Expression and characterization of recombinant soluble human CD3 molecules: presentation of antigenic epitopes defined on the native TCR–CD3 complex

Che-Leung Law1,3, Martha Hayden-Ledbetter1,2, Sonya Buckwalter1,2, Lisa McNeill1, Hieu Nguyen1, Phil Habecker1, Barbara A. Thorne1,4, Raj Dua1 and Jeffrey A. Ledbetter1,2

1 Xcyte Therapies, Inc., Seattle, WA 98104, USA 2 Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122, USA 3 Present address: Seattle Genetics, Inc., Bothell, WA 98021, USA 4 Present address: Targeted Genetics Corp., Seattle, WA 98101, USA

Correspondence to: J. A. Ledbetter, Pacific Northwest Research Institute, 720 Broadway, Seattle, WA 98122, USA; E-mail: jledbetter{at}pnri.org
Transmitting editor: E. A. Clark


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The TCR–CD3 complex consists of the clonotypic disulfide-linked TCR{alpha}ß or TCR{delta}{gamma} heterodimers, and the invariant CD3{delta}, {epsilon}, {gamma} and {zeta} chains. We generated plasmid constructs expressing the extracellular domains of the CD3{delta}, {epsilon} or {gamma} subunits fused to human IgG1 Fc. Recombinant fusion proteins consisting of individual CD3{delta}, {epsilon} or {gamma} subunits reacted poorly with anti-CD3 mAb including G19-4, BC3, OKT3 and 64.1. Co-expression of the CD3{epsilon}–Ig with either the CD3{delta}–Ig (CD3{epsilon}{delta}–Ig) or the CD3{gamma}–Ig (CD3{epsilon}{gamma}–Ig) resulted in fusion proteins with much increased binding to G19-4. A brief acid treatment of the purified CD3{epsilon}{delta}–Ig fusion protein substantially improved its binding to BC3, OKT3 and 64.1. Surface plasmon resonance analysis revealed that the dissociation constants for CD3{epsilon}{delta}–Ig and anti-CD3 mAb ranged from 10–8 to 10–9 M. Based on these results, a single-chain (sc) construct encoding the CD3{delta} chain linked to the CD3{epsilon} chain with a flexible linker followed by human IgG1 Fc was expressed. The sc CD3{delta}{epsilon}–scIg reacted with anti-CD3 mAb without requiring acid treatment. Moreover, anti-CD3 mAb bound CD3{epsilon}{delta}–Ig at a higher affinity than CD3{epsilon}{gamma}–Ig, suggesting potential structural differences between the CD3{epsilon}{delta} and CD3{epsilon}{gamma} subunits. In summary, we report the expression of soluble recombinant CD3 proteins that demonstrate structural characteristics of the native CD3 complex expressed on the T cell surface. These CD3 fusion proteins can be used to further analyze the structure of the TCR–CD3 complex, and to identify molecules that can interfere with TCR–CD3-mediated signal transduction by disrupting the interaction between CD3 and TCR subunits.

Keywords: author, to, supply,


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The TCR–CD3 complex consists of six subunits including the clonotypic disulfide-linked TCR{alpha}ß or TCR{delta}{gamma} heterodimers and the invariant CD3 complex. The invariant CD3 complex is made up of four relatively small polypeptides: CD3{delta} (25 kDa), CD3{epsilon} (20 kDa), CD3{gamma} (20 kDa) and CD3{zeta} (16 kDa). CD3{delta}, {epsilon} and {gamma} chains possess a significant degree of similarity to each other in their amino acid sequences. They are members of the Ig supergene family and each of them possesses a single extracellular (EC) Ig-like domain. In contrast, CD3{zeta} only has a 9-amino-acid EC domain and a longer cytoplasmic domain when compared to CD3 {delta}, {epsilon} and {gamma}. The cytoplasmic domains of the CD3 chains contain one to three copies of a conserved motif termed an immunoreceptor tyrosine-based activation motif (ITAM) that can mediate cellular activation. One consequence of TCR–CD3 complex ligation by peptide–MHC ligands is the recruitment of a variety of signaling factors to the ITAM of the CD3 chains. This initiates the activation of multiple signal transduction pathways, eventually resulting in gene expression, cellular proliferation and generation of effector T cell functions (14).

The assembly and surface expression of the TCR complex are tightly regulated processes. It is well established that surface expression of a TCR complex requires the presence of TCR{alpha}ß or TCR{gamma}{delta} plus CD3{delta}, CD3{epsilon}, CD3{gamma} and CD3{zeta} chains (5,6). Absence of any one chain renders the complex trapped in the endoplasmic reticulum (ER) and subjects them to rapid proteolytic degradation (711). The precise stoichiometry of a TCR–CD3 complex awaits complete elucidation. Initially, one copy of the clonotypic TCR{alpha}ß chains was proposed to be in complex with a CD3{epsilon}{delta} heterodimer, a CD3{epsilon}{gamma} heterodimer and a CD3{zeta}{zeta} homodimer (12). More recent findings suggest a bigger complex consisting of two copies of the clonotypic TCR{alpha}ß heterodimer in complex with the above six CD3 chains to form a decameric complex (1315). In this complex, the TCR heterodimers and CD3{zeta} homodimers are covalently linked by disulfide bonds, while the CD3{epsilon}{delta} and CD3{epsilon}{gamma} heterodimers are not covalently linked. Furthermore, the interaction among CD3{epsilon}{delta}, CD3{epsilon}{gamma}, CD3{zeta}{zeta} and TCR{alpha}ß or TCR{gamma}{delta} chains has been shown to be non-covalent.

