. Co-localization images were
obtained by selecting the pixels having 30100% intensities in each
channel using IMARIS co-localization software (bitplane, Zurich,
Switzerland).
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RESULTS |
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We next examined T1 CTLs degranulation, which reflects perforin-mediated cytotoxicity. T1 CTLs in suspension exhibited no esterase release upon incubation with monomeric and tetrameric Kd·PbCS(ABA) complexes. By contrast, T1 CTLs adhered to immobilized fibronectin efficiently degranulated in the presence of tetrameric and, slightly less efficiently, monomeric complexes (Fig. 2A). This response was abolished by cytochalasin D, the ZAP-70/Syk-specific inhibitor piceatannol (46), and by anti-CD8 mAb (Fig. 2C). No detectable esterase release was observed in response to irrelevant monomeric Kd·cw3 complexes when CTLs were adhered to immobilized fibronectin. Furthermore, T1 CTL degranulation induced by sensitized P815 cells was substantially stronger on fibronectin-adhered CTLs as compared with CTLs in suspension (Fig. 2B). Essentially the same findings were obtained for the related S14 CTLs clone (data not shown). Taken together these findings indicate that adhesion of T1 CTLs and T1 splenocytes to immobilized fibronectin promotes calcium flux and degranulation in response to soluble monomeric Kd·PbCS(ABA) complexes as well as recognition of sensitized target cells.
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Cloned CTLs are propagated by periodic re-stimulation and hence are
activated effector T cells, which express high levels of 1,
2, and
3 but not
7
integrins (Table I and data not
shown) (14,
24). The high expression of
1 and
3 integrins enables T1 CTLs to
spontaneously adhere to immobilized fibronectin
(Fig. 2D). This
adhesion was inhibited by the fibronectin-derived peptide GRGDS and its cyclic
variant, which selectively binds to
3 integrin
(47), as well as by
anti-
1 integrin antibody
(Fig. 2D). Nonspecific
adhesion of CTLs to immobilized BSA was 6-fold lower as compared with
fibronectin (data not shown). This adhesion was also inhibited by PP2,
cytochalasin D, and methyl-cyclodextrin, but not by piceatannol, indicating
that it requires src kinases activity, functional cytoskeleton, and lipid
rafts but not Zap-70/Syk or Syk kinase activity.
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CTLs Adhesion Induces Tyrosine Phosphorylation and Association of Pyk2 with Lck, Fyn, Paxillin, and TCR·CD3Upon adhesion of T1 CTLs to fibronectin, a dramatic increase in tyrosine phosphorylation of the focal adhesion kinase Pyk2, the cytoskeleton linker paxillin, and the src kinases Fyn and Lck was observed (Fig. 3, A and B). Because paxillin is a substrate for Pyk2 (23, 30, 48), its phosphorylation suggests that this CTL adhesion activates Pyk2. This adhesion also activates Fyn and Lck, which undergo autophosphorylation upon activation (49). By contrast, CTL adhesion caused no significant changes in tyrosine phosphorylation of ZAP-70, LAT, FAK, and CD3 (Fig. 3, A and B, and data not shown).
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Tyrosine-phosphorylated paxillin, Fyn, and Lck, were co-immunoprecipitated with Pyk2 from the lysate of adherent but not of non-adherent CTLs (Fig. 3C), indicating that, upon adhesion to fibronectin, these molecules associate with phosphorylated Pyk2. Similar findings have been reported for other systems (23, 2830). The scant co-precipitation of paxillin is most likely explained by its association with the cytoskeleton, i.e. was lost in the detergent-insoluble fraction. Importantly, CTL adhesion also promoted association of the TCR·CD3 complex with Pyk2; remarkably, however, without increasing phosphorylation of CD3. LAT also co-precipitated with Pyk2, but this was not induced by cell adhesion (Fig. 3C).
CTL Adhesion Induces Redistribution of TCR·CD3, CD8, Pyk2, and PaxillinBecause T cell activation involves redistribution of signaling molecules to lipid rafts (10, 11, 50), we examined what impact adhesion of T1 CTLs to fibronectin has on the distribution of TCR·CD3, CD8, Pyk2, and paxillin. In accordance with previous reports on cells in suspension, GM1 and Thy-1 were located predominantly in the detergent-insoluble rafts and CD45 in the detergent-soluble fractions (Fig. 4A and data not shown) (50, 51). CTL adhesion did not alter this distribution and did not change the distribution of Fyn and Lck. By contrast, the fraction of raft-associated TCR·CD3 increased substantially upon CTL adhesion. Moreover, Pyk2 and paxillin on cells in suspension were exclusively found in the detergent-soluble fraction but, upon adhesion, partitioned in rafts.
