From the Laboratory of Immunobiology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
Received for publication, October 19, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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Immune recognition of pMHCII ligands by a helper
T lymphocyte involves its antigen-specific T cell receptor (TCR) and
CD4 coreceptor. We have characterized the binding of both molecules to
the same pMHCII. The D10 The transmembrane CD4 glycoprotein expressed on the surface of a
majority of thymocytes as well as helper T lymphocytes is centrally
involved in MHC1 class
II-restricted differentiation and activation (1-6). The extracellular
rod-like segment of CD4 consists of four concatamerized Ig-like domains
(D1-D4), a single transmembrane-spanning segment, and a short
cytoplasmic tail (7-10). The membrane distal D1 and D2 domains bind to
the nonpolymorphic To date, biophysical parameters of CD4 interaction with pMHCII and TCR
ectodomain components have not been measured. Attempts to quantitate
TCR-independent binding between CD4 and pMHCII in human and mouse
systems using cell-based assays and soluble CD4 ectodomain constructs
have suggested a weak interaction (Kd > 100 µM) (15, 27, 28). However, the precise affinity and kinetics of binding are unknown. Furthermore, whether CD4 ectodomain interaction with pMHCII is modulated by the TCR or, alternatively, whether prior CD4-pMHCII interaction alters the MHC antigen-presenting platform, augmenting subsequent TCR affinity for its ligand is uncertain. In the present study, we have utilized highly purified recombinant TCR, pMHCII, and CD4 ectodomains to address these questions. The results offer insight into the fundamental nature of
CD4-pMHCII interaction, further elucidate the role of CD4 in class II
MHC-restricted TCR recognition, and indicate that if an interaction
exists between CD4 and TCR extracellular segments it must involve
components other than or in addition to the TCR Construction and Expression of D10 TCR in Escherichia
coli--
Plasmids pEE14-D10
Protein expression and inclusion body preparation of D10 Refolding and Purification of D10 TCR
The refolded material was filtered (Corning, 0.22 µm) and
immunoaffinity-purified using mAb 3D3 covalently coupled
The scD10 TCR consists of 237 residues and is organized from N to C
terminus as follows: V Eukaryotic Expression in Lec3.2.8.1 Cells and Purification of
hCD4 and CA/I-Ak Proteins--
The human CD4 ectodomain
(residues 1-371) was expressed in mammalian Chinese hamster
ovary-derived Lec3.2.8.1 cells (30) using the glutamine synthetase
vector pEE14 (31). To this end, 20 µg of linearized plasmid DNA
pEE14-hCD4 was transfected into Lec3.2.8.1 cells by a calcium phosphate
precipitation method using a transfection MBS kit (Stratagene)
as described (32). 48 h later, the transfected cells were
harvested and cultured on 96-well plates in Glasgow minimal essential
medium supplemented with 25 µM methionine sulfoximine.
After feeding the cells for 4 weeks, the growing clones were selected
and assayed by enzyme-linked immunosorbent assay. 4 µg/ml anti-D1
domain of hCD4 mAb, 19Thy5D7, was coated onto Immulon plates (Dynatech)
overnight at 4 °C and blocked by 1% bovine serum albumin at room
temperature for 1 h. Then 50 µl of supernatant from individual
clones was added to each well and incubated overnight at 4 °C. The
reaction was followed by adding 0.1 mg/ml biotinylated mAb OKT4
(anti-D3 domain of hCD4) and developed by horseradish
peroxidase-conjugated streptavidin. Once identified, positive clones
were transferred to 24-well plates and then to 6-well plates and
rescreened. Subsequently, the highest-expressing clone was picked and
amplified for large scale production as described previously (31).
Yields averaged 40 mg/liter culture supernatant. After each expression,
the supernatant was filtered (Corning, 0.22 µm) and
immunoaffinity-purified by mAb 19Thy5D7-coupled Affi-Gel 10 beads
(Bio-Rad). The hCD4 protein was eluted by 0.5 M acetic acid, pH 3.5, and neutralized to pH 7.0 immediately by adding 1 M Tris-HCl, pH 9.5. For BIAcore assay, the protein was
further purified by gel filtration on a Superdex 75 column and
concentrated to 20-50 mg/ml.
The mature I-Ak Determination of Protein Concentration and N-terminal Sequence
Analysis--
Protein concentrations were determined by
spectrophotometry at 280 nm using factors of 1.2 (scD10), 1.4 (D10),
1.5 (CA/I-Ak), and 1.4 ml/mg/cm (hCD4), respectively. The
factors were acquired from extinction coefficients that were calculated
based on the tryptophan, tyrosine, and cysteine contents of each
protein (33). N-terminal sequence was performed on each protein after
blotting to a polyvinylidene difluoride membrane using a 120A sequencer (Applied Biosystems).
Surface Plasmon Resonance Studies--
All binding studies were
performed on a BIAcore1000 surface plasmon resonance biosensor
(BIAcore) in HBS buffer (10 mM HEPES, 150 mM
NaCl, 3 mM EDTA, and 0.005% surfactant p-20, pH 7.4) at 25 °C. For indirect binding assay, an anti-LZ mAb, 13A12 (34), was
coupled to the CM5 sensorchip using a standard amine coupling kit
(BIAcore), resulting in ~8,000 RU coupled. 30 µl of 1 µM CA/I-Ak-LZ was injected over the surface
at a flow rate of 10 µl/min and followed by a dissociation period of
3 min. Different analytes (D10 or scD10 TCRs or hCD4) were then passed
through the13A12-CA/I-Ak-LZ surface either at a high flow
rate (50 µl/min for kinetic studies) or at a low flow rate (5 µl/min for equilibrium studies). The specific binding was taken after
subtracting the response on a control surface (the same 13A12
immobilized surface but without any CA/I-Ak-LZ captured).
