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*
Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, Epalinges, Switzerland;
Centre d’Immunologie, Institut National Scientifique et Recherche Medical-Centre National de Research Scientifique de Marseille-Luminy, Marseille, France; and
Basel Institute for Immunology, Basel, Switzerland
<. >
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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ß a more efficient coreceptor than homodimeric CD8, we
used various CD8 transfectants of T1.4 T cell hybridomas, which are
specific for H-2Kd, and a photoreactive derivative of the
Plasmodium berghei circumsporozoite
peptide PbCS 252–260 (SYIPSAEKI). We demonstrate that CD8 is
palmitoylated at the cytoplasmic tail of CD8ß and that this allows
partitioning of CD8ß, but not of CD8, in lipid rafts.
Localization of CD8 in rafts is crucial for its coreceptor function.
First, association of CD8 with the src kinase
p56lck takes place nearly exclusively in rafts,
mainly due to increased concentration of both components in this
compartment. Deletion of the cytoplasmic domain of CD8ß abrogated
localization of CD8 in rafts and association with
p56lck. Second, CD8-mediated cross-linking of
p56lck by multimeric Kd-peptide
complexes or by anti-CD8 Ab results in
p56lck activation in rafts, from which the
abundant phosphatase CD45 is excluded. Third, CD8-associated activated
p56lck phosphorylates CD3
in rafts and hence
induces TCR signaling and T cell activation. This study shows that
palmitoylation of CD8ß is required for efficient CD8 coreceptor
function, mainly because it dramatically increases CD8 association with
p56lck and CD8-mediated activation of
p56lck in lipid rafts. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and CD8ß chains, which are transmembrane proteins, containing
N-terminal Ig domains, extended and glycosylated hinge and stalk
regions, and transmembrane and cytoplasmic portions (1, 2). In contrast, NK cells or intestinal T cells express
homodimeric CD8, composed of disulfide-linked CD8 (1, 2). The Ig domain of CD8 interacts with the constant domain of
MHC class I molecules (1, 2), and the cytoplasmic tail of
CD8 can associate with the src kinase p56lck
(lck),3 by means of a
zinc cation, chelating vicinal cysteines on both molecules (3, 4). Therefore, this association is disrupted by EDTA or
iodoacetamide. The 19-residue-long cytoplasmic tail of CD8ß is not
known to interact with other molecules but has been reported to
increase CD8 association with lck (5, 6).
Several reports have shown that CD8ß is a much more
effective coreceptor than CD8. Cells expressing CD8ß are
able to recognize Ag at considerably lower concentrations than cells
expressing CD8 (7). This difference is particularly
striking for low affinity altered peptide ligands. Moreover, disruption
of the CD8ß gene or deletion of the cytoplasmic tail of CD8ß
results in severe reduction of positive selection of
CD8+ T cells (8, 9, 10, 11). The molecular
basis for these dramatic differences, in particular the role of CD8ß
in CD8 coreceptor function, remains enigmatic. Using TCR photoaffinity
labeling with soluble monomeric Kd-peptide
complexes, we have shown that on cells, CD8ß, by coordinate
binding to TCR-associated Kd molecules,
substantially increases TCR ligand binding (12). Similar
findings have been obtained using soluble multimeric MHC-peptide
complexes and flow cytometry (13). Surprisingly, however,
neither CD8 nor soluble CD8ß increase TCR ligand binding,
even though they bind to MHC class I molecules with similar affinities
(12, 14, 15). Another aspect of the role of CD8ß in CD8
coreceptor function emerged from studies by Hoeveler and Malissen
(4) and Irie et al. (5) showing that
CD8ß associates more efficiently with lck and induces higher lck
activation on cross-linking with anti-CD8 Ab, as compared with
CD8 or CD8ß lacking the cytoplasmic tail of CD8ß.
It is well established that lck plays a crucial role in T cell
activation. On one hand, on activation, it phosphorylates
immunoreceptor tyrosine-based activation motifs (ITAM) of CD3, which
permits recruitment of src homology domain 2 (SH2)-containing
molecules, such as ZAP-70, Syk, and p59fyn
(fyn), (16, 17). lck-mediated phosphorylation of
ITAM-associated kinases, e.g., ZAP-70, results in their activation
(16, 17). On the other hand, lck can interact with various
other signaling molecules via its SH2 and SH3 domains and thus support
their recruitment to activated TCR/CD3 (18). For example,
lck can bind via its SH2 domain to phosphorylated, CD3-associated
ZAP-70 and thus couple the coreceptor with the TCR (19).
Conversely, CD8 can associate with lck, as well as with the linker of
activation of T cells (LAT) (20). Thus, coordinate binding
of CD8 to TCR-associated MHC molecules brings these molecules to
TCR/CD3. LAT, on phosphorylation by ZAP-70, recruits a variety of
adapter and signaling molecules to TCR/CD3 and hence links initial TCR
activation to diverse downstream signaling cascades
(21).