Assembly of the TCR–CD3 complex begins with pairwise interactions between individual TCR{alpha} or TCRß chains with the CD3 chains in the ER. Intermediates consisting of a single TCR chain in association with the CD3 chains are detectable in the ER (16,17). Transfection studies conducted in non-lymphoid cells show that TCR{alpha} can associate with CD3{delta} and CD3{epsilon}, but not CD3{zeta}, whereas TCRß can associate with CD3{delta}, {epsilon} and {gamma}, but not CD3{zeta} (9,16). Incorporation of the CD3{zeta} chain appears to be the rate-limiting step for the formation of a mature TCR–CD3 complex. TCR{alpha}ß, and CD3{delta}, {epsilon} and {gamma} chains must be present in the ER before CD3{zeta} can assemble with the partial TCR–CD3 complex to form the final product for surface expression (18). Association between the TCR and CD3 chains depends largely on the charged amino acid residues in their transmembrane (TM) domains. Positively charged amino acid residues are present in the TM domains of the TCR{alpha}ß chains. Negatively charged amino acids are found in the TM domains of the CD3 chains. Formation of salt bridges due to these charged amino acid residues may be the main force driving the association between the TCR{alpha}ß and CD3 chains (19,20).

Even though the assembly and signaling properties of the TCR–CD3 complex have been extensively studied, the functions of the EC domains of the CD3{delta}, {epsilon} or {gamma} chains remain to be fully understood. The crystal structure of a TCR–anti-TCR complex provides evidence for the presence of a binding pocket in the TCRß chain large enough to accommodate the EC domain of CD3{epsilon} (21,22). Deletional analysis has also revealed an extracellular (EC) region proximal to the TM domains of CD3{delta}, {epsilon} or {gamma} with a conserved CXXCXE motif that may mediate hetero-dimerization CD3 chains (23).

In order to characterize further the structures and functions of the EC domains of the CD3 subunits, we sought to generate recombinant fusion protein constructs encoding the EC domains of human CD3{delta}, {epsilon} or {gamma} fused with the human IgG1 Fc fragment. We report the expression of soluble human CD3–Ig fusion proteins that contain either one or two of the CD3 chains, and demonstrate expression and processing conditions that allow these fusion proteins to exhibit antigenic epitopes that are defined on the native TCR–CD3 complex.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
mAb and cells
OKT3 was purchased from ATCC (Rockville, MD). BC3 was obtained from Dr Claudio Anasetti (Fred Hutchinson Cancer Research Center, Seattle, WA). 64.1 and 9.3 were obtained from Dr John Hansen (Fred Hutchinson Cancer Research Center). G19-4 has previously been described (24).

Normal peripheral blood mononuclear cells (PBMC) were activated by plate-immobilized G19-4 (anti-CD3) and 9.3 (anti-CD28) in RPMI 1640 supplemented with 10% FBS for 72 h before total RNA isolation. COS-7 cells were purchased from the ATCC and maintained in DMEM supplemented with 10% FBS.

CD3–Ig fusion protein construction and expression
For the generation of CD3–Ig fusion proteins, cDNAs encoding EC domains of CD3 were generated using total RNA from anti-CD3/anti-CD28-activated peripheral blood mononuclear cells (PBMC) as the template and RT-PCR using the Superscript II RNase H reverse transcriptase (Life Technologies, Gaithersburg, MD). The PCR primers used were as follows: CD3{delta} EC domain 5' primer, 5'-gcg aag ctt gcc acc atg gaa cat agc acg ttt ctc-3'; 3' primer, 5'-cgc gga tcc atc cag ctc cac aca gct ctg-3'; CD3{epsilon} EC domain 5' primer, 5'-gcg ata aag ctt gcc acc atg cag tcg ggc act cac tgg-3'; 3' primer, 5'-gcg gga tcc atc cat ctc cat gca gtt ctc aca-3'; CD3{gamma} EC domain 5' primer, 5'-gcg ata aag ctt gcc acc atg gaa cag ggg aag ggc ctg-3'; 3' primer, 5'- gcg gga tcc att tag ttc aat gca gtt ctg aga c-3'. Underlined nucleotides indicate HindIII and BamHI sites in the 5' and 3' primers respectively. All primer sets were designed to include the CXXCXE motifs present in the EC of the CD3{delta}, {epsilon} and {gamma} subunits. HindIII- and BamHI-cut PCR products were ligated in-frame 5' to a human IgG1 Fc fragment in a mammalian expression vector controlled by a CMV promoter similar to methods previously described (25).

The CD3 {delta}{epsilon}–single chain (sc) Ig fusion protein constructs were made by PCR amplification of the individual chains as cDNA cassettes, restriction digestion at compatible sites and ligation together into an Ig expression vector recipient plasmid. Oligonucleotides used to amplify the CD3{delta} and CD3{epsilon} EC domains included a leader peptide attached to the 5' end of the CD3{delta} domain cassette, with the first two repeats of the (Gly4Ser)3 linker at the 3' end of the cassette, including a BamHI site to simplify construction of the complete fusion gene. Similarly, the second domain cassette was amplified using a 5' oligonucleotide beginning with a BamHI site and adding the third (Gly4Ser) subunit to the linker, followed by the codons for the first 10 amino acids of the mature peptide and a 3' oligonucleotide with a BglII restriction site attached to the 3' end. The fragments were then assembled by three-way ligation of the HindIII–BamHI fragment, the BamHI–BglII fragment and HindIII–BglII digested vector already containing the BglII–XbaI human IgG1 fragment. A mammalian expression vector controlled by a CMV promoter was used to harbor the fusion protein expression cassette for expression in COS-7 cells. The PCR primers for construction of the CD3{delta}-linker-CD3{epsilon}–Ig are as follows: CD3{delta}103, 5'-ggc aag ctt atg gaa cat agc acg ttt ctc tct ggc ctg-3'; CD3{delta}104, 5'-tcc gga tcc gcc acc ccc aga ccc tcc gcc acc atc cag ctc cac aca gct ctg-3'; CD3{epsilon}103, 5'-ggc gga tcc gga ggt ggt ggc tca gat ggt aat gaa gaa atg ggt ggt att aca-3'; CD3{epsilon}104, 5'-ggc aga tct atc cat ctc cat gca gtt ctc aca cac tct-3'.