CD4 protein was coupled on the CM5 chip directly using an amine
coupling kit, ranging from 2,000 to 3,000 RU, and analytes were then
injected over the flow cell at a flow rate of 5 µl/min for affinity
studies. An irrelevant anti-gp140 mAb 116 Fab fragment was immobilized
at the same level as a control surface.
BIAevaluation 3.0 software (BIAcore) was used for data analysis.
Kinetic data fitting was performed using a Langmuir 1:1 binding model
(BIAcore), and equilibrium data were analyzed by nonlinear curve
fitting or Scatchard plotting RU/concentration versus RU followed by linear regression (Cricket-Graph software).
COS-7 Cell Transfection and Cell Binding Assay--
The COS-7
cell transfection of human CD4 and the inhibition effect of CD4 on
human MHC class II B-lymphoblastoid Raji cells binding were performed
as described previously (15). Briefly, 5 × 104 COS-7
cells were plated into each well of Falcon 6-well plates and
transfected by a calcium phosphate/chloroquine method (35) with 5 µg
of either full-length hCD4 DNA (residues 1-435 cloned in the CDM8
vector) or CDM8 vector DNA alone as a control. Two days later, the cell
binding assay was performed. 3 × 106 Raji cells were
pre-incubated in 1 ml of medium containing various concentrations of
affinity-purified hCD4 protein or bovine serum albumin (as negative
control) at 37 °C for 30 min and then added to transfected COS-7
cells. The 6-well plates were incubated at 37 °C for 1 h, and
the rosettes formed by B Raji cell-COS-7 cell binding were counted
under microscopic magnification. As a positive control for inhibition,
15 µg of 19Thy5D7 was added to transfected COS-7 cells and incubated
at 37 °C for 30 min before adding the B cells. The inhibition
percentage was calculated as 100 × [(Rc Expression and Purification of Recombinant D10 TCR, pMHCII, and
CD4 Ectodomain Proteins--
To study the interaction between a MHC
class II-restricted TCR, its ligands, and CD4 coreceptor, recombinant
ectodomains corresponding to each were expressed and purified. An
SDS-PAGE analysis of the purified protein shows that the
To investigate any possible function of the C module in antigen
recognition or CD4 interaction, we compared the above D10
A cDNA encoding the four extracellular domains of human CD4 (Fig.
1a) in the pEE14 plasmid was transfected and expressed in the glycosylation mutant Lec3.2.8.1 derivative of Chinese hamster ovary
cells, which produce glycoproteins with homogenous sugar adducts. In
these cells, N-linked carbohydrates are of the
Man5 form, and O-linked carbohydrates are
truncated to a single GalNac (30). The yield of hCD4 was more than 40 mg/liter culture supernatant, and the protein was readily purified by
affinity chromatography on an anti-CD4 D1 mAb 19Thy5D7-coupled Affi-Gel
10 column followed by gel filtration on a Superdex 75 column. The
protein shows high purity as well as size and charge homogeneity on
SDS-PAGE (Fig. 1b, lane 5 and 6) and
native gel (data not shown), respectively. As with the TCR constructs,
the faster mobility of the protein under nonreducing conditions (45 kDa
versus 47 kDa) indicates the formation of the expected
intrachain disulfide bonds. The ability of other mAbs such as OKT4 (a
CD4 D3-specific mAb) to react with the protein was verified by
enzyme-linked immunosorbent assay (data not shown).
The peptide-bound murine MHC class II molecule CA/I-Ak, the
D10 ligand, was produced in recombinant form from Lec3.2.8.1 cells as
well. This pMHCII protein consists of the extracellular domains of the
noncovalently associated Affinity Studies of D10 and scD10 TCRs Binding to
CA/I-Ak Using an Orientational Capture BIAcore
Method--
Surface plasmon resonance was used to examine the binding
affinity between the D10 TCR The Binding of the D10 CD4 Binds to pMHCII with Low Affinity--
The binding of
CA/I-Ak, D10 TCR
In the above BIAcore experiment, the interaction between mouse pMHCII
and hCD4 was examined. Given that hCD4 is known to interact with mouse
pMHCII in a fashion comparable with mCD4 in vitro and in vivo (6, 40-43), it was unlikely that the low affinity
was due to species differences. Nonetheless, to confirm the CD4-pMHCII binding affinity that we obtained from BIAcore assay and exclude any
effects arising from the proteins derived from heterologous species, we
examined the inhibitory effect of soluble hCD4 on the binding between
class II MHC-expressing human Raji B cells and hCD4-transfected COS-7
cells. Immunoaffinity-purified hCD4 was added into this
hCD4-MHCII-dependent cell-cell binding system at
concentrations ranging from 0 to 875 µM. Cell-cell
adhesion was then quantitated by enumerating rosettes formed between
hCD4-transfected COS cells and Raji B cells. As shown in Fig.
