©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Binding Stoichiometry of the Cytotoxic T Lymphocyte-associated Molecule-4 (CTLA-4)
A DISULFIDE-LINKED HOMODIMER BINDS TWO CD86 MOLECULES (*)

Peter S. Linsley (§) , Steven G. Nadler , Jürgen Bajorath , Robert Peach , Helios T. Leung , Julie Rogers , Jeff Bradshaw , Mark Stebbins , Gina Leytze , William Brady , Alison R. Malacko , Hans Marquardt , Shyh-Yu Shaw (1)

From the (1)Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121 and Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

CD28 and CTLA-4 are homologous T cell receptors of the immunoglobulin (Ig) superfamily, which bind B7 molecules (CD80 and CD86) on antigen-presenting cells and transmit important costimulatory signals during T cell activation. Here we have investigated the subunit structure of CTLA-4 and the stoichiometry of its binding to B7 molecules. We demonstrate CTLA-4 is a homodimer interconnected by one disulfide bond in the extracellular domain at cysteine residue 120. Each monomeric polypeptide chain of CTLA-4 contains a high affinity binding site for B7 molecules; soluble CTLA-4 and CD86 form complexes containing equimolar amounts of monomeric CTLA-4 and CD86 (i.e. a 2:2 molecular complex). Thus, CTLA-4 and probably CD28 have a receptor structure consisting of preexisting covalent homodimers with two binding sites. Dimerization of CTLA-4 and CD28 is not required for B7 binding, nor is it sufficient to trigger signaling.


INTRODUCTION

Intercellular interactions between T lymphocytes and antigen-presenting cells (APC)()generate T cell costimulatory signals which regulate T cell responses to antigen in an antigen nonspecific manner(1) . Costimulatory signals determine the magnitude of T cell responses, and whether an encounter with antigen activates or inactivates subsequent responses to antigen(2) . T cell activation in the absence of costimulation results in aborted or anergic T cell responses(3) . A key costimulatory signal(s) is provided by engagement of T cell receptors CD28 and CTLA-4 with B7 molecules on APC(4) . CD28 and CTLA-4 are both members of the immunoglobulin superfamily (IgSF) having single variable-like domains in their extracellular regions(5) . These molecules share sequence identity, most notably clustered in their CDR3-like regions(6) . Both CD28 and CTLA-4 bind the same natural ligands, CD80 (formerly known as B7-1) and CD86 (formerly known as B7-2 or B70)(7, 8) . Engagement of CD28 by B7 molecules delivers a strong costimulatory signal(4, 9) , whereas engagement of CTLA-4 in different systems triggers either a weak costimulatory signal (10, 11) or a negative signal(12) .

Binding interactions between CD28, CTLA-4, and B7 molecules have been studied with recombinant-soluble immunoglobulin fusion proteins of these molecules. CTLA4Ig bound to CD80 with 20-fold higher avidity than did the analogous CD28Ig(13) . Amino acid residues responsible for the increased binding activity of CTLA4Ig to CD80 were localized to the CDR-1 and COOH-terminally extended CDR3-like regions(6) . CD80 and CD86 share only 25% amino acid sequence identity in their extracellular domains, but show very similar equilibrium binding to CD28Ig or CTLA4Ig (14).

A fundamental uncertainty regarding the binding interactions of CTLA-4CD28 and B7 molecules is their binding stoichiometry. CD28 is a disulfide-linked homodimer, whereas the subunit structure of CTLA-4 has been controversial. Some studies have suggested that CTLA-4 is also a disulfide-linked homodimer(10, 13, 15) , but another suggested that it exists as a monomer(16) . It is currently unknown whether dimerization is required for binding activity of CD28 and or CTLA-4, as it is for many IgSF members(17) . In this study, we have determined the binding stoichiometry of CTLA-4 for B7 molecules. We show that CTLA-4 is a disulfide-linked homodimer containing two binding sites for its ligands.


MATERIALS AND METHODS

mAbs, Ig Fusion Proteins, and Flow Cytometry

Murine mAbs 9.3 (anti-CD28), G10-1 (anti-CD8), and G17-2 (anti-CD4) and F(ab`) fragments of mAb G19-4 (anti-CD3) were provided by Dr. J. Ledbetter of Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle. Murine mAb L6 (anti-human lung tumor antigen) and its chimeric derivative (human Fc) were from Drs. K. E. and I. Hellström also of Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle. mAb 11D4 (anti-CTLA-4) has been described previously(10) . mAb 7H4 (anti-CTLA-4) was prepared in the same manner as other anti-CTLA-4 mAbs(10) ; mAb 7H4 binds CTLA4Ig, but does not block its binding to CD80. Human CTLA4Ig, CD28Ig, CD80Ig, CD86Ig, CD80 mIg, CD86 mIg fusion proteins were prepared as described(14) . For immunostaining, cells were removed from their culture vessels by incubation in PBS containing 10 mM EDTA. CHO cells (1-10 10) were first incubated with mAbs or Ig fusion proteins in DMEM containing 10% fetal bovine serum (FBS), then washed and incubated with fluorescein isothiocyanate-conjugated goat anti-mouse or anti-human Ig second step reagents (Tago, Burlingame, CA). Cells were washed and analyzed on a FACScan (Becton Dickinson).

Cell Culture

Anti-CD3-activated T cell blasts were prepared essentially as described(10) , except that culture flasks were coated with F(ab`) fragments (10 µg/ml) of anti-CD3 mAb G19-4 instead of intact mAb. Peripheral blood mononuclear cells were isolated from heparinized blood and cultured on anti-CD3 F(ab`)-coated flasks for 3 days at 37 °C in RPMI 1640 containing 10% FCS, 2 mM glutamine, and pen-strep (100 units/ml penicillin, 75 units/ml streptomycin). Retroviral vectors encoding CD28 and C4-28 (extracellular and transmembrane regions of human CTLA-4 fused to the cytoplasmic domain of CD28) will be described elsewhere.()3T3 cells were transduced with these vectors, selected for neomycin resistance, and cultured in DMEM containing 10% FBS, 2 mML-glutamine, 50 µM 2-mercaptoethanol, and pen-strep. COS cells were maintained in DMEM containing 10% FBS.