Ligation products were transformed into DH5{alpha} bacteria and isolated colonies picked to screen for transformants containing the correct insertions. Constructs with the correct digestion pattern were then sequenced using the Big Dye terminator cycle sequencing kit reagents (PE Biosystems, Foster City, CA) on an ABI Prism 310 sequencer (PE Biosystems). The plasmids for positive clones with the correct sequence were then transfected into COS-7 cells.

Transient expression of CD3–Ig fusion protein constructs in COS cells was performed as reported (25). To purify Ig fusion proteins, COS cell supernatants were passed through Protein A–Sepharose (Pharmacia Biotech, Piscataway, NJ), and washed successively with 0.14 M potassium phosphate buffer (pH 8.0) and 0.1 M sodium acetate buffer (pH 4.5). Bound fusion proteins were then eluted with 0.1 M citric acid (pH 2) and immediately neutralized with Tris base. Fractions containing Ig fusion proteins were pooled and dialyzed against PBS before further analysis.

Acid treatment of CD3–Ig fusion proteins
Acid treatment of CD3–Ig was accomplished by incubating 9 parts of fusion protein in PBS with 1 part of 1 M citric acid at pH 2 or 1 M citrate buffers at pH 3, 4, 5 and 6. The resulting citric acid or citrate concentration was 0.1 M. Acidified fusion protein preparations were incubated at 4°C for 60 min. Fusion protein solutions were then neutralized with 1 M Tris base and dialyzed overnight at 4°C against PBS.

ELISA
Aliquots of 5 µg/ml of the capture anti-CD3 mAb in 0.054 M carbonate/bicarbonate buffer (pH 9.6) were adsorbed onto wells of ELISA plates overnight at 37°C. Wells were then blocked with 5% milk diluent/blocking solution concentrate (Kirkegaard & Perry, Gaithersburg, MD) in PBS at 37°C for 2 h. Before addition of the fusion proteins, wells were rinsed 3 times with wash buffer (0.5% Tween 20 in PBS). Fusion proteins diluted serially into 5% milk diluent/blocking solution concentrate in PBS and added to wells. Human IgG1 or irrelevant, similarly constructed Ig fusion protein was used as negative a control. After incubation at 37°C for 1 h, wells were rinsed 3 times with wash buffer. Horseradish peroxidase-conjugated donkey anti-human IgG (Jackson ImmunoResearch, West Grove, PA) diluted at 1:5000 in 5% milk diluent/blocking solution concentrate in PBS was added to wells and incubated for 1 h at 37°C to detect bound fusion proteins. After 3 rinses with wash buffer, 1 component TMB microwell peroxidase substrate (Kirkegaard & Perry) was added. Color was developed in the dark for 5–15 min, stopped by the addition of an equal volume of 0.1 M HCl, followed by reading on an ELISA reader using a 450 nm filter.

Anti-CD3 immunoblotting
CD3–Ig fusion proteins were resolved on SDS–PAGE under non-reducing conditions and they were then transferred onto either nylon or PVDF membranes. After blocking, membranes were immunoblotted with either horseradish peroxidase-conjugated goat anti-human IgG (Jackson ImmunoResearch) or anti-CD3 mAb. Binding of anti-CD3 mAb was detected by horseradish peroxidase-conjugated goat anti-mouse IgG Fc (Jackson ImmunoResearch). Blots were then developed using the enhanced chemiluminescence (ECL) kit from Amersham Pharmacia (Piscataway, NJ).

Binding affinity between CD3{epsilon}{delta}–Ig and anti-CD3 mAb
Measurements of affinity between CD3{epsilon}{delta}–Ig and anti-CD3 mAb were carried out with a BIAcore 1000 (Biacore, Piscataway, NJ) based on surface plasmon resonance (SPR) technology. Individual anti-CD3 mAb were immobilized onto dextran-based CM5 sensor chips (Biacore) in the presence of N-hydroxysuccinimide and N-ethyl-N'-[3-di-methyl-amino)propyl] carbodiimide hydrochloride (Biacore). Graded concentrations of CD3{epsilon}{delta}–Ig, ranging from 20 to 1.25 µg/ml, were injected over the mAb-coated CM5 sensor chip surface to initiate protein association. After maximal binding, dissociation between the bound CD3{epsilon}{delta}–Ig and anti-CD3 mAb was accomplished by the injection of the carrier buffer free of any Ig fusion protein. The association and dissociation kinetics between CD3{epsilon}{delta}–Ig and anti-CD3 mAb, and hence the on and off rates respectively, were detected as changes in SPR signals and expressed in sensograms as resonance units versus time. The on and off rates generated from graded doses of CD3{epsilon}{delta}–Ig interacting with the same anti-CD3 mAb-coated surface were then used to compile the dissociation constants, i.e. affinity, of interaction.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Construction and expression of soluble recombinant CD3–Ig fusion proteins
In order to characterize the CD3 complex in solution, cDNA encoding the EC domains of CD3{delta}, {epsilon} and {gamma} chains were individually fused at the N-terminus to a human IgG1 hinge–CH2–CH3 Fc fragment and cloned into a mammalian expression vector (Fig. 1A). Two cysteine residues, responsible for the formation of interchain disulfide bonds between the heavy chains of native human IgG1, are present in the hinge region of human IgG1 (Fig. 1A). Accordingly, we reasoned that expression of any individual CD3–Ig fusion plasmid construct in cells would result in the production of one species of disulfide-linked homodimer, hereinafter referred as CD3–Ig homodimers. On the other hand, the expression of two different CD3–Ig fusion plasmid constructs would result in the production of a mixture of two species of disulfide-linked homodimers and one species of disulfide-linked heterodimer (Fig. 1A), hereinafter referred as CD3–Ig heterodimers.