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Confocal microscopy provided further information on adhesion-induced redistribution of signaling molecules. Although TCR·CD3 lined the cell surface of T1 CTLs in suspension, it was mainly found in clusters in and near the adhesion zone of fibronectin-adhered T1 CTLs (Fig. 4B). The same adhesion-induced redistribution was observed for CD8 and Thy-1, except that these molecules were in small aggregates on CTLs in suspension, which most likely reflect rafts that contain these molecules (Fig. 4, A and B) (11, 34, 52). Pyk2 on cells in suspension was mainly cytosolic (Fig. 4A) (27) but, upon adhesion, was concentrated in bright patches in and near the adhesion zone. Finally, tyrosine-phosphorylated proteins on cells in suspension were evenly distributed at the cell membrane but, upon adhesion, were found in bright patches mainly at and near the contact site. Upon CTL adhesion, the amount of protein-tyrosine phosphorylation increased by about 4-fold, mostly in rafts (data not shown). Taken collectively these results indicate that CTL adhesion to immobilized fibronectin induces translocation of Pyk2, paxillin, and TCR·CD3 in rafts, visible as large aggregates at the adhesion site, where tyrosine phosphorylation mainly occurred.
Convergence of Adhesion- and TCR-mediated SignalsOn T1 CTLs
in suspension monomeric Kd·PbCS(ABA) complexes had no effect
on tyrosine phosphorylation (Fig.
5A). However, on fibronectin-adhered T1 CTLs they
increased the adhesion-induced phosphorylation of Lck/Fyn, paxillin, and Pyk2
and elicited phosphorylation of LAT, ZAP-70, and CD3. This is in
accordance with the finding that monomeric MHC·peptide complexes induce
intracellular calcium mobilization and esterase release on adherent, but not
on T cells, in suspension (Figs.
1 and
2). On T1 CTLs in suspension
tetrameric Kd·PbCS(ABA) complexes induced tyrosine
phosphorylation of Lck/Fyn, LAT, ZAP-70, and CD3
, and on adherent CTLs
the same phosphorylation was induced as for the monomeric complexes, but much
stronger, especially of paxillin and CD3
.
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Adhesion of T1 CTLs to immobilized fibronectin augmented the kinase activity of total Lck by about 2.3-fold (Fig. 5B). Upon incubation with monomeric Kd·PbCS(ABA) complexes, the kinase activity increased by about 1.6-fold in adherent CTLs, but remained unchanged on CTLs in suspension. Tetrameric Kd·PbCS(ABA) complexes caused a 2.7-fold increase in Lck kinase activity in adherent CTLs as compared with CTLs in suspension (Fig. 5B). Essentially the same changes in kinase activity were observed for CD8-associated Lck (data not shown).
As assessed by confocal microscopy, the tyrosine phosphorylation induced by Kd·PbCS(ABA) complexes on T1 CTLs in suspension occurred primarily at the cell membrane (Fig. 5C). By contrast, on adhered cells a dramatic increase in phosphorylation elicited by MHC·peptide complexes was observed throughout the cell with a maximal intensity in and near the adhesion zone (Fig. 5C, panel 2'). Taken together these findings indicate that MHC·peptide complexes and adhesion elicit tyrosine phosphorylation of various molecules and that their combination results in strong signal amplification at the contact zone.
Co-engagement of CD8 and TCR by MHC·peptide Monomers
Induces Co-aggregation of TCR, CD8, and Pyk2 in Adherent CTLsBased
on the observation that activation of T1 CTLs by soluble
Kd·PbCS(ABA) complexes requires that they co-engage CD8 and
TCR (Figs. 1 and
2), we examined whether they
induce proximity of CD8 and TCR. T1 CTLs stained in the cold with PE-labeled
anti-CD3 and Cy5-labeled anti-CD8 antibody exhibited substantial FRET
data when incubated with soluble Kd·PbCS(ABA) monomers but
not in their absence or presence of irrelevant Kd·Cw3
170179 complexes (Fig.