The recognition that cell membranes are compartmentalized, i.e.,
contain detergent-insoluble lipid rafts, has important implications for
cell activation studies. Rafts, also called detergent-insoluble
microdomains (DIM), detergent-insoluble glycolipid complex (DIG),
detergent-resistant membranes (DRM), or glycolipid-enriched membrane
domains (GEM) are formed primarily by cholesterol and sphingolipids,
which fail to integrate well in fluid phospholipid bilayers and hence
form microdomains (22). Rafts are stabilized by short
saturated fatty acids (e.g., palmitic and myristic acid), which
intercalate in spaces between bulky raft lipids. In the outer leaflet,
this is accomplished by integration of
glycosylphosphatidylinositol-linked proteins (e.g., Thy-1 and CD59)
(22), whereas palmitoylated and/or myristoylated proteins
(e.g., lck, fyn, and LAT) play the same role in the inner leaflet
(22). Other molecules, such as the abundant phosphatase
CD45, however, are excluded from DIM (23, 24). Thus, by
concentrating kinases and their substrates and excluding phosphatases,
rafts permit efficient phosphorylation reactions to proceed and hence
play a crucial role in TCR signaling. Importantly, on T cell activation
with anti-CD3 Abs, TCR/CD3 translocates from phospholipid fraction
to DIM (25, 26, 27, 28). Other signaling molecules (e.g., ZAP-70,
Syk, phospholipase C
) and adapters (e.g., Grb2 and Vav) follow this
trend, at least in part due to activation-induced molecular
interactions (25, 26, 27, 28).
In the present study, we examined the roles of CD8ß and CD8
in TCR signaling in lipid rafts. To this end, we used T1.4 T cell
hybridomas, which were obtained by fusing the T1 CTL clone with
TCR-BW5147 cells (29). The T1 TCR recognizes the
Plasmodium berghei circumsporozoite (PbCS) peptide 252–260
(SYIPSAEKI) containing photoreactive iodo-4-azidosalicylic acid (IASA)
in place of PbCS S252 and photoreactive 4-azidobenzoic acid (ABA) on
PbCS K259 in the context of Kd (30).
Selective photoactivation of IASA permits photo-cross-linking of the
peptide derivative to Kd and photoactivation of
ABA to TCR (30). However, the IASA, unlike the ABA group,
is not a part of the epitope and can be replaced by serine, which
increases peptide binding to Kd, but not Ag
recognition. We therefore refer to this peptide derivative as
PbCS(ABA). Moreover, various peptide derivative variants have been
tested previously on T1 and related CTL clones for Ag recognition, TCR
ligand binding, and TCR-ligand complex dissociation (31).
These studies showed, for example, that replacement of PbCS P255 with
serine (P255S) results in a low affinity peptide variant that is
inefficiently recognized by CTL, especially when CD8 is blocked
(31).
Here we show that CD8 is palmitoylated at the cytoplasmic tail of
CD8ß and therefore, unlike CD8, partitions in lipid rafts.
Association of CD8 with lck, which is also palmitoylated and partitions
in rafts, was greatly increased in this compartment. In addition,
CD8-mediated cross-linking of lck by Kd-PbCS(ABA)
complexes, or anti-CD8 Ab, resulted in substantial lck activation
in rafts and, in turn, in CD3 phosphorylation and induction of TCR
signaling. Thus, palmitoylation of the cytoplasmic tail of CD8ß
endows CD8ß with efficient coreceptor functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hybridomas expressing mAbs were from American Type Culture
Collection (Manassas, VA). Anti-CD8 mAb 53.6.72 and 19/178,
anti-CD8ß mAb H35-17, anti-CD3
145.2C11, and
anti-CD3 mAb HAM146 were purified from hybridoma supernatants by
affinity chromatography on protein G-Sepharose CL-4B (Amersham
Pharmacia Biotech, Piscataway, NJ). Anti-TCR Cß mAb H57 was purified
from hybridoma supernatants by affinity chromatography on protein
A-Sepharose CL-4B (Amersham Pharmacia Biotech). Anti-phosphotyrosine
mAb (clone 4G10) and anti-p56lck (clone 3A5)
were from Upstate Biologicals (Lake Placid, NY). Polyclonal
anti-p56lck was from Santa Cruz
Biotechnology (Santa Cruz, CA). Anti-LAT polyclonal antiserum was a
gift from Professor Dr. C. Bron (Institute of Biochemistry, University
of Lausanne, Lausanne, Switzerland).
Soluble H-2Kd-peptide complexes
Biotinylated H-2Kd/ß2 m/SYIPSAEK(ABA)I complexes were expressed and purified following standard protocols (32). Multimerization was achieved by reaction with PE-labeled streptavidin (Molecular Probes, Eugene, OR) or avidin (Molecular Probes) at a 4:1 molar ratio. Tetramers were purified by gel filtration chromatography on a Superdex 200 column.
Cell culture
Cells were maintained at 37°C in a humidified atmosphere
containing 5% CO2. T cell hybridomas were
cultured in DMEM (Life Technologies, Gaithersburg, MD) supplemented
with 5% (v/v) heat-inactivated FCS (HIFCS, Life Technologies),
penicillin-streptomycin-neomycin (PSN, Life Technologies), and 2-ME.
The T1.4.1 ß and T1.4.1 'ß ( tailless) and T1.4.1 ''ß
(CD8 lck binding site mutant) transfectants (33) were
cultured in the presence of 2 mg/ml G418 (Life Technologies). T1.4.2
transfectants were cultured in the presence of 2 mM histidinol
(Sigma), whereas T1.4.2 ß and T1.4.2 ß' (ß tailless)
transfectants were cultured in DMEM containing 1.5% (v/v) HIFCS
supplemented with 2 mM histidinol and 3 µg/ml puromycin (Calbiochem,
La Jolla, CA). P815 cells were maintained in DMEM supplemented with 5%
(v/v) HIFCS and PSN.