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Fig. 1. CD3–Ig fusion protein. (A) This schematic depicts cDNA constructs derived from the fusion of various CD{epsilon} EC domains to the human IgG1Fc (hinge–CH2–CH3) fragment and their expression to generate homo- and heterodimers of CD3–Ig. (B) COS cells were transiently transfected by single or two CD3–Ig fusion protein constructs. Spent supernatants were incubated with Protein A–Sepharose. Bound fusion proteins were eluted by either reducing or non-reducing sample buffer as indicated and resolved on SDS–PAGE. The gels were then stained by Coomasie brilliant blue.

 
Expression of these plasmids in COS cells, either individually or in combinations of two plasmids, resulted in the synthesis of recombinant proteins and their secretion into the culture medium (Fig. 1B). Soluble CD3–Ig proteins could be accumulated to 5–10 mg/l of spent COS cell medium. The sizes of different recombinant proteins, estimated by SDS–PAGE under reducing conditions, were all ~45–50 kDa (Fig. 1B). This range is consistent with the sizes predicted from the amino acid sequences of CD3 EC domains and human IgG Fc. On the other hand, multiple protein bands were detected when these fusion proteins were resolved on SDS–PAGE under non-reducing conditions (Fig. 2B). The lowest molecular weight bands were ~90–100 kDa, corresponding to the predicted size of a homo- or heterodimer with two CD3–Ig chains. Each higher mol. wt protein appeared to be ~45–50 kDa larger in size successively. Hence, each higher molecular protein might contain one more CD3–Ig chain disulfide-linked to the complex than the previous one.



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Fig. 2. Co-expression of CD3{epsilon}–Ig with CD3{delta}–Ig or CD3{gamma}–Ig in COS cells produced heterodimeric CD3–Ig fusion proteins that bound anti-CD3 mAb. COS cells were transiently transfected with constructs encoding CD3{delta}–Ig ({delta}), CD3{epsilon}–Ig ({epsilon}), CD3{gamma}–Ig ({gamma}), CD3{epsilon}–Ig + CD3{delta}–Ig ({epsilon}{delta}) or CD3{epsilon}–Ig + CD3{gamma}–Ig ({epsilon}{gamma}). COS cell supernatants were tested for binding of CD3–Ig fusion proteins to G19-4, OKT3, BC3 and 64.1 by ELISA. Supernatants containing CD3{epsilon}–Ig were either mixed with those containing CD3{delta}–Ig ({epsilon} + {delta}) or CD3{gamma}–Ig ({epsilon} + {gamma}) and tested for binding to the same panel of anti-CD3 mAb.

 
Binding of CD3–Ig fusion proteins to anti-CD3 mAb
Some anti-CD3 mAb have been reported to bind individual CD3 chains, whereas others recognize only CD3{epsilon} in complex with either CD3{delta} or CD3{gamma} (26). We applied four anti-CD3 mAb (G19-4, OKT3, BC3 and 64.1) to probe the structures of the CD3–Ig homo- and heterodimers. Plate-immobilized anti-CD3 mAb were used to capture CD3–Ig fusion proteins, which was then detected by antibodies directed specifically against the Fc fragment of human IgG. Figure 2 shows that supernatants from COS cells transfected with individual CD3–Ig constructs did not react with anti-CD3 mAb, with the exception of G19-4, which consistently showed low levels of binding to CD3{epsilon}{epsilon}–Ig-containing supernatants. Co-transfection of COS cells with CD3{epsilon}–Ig and CD3{delta}–Ig plasmids or CD3{epsilon}–Ig and CD3{gamma}–Ig plasmids generated supernatants that bound much better to G19-4. OKT3, BC3 and 64.1 did not show any consistent binding to CD3{epsilon}{delta}–Ig or CD3{epsilon}{gamma}–Ig supernatants. Mixing of CD3{epsilon}–Ig supernatants with CD3{delta}–Ig or CD3{gamma}–Ig supernatants did not increase the binding to G19-4, suggesting that CD3{epsilon}–Ig and CD3{delta}–Ig fusion proteins formed G19-4-binding complexes in COS cells before being secreted into the culture medium.

We then examined the binding of Protein A–Sepharose-purified CD3–Ig preparations to the same panel of anti-CD3 mAb (Fig. 3). All anti-CD3 mAb tested showed improved reactivity and saturable binding to purified CD3–Ig fusion proteins (Fig. 3A and B, upper panel). The concentration of CD3{epsilon}{delta}–Ig that half-saturated G19-4 was reproducibly 5- to 10-fold less than that required to saturate OKT3, BC3 and 64.1 (Fig. 3B, upper panel). The CD3{epsilon}{gamma}–Ig heterodimer interacted with the panel of anti-CD3 mAb at a much wider range of affinity. Saturable binding was observed between CD3{epsilon}{gamma}–Ig and G19-4 and CD3{epsilon}{gamma}–Ig and BC3, but the concentration of CD3{epsilon}{gamma}–Ig to half-saturate G19-4 was ~20-fold less than that needed to half-saturate BC3 (Fig. 3A and B, lower panel). In contrast to G19-4 and BC3, CD3{epsilon}{gamma}–Ig showed minimal binding to both OKT3 and 64.1. In addition, the CD3{epsilon}{delta}–Ig and CD3{epsilon}{gamma}–Ig heterodimers bound much better to anti-CD3 mAb than the CD3{delta}{delta}–Ig, CD3{epsilon}{epsilon}–Ig and CD3{gamma}{gamma}–Ig homodimers. Although detectable CD3{epsilon}{epsilon}–Ig/G19-4, CD3{gamma}{gamma}–Ig/G19-4, CD3{gamma}{gamma}–Ig/BC3 CD3{epsilon}{epsilon}–Ig/64.1 and CD3{gamma}{gamma}–Ig/64.1 binding was observed, such binding was not saturable even at fusion protein concentrations of 100 µg/ml (Fig. 3A). Finally, none of the four mAb used in this study recognized the CD3{delta}{delta}–Ig homodimer (Fig. 3A).