6A). Only background FRET was also observed in the
presence of D227KKd·PbCS(ABA) complexes, which are unable to
co-engage CD8 yet, at the high concentrations used (1 µM), bind
equally well to T1 CTLs (Ref.
40 and data not shown),
confirming that MHC·peptide induces proximity of CD8 and TCR. Similar
findings were obtained on CD8+ lymph node cells from TCR transgenic
mice but not on CD8-transfected T cell hybridomas, where CD8 association with
TCR is largely constitutive
(42,
52).
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Confocal microscopy showed that on fibronectin-adhered T1 CTLs, soluble Kd·PbCS(ABA) but much less D227KKd·PbCS(ABA) monomer induced co-localization of TCR and CD8 (Fig. 6B) similar as observed with non-adhered T1 CTLs (Fig. 6A). On adherent CTLs Kd·PbCS(ABA) monomer induced extensive co-localization of CD8 with Pyk2 and Thy-1 in aggregates at the adhesion site (Fig. 6C). Significantly less co-localization was observed in the presence of D227KKd·PbCS(ABA) complexes and almost none on adherent cells alone. Taken together these results demonstrate that Kd·PbCS(ABA) complexes induce proximity of TCR and CD8. Moreover they promote co-localization of TCR, CD8, Pyk2, and the raft marker Thy-1 in large clusters at the adhesion site. Because D227KKd·PbCS(ABA) complexes fail to do so, this implies a central role for CD8 in linking TCR- and adhesion-mediated activation events.
1 Integrin, Pyk2, and Paxillin Function in
Target Cell Recognition by CTLsWe next examined the role of
1and
3 integrins in the recognition of
sensitized target cells by T1 CTLs. The degranulation of T1 CTLs observed upon
incubation with PbCS(ABA) sensitized P815 cells was inhibited by about 50% by
anti-
1 integrin and nearly by 90% by anti-
2
integrin (LFA-1) antibody (Fig.
7A). Furthermore, the peptide GRGDS inhibited
degranulation by 70%, whereas the control peptide GRGES had no effect. The
cyclic GRGDS peptide, which selectively binds to
3 integrins
(47), caused only 15%
inhibition. Essentially the same findings were obtained when cloned S14 cells
were used as CTLs or A20 cells as targets (data not shown).
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As assessed by FACS, the surfaces of P815 mastocytoma, A20 B lymphoma
cells, L cells, macrophages, and splenic B cells, but not T cells, express
fibronectin (Table I). This is
consistent with the finding that most leukocytes secrete fibronectin and
retain it at the cell surface for processing before depositing it at the
extracellular matrix (53).
Moreover, T1 CTLs express the fibronectin-binding integrins 1
and
3 but not
5 and
7
(Table I and data not shown)
(14,
24). Taken together, this
implies that binding of
1 and
3 integrins of
the CTLs to cell-associated fibronectin on target cells greatly enhances
antigen recognition. Indeed, L-cells, which express no ICAM
(Table I), were well recognized
by T1 CTLs, and this response was greatly impaired by the GRGDS peptide and
anti-
1 integrin antibody but not affected by the GRGES
peptide (Fig. 7B).
Confocal microscopy of T1 CTLs conjugated with P815 cells sensitized with
PbCS(ABA) peptide showed in the contact site high enrichment of GM1, Pyk2, and
paxillin (Fig. 7C).
Co-localization of GM1 with Pyk2 and paxillin, respectively, were observed in
bright clusters at contact site. Moreover, on T1 CTLs conjugated with
Kd-transfected L-cells previously sensitized with Cy5-labeled
PbCS(ABA), paxillin and the peptide were strongly enriched at the contact zone
and co-localized with 1 integrins
(Fig. 7D). Taken
together these results demonstrate that
1 and
3 integrins, by interacting with fibronectin on target cells,
play an important role in target cell recognition and co-localize with Pyk2,
paxillin, and the antigenic peptide in the CTLs-target cell contact site,
similar to the role previously described for LFA1 and talin
(3,
5,
49,
54).
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DISCUSSION |
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It has been shown recently that soluble monomeric MHC·peptide complexes can activate CD8+ T cells by transfer of the peptide from soluble MHC to T cell-associated MHC molecules (38, 39). Although this mechanism does not account for our findings, because they were reproduced with covalent Kd·"IASA"-YIPSAEK(ABA) complexes (40, 55),2 these studies support the conclusion that activation of CD8+ T cells requires cell adhesion.