5, the number of rosettes was minimally
decreased when CD4 protein concentration reached 100 µM
but was abolished at 400 µM hCD4. The data are plotted
using percentage inhibition versus inhibitor concentration. The concentration of CD4 inhibiting 50% of rosette formation (157 µM) was taken as the dissociation constant
(Kd). This value is consistent with the
Kd calculated from BIAcore data (~200
µM). Note that the positive inhibition control, anti-CD4 mAb 19Thy5D7, completely inhibits the B cell binding at a concentration <0.1 µM, whereas the negative control, bovine serum
albumin, does not affect the binding even at a 1 mM
concentration.
TCR The present study provides the first direct quantitation by
BIAcore equilibrium analysis of the monomeric affinity of CD4 for
pMHCII (200 µM Kd). For these
measurements, we utilized highly purified, aggregate-free human CD4 and
mouse I-Ak ectodomains. We also observed a ~160
µM IC50 for human CD4 in blocking the
CD4-MHCII-based adhesion between COS cells transfected with human
transmembrane CD4 and human Raji B cells expressing class II MHC. Since
the Raji cells coexpress endogenous HLA-DR, -DP and -DQ
molecules, it is unlikely that greater binding affinities exist for any
of the polymorphic alleles present on the surface of Raji cells. These
results are also consistent with earlier measurements by cell-based
assays reported for hCD4-hpMHCII and mCD4-mpMHCII suggesting a
Kd >100 µM (15, 28).
In several regards, the interaction between CD4 and MHCII needs
to be compared and contrasted with the interaction between CD8 and
MHCI. CD8 and CD4 are both coreceptors for MHC molecules that bind to
the membrane proximal exposed loop on the Despite the similarities of CD4 and CD8 in terms of their target
ligands and TCR coreceptor function, substantial differences exist. For
example, the CD8 coreceptor is an Ig domain dimer that projects from
the T cell surface on a long, heavily O-glycosylated stalk
(reviewed in Ref. 47 and references therein). Interaction with pMHCI is
via the six antibody-like CDR loops, three from each subunit Ig domain
(47, 48). In contrast, CD4 interaction with pMHCII involves the D1-D2
module including residues outside of the CDR regions (13, 49, 50).
Perhaps more importantly, the membrane proximal CD4 segment is not a
flexible stalk but rather is composed of two Ig-like domains. Given the
structured nature of the CD4 D3-D4 module, it has been suggested that
the membrane proximal domain region may be involved in protein-protein interaction. In this regard, prior chimeric studies have shown that
although D3-D4 is not involved in pMHCII binding, the presence of this
module is necessary for mediating proper pMHCII ligation via D1-D2 (15,
51). Thus, its replacement with either the related CD4 D1-D2 module or
alternatively, CD2 D1-D2, fails to result in a CD4 chimera with class
II MHC binding activity. The potential for CD4 self-oligomerization via
D3-D4 might explain this result, accounting for the ability of the
non-MHC class II binding CD4 variant F43I to function as a dominant
negative mutant when cotransfected with wild-type CD4. Oligomerization
or other mechanisms of CD4 clustering offers a means to augment
avidity, thereby offsetting the weak monomeric CD4-pMHCII affinity.
Additional studies raise the possibility that the CD4 D3-D4 module can
interact with the T cell receptor (16, 17). In particular, CD4 mutants
that fail to interact with pMHCII (D2 mutations) or p56lck
(cytoplasmic tail mutants) surprisingly restore antigen responsiveness in the 3A9 T cell hybridoma system. Such restoration is dependent on
the D3-D4 module, as shown by additional mutagenesis studies (17).
Nevertheless, BIAcore analysis failed to reveal any measurable affinity
between the CD4 ectodomain and the TCR TCR heterodimer binds to
conalbumin/I-Ak with virtually identical kinetics and
affinity as the single chain V
V
domain module (scD10)
(Kd = 6-8 µM). The CD4 ectodomain
does not alter either interaction. Moreover, CD4 alone demonstrates
weak pMHCII binding (Kd = 200 µM),
with no discernable affinity for the
TCR heterodimer. Hence,
rather than providing a major contribution to binding energy, the
critical role for the coreceptor in antigen-specific activation likely results from transient inducible recruitment of the CD4 cytoplasmic tail-associated lck tyrosine kinase to the
pMHCII-ligated TCR complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 domain of MHC class II molecules, whereas the
membrane proximal D3-D4 module is thought to be involved in both CD4
oligomerization and TCR interaction (11-17). During antigen
recognition of peptides bound to MHC class II molecules (pMHCII), CD4
and TCR colocalize, interacting with the same pMHCII molecule (18-20).
Hence, CD4 has been termed a coreceptor (21). The cytoplasmic tail of
CD4 is noncovalently associated with p56lck, a Src-like
tyrosine kinase, and also contains membrane proximal palmitoylation
sites that direct CD4 to lipid rafts enriched in additional signaling
molecules (22-25). p56lck initiates CD4- and TCR-based signal
transduction and facilitates the physical coassociation of the TCR and
CD4 coreceptor (26).
heterodimer.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and pEE14-D10
A were used as
polymerase chain reaction templates to generate cDNAs encoding the
extracellular domains of D10
and
subunits. The 5' polymerase
chain reaction primers contained a XbaI restriction site, a
ribosome binding site, and a methionine initiation codon proximal to
the first amino acid residue of each mature subunit. The 3' primers
encoded a stop codon (TAA) and a BamHI restriction site. To
avoid cysteine mispairing and incorrect disulfide bond formation, both
and
subunit sequences were terminated before their respective
most-C-terminal cysteines utilized to form an interchain disulfide bond
in the native protein. Moreover, the unpaired cysteine at position 109 in the
chain was changed to serine (TGC to TCC) by overlapping polymerase chain reaction and restriction fragment replacement. The
and
subunit genes were inserted individually between
XbaI and BamHI sites within the expression vector
pET-11a (Novagen). After DNA sequences were confirmed, the expression
plasmids were transformed into E. coli strain BL21 (DE3) separately.