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Fig. 3. Binding of purified CD3–Ig fusion proteins to anti-CD3 mAb. (A) Various CD3–Ig homodimeric or heterodimeric fusion proteins were purified from spent medium collected from COS cells transiently expressing different CD3–Ig constructs, singly or in combinations of two. The binding of purified fusion proteins to anti-CD3 mAb was determined by ELISA. Anti-CD3 mAb was used to capture CD3–Ig fusion proteins as described in Methods. (B) CD3{epsilon}{delta}–Ig and CD3{epsilon}{gamma}–Ig binding curves from (A) were re-plotted to allow direct comparison of their binding activities to anti-CD3 mAb.

 
Acid-induced conformational changes of CD3{epsilon}{delta}–Ig
The ability of OKT3, BC3 and 64.1 to bind purified CD3{epsilon}{delta}–Ig (Fig. 3) was in stark contrast to the lack of binding of the same mAb to unpurified CD3{epsilon}{delta}–Ig fusion proteins present in the COS cell supernatants shown in Fig. 2. This prompted us to examine the possibility that changes in the conformation of CD3{epsilon}{delta}–Ig occurred during purification, which generated the epitope(s) recognized by OKT3, BC3 and 64.1. After binding of CD3–Ig fusion proteins to Protein A–Sepharose, we washed the matrix with pH 8 and 4.5 buffers successively, and eluted the bound fusion protein at pH 2. Eluted fusion proteins were then neutralized by Tris buffer. When CD3{epsilon}{delta}–Ig heterodimers were neutralized by Tris buffer immediately after elution, they showed extremely low binding activity to OKT3, BC3 and 64.1 (Fig. 4A and data not shown). However, the same fusion protein still bound to G19-4 in a saturable manner (Fig. 4A, untreated line). A brief, as short as 15 min, additional exposure of the CD3{epsilon}{delta}–Ig to pH 2 enabled it to bind OKT3 and BC3. Interestingly, this acid treatment did not change the binding activity of CD3{epsilon}{delta}–Ig to G19-4. This suggests that the G19-4 epitope on CD3{epsilon}{delta}–Ig may be different from those of the OKT3 and BC3 epitope(s). When the effect of different pH was examined, we found that pH 2 was most effective (Fig. 4B). Frozen preparations of acid-treated CD3{epsilon}{delta}–Ig retained its ability to bind OKT3, BC3 and 64.1 over a long period of time.




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Fig. 4. Acid-induced conformational changes in CD3{epsilon}{delta}–Ig. (A) Purified CD3{epsilon}{delta}–Ig was treated with pH 2 citric acid buffer at 4°C for the indicated lengths of time and its binding activity to G19-4, OKT3 and BC3 was determined by ELISA. The quantity of Ig fusion protein in each batch of acid-treated CD3{epsilon}{delta}–Ig was monitored by an anti-human IgG ELISA. (B) Purified CD3{epsilon}{delta}–Ig was treated by citric acid/citrate buffers at different pH values as indicated for 45 min at 4°C, and its binding activity to OKT3 and BC3 was determined by ELISA.

 
Non-reducing SDS–PAGE revealed the presence of multiple species of disulfide-linked fusion proteins in all of the purified CD3–Ig preparations (Fig. 1B). We examined which of the species could bind anti-CD3 mAb. Western blot analysis on CD3–Ig fusion proteins resolved under non-reducing conditions revealed that G19-4 could recognize CD3{epsilon}{epsilon}–Ig, CD3{gamma}{gamma}–Ig, CD3{epsilon}{delta}–Ig and CD3{epsilon}{gamma}–Ig, but not CD3{delta}{delta}–Ig (Fig. 5A). This is consistent with the failure of CD3{delta}{delta}–Ig to bind G19-4 on ELISA shown in Fig. 3. G19-4 preferentially bound the 90 kDa dimeric form of the CD3{gamma}{gamma}–Ig, CD3{epsilon}{delta}–Ig and CD3{epsilon}{gamma}–Ig, with the exception of CD3{epsilon}{epsilon}–Ig to which G19-4 appeared to bind better to the 150 kDa trimer. On the other hand, when OKT3 was used to probe similar blots, it specifically bound only to the 90 kDa heterodimer band in the CD3{epsilon}{delta}–Ig lane and it did not recognize any other forms of CD3–Ig fusion proteins (Fig. 4B). As OKT3 did not bind any of the three homodimers, it is most likely that the 90 kDa protein recognized by OKT3 on Western blot was the heterodimeric form consisting of one chain of CD3{epsilon}–Ig and one chain of CD3{delta}–Ig. Hence the fusion protein bound by OKT3 on ELISA shown in Fig. 2–4 might also be predominantly the same heterodimer.



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Fig. 5. Immunoblotting of CD3–Ig fusion proteins by G19-4 and OKT3. Purified CD3–Ig fusion proteins were resolved by non-reducing SDS–PAGE, transferred onto membranes, and then probed with G19-4 (A) and OKT3 (B).

 
High-affinity interaction between CD3{epsilon}{delta}–Ig and anti-CD3 mAb
The affinity of interaction between CD3{epsilon}{delta}–Ig and anti-CD3 was determined by SPR in which graded concentrations of CD3{epsilon}{delta}–Ig in liquid phase were allowed to bind anti-CD3 mAb immobilized on chips. Interactions between CD3{epsilon}{delta}–Ig and anti-CD3 showed on rates ranging from 3.2 x 105 to 17.1 x 105 M–1 s–1 and off rates ranging from 7.8 x 10–4 to 181 x 10–4 s–1, indicative of high-affinity interaction (Table 1). Whereas the on rates of CD3{epsilon}{delta}–Ig bound onto OKT3, BC3 and 64.1 were similar to each other, the off rate of CD3{epsilon}{delta}–Ig bound onto G19-4 was 6- to 20-fold lower. Thus, in two separate experiments G19-4 was found to bind CD3{epsilon}{delta}–Ig at the highest affinity. The average dissociation constant between CD3{epsilon}{delta}–Ig and G19-4 was 3.5- to 9-fold lower than those between CD3{epsilon}{delta}–Ig and the other three anti-CD3 mAb. These results are consistent with the ELISA data shown in Fig. 2.