Iodination and Immunoprecipitation

Cells were harvested and washed with PBS. Cell surface iodination using lactoperoxidase was performed as described(18) . Surface-labeled cells were washed three times with PBS and solubilized in lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1% Nonidet P-40, 0.25% deoxycholate, 10 µg/ml each of aprotinin and leupeptin) for 30 min at 4 °C. Detergent-insoluble material was then removed by centrifugation (13,000 g for 15 min at 4 °C) and the supernatant was used for immunoprecipitation. Solubilized membrane proteins were also iodinated using lactoperoxidase. Cells (2 10 for COS cells and 150 10 for peripheral blood lymphocytes) were resuspended in (5 or 10 ml, respectively) hypotonic buffer (10 mM Tris-HCl, pH 7.5, 0.5 mM MgCl, 10 µg/ml each of aprotinin and leupeptin) and incubated on ice for 10 min. Cells were then lysed (>95% lysis) using a tight fitting Dounce homogenizer. The homogenate was transferred to a clean tube containing 0.1 volume of 10 mM Tris-HCl, pH 7.5, 0.5 mM MgCl, 1.5 M NaCl, and debris was removed by centrifugation at 800 g for 5 min. The supernatant was removed, and EDTA was added to a final concentration of 5 mM. Membranes were collected by centrifugation at 125,000 g for 45 min. The membrane pellet was solubilized in lysis buffer on ice for 30 min, and detergent-insoluble material was removed by centrifugation. Soluble proteins were then iodinated using lactoperoxidase, and unreacted I was removed by passage over a column of Sephadex G25 (Pharmacia Biotech Inc.), equilibrated with lysis buffer. The labeled protein extract was then used for immunoprecipitation analysis.

Immunoprecipitation Analysis

I-Labeled lysates were incubated at 4 °C with an irrelevant antibody (2 µg) and 50-µl packed volume immobilized protein A (IPA, r-protein A-Sepharose, Repligen, Cambridge, MA). IPA was incubated before use for 1 h in a solution of 5% bovine serum albumin in PBS and washed in lysis buffer. Sepharose beads were removed by centrifugation, and aliquots of the supernatant (1-2 10 cpm) were mixed with mAbs or Ig fusion proteins and incubated at 4 °C for 1-2 h. Bovine serum albumin-blocked IPA was added (50 µl packed volume), and incubation at 4 °C was continued for an additional 1-2 h. Samples treated with mAb 11D4 or its isotype controls (mouse IgG1) were incubated with a ``bridging'' antibody (rabbit anti-mouse IgG, Sigma) at 50 µg/ml for 1-2 h at 4 °C before addition of protein A-Sepharose. Beads were collected by centrifugation, washed four times with PBS containing 0.5% Nonidet P-40, twice with PBS alone, and immunoprecipitated proteins solubilized in SDS-PAGE sample buffer. When labeled lysates from anti-CD3-stimulated T lymphocytes were used, CD28 was removed from solution by immunoprecipitation with anti-CD28 mAb 9.3 before addition of CD80Ig. For immunoblotting analysis, aliquots of conditioned medium or samples of purified protein (1 µg) were incubated with 5 µg of human Ig (negative control), CD80Ig, or CD86Ig for 1 h at 4 °C. Ig-containing proteins were then precipitated with 50 µl of IPA. Precipitates were washed three times with PBS, bound proteins were eluted by addition of concentrated SDS-PAGE sample buffer, and samples were analyzed by SDS-PAGE.

Preparation of Recombinant Fragments of CTLA-4

Soluble extracellular domain of CTLA-4 was expressed in COS cells by introducing a stop codon into CTLA-4 cDNA after Asp by polymerase chain reaction-directed mutagenesis(6) . For CTLA4X, the OMCTLA-4 (13) expression plasmid was used as template, the forward primer GAGGTGATAAAGCTTCACCAATGGGTGTACTGCTCACACAG was chosen to match sequences in the vector, and the reverse primer, GTGGTGTATTGGTCTAGATCAATCAGAATCTGGGCACGGTTC, corresponded to the last seven amino acids in the extracellular domain of CTLA-4 followed by a stop codon (TGA). CTLA4X was constructed in similar fashion, except that the reverse primer specified a C120S mutation. Polymerase chain reaction products were digested with HindIII/XbaI and directionally subcloned into the expression vector LN (a gift of Dr. A. Aruffo, Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle). Each construct was verified by DNA sequencing.

Expression and Purification of CTLA-4 Fragments

CTLA4X and CTLA4X were produced by transiently transfected COS cells(18) . Recombinant proteins were purified by CTLA-4 mAb affinity chromatography from transfected COS cell conditioned medium. Anti-CTLA-4 mAb 11D4 was coupled to cyanogen bromide (CNBr)-activated Sepharose 4B (8 mg of mAb/ml of resin) as suggested by the manufacturer (Pharmacia Biotech Inc.). COS cell conditioned medium was collected, filter-sterilized, and applied to the affinity resin by gravity flow. Following application of conditioned medium, the resin was washed with 5 bed volumes of PBS. Bound CTLA-4 fragments were eluted with 0.05 M trisodium phosphate, pH 11.5. Fractions (0.5 ml) were collected and immediately neutralized by addition of 0.2 ml of 2 M Tris-HCl, pH 6.8. CTLA-4-containing fractions were identified by capture immunoassay (10) and/or A. Peak fractions were pooled, concentrated using a Centriprep 10 (Amicon, Beverly, MA), and filter-sterilized. Recoveries of recombinant proteins were 50-90% based on immunoreactivity in the capture immunoassay. Extinction coefficients of 1.0 and 1.1 ml/mg were experimentally determined by amino acid analysis for purified CTLA4X and CTLA4X preparations, respectively. M = 13,400, corresponding to the predicted peptide mass of CTLA4X, was assumed for calculation of molar concentrations. Where indicated, concentrated conditioned medium from transfected COS cells was used as a source of recombinant CTLA-4X. This was concentrated 30-fold using a Centriprep 10 before fractionation.