Molecular biology
cDNAs coding for the wild-type CD8 gene, for the naturally
occurring tailless CD8' isoform (34), and for a mutant
CD8 chain (CD8'') in which the cysteine residues at positions 200
and 202 (p56lck binding site) have been mutated
to alanine (4) were cloned into the expression vector
pHbAPr-1 neo.
The CD8ß cDNA in the LXSH vector was mutated at position 178 (TAC (Tyr)->TAG (Stop)) by site-directed mutagenesis using the Quick-Change Mutagenesis Kit (Stratagene, La Jolla, CA). The open reading frame of the resulting construct encoding CD8ß tailless (ß') was checked by sequencing both strands. All sequencing was performed at Microsynth (Balgach, Switzerland). The construct was then subcloned into the LXSP vector and used to transfect the BOSC 23 packaging cell line (35).
Transfection and infection
Transfections of T1.4 hybridomas with protoplasts and selection
in the presence of G418 were performed as described (36)
to generate the T1.4.1 ß, T1.4.1 'ß, and T1.4.1 ''ß cell
lines. Alternatively, a clone that lost CD8ß surface expression was
obtained (T1.4.2 ) from T1.4 hybridoma transfected with CD8.
To obtain the T1.4.2 ß and T1.4.2 ß' clones, BOSC 23
packaging cells were transfected with the CD8ß or CD8ß' constructs
in LXSP using a standard calcium-phosphate protocol (35).
After transfection, the retroviral supernatant was used to infect
T1.4.2 hybridomas. Puromycin-resistant populations were sorted
for CD8ß expression by FACS.
Flow cytometry
T cell hybridomas (5 x 105) were stained with H-2Kd/ß2m/SYIPSAEK(ABA)I/PE-labeled streptavidin tetramers in 50 µl FACS buffer (Optimem supplemented with 0.5% (w/v) BSA and 0.02% (w/v) NaN3) for 1 h at 26°C. The cell-associated fluorescence was measured using a FACScaliber (Becton Dickinson, Mountain View, CA).
Calcium measurements
P815 cells (106/ml) were loaded with varying concentrations of IASA-YIPSAEK(ABA)I or of IASA-YISSAEK(ABA)I. The peptide was photo-cross-linked with Kd by UV irradiation as described (30, 31). T cell hybridomas (106/ml in DMEM) were incubated with 5 µM Indo-1/AM at 37°C for 45 min, washed in DMEM, and resuspended at 106 cells/ml. The hybridomas were then mixed with P815 cells at an E:T ratio of 1:3, sedimented by centrifugation at 1,200 x g for 2 min, and incubated at 37°C for 1 min. Intracellular calcium mobilization was measured on a FACStar cytofluorometer (Becton Dickinson).
Metabolic labeling
T1.4.1 ß hybridomas (107) were
labeled with [3H]palmitic acid (0.5 mCi/ml,
NEN, Boston, MA) as described (37). The cells were lysed
in 1 ml lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1%
(w/v) Brij 96, 1 mM PMSF, 10 µg/ml leupeptin, 10 µM pepstatin A)
for 60 min on ice. After centrifugation for 30 min at 12,000 x
g and 4°C, the supernatants were incubated with 10 µg/ml
anti-CD8 mAb 19/178, anti-CD8ß mAb H35-17, or anti-LAT
antiserum and protein G-Sepharose. The immunoprecipitates were then
analyzed by SDS-PAGE and fluorography.
Isolation of DIM, immunoprecipitation, and Western blotting
T cell hybridomas were lysed in 1% (w/v) cold Triton X-100 and
fractionated by ultracentrifugation on discontinuous sucrose gradients,
as described (37). After centrifugation, 400-µl
fractions were collected from the top of the gradients and analyzed by
SDS-PAGE and Western blotting. Surface biotinylation was performed as
described (26). After sucrose gradient fractionation, the
fractions were immunoprecipitated with 10 µg/ml anti-CD8 mAb
19/178 or anti-CD8ß mAb H35-17 and protein G-Sepharose. The
immunoprecipitates were analyzed by SDS-PAGE and immunoblotted with
streptavidin-HRP (Life Technologies). Alternatively, T cell hybridomas
(5 x 107) were lysed in 1 ml lysis buffer
(50 mM HEPES (pH 7.4), 150 mM NaCl, 1% (w/v) Triton X-100, 1 mM
Na3VO4, 50 µM
(4-amidinophenyl)methanesulfonyl fluoride (Boehringer Mannheim,
Mannheim, Germany), 10 µg/ml leupeptin, 10 µM pepstatin A, 2
µg/ml aprotinin) for 30 min on ice. The samples were centrifuged at
100,000 x g for 60 min at 4°C, and the supernatant
was collected (membrane (M) fraction). The pellet was homogenized in 1
ml Brij lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1% (w/v) Brij
96 (Fluka, Buchs, Switzerland), and the same inhibitors) and incubated
for 60 min on ice. After centrifugation for 20 min at 12,000 x
g and 4°C, the supernatant was collected (DIM fraction).