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Table 1. Affinity between CD3{epsilon}{delta}–Ig and anti-CD3 mAb determined by surface plasmon resonance
 
Construction and expression of CD3{delta}{epsilon}–scIg fusion protein
Co-expression of plasmid constructs encoding CD3{epsilon}–Ig and CD3{delta}–Ig in COS cells clearly generated disulfide-bonded heterodimeric proteins bearing apparently native epitopes for anti-CD3 mAb. However, the molecular composition of CD3{epsilon}{delta}–Ig was likely to be a complex mixture of homo- and heterodimers and oligomers (Figs 1 and 5). Acid treatment of the purified CD3{epsilon}{delta}–Ig protein was also required to generate the BC3, OKT3 and 64.1 epitope(s) (Fig. 4). We sought to resolve some of these problems by re-engineering the CD3–Ig fusion protein expression constructs. Two major changes were incorporated as depicted in Fig. 6. First, a cDNA fragment encoding the leader plus the EC domain of CD3{delta} was placed N-terminal to a cDNA fragment encoding the EC domain of CD3{epsilon}. A Gly/Ser linker was used to separate the two fragments, which may also add flexibility to the expressed protein and enhance association between the CD3{epsilon} and CD3{delta} subunits. Second, we substituted serine residues for the two cysteine residues in the hinge region of the human IgG1 Fc fragment. We reasoned that since the CD3{delta} and CD3{epsilon} subunits were present on the same polypeptide they should be able to associate with each other to form the epitope(s) recognized by anti-CD3 mAb. In the absence of the cysteine residues in the Fc fragment, monomeric forms of the CD3{delta}{epsilon}–scIg fusion protein might be produced.



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Fig. 6. CD3{delta}{epsilon}–scIg fusion protein construct. The coding region for the leader and EC domain of CD3{delta} was joined to the 5' end of the coding sequence for the EC domain of CD3{epsilon} via a flexible linker. This fragment was in turn fused 5' to a cDNA encoding the human IgG1 Fc fragment. The two cysteine residues in the hinge region of the human IgG Fc fragment were replaced by serine residues by site-directed mutagenesis.

 
The re-engineered CD3{delta}{epsilon}–scIg plasmid construct was transiently expressed and purified fusion proteins were analyzed by Western blotting. An anti-human IgG reagent detected three major species of proteins of ~60, 120 and >200 kDa, the predominant forms being the 120 kDa species (Fig. 7A). The 60 kDa protein corresponded to the predicted size of a monomeric form of CD3{delta}{epsilon}–scIg. Accordingly, the 120 and >200 kDa proteins might be disulfide-linked dimeric and tetrameric forms of CD3{delta}{epsilon}–scIg respectively. Four unpaired cysteine residues were present in the EC domains of CD3{delta} and CD3{epsilon} in this construct (Fig. 6); they were probably involved in the disulfide linking of CD3{delta}{epsilon}–scIg monomers to give dimers and tetramers. When G19-4 was used to probe Western blotted CD3{delta}{epsilon}–scIg, it bound predominantly to the 60 kDa monomers and reacted very poorly with the 120 kDa dimer (Fig. 7B). On the other hand, OKT3 bound exclusively to the 60 kDa protein with virtually no binding to the 120 kDa dimer. These results suggest that when the CD3{delta} and CD3{epsilon} subunits were expressed as a single translational unit separated by a flexible linker they were able to associate with each other to regenerate epitopes recognizable by anti-CD3 mAb. It is also noteworthy that, unlike the CD3{epsilon}{delta}–Ig used in Fig. 5, the CD3{delta}{epsilon}–scIg fusion protein was directly eluted from Protein A–Sepharose using SDS sample buffer without any acid treatment before SDS–PAGE and Western blotting.



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Fig. 7. Immunoblotting of CD3{delta}{epsilon}–scIg fusion proteins by G19-4 and OKT3. Purified CD3{delta}{epsilon}–scIg fusion proteins were resolved by non-reducing SDS–PAGE, transferred onto membranes, and then probed with anti-human IgG (A), G19-4 (B) and OKT3 (C).

 
Binding of CD3{delta}{epsilon}–scIg fusion protein to anti-CD3 mAb
In order to confirm that an OKT3 epitope was already present on the CD3{delta}{epsilon}–scIg fusion protein as secreted from COS cells, we compared the binding of purified CD3{delta}{epsilon}–scIg and purified acid-treated CD3{epsilon}{delta}–Ig to G19-4 and OKT3 by ELISA. Purified CD3{delta}{epsilon}–scIg was eluted from Protein A–Sepharose and immediately neutralized by Tris buffer and dialyzed against PBS. No additional acid treatment was performed. Figure 8 shows that both CD3{epsilon}{delta}–Ig and CD3{delta}{epsilon}–scIg bound to G19-4. The concentration of CD3{delta}{epsilon}–scIg needed to half-saturate G19-4 was ~3-fold higher than that needed by CD3{epsilon}{delta}–Ig. Interestingly, unlike untreated CD3{epsilon}{delta}–Ig (Fig. 4), CD3{delta}{epsilon}–scIg showed saturable binding to OKT3. The concentration needed to half-saturate OKT3 was ~6-fold less than that needed by CD3{epsilon}{delta}–Ig. Supernatants from COS-7 or CHO cells expressing CD3{delta}{epsilon}–scIg also bound OKT3 by ELISA without the need of any acid treatment (data not shown). Hence, acid treatment of CD3{delta}{epsilon}–scIg was not required to generate the OKT3 epitope.



