T Cell Receptor Binding to a pMHCII Ligand Is Kinetically Distinct from and Independent of CD4*

Yi Xiong, Petra Kern, Hsiu-Ching Chang, and Ellis L. ReinherzDagger

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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha beta TCR heterodimer binds to conalbumin/I-Ak with virtually identical kinetics and affinity as the single chain Valpha Vbeta 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 alpha beta 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

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 beta 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).

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 alpha beta heterodimer.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction and Expression of D10 TCR in Escherichia coli-- Plasmids pEE14-D10alpha B and pEE14-D10beta A were used as polymerase chain reaction templates to generate cDNAs encoding the extracellular domains of D10 alpha  and beta  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 alpha  and beta  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 alpha  chain was changed to serine (TGC to TCC) by overlapping polymerase chain reaction and restriction fragment replacement. The alpha  and beta  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.

Protein expression and inclusion body preparation of D10 alpha  and beta  subunits were performed by the Cell Production and Recovery Facility, Rutgers University. Briefly, bacterial cells transformed with either pET-D10alpha or pET-D10beta 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-beta -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.

Refolding and Purification of D10 TCR alpha beta Heterodimer and Single Chain D10 (scD10)-- D10 TCR alpha beta heterodimer was refolded as described previously (29) with some modifications. The alpha  and beta  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 alpha  and beta  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 alpha /beta mixture was added. A third mixture was added 6-12 h later, and a further 24-h incubation was performed.

The refolded material was filtered (Corning, 0.22 µm) and immunoaffinity-purified using mAb 3D3 covalently coupled gamma  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.

The scD10 TCR consists of 237 residues and is organized from N to C terminus as follows: Vbeta 8.2 (residues 3-116)-linker (GSADDAKKDAAKKDG)-Valpha 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.

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 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 beta  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 alpha  (residues -3-192) and beta  (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.

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 - 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

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 alpha beta 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 alpha  and beta  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, alpha  chain cysteine 109 was replaced by a serine. The alpha  and beta  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 alpha beta 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.



View larger version (35K):
[in this window]
[in a new window]
 
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 Vbeta is connected to Valpha by a 15-residue linker. D10 represents the extracellular Ig-like variable (V) and constant (C) domains of D10 TCR, with alpha  and beta  chains noncovalently associated to form an alpha beta 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 beta -chain via a flexible linker (short line) and the 37-residue LZ peptides (coil) attached to both the alpha  and beta  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.

SDS-PAGE analysis of the purified protein shows that the alpha  and beta  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 alpha  and beta  bands on SDS-PAGE under nonreducing relative to reducing conditions (Fig. 1b, lane 2 versus 1; 23 versus 26 kDa for alpha  and 27 versus 30 kDa for beta ) 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 alpha  and beta  chains were confirmed by Edman degradation after transfer to a polyvinylidene difluoride membrane. To assess the integrity of the alpha  and beta  constant domains, the TCR protein was immunoprecipitated with TCR Calpha -specific mAb H28 and Cbeta -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 Valpha and Vbeta domains, the above results indicate that the purified D10 TCR assumes a correctly folded conformation in both V- and C-region modules.

To investigate any possible function of the C module in antigen recognition or CD4 interaction, we compared the above D10 alpha beta TCR heterodimer with a scD10 TCR Valpha Vbeta module. As shown in Fig. 1a, the latter contains the D10 TCR Vbeta 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 Valpha 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).

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 alpha  and beta  subunits, with a 13-residue peptide corresponding to residues 153-165 of hen CA attached to the N terminus of the beta  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 alpha  chain and the lower one is the CA peptide fused beta  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 (alpha  = 27,295 daltons and beta  = 30,908 daltons). These differences are due to post-translational modification (there are two N-linked glycosylation sites in the alpha  chain and one in the beta  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).

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 alpha beta 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 alpha beta 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).



View larger version (35K):
[in this window]
[in a new window]
 
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.

The Binding of the D10 alpha beta 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 alpha beta heterodimer protein was mixed with different concentrations of soluble hCD4 and incubated overnight at 4 °C. In these experiments, the concentration of D10 TCR alpha beta 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 alpha beta 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):
[in this window]
[in a new window]
 
Fig. 3.   CD4 does not alter interactions between the TCR alpha beta 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.