Membrane and DIM fractions were immunoprecipitated with anti-CD8
mAb 53.6.72 or H35-17 (20 µg) coupled to Sepharose. The samples were
analyzed by SDS-PAGE and Western blotting with
anti-p56lck Abs.
CD8-lck association and protein kinase assays
For protein kinase assays, T cell hybridomas (1 x
107) were resuspended in 1 ml DMEM, 5% (v/v)
HIFCS, and PSN and incubated with 500 ng/ml anti-CD8 mAb 53.6.72
for 10 min on ice. A secondary anti-mouse IgG Ab (10 µg/ml,
Sigma, St. Louis, MO) was then added for 10 min, and the cells were
subsequently incubated for 3 min at 37°C. Alternatively, the cells
were incubated for 60 min at 4°C with 25 µg/ml monomeric
H-2Kd-SYIPSAEK(ABA)I, UV irradiated for 90 s
at 350 nm, and washed. Monoclonal antibiotin (1 µg/ml, Sigma) was
added, followed by anti-IgG Ab as above, and the cells were
subsequently incubated for 3 min at 37°C. The samples were then
placed on ice, washed twice with 1x PBS containing 1 mM
Na3VO4, and lysed in Brij
96 lysis buffer as above. After centrifugation, the supernatants were
immunoprecipitated as indicated with protein G-Sepharose or
anti-lck Ab. The immunoprecipitates were washed twice in lysis
buffer and once in kinase buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 10
mM MnCl2, 10 mM MgCl2,
0.1% (w/v) Brij 96, 0.1 mM
Na3VO4, 10 µg/ml
leupeptin, 10 µg/ml aprotinin), resuspended in 100 µl kinase buffer
containing 100 µM ATP, 10 µCi [-32P]ATP
(3000 Ci/mmol, NEN), and 10 µg/ml biotinylated CD3 c-ITAM peptide
(38) and incubated for 5 min at 37°C. After
centrifugation, the supernatant was collected, and the beads were
washed twice with lysis buffer. The supernatants were pooled and
immunoprecipitated with streptavidin-agarose (Pierce, Rockford, IL).
After washing, the radioactivity bound to the immunoprecipitates was
measured in a beta counter.
Lck and CD3 phosphorylation in tetramer-stimulated cells
T1.4 or T1.4.1 ß hybridomas (5 x
107) were incubated under agitation at
107/ml in DMEM (0.5% HIFCS, PSN, and 2-ME) with
either 25 µg/ml Kd-SYIPSAEK(ABA)I tetramers or
1 µg/ml anti-CD3 145.2C11 mAb, followed by anti-IgG (10
µg/ml) for 2 h at 4°C. The cells were then washed once in
medium, resuspended at 107/ml, and incubated at
37°C for 3 min. The samples were then placed on ice and lysed in 1%
(w/v) Triton X-100, and Triton-soluble (M fraction) and -insoluble (DIM
fraction) fractions were prepared essentially as described
(27). M and DIM fractions were immunoprecipitated with
anti-CD3 HAM146 (20 µg/ml) or
anti-p56lck polyclonal antiserum (5 µg/ml)
and protein G-Sepharose. After two washes with the respective lysis
buffers, the samples were analyzed by SDS-PAGE and Western blotting
with anti-phosphotyrosine, anti-p56lck
or anti-CD3 Abs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß are readily activated
by cognate Kd-PbCS(ABA) complexes
To investigate the role of CD8ß in T cell activation,
CD8- T1.4 T cell hybridomas were transfected
with CD8 and CD8ß genes. The resulting transfectant T1.4.1 ß
expressed homodimeric CD8, and to a lesser degree heterodimeric
CD8ß (Table I
). When incubated with
P815 cells, pulsed with graded concentrations of PbCS(ABA), T1.4.1
ß cells exhibited high intracellular calcium mobilization (Fig. 1A), which was sustained for
the 10 min of recording. In the presence of anti-CD8ß mAb H35-17,
this calcium mobilization was reduced nearly to basal levels (Fig. 1B). Because T1.4.1 ß cells express high levels of
CD8, which does not bind H35-17 mAb, this implies that CD8,
unlike CD8ß, essentially fails to promote efficient calcium flux.
Pretreatment of T1.4.1 ß cells with 2-bromopalmitate, an inhibitor
of protein palmitoylation (39), reduced the calcium flux
to basal levels in most, but not all cells (Fig. 1C). At
higher concentrations of 2-bromopalmitate, this fraction decreased, but
also the cell viability (data not shown).
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, D–F). In contrast, CD8- T1.4 cells
exhibited only a marginal calcium flux on incubation with
PbCS(ABA)-pulsed APC (Fig. 1G). Moreover, this calcium flux
was transient, i.e., decreased gradually after 5 min of incubation. No
increase in intracellular calcium concentration was observed when the
peptide variant P255 was used (Fig. 1H). Similarly,
incubation of T1.4.1 ß cells with soluble
Kd-PbCS(ABA) tetramers elicited intracellular
calcium mobilization (Fig. 2A). However, in contrast to
the previous experiment (Fig. 1A), this calcium flux was
transient and gradually decreased after 3–5 min. Again CD8ß was
needed for this activation, because in the presence of mAb H35-17, or
on CD8- T1.4 cells, no calcium mobilization was
observed (Fig. 2B and data not shown). We then assessed
binding of PE-labeled Kd-PbCS(ABA) tetramers to
T1.4 and T1.4.1 ß cells by FACS (Fig. 2C). Half-maximal
binding was observed at 
8 nM tetramer concentration. In the presence
of anti-CD8 mAb H35-17, the staining of T1.4.1 ß cells was
reduced by 20–25%, to the same levels as observed on T1.4 cells.