The role of 1 and
3 integrins in CTLs
function is 2-fold. First, adhesion of CTLs to immobilized ECM proteins, such
as fibronectin, provides co-stimulation for the recognition of sensitized
target cells and soluble MHC·peptide complexes (Figs.
1 and
2)
(13,
46). Second,
1 and
3 integrins are directly involved in
target cell recognition by CTLs (Fig.
7). We demonstrate that most cells express fibronectin at their
surface and that recognition of vastly different target cells (e.g.
P815 mastocytoma, A20 B lymphoma, and L cells) by CTLs is greatly impaired in
the presence of the RGD peptide or anti-
1 antibody
(Fig. 7 and
Table I). Similar observations
were made for other ECM proteins binding to
1 and
3 integrins
(17,
56). Although the
2 integrin LFA-1 plays an important role in antigen
recognition (3,
8), our findings indicate that
1 and
3 integrins play a hitherto
unappreciated important role as well. In particular, the ECM binding integrins
allow antigen recognition in the absence of LFA-1 or ICAMs
(Fig. 7)
(57,
58).
The use of soluble MHC·peptide complexes and spontaneous adhesion to
immobilized fibronectin allowed us to conclusively investigate
1/
3 integrin- and TCR/CD8-mediated signals
separately and how they elicit CTL degranulation when they are combined. The
hallmark of
1/
3 integrin-mediated adhesion
is the formation of tyrosine-phosphorylated molecular aggregates containing
Pyk2, Lck, Fyn, and paxillin in and near the cell adhesion zone (Figs.
3 and
4). The strong tyrosine
phosphorylation of Pyk2, Lck, and Fyn argues that adhesion activates these
tyrosine kinases. For Lck the increase in kinase activity was directly
assessed (Fig. 3), and the
activation of Pyk2 and Fyn is deduced from the phosphorylation of their
substrates, paxillin and Pyk2, respectively (Figs.
3A and
5A)
(23,
2830).
Several studies indicate that these complexes are raft-associated as follows.
1) They contain LAT, Fyn, Lck, and CD8, which are palmitoylated and partition
in rafts (Fig. 4)
(10,
11,
34,
42,
52,
59). 2) CTL adhesion induced
translocation of Pyk2 and paxillin to rafts
(Fig. 4). 3) The strong
tyrosine phosphorylation of paxillin, Pyk2, Lck, and Fyn is indicative for
localization in raft, where phosphatases are excluded (Figs.
3 and
4)
(1,
11,
60). Moreover, because
1 integrins, as well as phosphorylated paxillin and Lck,
associate with the cytoskeleton, these rafts are cytoskeleton-associated
(20,
23,
31).
Conversely, on CTLs in suspension soluble MHC·peptide complexes by
co-engaging TCR and CD8 promote their association
(Fig. 5)
(42,
61). Cross-linking of the
resulting TCR·CD3-CD8/Lck adducts resulted in Lck activation and
phosphorylation of CD3 (Fig.
5A) (34).
ZAP-70 is then recruited to phosphorylated CD3 and, upon activation by Lck,
phosphorylates LAT (Fig. 5, A and
B) (11,
62). Phosphorylated LAT in
turn interacts with various adaptors and signaling molecules such as Vav,
SLP-76, PLC, Grb2, and SOS, which are involved in various downstream
signaling events, including actin polymerization and mobilization of
intracellular calcium (12,
6365).
Because the ZAP-70/Syk-specific inhibitor piceatannol
(46) had no effect on CTL
adhesion (Fig. 2D),
but blocked the activation of adherent CTLs by MHC·peptide complexes
(Fig. 2C), the
recruitment and phosphorylation of ZAP-70 and in turn of LAT is induced by
MHC·peptide complexes and not by cell adhesion. Thus, although CTL
adhesion elicited strong tyrosine phosphorylation of Pyk2, Lck, Fyn, and
paxillin, phosphorylation of TCR·CD3, ZAP-70, and LAT was induced only
by MHC·peptide complexes (Figs.
3 and
5). However, TCR·CD8
triggering by MHC·peptide and
1/
3
integrin-mediated adhesion elicited clearly different activation events, and,
when combined, they provide the powerful signaling, resulting in sustained
calcium flux and CTL degranulation (Figs.