Preparation of Thrombin Fragments

CTLA4Ig or CD86Ig fusion proteins (0.5 mg/ml in Tris-HCl buffer, pH 7.4-7.5) were digested with purified bovine thrombin (Armour Pharmaceutical Corp., Kankakee, IL) at a final concentration of 100 units/ml, at 37 °C for 16-24 h. After cleavage, thrombin was removed by twice incubating the protein solution with 0.10 packed bed volume of washed Affi-Gel Blue (Bio-Rad). Ig Fc-containing fragments were removed by twice incubating the protein solution with 0.20 packed bed volume of washed IPA. The resulting solution contained protein fragments CTLA4t and CD86t. The final recovery of purified fragments was 10-40%. Extinction coefficients of 1.3 and 1.2 ml/mg were experimentally determined by amino acid analysis for purified CTLA4t and CD86t preparations, respectively. M values of 13,900 and 25,800, corresponding to the predicted peptide mass of a single chain of CTLA4t and CD86t, respectively, were assumed for calculation of molar concentrations.

Gel Permeation Chromatography

Two columns were used for gel permeation chromatography. CTLA4X preparations were analyzed using a TSK-GEL G2000 SW column (7.8 300 mm, Tosohaas, Montgomeryville, PA). The column was equilibrated in PBS containing 0.02% NaN at a flow rate of 1 ml/min. Concentrated COS cell medium containing CTLA4X (0.2 ml) was fractionated by gel permeation chromatography, and fractions of 0.5 ml were collected and used for analysis. In other experiments, purified protein fragments were analyzed on a Waters 300 SW column equilibrated with PBS, containing 10 mM Tris-HCl, pH 7.4, and 0.01% NaN, at a flow rate of 0.35 ml/min. Essentially the same results were obtained with both systems.

Protein Sequencing

Protein sequencing methods have been described previously(21) . CTLA4t was first cleaved with cyanogen bromide (CNBr), and the peptides were purified. CNBr peptides were further digested with trypsin or chymotrypsin in the presence of 1 mM iodoacetamide and purified by reversed-phase HPLC. Automated sequence analysis was performed in a pulsed-liquid protein sequencer, and mass spectra were recorded on a Biolon 20 plasma desorption mass spectrometer (Applied Biosystems, Inc., Foster City, CA).

SDS-PAGE and Immunoblotting

SDS-PAGE was performed on Tris/glycine gels. In some cases, performed gels (Novex, San Diego, CA) were used. Analytical gels were stained with Coomassie Blue, and images of wet gels were obtained by digital scanning. Gels containing I-labeled proteins were dried and subjected to autoradiography. For immunoblotting, separated proteins were electrophoretically transferred to nitrocellulose membranes. Membranes were blocked with Specimen Diluent (Genetic Systems Corp., Redmond, WA) and probed with anti-CTLA-4 mAb 7H4 diluted in Specimen Diluent. Bound mAb was detected using horseradish peroxidase-conjugated goat anti-mouse Ig (Biosource International, Camarillo, CA) and ECL reagents (Amersham Corp.).

Competitive Binding Assays

Binding assays were performed on immobilized recombinant B7Ig fusion proteins(14) . Individual wells in 96-well plates (Immulon 2; Dynatech laboratories, Inc., Chantilly, VA) were coated for 16-24 h with 50 ng/well goat F(ab`) anti-mouse immunoglobulin antibodies (Tago). Wells were then washed, blocked with Ig binding medium (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 10% FBS, 0.1% NaN), and coated with CD80 mIg or CD86 mIg at 1 µg/ml. CTLA4Ig was radiolabeled with I using Bolton-Hunter reagent(14) , diluted in Ig binding medium, and mixed with solutions of competitor proteins diluted in PBS. Aliquots of the mixtures (50 µl) were then added to duplicate B7Ig-coated wells. Binding was allowed to proceed for 4 h at 23 °C, wells were washed, and bound radioactivity was measured by counting.


RESULTS

Cell Surface CTLA-4 Is a Disulfide-linked Homodimer

Various studies on the subunit structure of CTLA-4 have employed different serological reagents and different cell types(10, 13, 15, 16) . It was unclear how these differences contributed to the reported variation in the subunit structure of CTLA-4. We wished to determine the subunit structure of biologically active CTLA-4 transfected COS cells and on activated T cells. As shown in Fig. 1, both specific anti-CTLA-4 mAb and CD80Ig immunoprecipitated from surface-labeled COS cells a M 46,000 protein under nonreducing conditions and a M 30,000 protein under reducing conditions. Similar results were obtained with activated T cells; a M 50,000 protein was obtained under nonreducing conditions and a M 33,000 protein under reducing conditions. Thus, regardless of the cell source, CTLA-4, as recognized by specific mAb or soluble natural ligand, migrated more rapidly under reducing conditions. This was consistent with CTLA-4 being a disulfide-linked homodimer or being linked to an unlabeled protein. Evidence supporting the first of these possibilities was provided in subsequent experiments (see ).


Figure 1: CTLA-4 contains an intermolecular disulfide bond. Left panel, immunoprecipitation from COS cells. COS cells were transfected with the OMCTLA-4 expression plasmid. Forty-eight h post-transfection, cells were removed from their culture dishes and cell surface-labeled with I using lactoperoxidase. I-Labeled cells were then solubilized with non-ionic detergent solution, subjected to immunoprecipitation analyses with: CD5Ig (4 µg) (lanes 1), control mAb G17-2 (anti-CD4, 2 µg) (lanes 2), control mAb G10-1 (anti-CD5, 2 µg) (lanes 3), CD80Ig (4 µg) (lanes 4), and mAb 11D4 (anti-CTLA-4, 2 µg) (lanes 5). Samples were analyzed by SDS-PAGE (10% gel) under nonreducing (--mercaptoethanol (ME)) or reducing +-mercaptoethanol (+ME) conditions. Migration positions of standards are indicated. Right panel, immunoprecipitation from anti-CD3-activated peripheral blood T cells. Membranes were prepared from anti-CD3-activated human mononuclear peripheral blood cells. Detergent extracts of these membranes were labeled with I and subjected to immunoprecipitation analysis. Labeled extracts were precleared of CD28 by immunoprecipitation with mAb 9.3 before addition of the following (4 µg each): control mAb L20 (lanes 1); mAb 11D4 (anti-CTLA-4) (lanes 2); control Ig, chimeric mAb (lanes 3); CD80Ig (lanes 4). SDS-PAGE (10% gel) was run under nonreducing (-ME) or reducing (+ME) conditions.