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ß, but not
CD8, is required for efficient calcium mobilization in response
to APC pulsed with either PbCS(ABA), the variant PbCS(ABA) P255S, or
soluble Kd-PbCS(ABA) tetramers. Similar results
were obtained when IL-2 production was measured. When incubated with
PbCS(ABA)-pulsed P815 cells, T1.4.1 ß cells produced IL-2 more
efficiently than T1.4 cells. When the variant peptide PbCS(ABA) P255S
was used, only T1.4.1 ß cells displayed detectable IL-2
production, and no response was observed on T1.4 cells. Pretreatment of
T1.4.1 ß cells with 2-bromopalmitate abolished the IL-2 production
(data not shown).
CD8ß, but not CD8, partitions in DIM
To assess the distribution of CD8ß and CD8 in DIM,
T1.4.1 ß cells were surface biotinylated and lysed in cold Triton
X-100; the lysates were fractionated on sucrose density gradients. As
shown in Fig. 3A, CD8ß
partitioned to a substantial degree in the light fractions (two and
three), which contain DIM. In contrast, CD8 was nearly
exclusively found in the dense fractions, which contain Triton-soluble
components (M fractions) (Fig. 3B). Furthermore, lck was
found in both fractions, although more in M than in DIM fractions (Fig. 3C). This enzyme as well as fyn can be reversibly
palmitoylated on two cysteine residues near its N terminus, which
affects their distribution in rafts (40, 41). Because in
this experiment the lysis buffer contained EDTA, which disrupts the
association of CD8 with lck, the observed distributions of CD8 and lck
reflect those of the nonassociated molecules. Conversely, LAT, which
can be palmitoylated at two membrane-proximal cysteines
(37), was found predominantly in the DIM fractions (Fig. 3D). In contrast, the chain of the CD3 complex was found
predominantly in M fractions (Fig. 3E). A small fraction of
-chain, however, was consistently observed in DIM fractions.
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Because it is known that partitioning of LAT, CD4, lck, and fyn in
DIM requires that they be palmitoylated (37, 40, 41, 42), we
examined whether CD8 was palmitoylated. As shown in Fig. 4A, CD8ß shares with LAT and
CD4 the membrane proximal sequence CVR, which has been shown to be
palmitoylated in these molecules (37, 42). CD8 also has
a free membrane-proximal cysteine, but there is no consensus
palmitoylation sequence with CD4 or LAT. To assess CD8 palmitoylation,
T1.4.1 ß cells were metabolically labeled with
[3H]palmitate and lysed in Brij 96 which
solubilizes DIM at the detergent to lipid ratio used here; the
detergent-soluble fraction was immunoprecipitated with Abs specific for
CD8ß, CD8, or LAT. As shown in Fig. 4B, both CD8
immunoprecipitates displayed a major 3H-labeled
protein with an apparent molecular mass of 29 kDa, corresponding to
CD8ß. These results indicate that CD8 is selectively palmitoylated at
CD8ß. The anti-LAT immunoprecipitate exhibited two palmitoylated
species of 36–38 kDa corresponding to LAT (37). Only a
scant amount of palmitoylated LAT was present in anti-CD8 or
anti-CD8ß immunoprecipitates (Fig. 4B), suggesting
that there was no significant association of palmitoylated LAT
with CD8.
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Because both CD8 and lck were found in DIM and M fractions, we
examined in which compartment they associated. For these experiments,
T1.4.2 cells, which do not express CD8ß, were compared with
T1.4.2 ß. In T1.4.2 ß cells, efficient association of CD8
with lck was observed only in rafts (Fig. 5A). In T1.4.2 ,
CD8-associated lck was barely detectable. Because CD8
hardly partitions in rafts (Fig. 3), we examined whether raft
localization was required for efficient CD8-lck association. This was
indeed the case, because pretreatment of cells with methylcyclodextrin
(MCD), which destroys rafts (25), greatly reduced
association of CD8ß with lck (Fig. 5A). Pretreatment of
cells with 2-bromopalmitate, which inhibits protein palmitoylation
(39), had the same effect (Fig. 5B).
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ß'),
which contains the palmitoylation site cysteine 179, abolished
significant partitioning of CD8ß in DIM and effective association
of CD8ß with lck (Fig. 5, C and D). As
expected, association of CD8ß with lck was abrogated on deletion
of the cytoplasmic tail of CD8 (CD8') or mutation of its two
cysteines (CD8''), known to mediate lck binding (Fig. 5D
and Refs. 3 and 4). Our results demonstrate
that efficient association of CD8 with lck requires colocalization of
both components in rafts and that the tail of CD8ß increases CD8-lck
association by conveying CD8 localization in rafts. Cross-linking of CD8-associated p56lck induces p56lck activation
As it is established that cross-linking of CD8-associated lck
results in activation of lck (5, 6), we examined
anti-CD8 Ab-mediated lck activation on T1.4, T1.4.2 , and
T1.4.2 ß. As shown in Fig. 6A, cross-linking of CD8 on
T1.4.2 cells increased the kinase activity 2-fold above
background. In contrast, on T1.4.2 ß cells, which express about
the same level of CD8, in addition to CD8ß (Table I),
cross-linking of CD8 with anti-CD8 mAb 53.6.72 caused a 5-fold
increase in lck activity. Because mAb 53.6.72 cross-links CD8 and
CD8ß, this increase in lck activity was essentially accounted for
by cross-linking of CD8ß.