and
subunits were performed by the Cell Production and Recovery Facility,
Rutgers University. Briefly, bacterial cells transformed with either
pET-D10
or pET-D10
expression vectors were grown in a 50-liter
bioreactor at 37 °C in 4 × YT medium (32 g/liter bactotryptone, 20 g/liter yeast extract, 5 g/liter NaCl)
containing 50 µg/ml ampicillin and 2% glycerol. 1 mM
isopropyl-
-D-thiogalactopyranoside was added to induce
protein expression at A600 = 8-12, and cells were harvested 4 h after the induction. Cells were resuspended in
a buffer containing 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, and 1 mM
phenylmethylsulfonyl fluoride at 10 ml/g cell wet weight and lysed.
Lysate was then incubated at room temperature for 1 h with 5 µg/ml DNaseI and 4 mM MgCl2 added. Inclusion
bodies were spun down and washed twice with washing buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 0.02% sodium
azide) containing 0.5% Triton X-100 followed by two washes with
washing buffer alone. The inclusion body pellets were frozen at
80 °C and used for subsequent purification.
Heterodimer and
Single Chain D10 (scD10)--
D10 TCR
heterodimer was refolded
as described previously (29) with some modifications. The
and
inclusion bodies were dissolved separately in 50 mM
Tris-HCl, pH 6.8, 8 M urea, 10 mM EDTA and 0.1 mM dithiothreitol and centrifuged at high speed. The
protein concentration of the supernatant was determined by Bio-Rad
protein assay. The denatured
and
inclusion bodies (1 µmol of
each) were added together to 10-12 ml of 6 M
guanidine-HCl, 10 mM sodium acetate, and 10 mM
EDTA, pH 4.2. The mixture was then diluted dropwise into 1 liter of
cold refolding buffer (100 mM Tris-HCl, pH 8.0, 400 mM L-arginine-HCl, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized
glutathione, and 0.5 mM phenylmethylsulfonyl fluoride) with
vigorous stirring. After the refolding solution was incubated at
4 °C with slow stirring for 6-12 h, another
/
mixture was
added. A third mixture was added 6-12 h later, and a further 24-h
incubation was performed.
bind plus
Sepharose beads (Amersham Pharmacia Biotech) at 5 mg of mAb/ml of
beads. The correctly refolded TCR protein was eluted with low pH buffer
(0.5 M acetic acid, 10% glycerol, pH 3.0) and adjusted to
pH 5.0-6.0 immediately using 1 M Tris-HCl, pH 9.5. The
protein was then concentrated using a Centriprep-10 concentrator
(Amicon) and sized on a Superdex 75 gel filtration column (Amersham
Pharmacia Biotech) equilibrated with 20 mM sodium
acetate/acetic acid, pH 5.0, and 100 mM NaCl. The purified
protein was finally concentrated, and the buffer was changed to 20 mM sodium acetate/acetic acid, pH 5.0.
8.2 (residues 3-116)-linker
(GSADDAKKDAAKKDG)-V
2 (residues 1-117), with a mutation at C235S.
Bacterial expression and inclusion body purification were the same as
that of the D10 TCR (described above). An efficient refolding was
achieved by diluting rapidly into a refolding buffer (50 mM
Tris-HCl, pH 8.0, 400 mM arginine, 2 M urea, 2 mM EDTA, 4 mM reduced glutathione, and 0.4 mM oxidized glutathione). The refolded material was then applied to a 3D3 affinity column followed by gel filtration on Superdex
75, and buffer was changed to 20 mM sodium acetate, pH 5.0, with 0.025% sodium azide.
chain was fused at its N terminus via a
flexible linker with a 13-residue hen egg conalbumin (CA) peptide (residues 153-165) that is recognized by D10 TCR. The 37-residue leucine zipper (LZ) sequences were appended to both C termini of the
(residues -3-192) and
(residues 3-198) chains by flexible thrombin-cleavable spacers. The cDNA constructions were subcloned into the pEE14 vector and expressed in Lec3.2.8.1 cells as described above. The screening of secreted recombinant protein in the culture supernatant was performed by both Sandwich enzyme-linked immunosorbent assay and BIAcore using antibodies specific for either the
CA/I-Ak (10.2.16) or the LZ epitope (2H11 or 13A12). The
yield was ~0.7 mg of CA/I-Ak/liter of supernatant.
Protein in production supernatant was purified by 2H11 affinity
chromatography followed by Superdex 75 gel filtration.
RI)/ Rc], where
Rc and RI represent the rosette
numbers in the absence or presence of inhibitor, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TCR heterodimer derived from the mouse T cell clone D10 (D10
TCR), specific for the hen egg CA foreign peptide (residues 153-165)
associated with the self-MHC class II I-Ak molecule
(pMHCII), was constructed (Fig.
1a). The
and
subunits of the D10 TCR were truncated at residues 200 and 238 within their respective extracellular segments proximal to the cysteines involved in
the interchain disulfide bond formation. Furthermore, to avoid cysteine
mispairing,
chain cysteine 109 was replaced by a serine. The
and
subunits were expressed separately as inclusion bodies in
E. coli and refolded together at a 1:1 molar ratio as
described under "Experimental Procedures." The refolded
TCR
was purified by immunoaffinity chromatography using the 3D3 anti-D10
TCR clonotypic mAb followed by gel filtration on a Superdex 75 column.