Preparation and Characterization of Soluble Recombinant Fragments of the Extracellular Domains of CTLA-4 and CD86

Since biologically active CTLA-4 was a homodimer, we wished to determine whether one or two CTLA-4 chains comprise a binding site for B7 molecules. We therefore prepared soluble fragments of CTLA-4 and CD86 extracellular domains. Two strategies were employed, as shown schematically in Fig. 2. The characterization of these fragments is presented below ( Fig. 3and ).


Figure 2: Schematic representation of Ig fusions and proteolytic and recombinant fragments used in this study. Shown are cartoon structures of CTLA4Ig, CD86Ig, CTLA4t, CD86t, CTLA4X, and CTLA4X. Preparation of these molecules and elucidation of their subunit structures are discussed in the text. The relative positions of inter- and intracellular disulfide bonds in CTLA-4-related structures was determined as described in Table I; the disulfide bonds in CD86 were inferred from homology to other members of the IgSF. The Fc domains of CTLA4Ig and CD86Ig are indicated. The arrow indicates the site of thrombin cleavage in CTLA4Ig. The relative proportions of monomeric and dimeric CTLA4X were estimated from gel permeation chromatography profiles of purified preparations.




Figure 3: Characterization of Ig fusions and proteolytic and recombinant fragments. Purified recombinant Ig fusion proteins, thrombin fragments and soluble receptors were analyzed by SDS-PAGE (A) or gel permeation chromatography (B). A, SDS-PAGE analysis. Samples (1 µg) of each of the indicated proteins were analyzed on 4-20% (lanes 1-9) or 5-15% (lanes 10-12) acrylamide gels under nonreducing (lanes 1-7, 10-12) or reducing (lanes 8, 9) condition: lane 1, CTLA4Ig; lane 2, CD86Ig; lane 3, CTLA4t; lane 4, CTLA4X; lane 5, CTLA4X, which had been enriched for monomeric protein by gel permeation chromatography; lane 6, CD86t; lane 7, molecular weight markers; lane 8, CTLA4t; lane 9, CTLA4X; lane 10, molecular weight markers; lane 11, CTLA4t (a different preparation from that shown in lane 1); and lane 12, CTLA4X. B, gel permeation chromatography. Samples (3-4 µg each) of the indicated proteins were analyzed by gel permeation chromatography on a Waters 300 SW column. Panel a1, CTLA4t; panel a2, CTLA4t, following reduction with 10 mM dithiothreitol at 23 °C for 30 min; panel a3, CTLA4X; panel a4, CD86t. Panel b1, CTLA4t (a different preparation than panel a1); panel b2, CTLA4t following reduction with dithiothreitol; panel b3, CTLA4X; panel b4, CTLA4X following reduction. Arrows indicate elution positions of standards having the indicated molecular weights: V, void volume; catalase, M = 232, 000; aldolase, M = 158,000; bovine serum albumin, M = 67,000; ovalbumin, M = 43,000; carbonic anhydrase, M = 31,000.



We first prepared proteolytic fragments of CTLA4Ig and CD86Ig which retained binding activity. Preliminary examination of the Ig hinge sequence showed that it contained the sequence 126E-P-K128 which is similar to a thrombin cleavage site in actin(22) . We determined that this nonclassical thrombin site was suitable for producing active fragments of CTLA-4, CD86, CTLA4t, and CD86t. As a second strategy, we introduced an in frame stop codon into a cDNA construct encoding CTLA-4, proximal to the transmembrane domain. The resulting cDNA was then expressed in COS cells and, yielding a mixture of monomeric and dimeric proteins, CTLA4X. These proteins were purified on an anti-CTLA-4 mAb affinity column as described under ``Materials and Methods.'' Analysis of purified preparations by gel permeation chromatography indicated that purified preparations contained 80% monomeric CTLA4X and 20% dimeric CTLA4X. To improve the yield of monomeric CTLA4X, we later mutated the cysteine residue which mediates the interchain disulfide bond in CTLA-4 to a serine (see below). This mutant protein, CTLA4X, was expressed in COS cells and purified.

These fragments of CTLA-4 and CD86 were characterized by SDS-PAGE (Fig. 3A). Under nonreducing conditions, CTLA4Ig gave a major M 100,000 protein and a minor protein of M 50,000 (lane 1). The minor protein was not seen in all preparations and comigrated with the M 50,000 protein found under reducing conditions(13) . CD86Ig migrated as a diffuse M 75,000 protein (lane 2) which was not affected by reduction (data not shown). Thus, as shown previously, CTLA4Ig migrated during SDS-PAGE as a disulfide-linked homodimer of M 50,000 subunits, whereas CD86Ig did not contain an interchain disulfide bond. Since the Ig hinge disulfides were mutated to serine residues, dimerization of CTLA4Ig results from a disulfide bond in CTLA-4(13) . CD86 does not contain an interchain disulfide bond, hence, CD86Ig was not disulfide linked.

An intermolecular disulfide bond was also present in CTLA4t. CTLA4t (prepared from CTLA4Ig analyzed in lane 1) contained a major dimeric M 38,000 protein and a minor monomeric M 22,000 protein (lane 3). The presence of monomeric CTLA4t reflects the presence of monomeric CTLA4Ig in the starting preparation (lane 1). Another preparation of CTLA4t contained only the dimeric M 38,000 protein when analyzed under nonreducing conditions (lane 11). Analysis of CTLA4t preparations after reduction revealed only the monomeric M 22, 000 protein (lane 8). CTLA4X preparations contained a minor dimeric M 38,000 protein and a major monomeric M 22,000 protein (lane 4); reduction of this preparation gave a M 22,000 protein (lane 9). The proportion of monomeric CTLA4X in these preparations could be enriched by gel permeation chromatography (lane 5). This enriched preparation was stable for several weeks when reanalyzed, indicating that monomeric CTLA4X did not readily reform dimeric molecules. CTLA4X migrated as a monomeric M 22,000 protein (lane 12). CD86t migrated as a M 57,000 protein (lane 6); as with the parental molecule, CD86Ig, the mobility of CD86t was unaffected by reduction (data not shown).