CD4 Binds to pMHCII with Low Affinity-- The binding of CA/I-Ak, D10 TCR alpha beta 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 alpha beta 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 alpha beta heterodimer.



View larger version (27K):
[in this window]
[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.

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.



View larger version (17K):
[in this window]
[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.

TCR alpha beta Heterodimer Interaction with pMHCII Does Not Augment Subsequent CD4 Binding-- The presence of CD4 does not influence the interaction between D10 alpha beta and CA/I-Ak ectodomains, indicating independent binding of TCR and CD4 to CA/I-Ak. We next examined whether the D10 alpha beta 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 this window]
[in a new window]
 
Fig. 6.   TCR alpha beta 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

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 alpha 3 and beta 2M domains of class I MHC and homologous beta 2 and alpha 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 CD8alpha alpha or CD8alpha beta 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 CD8alpha alpha and HLA-A2 (46).

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 alpha beta heterodimer. In fact, sensorgrams investigating the CD4 interaction with the D10 TCR alpha beta 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 alpha beta -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 alpha beta heterodimer. Recent structural analysis of scD10 in complex with CA/I-Ak indicates that the alpha -helix of the I-Ak beta -chain is contacted by the TCR Valpha domain (36). Furthermore, mAb mapping studies suggest that the CD3epsilon delta heterodimer is adjacent to the TCR alpha  subunit (52). Other potential contacts may involve CD3epsilon delta as shown in Fig. 7. CD3epsilon delta association with TCR alpha beta may alter the structure of the outer face of the Calpha domain as well (53, 54).



View larger version (43K):
[in this window]
[in a new window]
 
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 Valpha -Vbeta module of the TCR alpha beta 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 (gamma , delta , epsilon , and zeta ). CD4 binds to the beta 2 domain of MHCII and other possible contacts (not shown). Note how the TCR Valpha domain binds to the beta 1-helix of the antigen-presenting platform containing a peptide (p). Hence, the CD4 D3-D4 module may interact with or lie near the CD3epsilon delta 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 CD3gamma , delta , epsilon , and zeta  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 CD3zeta 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 alpha beta 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 CD8alpha alpha -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 CD8alpha alpha 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 alpha beta 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.


    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.


    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.