A yield of 6-8 mg of purified protein/liter of starting material
(~150 mg of inclusion bodies) was obtained. The purified protein can be stored at 4 °C at high concentration (up to 1 mM)
with little tendency to form aggregates.
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Fig. 1.
Schematic diagrams and SDS-PAGE analysis of
soluble recombinant scD10, D10, CA/I-Ak, and hCD4
ectodomain constructs. a, protein constructs. The scD10 TCR
consists of the variable domain module of D10 TCR, where V is
connected to V
by a 15-residue linker. D10 represents the
extracellular Ig-like variable (V) and constant
(C) domains of D10 TCR, with
and
chains
noncovalently associated to form an
heterodimer.
CA/I-Ak consists of the extracellular domains of MHC class
II molecule with a 13-residue hen egg CA peptide(p) fused to
the N terminus of the
-chain via a flexible linker (short
line) and the 37-residue LZ peptides (coil)
attached to both the
and
chains. hCD4 consists of the 4 extracellular Ig-like domains of human CD4. b, 15% SDS-PAGE
of the purified proteins stained by Coomassie Blue. 1, D10
TCR, under reducing conditions; 2, D10 TCR, under
nonreducing conditions; 3, scD10 TCR, under reducing
conditions; 4, CA/I-Ak, under reducing
conditions; 5, hCD4, under reducing conditions;
6, hCD4, under nonreducing conditions. Approximately 5 µg
was loaded per lane. Molecular weight markers are from
Bio-Rad.
and
chains are present in equal molar amounts (Fig. 1b,
lane 1 and 2). Since the interchain disulfide
bond was mutated, no covalently linked heterodimer band is seen under
nonreducing conditions (Fig. 1b, lane 2). The
faster mobility of the
and
bands on SDS-PAGE under nonreducing
relative to reducing conditions (Fig. 1b, lane 2 versus 1; 23 versus 26 kDa for
and
27 versus 30 kDa for
) is a consequence of the two
intra-chain disulfide bonds in each subunit leading to a more compact
structure. Expected N-terminal sequences of both
and
chains
were confirmed by Edman degradation after transfer to a polyvinylidene
difluoride membrane. To assess the integrity of the
and
constant domains, the TCR protein was immunoprecipitated with TCR
C
-specific mAb H28 and C
-specific mAb H57. SDS-PAGE showed that
the TCR was precipitated by both mAbs. The TCR-mAb complexes were
readily observed by native gel and equivalent to D10 expressed in the mammalian Chinese hamster ovary derivative Lec 3.2.8.1 cells as a
glycosylated protein with the intact interchain disulfide bonds (data
not shown). Given that the 3D3 mAb used for purification recognizes a
conformational epitope dependent on the proper juxtaposition of V
and V
domains, the above results indicate that the purified D10 TCR
assumes a correctly folded conformation in both V- and C-region modules.
TCR
heterodimer with a scD10 TCR V
V
module. As shown in Fig. 1a, the latter contains the D10 TCR V
domain connected
from its C terminus by a flexible linker, a 15-amino acid residue
peptide (GSADDAKKDAAKKDG), to the N terminus of the D10 TCR V
domain. Like the D10 TCR, the scD10 protein was expressed in E. coli, refolded in vitro, and purified by 3D3 affinity
column followed by gel filtration on Superdex 75. The scD10 protein
appears as a single band migrating at 31 kDa under reducing conditions
(Fig. 1b, lane 3).
and
subunits, with a 13-residue peptide corresponding to residues 153-165 of hen CA attached to the N
terminus of the
chain and the leucine zipper sequences appended to
the C termini of both chains (Fig. 1a). Although the linkage
of the peptide to the class II MHC is distinct from that found under
natural circumstances, crystallographic analysis of the pMHCII protein
demonstrates a native structure (36). The yield of the
CA/I-Ak is ~0.7 mg/liter culture supernatant after
affinity purification. On SDS-PAGE (Fig. 1b, lane
4), the purified CA/I-Ak migrates as two closely
spaced bands, with apparent molecular masses of ~35 kDa. N-terminal
amino acid sequence determination verified that the upper band is the
chain and the lower one is the CA peptide fused
chain. Unlike
with the other recombinant proteins, the apparent molecular mass
mobility on SDS-PAGE of the I-Ak subunits is inconsistent
with those predicted from amino acid sequences (
= 27,295 daltons and
= 30,908 daltons). These differences are due to
post-translational modification (there are two N-linked glycosylation sites in the
chain and one in the
chain) as well
as the highly charged leucine zipper sequences, resulting in anomalous
migration on SDS-PAGE (32, 34). All four proteins can be concentrated
to 1-2 mM with no aggregates observed by either light
scattering or gel filtration (data not shown).
heterodimer and CA/I-Ak
and, as a comparison, between the scD10 TCR and CA/I-Ak. To
this end, the anti-LZ mAb 13A12 was immobilized on a CM5 sensorchip to
a level of ~8000 RU. The CA/I-Ak protein was captured
(~800 RU) through binding of the C-terminal LZ appended to the
pMHCII, facilitating an orientation that exposes the pMHCII
antigen-presenting platform to the TCR. pMHCII accessibility to immune
recognition was first verified by injecting 0.5 µM
anti-I-Ak mAb 10.2.16 (data not shown). Subsequently, the
D10
TCR and scD10 were individually injected as the analyte over
the pMHC surface (Fig. 2, a
and b, insets). Sensorgrams of the indicated concentrations of D10 and scD10 TCR binding to captured
CA/I-Ak are shown in Fig. 2, a and b
(main plots), respectively. For kinetic studies, data were fitted using
a Langmuir 1:1 binding model, and similar dissociation constants
(Kd) of the two D10 TCRs binding to
CA/I-Ak were acquired (see below). Moreover, consistent
results were obtained independently by equilibrium studies (Fig. 2,
c and d). Both direct nonlinear curve fitting and
Scatchard plot analysis gave similar Kd values. Fig.