Fragments of CTLA-4 and CD86 were further characterized by gel permeation chromatography (Fig. 3B). The elution times of CTLA-4 fragments corresponded closely to M values obtained by SDS-PAGE. The CTLA4t preparation analyzed by SDS-PAGE in Fig. 3A, lane 1, gave a major protein of M 40,000 and a minor high molecular weight protein when analyzed by gel permeation chromatography (Fig. 3B, panel a1). The minor high molecular weight protein was not observed in another preparation which had not been concentrated after final purification (panel b1); reduction of both preparations before chromatography gave a M 30,000 protein (panels a2 and b2). CTLA4X eluted primarily as a M 30,000 peak, with a shoulder (panel a3). CTLA4X eluted as a M 30,000 protein (panel b3) whose elution time was unaffected by reduction (panel b4). Thus, CTLA4t behaved as a disulfide-linked dimer in solution, whereas CTLA4X and the major protein in CTLA4X were monomeric. As shown in panel a4, CD86t eluted larger (M 140,000) than its M determined by SDS-PAGE (M 57,000). The elution time of CD86t was unaffected by reduction and denaturation with 4 M guanidine HCl (data not shown and Fig. 6). This treatment disrupted the high avidity interaction between CTLA-4 and CD86, suggesting that it likewise would have disrupted interactions between subunits of CD86. Therefore, the larger apparent size of CD86t in solution is probably not due to oligomerization, but to aberrant migration during gel permeation chromatography, possibly resulting from its high degree of glycosylation(19, 20) .


Figure 6: CTLA-4 homodimer binds two CD86 molecules. A, formation of complexes between monomeric CTLA4X and CD86t. Aliquots (3 µg, 200 pmol) of CTLA4X, which had been enriched for monomeric protein by preparative gel permeation chromatography (see Fig. 3A), were incubated in a final volume of 0.1 ml with CD86t (7.4 µg, 300 pmol, dotted line, or 15 µg, 600 pmol, solid line) for 30 min at 23 °C before fractionation by gel permeation chromatography as in Fig. 3. The arrow indicates the elution position of CD86t and the vertical dotted line the elution position of CTLA4t/CD86t complexes. Monomeric CTLA4X elutes at 26 min. B, formation of complexes between CTLA4t and CD86t. Aliquots of CTLA4t (3.4 µg, 250 pmol) were incubated in a final volume of 0.1 ml with CD86t (3.7 µg, 140 pmol, dotted line, or 7.4 µg, 300 pmol, solid line) for 30 min at 23 °C before fractionation by gel permeation chromatography. CTLA4t elutes at 25 min. C, preparative chromatography of complexes between CTLA4t and CD86t. Aliquots of CTLA4t (22 µg, 1600 pmol) were incubated in a final volume of 0.2 ml with CD86t (44 µg, 1700 pmol) for 30 min at 23 °C before fractionation by gel permeation chromatography. Complexes eluting with the horizontal bar (19-20 min) were collected and used in D. The arrow indicates the elution position of CD86t. The peak eluting at >26 min was not observed in other experiments in which lower concentrations were used. D, composition of complexes between CTLA4t and CD86t. An aliquot ( the total) of complexes eluting at the position of the horizontal bar in C was dissociated by treatment for 30 min at 23 °C with 4 M guanidine HCl and 10 mM dithiothreitol and reanalyzed as in B. This experiment was repeated twice with identical results.



Determination of the Disulfide Bonding Pattern in CTLA4t

CTLA4t was further characterized by protein sequencing to identify amino and carboxyl termini and disulfide bonding patterns. The amino terminus of CTLA4t was determined by automated Edman degradation (21). Sequence analysis revealed an amino terminus corresponding to M1 (M27 of the precursor sequence), in agreement with the predicted site of signal peptide processing(13) . Carboxyl-terminal sequence analysis was performed to determine the site of thrombin cleavage. CTLA4t was first fragmented with CNBr, and the peptides were purified by gel permeation chromatography. The carboxyl-terminal CNBr fragment was identified by sequence analysis. This peptide CNBr-(98-X) was subsequently subfragmented with chymotrypsin (Ch), and the amino acid sequence of the most carboxyl-terminal fragment (Ch-(114-X)) was determined. The sequence of this peptide terminated at residue Lys, suggesting that this residue was the carboxyl terminus. Fragment Ch-(114-128) was subjected to mass spectral analysis which confirmed the carboxyl-terminal residue as K128. This was the predicted thrombin cleavage site.

The disulfide bonding pattern of CTLA4t was determined (). The position of the intermolecular disulfide bridge in CTLA4t was determined by further analysis of fragment Ch-(114-128). Mass spectral analysis of the peptide was performed in the presence or absence of dithiothreitol. This analysis revealed that the mass ((M + H)) of Ch-(114-128) under nonreducing and reducing conditions was 3336.4 and 1669.2, respectively. The change in mass of the peptide following reduction demonstrates that this peptide contained one intermolecular bond at Cys. The experimental mass values were in good agreement with the predicted masses of this peptide under nonreducing and reducing conditions, 3336.6 and, 1668.8, respectively.

The intramolecular disulfide bonding pattern was determined by Edman degradation of cystine-containing peptides. Sequence analysis located the positions of the purified CNBr peptides in the CTLA-4 sequence. Edman degradation of a large molecular weight CNBr fragment gave simultaneously three amino-terminal sequences beginning at Met (or His), Gly, and Asp in equimolar amounts. These results suggested that the large molecular weight CNBr fragment consisted of three peptides interlinked by two disulfide bonds: CNBr-(1(2)-53)-CNBr-(55-85)-CNBr-(86-97). To confirm this, the large molecular weight peptide was subfragmented with trypsin, and the disulfide-containing peptides were purified by reversed-phase HPLC and identified by Edman degradation. Two major cystine-containing tryptic peptides (T) were obtained: T-(15-28)-T-(86-97) and T-(39-53)-T-(55-83). These results demonstrate the existence of two intramolecular disulfide bonds between Cys-Cys and Cys-Cys. The first of these pairs is the disulfide bond characteristic of the Ig fold. The C48-C66 disulfide bond is unusual in the Ig superfamily, but did not appear to be artifactual, since disulfide interchange during purification of fragments could be excluded by the lack of carboxamidomethylated cysteines in the fragments. This bond was also predicted in molecular modeling studies of CTLA-4, since these residues were adjacent in the model(6) .