1 and
2).
What is the molecular basis for this signal integration and amplification?
Our finding that Pyk2 associates with TCR·CD3 and LAT
(Fig. 3) probably explains the
dramatic changes in TCR signaling observed upon 1 and
3 integrin-mediated CTL adhesion (Figs.
1,
2, and
5). For example, in view of the
importance of Lck activation in CTL activation driven antigen-specifically, it
is interesting to note that adhesion of CTLs to fibronectin more strongly
activates Lck than does the MHC·peptide on CTLs in suspension (Figs.
3 and
5)
(34). However, even though in
adherent CTLs TCR·CD3 is part of the adhesion-induced molecular
aggregates, adhesion-activated Lck only becomes effective upon cross-linking
of TCR and CD8 by MHC·peptide (Figs.
5 and
6). Thus CD8 plays a central
role in converging of adhesion and TCR-mediated signals, namely by bringing
adhesion-activated Lck to TCR·CD3.
Furthermore, adhesion of CTLs also efficiently activates Fyn
(Fig. 3). Because Fyn
associates with Pyk2 (29),
this probably explains the strong tyrosine phosphorylation of Pyk2 (Figs.
3 and
5). Pyk2 in cytotoxic cells is
recruited to the contact site with target cells and, upon activation, plays a
critical role in the re-orientation of the microtubule-organizing center
(27,
66). Consistent with this is
the finding that, in degranulating CTLs, Pyk2 and its cytoskeleton linker
paxillin (Figs. 4 and
6) are focused in the contact
site with target cells, irrespective of whether these cells express ICAM or
not (Fig. 7). In addition, Fyn
associates with the Fyn-binding protein Fyb, also known as SLAP-130
(SLP-76-associated phosphoprotein) or ADAP (adhesion and degranulation adaptor
protein), which regulates 1/
3
integrin-mediated adhesion and their cross-talk with the TCR
(6769).
In conclusion, the present study shows that 1 and
3 integrins play an important role in the function of CTLs,
both in sensing changes in the extracellular environment and in target cell
recognition. These ECM-binding integrins directly implicate Pyk2, which is
important for CTL degranulation. This focal adhesion kinase is involved in
re-localizing the microtubule-organizing center and, together with paxillin
and Fyn, forms cytoskeleton and raft-associated molecular aggregates. Such
aggregates, including TCR·CD3, CD8, and LAT, are capable of integrating
and amplifying adhesion and MHC·peptide-mediated signals, thus
eliciting CTL effector functions.
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FOOTNOTES |
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g Supported by grants from La Ligue contre le Cancer, l'Association de la
Recherche contre le Cancer, and la Fondation pour la Recherche
Médicale.
h Supported by a grant of the Gabriella Giorgi-Cavaglieri Foundation and by
the Swiss National Science Foundation (Grant 31-061960.00).
i Supported by the Swiss National Science Foundation (Grant
3100-061946.00).
b To whom correspondence should be addressed. Tel.: 41-21-692-5745; Fax: 41-21-692-5705; E-mail: ma_doucey{at}hotmail.com.
1 The abbreviations used are: CTLs, cytotoxic T lymphocytes; TCR, T cell
receptor; MHC, major histocompatibility complex; APCs, antigen-presenting
cells; LFA-1, lymphocyte function associated antigen-1; ICAMs, intracellular
adhesion molecule; ECM, extracellular matrix; PbCS, P. berghei
circumsporozite; ABA, 4-azidobenzoic acid; DMEM, Dulbecco's modified Eagle's
medium; FACS, fluorescence-activated cell sorting; mAb, monoclonal antibody;
FRET, fluorescence resonance energy transfer; PBS, phosphate-buffered saline;
BSA, bovine serum albumin; pY, phosphotyrosine; PP2,
4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine;
PE, phycoerythrin; GM1,
Gal1,3GalNac
1,4NeuAc
2,3Gal
1,4Glc-ceramide.
2 M.-A. Doucey, D. F. Legler, M. Faroudi, N. Boucheron, P. Baumgaertner, D.
Naeher, M. Cebecauer, D. Hudrisier, C. Rüegg, E. Palmer, S. Valitutti, C.
Bron, and I. F. Luescher, unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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