2e summarizes the fitted kinetic and equilibrium data. Both
D10 and scD10 TCRs show relatively fast association and dissociation
rates when binding to CA/I-Ak. D10 binds to
CA/I-Ak with a Kd of 6.7 µM and a half-life of about 17.5 s. scD10 TCR shows
a similar binding affinity to CA/I-Ak with a
Kd of 7.8 µM and a half-life of
12.5 s. The similarities in these binding parameters imply that
the V domain module of the TCR alone is sufficient for pMHCII
recognition. These values are within the range of those reported for
other TCR-pMHCI and pMHCII interactions at 25 °C (37) as well as
different D10 TCR-related constructs (38, 39).
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Fig. 2.
Kinetic and equilibrium binding studies of
D10 TCRs with CA/I-Ak. BIAcore sensorgrams of D10
(a) or scD10 (b) binding to
CA/I-Ak-LZ captured by surface-immobilized anti-LZ-specific
mAb 13A12 at a flow rate of 50 µl/min. The sensorgrams shown have had
the corresponding background responses subtracted. The latter were
obtained by injecting the analyte over the same mAb 13A12 surface in
the absence of prior CA/I-Ak-LZ capture. Concentrations of
injected proteins are indicated. The insets in panels
a and b represent the schematic interaction between D10
receptor (shaded) and CA/I-Ak
(dotted), as captured by surface-bound 13A12 mAb
(open). In panels c and d, the
specific equilibrium binding responses of CA/I-Ak-LZ with
D10 (c) and scD10 (d) are plotted for each
analyte concentration. The corresponding background responses have been
subtracted. The insets show that Scatchard plots of the same
data were linear, giving Kd values
(Kd= 1/slope) of 6.67 µM (D10)
and 7.81 µM (scD10), respectively. In panel e,
fitted parameters from kinetic and equilibrium binding to
CA/I-Ak are given for both D10 and scD10 TCRs.
Heterodimer to CA/I-Ak Is
Not Influenced by the CD4 Ectodomain--
To determine whether CD4
could influence the binding between the D10 TCR and its pMHCII ligand,
the D10 TCR
heterodimer protein was mixed with different
concentrations of soluble hCD4 and incubated overnight at 4 °C. In
these experiments, the concentration of D10 TCR
protein was
constant (20 µM), whereas the hCD4 concentration was
varied from 0 to 100 µM, generating a series of
"D10/CD4" molar ratios. The individual D10 or hCD4 components or
alternatively, a mixture of the two at a given molar ratio was injected
separately over the same 13A12 mAb-captured CA/I-Ak surface
as described above. As shown in Fig.
3a, the response of the 20 µM D10 plus 50 µM hCD4 mixture (D10 + CD4)
binding to CA/I-Ak simply appears to be the addition of the
individual D10 and hCD4 binding responses. Furthermore, the
dissociation phases of the D10 + CD4 mixture and the D10-alone sample
overlay quite well, indicating that the addition of CD4 does not affect
the kinetics of D10 TCR binding to CA/I-Ak. Fig.
3b shows that the specific D10 binding responses in the presence of a range of hCD4 concentrations (computed by subtracting the
hCD4 response from the response of the corresponding D10 + CD4 mixture)
were virtually identical to that in the absence of hCD4. These data
suggest that the affinity of the D10 TCR
clonotype for its
ligand CA/I-Ak is not altered by CD4. Such identity would
not be observed if interaction between CD4 and pMHCII conformationally
affected the antigen-presenting platform, thereby modulating TCR
binding. Similar results were observed at 37 °C (data not
shown).
View larger version (15K):
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Fig. 3.
CD4 does not alter interactions between the
TCR heterodimer and its pMHCII
ligand. a, sensorgrams of 20 µM D10 TCR
binding to surface-captured CA/I-Ak in the presence
(thin line) or absence (thick line) of 50 µM hCD4. The sensorgram of 50 µM hCD4
binding alone to the surface-captured CA/I-Ak-LZ
(i.e. in the absence of D10 TCR) is also shown (dashed
line). b, composite plot of binding responses of D10
with CA/I-Ak in the presence of varying concentrations of
hCD4 (open circles). The response of injection of hCD4 alone
over the same surface is also plotted at each concentration
(filled circles). The difference in the hCD4 binding seen
with or without D10 is taken as specific D10 binding response and is
plotted as open squares. The responses range between 130 and 160 RU. In
the absence of hCD4, 20 µM D10 TCR shows a binding
response of 140 RU.
heterodimer, or scD10 TCR to CD4
was studied by passage of these individual protein analytes over a CM5
sensor chip onto which the hCD4 coreceptor ectodomain was directly
immobilized. Successive injections of mAb 19Thy5D7 (anti-D1 domain) and
OKT4 (anti-D3 domain) verified the orientation and immunoreactivity of
the coupled hCD4. Both mAbs showed identical binding responses,
indicating that the overall exposure of the various segments of the CD4
molecules is equivalent (data not shown). The insets of Fig.