Monomeric CTLA-4 Binds CD80 and CD86

To determine if monomeric CTLA-4 contained an intact binding site for B7 molecules, we compared B7 binding properties of monomeric and dimeric forms of CTLA-4. Concentrated conditioned medium from CTLA4X-transfected COS cells was subjected to immunoprecipitation analysis with CD80Ig and CD86Ig. Immunoprecipitated proteins were separated by SDS-PAGE and different forms of CTLA-4 were identified by immunoblotting analysis. As shown in Fig. 4, CD80Ig and CD86Ig immunoprecipitated both monomeric and dimeric forms of CTLA-4 from solution. Fractionation of CTLA4X preparations by gel permeation chromatography separated monomeric and dimeric forms of CTLA-4. Both forms were immunoprecipitated with CD80Ig and CD86Ig. Identical results were obtained when purified CTLA4X was used for analysis. Thus, both monomeric and dimeric forms of CTLA-4 bound CD80 and CD86. However, the relative amounts of monomeric and dimeric forms of CTLA-4 precipitated by CD80Ig and CD86Ig were different (Fig. 4). CD80Ig precipitated more monomeric CTLA-4, whereas CD86Ig precipitated more dimeric CTLA-4.


Figure 4: Monomeric extracellular domain of CTLA-4 binds CD80 and CD86. COS cells were transfected with the CTLA4x expression plasmid, conditioned medium was collected and concentrated approximately 30-fold. A sample of concentrated conditioned medium (0.2 ml) was fractionated by gel permeation chromatography on a TSK-Gel G2000 SW column at 1 ml/min and fractions (0.5 ml) were collected. In parallel column runs, aldolase, M = 158,000, eluted at 6.5 min. (fraction 13); bovine serum albumin, M = 67,000, at 6.8 min. (fraction 14); and ovalbumin, M = 43,000, at 7.4 min (fraction 15). Aliquots of concentrated conditioned medium (0.02 ml) or the indicated fractions (0.25 ml) were then subjected to immunoprecipitation analysis with human Ig (HIg, negative control), CD80Ig, or CD86Ig. Precipitates were analyzed by SDS-PAGE (8-16% gel) under nonreducing conditions and CTLA-4-related proteins were detected by immunoblotting analysis. Migration positions of molecular weight (M) standards are indicated on the left and on the right, migration positions of HIg, CD80Ig and/or CD86Ig, and CTLA4x dimer and monomer. HIg, CD80Ig, and CD86Ig reactivities in this assay represent cross-reaction of the horseradish peroxidase conjugate with human Ig. This figure is representative of three experiments using concentrated culture medium and two experiments using affinity-purified CTLA4x.



The relative avidities of monomeric and dimeric CTLA-4 were compared by competitive binding assay in the experiment shown in Fig. 5. CTLA4Ig was labeled with I and bound to CD80Ig and CD86Ig which had been immobilized on plastic wells. CTLA4t competed similar to CTLA4Ig (EC 4 versus 8 nM for CTLA4Ig versus CTLA-t inhibition of CD80Ig binding and 8 versus 10 nM for CD86Ig). CTLA4X competed for I-CTLA4Ig binding with an EC 300 nM for CD80Ig and 900 nM for CD86Ig. Monomeric CTLA-4 was a more effective competitor than multimeric (18) CD28Ig (EC >10,000 nM). The apparent plateau in binding inhibition by CD28Ig was not seen in other experiments. Control Ig did not show significant competition in this assay at concentrations >13,000 nM. Thus, monomeric CTLA-4 was a less avid competitor for I-CTLA4Ig binding than was dimeric CTLA-4, but was more effective than multivalent CD28Ig.


Figure 5: Comparison of the avidities of dimeric and monomeric CTLA-4. Ninety-six-well plates were coated with CD80Ig (A) or CD86Ig (B). I-Labeled CTLA4Ig (final concentration, 0.6 nM) was mixed with unlabeled CTLA4Ig (circles), CTLA4t (squares), CTLA4X (triangles), or CD28Ig (inverted triangles), to the indicated final concentrations. The mixtures were then added to B7-coated wells and binding was measured. Binding in the presence of competitor is expressed as a percentage of binding in wells containing no competitor (30,000 cpm for CD80Ig and 16,000 cpm for CD86Ig). Concentrations of competitors were determined assuming the following M values per chain or binding site): CTLA4Ig, M = 39,000; CTLA4t, M = 13,900; CTLA4X, 13,400; CD28Ig, M = 42,000; and chimeric L6 mAb, M = 75,000. Addition of chimeric mAb L6 inhibited I-CTLA4Ig binding by <10% over a concentration range of 5-13,000 nM. This experiment was repeated twice with identical results, and similar results were also observed using partially purified monomeric preparations of CTLA4X.



CTLA-4 Homodimer Binds Two CD86 Molecules

To determine the stoichiometry of CTLA-4B7 complexes, we analyzed the size of complexes formed with monomeric and dimeric CTLA-4 and the composition of the complex formed with dimeric CTLA-4 (Fig. 6). Addition of increasing concentrations of CD86t to a fixed concentration of CTLA4X monomer (A) or CTLA4t (B) resulted in formation of larger complexes. Higher concentrations of CD86t were required to force complete complex formation with monomeric CTLA-4X than with dimeric CTLA4t, consistent with the lower avidity of monomeric CTLA-4 (Fig. 5). Complexes formed with monomeric CTLA-4X eluted 1 min later than complexes formed with dimeric CTLA4t. The elution times of these complexes were faster than the largest molecular weight standard, so it was not possible to accurately estimate their molecular mass; however, a change in elution time of 1 min in the range of standards analyzed corresponded to M 70,000. To determine the composition of complexes between dimeric CTLA4t and CD86t, larger amounts of these molecules were mixed and complexes were subjected to preparative gel permeation chromatography (C). The elution profile of this mixture gave a more clearly resolved complex peak than seen in the experiment shown in B, and in addition, another late eluting peak. The identity of the late eluting peak was not determined (it was not seen when smaller amounts of these proteins were analyzed, see Fig. 3), although it eluted similar to monomeric CTLA4t. A peak corresponding to free CD86t was observed, indicating that it was present in excess. The leading edge (19-20 min elution time) of the high molecular weight complex peak was collected and analyzed for the relative amounts of CD86t and CTLA4t in two ways. In the first, an aliquot of the complex was dissociated with 4 M guanidine HCl and 10 mM dithiothreitol and reanalyzed by gel permeation chromatography (D). Peaks corresponding to CD86t and monomeric CTLA-4 were clearly resolved. Absorbance at 214 nm is proportional to the number of peptide bonds, and hence, to peptide mass. Thus, comparison of integrated peak areas divided by calculated molecular masses permits an estimate of the relative molecular ratios of CD86t and CTLA4t in the complex. This analysis gave a ratio of CD86t to CTLA4t of 1.2:1. The same value was obtained when a separate aliquot of the complex was fractionated and analyzed.