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Fig. 8. Binding of purified CD3{delta}{epsilon}–scIg fusion protein to G19-4 and OKT3. CD3{delta}{epsilon}–scIg fusion protein was purified from spent medium collected from COS cells transiently expressing the CD3{delta}{epsilon}–scIg construct. The binding of purified CD3{delta}{epsilon}–scIg to G19-4 and OKT3 was compared to that of CD3{epsilon}{delta}–Ig by ELISA.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The role of the CD3 subunits in the TCR complex has been the subject of intense research. Various gene deletion experiments unequivocally demonstrated the importance of the CD3{delta} (27,28), {epsilon} (29), {gamma} (28,30) and {zeta} (3133) in T cell differentiation and functions. Assembly and trafficking of the TCR–CD3 complex (510,12,1618), interactions between charged amino acid residues in the TM domains of the CD3 and TCR {alpha}ß subunits (19,20) and signaling transduction functions of CD3 cytoplasmic domains (24) are the other well-characterized aspects of the CD3 complex. Recently, the structure of a murine CD3{epsilon}{gamma} complex in solution has become available (34). The construction and expression of soluble human CD3 complexes will certainly facilitate elucidation of the human CD3 structures and functional interaction with the clonotypic subunits of the human TCR.

In this study, we report the expression and secretion of soluble Ig fusion proteins consisting of the EC domains of human CD3. Previous studies both in T cells and non-lymphoid cells have shown that partial TCR complexes are invariably trapped in ER (17,26,35). TCR {alpha}ß and CD3{delta} chains are further directed to pre-Golgi proteolytic degradation pathways, presumably due to the exposure of peptide sequences in their TM domains signaling for proteolysis. We have successfully expressed the soluble CD3–Ig fusion proteins by simply fusing the CD3{delta}, {epsilon} and {gamma} EC domains to the N-terminus of human IgG1. We have also produced stable CHO cell transfectants secreting comparable levels of CD3{epsilon}{delta}–Ig (data not shown). Expression of murine CD3{epsilon} and CD3{epsilon}{gamma} sc proteins in a bacterial expression system has been reported. These proteins were expressed as inclusion bodies in bacteria. Detergent solubilization and refolding could successfully regenerate a soluble murine CD3{epsilon}{gamma} protein that bound anti-CD3 mAb (36).

One characteristic of the CD3–Ig and CD3{delta}{epsilon}–scIg fusion proteins is their tendency to oligomerize via disulfide linkages (Figs 1, 5 and 7A). Although the presence of two cysteine residues in the hinge region of human IgG1 can in theory explain the oligomeric structures, we consider this unlikely. First, most recombinant Ig fusion proteins reported by various groups, including us, exist predominantly as dimers. Second, as shown in Figs 6 and 7, oligomeric structures were still evident even when both cysteine residues in the IgG hinge of the CD3{delta}{epsilon}–scIg were removed. The unpaired, membrane-proximal cysteine residues in the CXXCXE motifs of CD3 may be one reason for oligomerization of the CD3–Ig fusion proteins. Although most CD3{epsilon}{delta} and CD3{epsilon}{delta} heterodimers do not appear to be covalently linked in their native state in the TCR complex, disulfide-bonded CD3{epsilon} homodimers have been reported on human and mouse T cells before (37,38). Disulfide-linked oligomeric CD3{epsilon}, {delta} and {gamma} have also been detected both in murine T cells (39), and in an experimental system designed for in vitro translation of the TCR–CD3 complex (40). The cysteine residues in CXXCXE motifs have been proposed to be responsible for disulfide bonding of CD3{epsilon} and CD3{gamma} (40). A sc recombinant murine CD3{epsilon}{gamma} heterodimer without any CXXCXE motifs expressed by bacteria bound to anti-mCD3 mAb (36). Experiments to substitute the corresponding cysteine residues in the CD3{epsilon}{delta}–scIg construct (Fig. 6) with serine residues should be able to test the possibility that expression of human CD3{epsilon} and CD3{delta} in a co-linear translational unit may permit proper pairing even in the absence of the CXXCXE motifs.

Most anti-human CD3 mAb recognize the CD3{epsilon} subunit (26,41,42). A subset of them requires the presence of either CD3{delta} or CD3{gamma} before they can recognize CD3{epsilon}, suggesting that the conformation of CD3{epsilon} is modified when it is in association with CD3{delta} or CD3{gamma} (26,40). Binding results obtained from this study largely agree with previously published studies. Thus, G19-4, OKT3, BC3 and 64.1 demonstrated saturable high-affinity interaction with CD3{epsilon}{delta}–Ig (Figs 3 and 4) (Table 1), while G19-4 and OKT3 also bound at high affinity to CD3{delta}{epsilon}–scIg (Fig. 8). When G19-4 and OKT3 were used to Western blot CD3{epsilon}{delta}–Ig (Fig. 5) and CD3{delta}{epsilon}–scIg (Fig. 7), both mAb recognized almost exclusively the dimeric form of CD3{epsilon}{delta}–Ig and the monomeric form of CD3{delta}{epsilon}–scIg. These results are consistent with a model in which Ig fusion protein molecules consisting of only one CD3{epsilon} chain and one CD3{delta} chain are most efficient in generating an epitope for anti-CD3 mAb binding. The ability of CD3{epsilon}{delta}–Ig and CD3{delta}{epsilon}–scIg to be bound by anti-CD3 mAb suggests that both CD3–Ig fusion proteins are structurally similar to the native TCR–CD3 complex.