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C and Ref. 29), activation of
CD8-associated lck should also be induced by cross-linking of
TCR-associated Kd-PbCS(ABA) complexes. To verify
this, we photoaffinity labeled T1.4 and T1.4.1 ß cells with
biotinylated soluble Kd-PbCS(ABA). As shown in
Fig. 6B, cells that were TCR photoaffinity labeled exhibited
the same low kinase activity as untreated cells. Cross-linking of
TCR/CD8 by antibiotin and anti-IgG Abs increased the kinase
activity substantially on T1.4.1 ß, but not T1.4 cells. Antibiotin
Ab alone gave only small increase in lck activity (data not shown).
These findings indicate that CD8-associated lck is activated by
cross-linking of CD8 by anti-CD8 mAb and, more physiologically, by
multimeric Kd-PbCS(ABA) complexes.
CD8ß promotes lck activation and CD3 phosphorylation in
T1.4.1 ß cells incubated with Kd-PbCS(ABA) tetramers
We next examined lck activation and -chain phosphorylation in M
and DIM fractions of T1.4.1 ß cells activated with tetramers. For
simplicity, in this experiment lck activation was assessed by lck
tyrosine phosphorylation, which typically increases on
receptor-mediated lck activation. As shown in Fig. 7, T1.4.1 ß and T1.4 hybridomas,
which are rapidly cycling cells, exhibit significant basal levels of
tyrosine-phosphorylated -chain (pp21) and lck phosphorylation, i.e.,
are constitutively activated to some degree. Incubation of T1.4.1
ß cells with tetramers resulted in translocation of lck and
-chain to DIM (Fig. 7A). This was also observed when
anti-CD3 and anti-Ig were used instead of tetramers, in
agreement with the results reported in other systems
(25, 26, 27, 28). After incubation with tetramer and, to a lesser
extent, anti-CD3 Ab, a dramatic increase in lck tyrosine
phosphorylation was observed in the DIM fraction. At the same time, the
phosphorylation of -chain increased, as seen by the higher amount of
the pp23 phospho form. The tetramer (and anti-CD3-)-induced
phosphorylation of lck and -chain is in accordance with
intracellular calcium mobilization observed under these
conditions (Fig. 2). The pp21 and pp23 phosphoforms of the
-chain were observed only in DIM fractions, whereas in the M
fractions the unphosphorylated p18 form prevailed.
|
-chain were reduced to levels slightly
higher than on untreated cells (Fig. 7A). Thus, CD8,
although more abundant on T1.4.1 ß cells than CD8ß, was quite
unable to elicit these crucial steps of T cell activation, and
accordingly no calcium mobilization occurred (data not shown). On
CD8- T1.4 cells, only anti-CD3 in
combination with anti-IgG induced translocation and phosphorylation
of lck and -chain (Fig. 7B). Together, these results
demonstrate that CD8ß, but not CD8, promotes raft
translocation and phosphorylation of lck and CD3 and that higher
-chain phosphoforms are found only in the DIM fraction.
Our data thus far indicated that CD8ß greatly promotes Ag-specific
activation of T1.4 hybridomas and that palmitoylation of CD8ß is
essential for this, because it mediates efficient CD8-lck association
in rafts. To verify this, we examined the effect of inhibition of
protein palmitoylation on Kd-PbCS(ABA)
tetramer-induced activation of T1.4.1 ß cells. To this end, T1.4.1
ß and T1.4 cells were treated with 2-bromopalmitate and analyzed
the same way as described in the previous section. As shown in Fig. 8, treatment of T1.4.1 ß cells with
2-bromopalmitate abrogated tetramer-induced translocation to DIM and
tyrosine phosphorylation of lck and -chain. These results are
consistent with the findings that, as a result of 2-bromopalmitate
treatment, CD8ß was excluded from rafts (Fig. 5) and calcium
mobilization impaired (Fig. 1).
|
|
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß
is a much more effective coreceptor than CD8. Because
CD8+ CTL express only CD8ß, we took
advantage of T cell hybridomas, which, when obtained by fusion of
cloned CTL with the TCR- cell line BW5147, lack
CD8 expression and hence can be transfected with CD8 genes
(29). Unlike CTL, CD8 transfectants of such hybridomas
typically express CD8 homodimers and to a lesser extent CD8ß
heterodimers (Table I and Ref. 12). Because CD8ß can
be blocked selectively by anti-CD8ß mAb, this permits comparative
functional and biochemical analysis of CD8 and CD8ß
coreceptor function.