The composition of the CD86tCTLA-4 complex was also determined by subjecting it to NH-terminal amino acid sequencing. An aliquot corresponding to of the total isolated CD86tCTLA4t complex was subjected to automated Edman degradation. Two sequences were obtained simultaneously, corresponding to the NH termini of CD86 and CTLA-4. The initial yield of M1 from CTLA-4 was 38 pmol and L1 from CD86, 41 pmol. At position 3, 34 pmol of valine (CTLA-4) and 37 pmol of isoleucine (CD86) were determined. Thus, the average ratio of CD86t to CTLA4t determined by amino acid sequence analysis was 1.1:1, similar to the ratio determined by comparison of absorbance peaks.

It was important to determine if some of the CD86t present in the complex peak was due to contamination with free CD86, which was present in excess. Extrapolation of the free CD86t A214 peak (C; see also Fig. 3B) indicated that free CD86t in the 19-20-min fraction contributed <3% of initial CD86t. Since 1700 pmol of CD86t were used, then <50 pmol were present in fraction 19-20. The total amount of CD86t recovered was 320 pmol (40 pmol at position 1 in 1/8 total sample). Thus, <16% (<50/320) of recovered CD86t could be attributed to excess free CD86t based on sequence analysis. This was similar to the 10-20% of CD86t recovered in excess of a 1:1 ratio, as measured by both absorbance and NH-terminal sequencing.


DISCUSSION

CTLA-4 expressed in transfected COS cells or in activated T cells exists primarily as a protein of M 45,000-50,000 under nonreducing conditions and M 30,000-33,000 under reducing conditions. The native dimeric form of CTLA-4 is preserved in soluble fragments of its extracellular domain by an intermolecular disulfide bond at C120. Mutation of this cysteine to serine abolished residual dimeric protein in CTLA4X preparations. Our data thus show that CTLA-4, like its homologue CD28, exists primarily as a disulfide linked homodimer.

Residue Cys is conserved in most CTLA-4CD28 family homologues (6) but a recent report (23) showed that chicken CD28 lacks this cysteine residue and does not contain an intermolecular disulfide bond. This led to the proposal that chicken CD28 differs from other family members in being monomeric. However, it cannot be ruled out that chicken CD28 exists as a non-disulfide-linked dimer. Our data show that although full-length CTLA-4 is predominantly homodimeric, removal of the transmembrane and cytoplasmic domains (CTLA4X) leads to predominantly monomeric protein. Thus, the presence of C120 does not ensure dimerization. This suggests that dimerization of CTLA-4 and formation of the disulfide bond at Cys requires the transmembrane and/or cytoplasmic domains of the two chains.

Each chain of a CTLA-4 homodimer has a binding site for CD80 and CD86. Monomeric CTLA-4X has binding activity for CD80Ig and CD86Ig in immunoprecipitation analysis and monomeric CTLA4X competes for binding of I-labeled CTLA4Ig to CD80Ig and CD86Ig. Monomeric CTLA-4 had reduced binding activity when compared with dimeric CTLA-4. CTLA4X was 30-90-fold less active at competing for I-CTLA4Ig than dimeric CTLA4t. CD80Ig and CD86Ig differed in their preferences for monomeric and dimeric forms of CTLA-4. Monomeric CTLA-4 was preferentially immunoprecipitated by CD80Ig, whereas dimeric CTLA-4 was preferentially precipitated by CD86Ig. In competitive binding assays, dimeric CTLA4t was approximately equivalent at competing for I-CTLA4Ig binding to CD80Ig and CD86Ig (EC 8 versus 10 nM, respectively), whereas monomeric CTLA4X was 3-fold less effective at competing for binding to CD86Ig than to CD80Ig (EC 300 versus 900 nM). Thus, CD86 binds less well to monomeric CTLA-4.

This difference may be due to differences in binding kinetics of CD80 and CD86. We previously showed that the equilibrium dissociation constants for CTLA4Ig binding to CD80 and CD86 were very similar (within 2-fold) but that CTLA4Ig dissociated more rapidly from CD86 than from CD80. We proposed that CD86 is a faster on/faster off counter receptor for CTLA-4 than is CD80. Since monomeric CTLA-4 binds less avidly than dimeric CTLA-4, dissociation differences between CD80 and CD86 may be more pronounced with monomeric CTLA-4. In vivo, this difference may be important where CD80 or CD86 expression levels are a limiting factor for interaction with CTLA-4. Occupancy of CTLA-4 would be suboptimal, and on average, less than two binding sites per molecule occupied. Since complexes of CD86 with a single CTLA-4 binding site would dissociate faster, the time of CTLA-4 receptor occupancy by CD86 would be less, and hence, effects on T cell costimulation would also be less. Thus, differences in costimulatory signals by CD80 and CD86 would be exaggerated at low expression levels. We previously noted that CHO cells transfected with CD80 and CD86 differed more in their adhesion to CD28 and in T cell costimulation when added at low numbers (14).

The complex of CD86tCTLA4t formed in the presence of excess CD86t was isolated and determined to contain 1.1-1.2 mol of CD86t/mol of CTLA-4. Thus, one molecule of CD86t fragment bound per chain of CTLA4t or two molecules of CD86t per CTLA4t homodimer (i.e. a 2:2 stoichiometry). Since CD28 is homologous to CTLA-4, and is homodimeric, it likely also binds two B7 molecules.