4, a, c, and
d, show the schematic interaction of CA/I-Ak
(Fig. 4a), D10 (Fig. 4c), or scD10 (Fig.
4d) to the immobilized hCD4 (only one hCD4 orientation is
shown here). The sensorgrams of CA/I-Ak binding to CD4 and
Fab116 (an anti-SIV gp140 mAb fragment used as the control surface) at
indicated concentrations are shown in Fig. 4a, main plot
(solid and dashed lines, respectively). The
binding of CA/I-Ak to CD4 shows weak affinity with on- and
off-rates too rapid to measure. The sustainable responses on the
control surface are mainly due to the bulk effect from the high
CA/I-Ak protein concentrations (up to 250 µM). The specific CA/I-Ak binding to CD4 is
taken as the difference between responses in RU on the CD4 and Fab116
surfaces. These data are plotted using Scatchard analysis as shown in
Fig. 4b, giving an affinity of ~200 µM
(Kd) for the hCD4-CA/I-Ak interaction.
The affinity between hCD4 and the D10 or scD10 TCR is much lower than
that between hCD4 and CA/I-Ak (Fig. 4, c and
d). In fact, no specific binding could be detected between
CD4 and the D10 TCR
heterodimer or between CD4 and the scD10.
The responses on the Fab control surface (Fig. 4, c and
d) and the intact, unrelated 13A12 IgG control surface (data not shown) were slightly higher than that on the CD4 surface. Comparison of the sensorgrams (Fig. 4, c and d)
shows very similar results, except that the bulk effect differs,
resulting from the distinct molecular masses of D10 and scD10 TCRs.
Hence, we detect no binding between the TCR and CD4 ectodomains,
implying that either the interaction, if it exists, is too low to
measure or involves other TCR components in addition to or distinct
from the
heterodimer.
View larger version (27K):
[in a new window]
Fig. 4.
Low affinity interaction between hCD4 and
CA/I-Ak. Interaction studies are shown on the
individual binding of CA/I-Ak, D10, or scD10 to directly
immobilized hCD4. The main plots show the sensorgrams of
CA/I-Ak (a), D10 (c), and scD10
(d) passing through the hCD4 surface (~3000 RU
immobilized, solid line) and the irrelevant
Fab116-immobilized control surface (~2800 RU immobilized,
dashed line). The insets show schematically the
potential interactions between immobilized hCD4 and
CA/I-Ak, D10, and scD10. Panel b shows a
Scatchard plot of the CD4-CA/I-Ak interaction studied in
panel a.
View larger version (17K):
[in a new window]
Fig. 5.
Comparatively weak affinity interaction
between hCD4 and human MHCII alleles. The inhibition curve of
soluble hCD4 on the interaction between class II MHC+ B
Raji cells and COS-7 cells transfected with surface-expressed
full-length hCD4 (open circles). The concentration of
soluble hCD4 required to inhibit cell-cell interaction by 50% was
taken as the dissociation constant (Kd). The effects
of 0.1 µM D1-specific anti-CD4 mAb 19Thy5D7
(asterisk) and various concentrations of bovine serum
albumin (filled circles) were also plotted as positive and
negative controls, respectively. Results are representative of two
independent titration experiments.
Heterodimer Interaction with pMHCII Does Not Augment
Subsequent CD4 Binding--
The presence of CD4 does not influence the
interaction between D10
and CA/I-Ak ectodomains,
indicating independent binding of TCR and CD4 to CA/I-Ak.
We next examined whether the D10
heterodimer ligation to CA/I-Ak could affect binding of each component to the
immobilized CD4. Since the D10 TCR binds to CA/I-Ak at a
1:1 molar ratio (36), the two ectodomain proteins were mixed in
equivalent amounts and injected over the CD4 surface. As shown in Fig.
6, a-c, the sensorgrams of
the binding of the D10-CA/I-Ak pre-mixture to CD4 are
clearly no greater than the sum of the individual components in each
pre-mixture. Furthermore, no change is observed in either association
or dissociation phases. Collectively, these data show that the D10 TCR
and CA/I-Ak do not affect each other's binding to CD4.
View larger version (16K):
[in a new window]
Fig. 6.
TCR
heterodimer interaction with pMHCII does not increase the
affinity for CD4. Sensorgrams of the binding of immobilized hCD4
to CA/I-Ak (bold line), D10 (dashed
line), and the pre-mixed combination of CA/I-Ak and
D10 (solid line). Indicated concentrations of
CA/I-Ak, D10, and the mixture are shown in separated panels
(a, b, and c).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3 and
2M domains of
class I MHC and homologous
2 and
2 domains of class II MHC
molecules, respectively (11, 12, 14, 44). Furthermore, to mediate
physiologic interactions, each coreceptor binds to the same MHC as the
TCR- ligated MHC molecules. In the case of murine CD8
or
CD8
interaction with H-2Kb molecules, the affinity is
~30-75 µM (32, 45). A severalfold lower affinity has
been reported for the interaction between human CD8
and
HLA-A2 (46).
heterodimer. In fact,
sensorgrams investigating the CD4 interaction with the D10 TCR
heterodimer were equivalent to those of CD4 and the scD10 module.