To be able to correlate cell activation with biochemical analysis of
TCR-proximal signaling events, we assessed intracellular calcium
mobilization in this study, which is a much more rapid cellular
response than IL-2 production. This also made possible direct
comparison of T cell activation elicited by sensitized APC and soluble
MHC-peptide complexes, respectively. Because activation of T cell in
suspension by soluble tetramers is dependent on CD8, but not on cell
polarization or auxiliary molecules, it is suitable for stringent
analysis of TCR- and CD8-mediated T cell activation. Importantly, in
suspension, T cells are activated by soluble multimeric, but not
monomeric MHC-peptide complexes. This is true for
CD4+ and CD8+ T cells (Fig. 6 and Refs. 13 and 43). Monomeric
Kd-peptide complexes recruit CD8/lck to TCR/CD3,
but for T cell activation CD8-associated lck needs to be activated
first (M.-A. Doucey, D. F. Legler, N. Boucheron, J.-C. Cerottini, C.
Bron, and I. F. Luescher, manuscript in preparation). It has been
reported that CD8+ T cells are activated by
monomeric Kd-peptide complexes, but in this study
the cells were adhered and not in suspension (33).
Our finding that CD8ß, but not CD8, enables T1.4 cells to
efficiently recognize APC pulsed with PbCS(ABA), or the weak agonist
PbCS(ABA) P255S, is in accordance with numerous previous studies
showing that although CD8ß is a most effective coreceptor,
CD8 is not (1, 12). The reason for this difference
is unknown, because in solution CD8 and CD8ß bind to MHC
class I molecules with similar affinities (15). Moreover,
the binding site of CD8 for lck resides in the cytoplasmic tail of
CD8 (3, 4). We have previously observed that on cells,
CD8ß strengthens TCR-ligand binding more than CD8
(12). Although these studies were performed with soluble
monomeric Kd-peptide complexes (12),
the CD8ß-mediated increase in tetramer binding observed here is
modest (Fig. 2) and seems unlikely to account for the dramatic effects
observed on cell activation.
A key observation of this report is that CD8 is selectively
palmitoylated at the tail of CD8ß and that therefore CD8ß, but
not CD8, partitions effectively in rafts (Figs. 3 and 4). It is
well established that protein palmitoylation is reversible and
determines the partitioning of signaling molecules in DIM (37, 40, 41, 42). In contrast to CD8, CD4, as well as LAT, lck and fyn,
have two membrane-proximal cysteines that can be palmitoylated
(37, 40, 41, 42). In particular, for lck and fyn, it has been
shown that the state of palmitoylation, hence their partitioning in
rafts, can vary among cell types and change during cell differentiation
(44). For example, the distribution of lck in rafts in
T1.4.1 ß cells is considerably lower as compared with CTL or
Jurkat cells (37), suggesting that lck palmitoylation is
quite partial in CG72.1 hybridomas. The higher distribution of LAT in
rafts, as compared with CD8ß, is most likely due to
dipalmitoylation of LAT, whereas CD8ß is monopalmitoylated.
For T cell activation, a crucial consequence of CD8ß and lck
colocalization in rafts is the resulting highly increased association
of CD8 with lck. This conclusion is based on several complementary
findings: 1) CD8 or CD8ß', which are largely excluded from
rafts, fail to effectively associate with lck (Figs. 3 and 5); 2)
inhibition of protein palmitoylation by 2-bromopalmitate dramatically
reduces CD8-lck association (Fig. 5); and 3) destabilization of rafts
by MCD has the same effect. Because association of CD8 with lck is weak
and rafts constitute only a small fraction of the cell membrane, the
preferential association of the two proteins in rafts is probably
mainly explained by increased concentration of both components in this
compartment. Similar findings have been obtained for fyn, which
associated with CD3 preferentially in rafts (41). We
expect that the same findings apply to CD4. Because CD4 is
dipalmitoylated (42), it is expected to partition in rafts
more efficiently than CD8, which probably explains why CD4 associates
with lck more extensively than CD8 (45).
It has been reported that CD8 (and CD4) can also associate with LAT and
hence bring LAT to TCR/CD3, much the same way as lck (20).
Consistent with this is the weak coimmunoprecipitation of LAT with
anti-CD8 Abs (Fig. 4B). According to the study by
Bosselut et al. (20), however, LAT interact with the
coreceptor via its two membrane-proximal cysteines, which are the
palmitoylation sites. If this is correct, association of CD8 with LAT
is not expected to increase in rafts, at least not in the way observed
with lck.
The preferential formation of coreceptor-lck adducts in rafts has an
important implication for T cell activation. Rafts, by excluding
phosphatases, namely, the abundant CD45, and by concentrating kinases
and their respective substrates, allow kinase-mediated phosphorylation
reactions to proceed and cell activation to occur (23, 24). Because src kinases are activated by cross-linking, namely,
by trans-phosphorylation of the regulatory A loop tyrosine
394 (24), CD8-associated lck is readily activated in rafts
by cross-linking of CD8. This can be accomplished by anti-CD8 Abs
and, more physiologically, by multimeric
Kd-peptide complexes, which coengage TCR and CD8
(
Figs. 6–8 and 13). Taking advantage of our unique experimental
system, we were able to demonstrate that the initial and essential
activation of lck takes place in rafts and is induced by cross-linking
of TCR/CD3-CD8/lck complexes (Figs. 6 and 7; M.-A. Doucey, D. F.
Legler, N. Boucheron, J.-C. Cerottini, C. Bron, and I. F. Luescher,
manuscript in preparation). This conclusion is further supported by the
observations that 1) monomeric Kd-PbCS(ABA)
complexes failed to activate lck (Fig. 6B); 2) inhibition of
protein palmitoylation by 2-bromopalmitate impaired CD8-lck association
and lck activation in rafts (Figs. 5 and 8); and 3) CD8 failed to
associate with lck and to mediate lck activation in rafts (Figs. 5 and 7).