The Ig domains of CD28 and CTLA-4 display little sequence similarity with other members of the IgSF, limited to a few conserved residues characteristic of the Ig fold(24) . Despite sharing a common structural scaffold, members of the IgSF have diverse structures, utilize different combinations of Ig domains to form binding sites, and have distinct binding stoichiometries per molecule. In antibodies, variable regions from two different Ig domains combine to form an antigen binding site(25) , and on per molecule basis, each Ig molecule contains two antigen binding sites (a 2:2 stoichiometry). With human growth hormone receptor, two molecules (two Ig domains) bind a single growth hormone molecule (human growth hormone receptor); the complex has a 2:1 stoichiometry(26) . Thus, the number of Ig domains per binding site of human growth hormone receptor is the same as a Fab fragment, but the stoichiometries of the intact molecules is different. A single domain of CD2 binds its CD58 ligand and the binding stoichiometry on a per molecule basis is thought to be 1:1(27, 28) . Finally, with CD8, a single Ig domain of each chain of a homodimer is believed to bind a major histocompatibility complex class I molecule; thus, on a per molecule basis, CD8/MHC class II complexes have a 2:2 stoichiometry (29, 30). The stoichiometry of CD8 has not been directly demonstrated, but is inferred from mutagenesis studies(29) .

The binding stoichiometries of CTLA-4 and CD28 most closely resemble CD8. With both molecules each chain of a homodimer contains a binding site and the intact molecule has a 2:2 stoichiometry. However, there are significant differences in the way CD8 and CTLA-4CD28 bind ligand. Different areas of the V(ariable)-like domains are utilized for binding, with the CDR-1 and CDR-2-like loops and -strands A and B being involved in the binding site of each CD8 subunit(29) , and the CDR-1 and CDR-3-like loops and -strand G, at least in part, being involved in binding by CTLA-4(26) . Also, CD8 forms an Fv-like homodimer, which is stable even in the absence of a disulfide bond (30). In contrast, the two subunits of a homodimer of CTLA-4 have no particular affinity for each other, and monomeric CTLA-4 is stable in solution.

Ligand-induced receptor dimerization is a common theme of many signaling pathways. Dimerization of growth factor receptors permits receptor transphosphorylation (31) and facilitates recruitment of SH2-containing effector molecules(32, 33) . The CD28 signaling pathway also involves tyrosine kinase activation and binding of SH2-containing effector molecules(9) . However, CTLA-4 or CD28 signaling differ from growth factor receptor signaling in two ways: their dimeric states are preformed and they are inactive. Thus, with CTLA-4CD28, dimerization per se is not sufficient to trigger signal transduction.

Why then are CTLA-4 and CD28 covalent homodimers having two binding sites? One possible clue to the answer is the fast kinetic off rates of B7 binding(14) . Recent data()indicate that the t of CTLA4Ig occupancy by CD80Ig and CD86Ig is 11 and 3 s, respectively, and that the t of CD28Ig occupancy is also very short. These off rates are rapid compared with those known for growth factor/growth factor receptor interactions(34, 35, 36) . Rapid receptor kinetic off rates favor disengagement of CD28/CTLA-4/B7 intercellular interactions, but discourage productive intracellular signaling. Homodimerization of CTLA-4 and CD28 should enhance signaling by increasing the time of receptor occupancy by ligand. Homodimeric CTLA-4 has increased binding avidity for B7 molecules, since the probability of rebinding ligand after its dissociation is increased. The effective time of receptor-ligand interactions would thereby be prolonged and receptor activation may be increased.

It is currently unknown whether signal transduction requires occupancy of one or both subunits of CTLA-4 and/or CD28 by B7 molecules. If occupancy of both subunits is required for signal transduction, monomeric B7 molecules on the APC must be brought into proximity such that both subunits of CTLA-4CD28 can be occupied. However, unlike CTLA-4 and CD28, B7 molecules are not disulfide-linked. Thus for full occupancy of CTLA-4CD28 to occur, B7 molecules must either preexist as noncovalent oligomers on the APC, or they must oligomerize as the result of binding to CTLA-4CD28. Evidence for the former possibility was provided by immunofluorescence microscopy studies showing that that B7 molecules on human Langerhans cells assume a non-uniform distribution(37) .

It is also possible that signaling through CTLA-4CD28 requires occupancy of only one receptor subunit by a B7 molecule. If so, oligomers of B7 on APC (37) could cross-link different CTLA-4CD28 homodimers. Early studies (4) showed that CD28 receptor cross-linking with mAbs enhanced signaling through this receptor, but that Fab fragments did not, suggesting that cross-linking of CTLA-4CD28 receptors is an essential feature of their signaling pathways. The binding stoichiometry of CTLA-4CD28 receptors may enhance CTLA-4CD28 receptor cross-linking by B7 aggregates on APC.

  
Table: Disulfide-linked peptides from CTLA4t

CTLA4t was fragmented with CNBr and peptides were isolated by gel permeation chromatography and identified by amino acid sequence analysis. CNBr peptide M-(98-128) was subfragmented with chymotrypsin (Ch) and the peptides were purified by reversed-phase HPLC. An intermolecular disulfide bond (C120-C120) in one of these fragments was confirmed by mass spectral analysis as described in the text. A high molecular weight CNBr fragment was subfragmented with trypsin (T), cystine-containing peptides were isolated and identified by amino acid sequence analysis. Peptide T-(55-83) was sequenced only as indicated. These analyses permitted determination of two intramolecular disulfide bonds (Cys-Cys and Cys-Cys) as described in the text. Numbers are from the amino terminus of mature CTLA4t.



FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 206-727-3675; Fax: 206-727-3501; e-mail: linsley@bms.com.

The abbreviations used are: APC, antigen-presenting cell; IgSF, immunoglobulin superfamily; mAb, monoclonal antibody; DMEM, Dulbecco's modified Eagle's medium; Ig, immunoglobulin; FBS, fetal bovine serum; IPA, immobilized protein A; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; Ch, chymotrypsin.

H. Leung and P. S. Linsley, manuscript in preparation.

W. Cosand, J. Emswiler, G. Leytze, J. Greene, and P. S. Linsley, unpublished data.


ACKNOWLEDGEMENTS

We thank Gary R. Matsueda for valuable discussions in preparing CTLA4t, William Fenderson for his excellent technical assistance, and Drs. J. Ledbetter and A. Aruffo for comments on the manuscript.


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