Dagger 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


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Reinherz, E. L., Kung, P. C., Goldstein, G., and Schlossman, S. F. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4061-4065[Abstract]
2. Reinherz, E. L., Kung, P. C., Goldstein, G., Levey, R. H., and Schlossman, S. F. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 1588-1592[Abstract]
3. Janeway, C. A., Jr. (1992) Annu. Rev. Immunol. 10, 645-674[CrossRef][Medline] [Order article via Infotrieve]
4. Kruisbeek, A. M., Mond, J. J., Fowlkes, B. J., Carmen, J. A., Bridges, S., and Longo, D. L. (1985) J. Exp. Med. 161, 1029-1047[Abstract]
5. Rahemtulla, A., Fung-Leung, W. P., Schilham, M. W., Kündig, T. M., Sambhara, S. R., Narendran, A., Arabian, A., Wakeham, A., Paige, C. J., Zinkernagel, R. M., Miller, R. G., and Mak, T. W. (1991) Nature 353, 180-184[CrossRef][Medline] [Order article via Infotrieve]
6. Killeen, N., Sawada, S., and Littman, D. R. (1993) EMBO J. 12, 1547-1553[Abstract]
7. Wang, J., Yan, Y., Garrett, T. P. J., Liu, J., Rodgers, D. W., Garlick, R. L., Tarr, G. E., Husain, Y., Reinherz, E. L., and Harrison, S. C. (1990) Nature 348, 411-418[CrossRef][Medline] [Order article via Infotrieve]
8. Ryu, S. E., Kwong, P. D., Truneh, A., Porter, T. G., Arthos, J., Rosenberg, M., Dai, X., Xuong, N., Axel, R., Sweet, R. W., and Hendrickson, W. A. (1990) Nature 348, 419-426[CrossRef][Medline] [Order article via Infotrieve]
9. Lange, G., Lewis, S. J., Murshudov, G. N., Dodson, G. G., Moody, P. C., Turkenburg, J. P., Barclay, A. N., and Brady, R. L. (1994) Structure (Lond.) 2, 469-481[Medline] [Order article via Infotrieve]
10. Wu, H., Kwong, P. D., and Hendrickson, W. A. (1997) Nature 387, 527-530[CrossRef][Medline] [Order article via Infotrieve]
11. Potter, T. A., Rajan, T. V., Dick, R. F., and Bluestone, J. A. (1989) Nature 337, 73-75[CrossRef][Medline] [Order article via Infotrieve]
12. Salter, R. D., Benjamin, R. J., Wesley, P. K., Buxton, S. E., Garrett, T. P. J., Clayberger, C., Krensky, A. M., Norment, A. M., Littman, D. R., and Parham, P. (1990) Nature 345, 41-46[CrossRef][Medline] [Order article via Infotrieve]
13. Moebius, U., Pallai, P., Harrison, S. C., and Reinherz, E. L. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8259-8263[Abstract/Free Full Text]
14. Konig, R., Huang, L. Y., and Germain, R. (1992) Nature 356, 796-798[CrossRef][Medline] [Order article via Infotrieve]
15. Sakihama, T., Smolyar, A., and Reinherz, E. L. (1995) Immunol. Today 16, 581-587[CrossRef][Medline] [Order article via Infotrieve]
16. Vignali, D. A., Carson, R. T., Chang, B., Mittler, R. S., and Strominger, J. L. (1996) J. Exp. Med. 183, 2097-2107[Abstract]
17. Vignali, D. A., and Vignali, K. M. (1999) J. Immunol. 162, 1431-1439[Abstract/Free Full Text]
18. Saizawa, K., Rojo, J., and Janeway, C. A., Jr. (1987) Nature 328, 260-263[CrossRef][Medline] [Order article via Infotrieve]
19. Kupfer, A., Singer, S. J., Janeway, C. A., Jr., and Swain, S. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5888-5892[Abstract]
20. Janeway, C. A., Jr. (1992) Immunol. Today 13, 11-16[CrossRef][Medline] [Order article via Infotrieve]
21. Janeway, C. A., Jr. (1989) Immunol. Today 10, 234-238[Medline] [Order article via Infotrieve]
22. Rudd, C. E., Trevillyan, J. M., Dasgupta, J. D., Wong, L. L., and Schlossman, S. F. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 5190-5194[Abstract]
23. Veillette, A., Bookman, M. A., Horak, E. M., and Bolen, J. B. (1988) Cell 55, 301-308[Medline] [Order article via Infotrieve]