Retention of antibody epitopes on key TCR, pMHCII, and CD4 domains
suggests that the recombinant proteins utilized herein are native.
Although mAbs to all protein domains were not available for testing,
the fact that each protein has been crystallized and diffracts to
near-atomic resolution argues for the native structures of the
components analyzed above (36). Hence, we conclude that there is no TCR
-CD4 interaction. These findings suggest that either such a
CD4-TCR interaction doesn't exist or, if it does, that the CD4
membrane proximal region must contact other TCR components in addition
to or distinct from the
heterodimer. Recent structural analysis
of scD10 in complex with CA/I-Ak indicates that the
-helix of the I-Ak
-chain is contacted by the TCR
V
domain (36). Furthermore, mAb mapping studies suggest that the
CD3
heterodimer is adjacent to the TCR
subunit (52). Other
potential contacts may involve CD3
as shown in Fig.
7. CD3
association with TCR
may alter the structure of the outer face of the C
domain as well
(53, 54).
View larger version (43K):
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Fig. 7.
Coordinated immune recognition of pMHCII
ligands by the TCR and CD4 (a model). Recognition is initiated by
TCR ligation of pMHCII via the V -V
module of the TCR
heterodimer. Any transient interaction is subsequently stabilized and
facilitated by CD4-associated p56lck binding to ITAM
motifs (open rectangle) in the cytoplasmic tail of the CD3
components (
,
,
, and
). CD4 binds to the
2 domain of
MHCII and other possible contacts (not shown). Note how the TCR V
domain binds to the
1-helix of the antigen-presenting platform
containing a peptide (p). Hence, the CD4 D3-D4 module
may interact with or lie near the CD3
heterodimer.
Aside from interactions between their respective ectodomains,
additional mechanisms exist to juxtapose the TCR and CD4 molecules. Upon cross-linking, the TCR redistributes into lipid rafts where CD4
and p56lck are resident (55, 56). TCR ligation by pMHC results
in phosphorylation of ITAMs in the cytoplasmic tail of the
CD3,
,
, and
components (57-61). These segments serve as
substrates for p56lck-mediated tyrosine phosphorylation as well
as p56lck SH2 interaction, both of which contribute to
TCR-based signaling (26, 62). Coassociation of a ligated TCR and CD4
could result from an "adaptor" role for p56lck given its
noncovalent linkage to CD4, as demonstrated from the studies of Xu and
Littman (26). Consistent with this suggestion, only partial CD3
phosphorylation is induced by antagonist peptide ligands
versus more complete phosphorylation by agonist peptide ligands (63-65). CD4 selectively enhances T cell recognition of the
agonists but not the antagonists (66).
In these current studies, we show that the binding of the D10
TCR heterodimer to its pMHCII ligand is not affected by prior contact
with the CD4 ectodomain. Thus, no conformational change and/or
alteration in the peptide antigen-presenting platform or pMHC
interdomain disposition affecting TCR binding is induced. This result
is analogous to earlier findings involving mouse as well as human
CD8
-pMHCI complexes (47, 48). Coreceptor function per
se does not modify monomeric TCR affinity for its pMHC ligand (32,
46). Consistent with these functional results, crystallographic studies
of unligated human and mouse MHC class I molecules show no detectable
differences in the MHC antigen-resenting platform in the presence or
absence of the CD8
interaction. The common theme for both CD4 and
CD8 coreceptor is the bidentate interaction involving separate sites
for TCR and coreceptor on the pMHC and the attendant recruitment of
p56lck.
Collectively, our results suggest that on helper T cells, a given TCR
first interacts with the relevant pMHC class II ligand. The 30-fold
higher affinity of the D10 TCR heterodimer relative to CD4 for
the same pMHCII ligand argues that the TCR is the major contributor of
the binding energy. TCR ligation then initiates a signaling cascade
that includes and is critically dependent upon p56lck in
association with CD4. CD4 is directed toward the TCR-ligated pMHCII
molecule, augmenting binding and perhaps facilitating TCR cross-linking
via CD4 and/or TCR clustering. Consistent with this notion, anti-CD4
mAbs directed against a CD4 ectodomain fail to block pMHCII tetramer
binding to antigen-specific T cells yet inhibit activation of those
cells (67, 68). Videomicroscopy results also support the notion of a
primary binding role for the TCR rather than CD4 in T cell recognition
of pMHCII on antigen-presenting cells (69). This bidentate interaction
will increase contact of the T cell with pMHCII, permitting a
sufficient dwell time to activate gene programs required for cytokine
production and to mediate helper T cell activity.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Jia-huai Wang, Linda Clayton, and Catherine Zhang for critical reading and helpful comments. Technical support from Rebecca Hussey and Michelle Neben is gratefully acknowledged.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants AI19807 and AI43649.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Laboratory of
Immunobiology, Dana-Farber Cancer Institute, 44 Binney St., Boston, MA
02115. Tel.: 617-632-3412; Fax: 617-632-3351; E-mail:
ellis_reinherz@dfci.harvard.edu.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M009580200
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ABBREVIATIONS |
---|
The abbreviations used are: MHC, major histocompatibility complex; TCR, T cell receptor; pMHC, peptide-MHC complex; CA, conalbumin A; mAb, monoclonal antibody; LZ, leucine zipper; RU, relative units; PAGE, polyacrylamide gel electrophoresis; scD10, single chain D10; ITAM, immunoreceptor tyrosine activation motif.
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