As a result of this initial lck activation, tyrosine phosphorylation of
CD3 ITAM occurs mainly, if not exclusively, in rafts (Figs. 7 and 8). In M and DIM fractions obtained from Triton X-100 lysates of
resting T cells, only a small fraction of TCR/CD3 is found in the M
fraction (Fig. 3 and 26). However, after activation of T cells
with anti-CD3 Abs (25, 26, 27, 28) or with MHC-peptide
tetramers (Fig. 7), this fraction increases substantially, what has
been referred to as translocation of TCR/CD3 to rafts
(25, 26, 27, 28). It is well established that phosphorylated ITAM
are binding sites for molecules containing SH2 domains, such as ZAP-70,
Syk, and fyn (16, 17). Phosphorylation of recruited ZAP-70
by lck stimulates its protein tyrosine kinase activity and promotes
binding of lck to ZAP-70, thus strengthening CD8/lck association with
TCR/CD3 (46). Moreover, LAT becomes firmly recruited to
TCR/CD3, which is crucial because LAT, on phosphorylation by ZAP-70,
recruits various adapter and signaling molecules to TCR/CD3, thus
linking the initial TCR activation to diverse downstream signaling
cascades (21).
Several observations indicate that these molecular interactions take
place inside or at the rims of rafts. For example, the phosphatase CD45
is excluded from rafts (23, 24). In fact, due to its high
abundance and high enzymatic activity, and its ability to associate
with various signaling molecules, activation-induced, i.e.,
phosphorylation-mediated molecular interactions, can persist only when
segregated from CD45. Consistent with this is our observation that
activation of CD8-associated lck and phosphorylation of CD3 take
place in rafts (
Figs. 6–8). Furthermore, in a study using confocal
microscopy, rafts were shown to be lined with TCR/CD3
(28). The use of cold Triton X-100 to isolate
detergent-insoluble DIM is likely to disrupt weak molecular
interactions that allow TCR/CD3 to interact with raft-associated
molecules, such as src kinases and coreceptor. Because both lck and
fyn, as well as CD8 and CD4, have been reported to weakly associate
with TCR/CD3 (47), lck-coreceptor complexes, which are
formed in rafts (Fig. 5), seem likely sites for TCR/CD3 association
with rafts. This would explain why coengagement of TCR and CD8 by
multimeric MHC-peptide complexes induces lck activation and CD3
phosphorylation in rafts. The subsequent activation-induced molecular
interactions, resulting in the recruitment of other molecules (e.g.
ZAP-70 and LAT), can be expected to further strengthen the initially
weak association of TCR/CD3 with raft-associated src kinases and
coreceptor, e.g., by linking coreceptor-associated lck with CD3 via
ZAP-70 (19, 46). Thus, activation-induced translocation of
TCR/CD3 and other signaling molecules to rafts (Figs. 7 and 8 and Refs.
25–28) probably reflects, at least in part, an increased stability of
raft-associated molecular complexes in Triton X-100.
Palmitoylation of CD8ß plays a crucial role for CD8 coreceptor function, because it allows partitioning of CD8 in rafts. This on one hand greatly favors its association with lck and on the other hand permits efficient activation of lck by cross-linking of CD8. This initial CD8-mediated lck activation is crucial for induction of TCR signaling and T cell activation, especially when Ag is limited or presented as low affinity variants.
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Acknowledgments |
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Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Immanuel F. Luescher, Ludwig Institute for Cancer Research, Chemin des Boveresses 155, 1066 Epalinges, Switzerland,
3 Abbreviations used in this paper: lck, p56lck; ABA, 4-azidobenzoic acid; CD3 C-ITAM, third ITAM of CD3; DIM, detergent-insoluble microdomain; IASA, iodo-4-azidosalicylic acid; ITAM, immunoreceptor tyrosine-based activation motif(s); LAT, linker of activation of T cells; M, membrane fraction; MCD, methyl-ß-cyclodextrin; PbCS, Plasmodium berghei circumsporozoite; SH2, src homology 2; HIFCS, heat-inactivated FCS; PSN, penicillin-streptomycin-neomycin.<. >
Received for publication March 20, 2000. Accepted for publication June 6, 2000.
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M. Higuchi, K. M. Izumi, and E. Kieff Epstein-Barr virus latent-infection membrane proteins are palmitoylated and raft-associated: Protein 1 binds to the cytoskeleton through TNF receptor cytoplasmic factors PNAS, April 10, 2001; 98(8): 4675 - 4680. [Abstract] [Full Text] [PDF]
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T. Harder and M. Kuhn Selective Accumulation of Raft-Associated Membrane Protein Lat in T Cell Receptor Signaling Assemblies J. Cell Biol., October 16, 2000; 151(2): 199 - 208. [Abstract] [Full Text] [PDF]
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) or T1.4.1 
), or presence of anti-CD8ß mAb H35-17 (•)
were incubated for 1 h at 26°C with graded concentrations of
PE-labeled Kd-SYIPSAEK(ABA)I tetramers and
analyzed by FACS. Mean values and SD were calculated from two
independent experiments.<. >