24. Turner, J. M., Brodsky, M. H., Irving, B. A., Levin, S. D., Perlmutter, R. M., and Littman, D. R. (1990) Cell 60, 755-765[Medline] [Order article via Infotrieve]
25. Crise, B., and Rose, J. K. (1992) J. Biol. Chem. 267, 13593-13597[Abstract/Free Full Text]
26. Xu, H., and Littman, D. R. (1993) Cell 74, 633-643[Medline] [Order article via Infotrieve]
27. Hussey, R. E., Richardson, N. E., Kowalski, M., Brown, N. R., Chang, H. C., Siliciano, R. F., Dorfman, T., Walker, B., Sodroski, J., and Reinherz, E. L. (1988) Nature 331, 78-81[CrossRef][Medline] [Order article via Infotrieve]
28. Weber, S., and Karjalainen, K. (1993) Int. Immunol. 5, 695-698[Abstract]
29. Garboczi, D. N., Utz, U., Ghosh, P., Seth, A., Kim, J., Van Tienhoven, E. A., Biddison, W. E., and Wiley, D. C. (1996) J. Immunol. 157, 5403-5410[Abstract]
30. Stanley, P. (1989) Mol. Cell. Biol. 9, 377-383[Medline] [Order article via Infotrieve]
31. Liu, J., Tse, A. G. D., Chang, H.-C., Liu, J.-H., Wang, J., Hussey, R. E., Chishti, Y., Reinhold, B., Spoerl, R., Nathenson, S. G., Sacchettini, J. C., and Reinherz, E. L. (1996) J. Biol. Chem. 271, 33639-33646[Abstract/Free Full Text]
32. Kern, P., Hussey, R. E., Spoerl, R., Reinherz, E. L., and Chang, H.-C. (1999) J. Biol. Chem. 274, 27237-27243[Abstract/Free Full Text]
33. Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326[Medline] [Order article via Infotrieve]
34. Chang, H.-C., Bao, Z.-Z., Yao, Y., Tse, A. G. D., Goyarts, E. C., Madsen, M., Kawasaki, E., Brauer, P. P., Sacchettini, J. C., Nathenson, S. G., and Reinherz, E. L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 11408-11412[Abstract/Free Full Text]
35. Landau, N. R., and Littman, D. R. (1992) J. Virol. 66, 5110-5113[Abstract]
36. Reinherz, E. L., Tan, K., Tang, L., Kern, P., Liu, J.-H., Xiong, Y., Hussey, R. E., Smolyar, A., Hare, B., Zhang, R., Joachimiak, A., Chang, H.-C., Wagner, G., and Wang, J.-H. (1999) Science 286, 1913-1921[Abstract/Free Full Text]
37. Davis, M. M., Boniface, J. J., Reich, Z., Lyons, D., Hampl, J., Arden, B., and Chien, Y. (1998) Annu. Rev. Immunol. 16, 523-544[CrossRef][Medline] [Order article via Infotrieve]
38. Khandekar, S. S., Brauer, P. P., Naylor, J. W., Chang, H. C., Kern, P., Newcomb, J. R., Leclair, K. P., Stump, H. S., Bettencourt, B. M., Kawasaki, E., Banerji, J., Profy, A. T., and Jones, B. (1997) Mol. Immunol. 34, 493-503[CrossRef][Medline] [Order article via Infotrieve]
39. Khandekar, S. S., Bettencourt, B. M., Wyss, D. F., Naylor, J. W., Brauer, P. P., Huestis, K., Dwyer, D. S., Profy, A. T., Osburne, M. S., Benerji, J., and Jones, B. (1997) J. Biol. Chem. 272, 32190-32197[Abstract/Free Full Text]
40. von Hoegen, P., Miceli, M. C., Tourvieille, B., Schilham, M., and Parnes, J. R. (1989) J. Exp. Med. 170, 1879-1886[Abstract]
41. Glaichenhaus, N., Shastri, N., Littman, D. R., and Turner, J. M. (1991) Cell 64, 511-520[Medline] [Order article via Infotrieve]
42. Law, Y. M., Yeung, R. S. M., Mamalaki, C., Kioussis, D., Mak, T. W., and Flavell, R. A. (1994) J. Exp. Med. 179, 1233-1242[Abstract]
43. Sakihama, T., Hunsicker, M. E., Hussey, R. E., and Reinherz, E. L. (2000) Eur. J. Immunol. 30, 279-290[CrossRef][Medline] [Order article via Infotrieve]
44. Connolly, J. M., Hansen, T. H., Ingold, A. L., and Potter, T. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2137-2141[Abstract]
45. Garcia, K. C., Scott, C. A., Brunmark, A., Carbone, F. R., Peterson, P. A., Wilson, I. A., and Teyton, L. (1996) Nature 384, 577-581[CrossRef][Medline] [Order article via Infotrieve]
46. Wyer, J. R., Wilcox, B. E., Gao, G. F., Gerth, U. C., Davis, S. J., Bell, J. I., van der Merwe, P. A., and Jakobsen, B. K. (1999) Immunity 10, 219-225[CrossRef][Medline] [Order article via Infotrieve]
47. Kern, P., Teng, M.-K., Smolyar, A., Liu, J.-H., Liu, J., Hussey, R. E., Chang, H.-C., Reinherz, E. L., and Wang, J.-H. (1998) Immunity 9, 519-530[Medline] [Order article via Infotrieve]
48. Gao, G. F., Tormo, J., Gerth, U. C., Wyer, J. R., McMichael, A. J., Stuart, D. I., Bell, J. I., Jones, E. Y., and Jakobsen, B. K. (1997) Nature 387, 630-634[CrossRef][Medline] [Order article via Infotrieve]
49. Moebius, U., Clayton, L. K., Abraham, S., Diener, A., Yunis, J. J., Harrison, S. C., and Reinherz, E. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 12008-12012[Abstract]
50. Fleury, S., Lamarre, D., Meloche, S., Ryu, S. E., Cantin, C., Hendrickson, W. A., and Sekaly, P. (1991) Cell 66, 1037-1049[Medline] [Order article via Infotrieve]
51. Sakihama, T., Smolyar, A., and Reinherz, E. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6444-6448[Abstract]
52. Ghendler, Y., Teng, M.-K., Liu, J.-H., Witte, T., Liu, J., Kim, K. S., Kern, P., Chang, H.-C., Wang, J.-H., and Reinherz, E. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10061-10066[Abstract/Free Full Text]
53. Garcia, K. C., Degano, M., Stanfield, R. L., Brunmark, A., Jackson, M. R., Peterson, P. A., Teyton, L., and Wilson, I. A. (1996) Science 274, 209-219[Abstract/Free Full Text]
54. Wang, J., Lim, K., Smolyar, A., Teng, M.-K., Liu, J.-H., Tse, A. G. T., Liu, J., Hussey, R. E., Chishti, Y., Thomson, C. T., Sweet, R. M., Nathenson, S. G., Chang, H.-C., Sacchettini, J. C., and Reinherz, E. L. (1998) EMBO J. 17, 10-26[Abstract/Free Full Text]
55. Montixi, C., Langlet, C., Bernard, A. M., Thimonier, J., Dubois, C., Wurbel, M. A., Chauvin, J. P., Pierres, M., and He, H. T. (1998) EMBO J. 17, 5334-5348[Abstract/Free Full Text]
56. Xavier, R., Brennan, T., Li, Q., McCormack, C., and Seed, B. (1998) Immunity 8, 723-732[Medline] [Order article via Infotrieve]
57. Reth, M. (1989) Nature 338, 383-384[Medline] [Order article via Infotrieve]
58. Irving, B. Q., and Weiss, A. (1991) Cell 64, 891-901[Medline] [Order article via Infotrieve]
59. Letourneur, F., and Klausner, R. D. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8905-8909[Abstract]
60. Romeo, C., Amiot, M., and Seed, B. (1992) Cell 68, 889-897[Medline] [Order article via Infotrieve]
61. Wegener, A. M., Letourneur, F., Hoeveler, A., Brocker, T., Luton, F., and Malissen, B. (1992) Cell 68, 83-95[Medline] [Order article via Infotrieve]
62. Straus, D. B., and Weiss, A. (1992) Cell 70, 585-593[Medline] [Order article via Infotrieve]
63. Kersh, E. N., Shaw, A. S., and Allen, P. M. (1998) Science 281, 572-575[Abstract/Free Full Text]
64. Kersh, E. N., Kersh, G. J., and Allen, P. M. (1999) J. Exp. Med. 190, 1627-1636[Abstract/Free Full Text]
65. Sloan-Lancaster, J., Shaw, A. S., Rothbard, J. B., and Allen, P. M. (1994) Cell 79, 913-922[Medline] [Order article via Infotrieve]
66. Hampl, J., Chien, Y. H., and Davis, M. M. (1997) Immunity 7, 379-385[Medline] [Order article via Infotrieve]
67. Boniface, J. J., Rabinowitz, J. D., Wulfing, C., Hampl, J., Reich, Z., Altman, J. D., Kantor, R. M., Beeson, C., McConnell, H. M., and Davis, M. M. (1998) Immunity 9, 459-466[Medline] [Order article via Infotrieve]
68. Crawford, F., Kozono, H., White, J., Marrack, P., and Kappler, J. (1998) Immunity 8, 675-682[Medline] [Order article via Infotrieve]
69. Krummel, M. F., Sjaastad, M. D., Wülfing, C., and Davis, M. M. (2000) Science 289, 1349-1352[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.