An acid treatment of CD3{epsilon}{delta}–Ig was needed before it could demonstrate high-affinity binding to OKT3 and BC3, whereas the interaction between CD3{epsilon}{delta}–Ig and G19-4 was independent of this acid treatment (Figs 2, 3 and 4). This may reflect that the epitope recognized by OKT3 and BC3 is distinct from that recognized by G19-4. Thus, in the presence of CD3{delta} chain acid treatment might induce proper folding of the OKT3/BC3 epitope for antibody recognition. On the other hand, as long as CD3{epsilon} is in complex with CD3{delta} chain a high-affinity epitope for G19-4 binding was already formed. The ability of G19-4 to recognize CD3{epsilon} alone while OKT3 only bound CD3{epsilon}{delta}–Ig or CD3{delta}{epsilon}–scIg (Figs 2 and 5) is consistent with the idea that there are separate epitopes on the CD3{epsilon} chain for G19-4 and OKT3 or BC3. Molecular refolding induced by acid treatment also seemed to be a specific property of CD3{epsilon}{delta}–Ig; acid treatment of CD3{epsilon}{gamma}–Ig, CD3{delta}{delta}–Ig, CD3{epsilon}{epsilon}–Ig and CD3{gamma}{gamma}–Ig did not significantly alter their binding affinities to any of the four anti-CD3 mAb (data not shown). A number of molecular chaperones have been identified or proposed to regulate the assembly of TCR and CD3 chains into native TCR–CD3 complexes in the ER. These include CD3{omega} (6), calnexin (39,40,43,44) and calreticulin (45). Through their binding to the TCR and CD3 chains, these chaperones may facilitate CD3 folding and subunit association. The acid treatment might substitute some functions performed by the chaperones to generate a conformational epitope on CD3{epsilon}{delta}–Ig. The CD3{delta}{epsilon}–scIg bound G19-4 and OKT3 at high affinity without any acid treatment (Figs 7 and 8), suggesting that a co-linear CD3{delta} and CD3{epsilon} polypeptide unit may be more autonomous in forming the OKT3/BC3 epitope.

The binding affinity between various anti-CD3 mAb and CD3–Ig fusion proteins was assessed by ELISA and SPR (Figs 3 and 8) (Table 1). Among the four anti-CD3 mAb studied, G19-4 consistently gave the highest affinity in binding to CD3{epsilon}{delta}–Ig, CD3{epsilon}{gamma}–Ig and CD3{delta}{epsilon}–scIg (Figs 3 and 8) (Table 1). Anti-CD3 mAb also showed detectable binding to CD3 homodimers at concentrations >10 µg/ml. The concentrations of CD3{epsilon}{delta}–Ig or CD3{epsilon}{gamma}–Ig that half-saturated ant-CD3 mAb were at least 2 orders of magnitude lower than those of the CD3 homodimers (Fig. 3A). Hence, it is unlikely that the presence of CD3{delta}{delta}–Ig, CD3{epsilon}{epsilon}–Ig or CD3{gamma}{gamma}–Ig in the CD3–Ig heterodimer preparations could substantially modify the apparent affinity between anti-CD3 mAb and CD3{epsilon}{delta}–Ig or CD3{epsilon}{gamma}–Ig using ELISA or SPR. The affinity between OKT3 and TCR–CD3 expressed on T cells was determined to be 8.3 x 10–10 M (46,47). The affinity for OKT3 binding to CD3{epsilon}{delta}–Ig was found to be ~2 x 10–9 M in this study, ~ 14-fold less than 8.3 x 10–10 M (Table 1). Several possibilities may account for this apparent lower affinity of OKT3. First, the affinity of OKT3 previously reported was determined by the binding of OKT3 to CD3 molecules expressed on the T cell surface (46,47). Hence, it was a measure of overall avidity. We have shown that OKT3 binds exclusively to the 90 kDa form of CD3{epsilon}{delta}–Ig containing one chain each of CD3{epsilon} and CD3{delta} (Fig. 5). Accordingly, the affinity measured in this study most likely reflects a monovalent interaction between a single CD3{epsilon}{delta} dimer and one F(ab) arm of an OKT3 molecule. Second, we cannot exclude the possibility that CD3{epsilon}{delta}–Ig did not fully resemble its native form in the TCR–CD3 complexes expressed on T cells.

Only G19-4 and BC3, but not OKT3 or 64.1, demonstrated saturable binding to CD3{epsilon}{gamma}–Ig (Fig. 3). However, they bound at affinities that were 1–2 orders lower than their binding to CD3{epsilon}{delta}–Ig (Fig. 3). Acid treatment did not improve the binding of CD3{epsilon}{gamma}–Ig to the anti-CD3 mAb tested in this study (data not shown). These results suggest that structural and possibly functional differences may exist between the CD3{epsilon}{delta} and CD3{epsilon}{gamma} dimers in the TCR complex. Structural determination of the CD3{epsilon}{delta}–Ig and CD3{delta}{epsilon}–scIg should provide valuable information on this question, and on how CD3{epsilon}{delta} and CD3{epsilon}{gamma} may function differently in TCR-mediated signal transduction.

Members of the Ig supergene family are well known for their functions as adhesion molecules. Therefore it would not be surprising if there were ligands for the EC domains of CD3 of Ig-like domains. During TCR–CD3 synthesis in the ER, formation of CD3{epsilon}{delta} and CD3{epsilon}{gamma} pairs takes place first and involves the specific interaction between their EC domains (48). CD3{delta} and CD3{gamma} compete for binding to the same site on CD3{epsilon} (17,49). Besides interacting with CD3{delta} and CD3{gamma} chains, the EC domain of CD3{epsilon} may also interact with the TCR{alpha}ß chains via a pocket formed by the FG loop of the TCR Cß domain (21,22). This interaction is mirrored in the B cell receptor complex in which the EC domains of Ig{alpha}/Igß interact with membrane IgM and IgD molecules (50,51). Mouse CD3{epsilon} may also interact with CD4 via their EC domains during T cell activation (52). A possible role played by the CD3{epsilon} chain in T cell–B cell interaction is also implicated by the binding involving the EC domains of CD3{epsilon} and Igß (53). We investigated whether CD3–Ig fusion proteins can displace the native CD3 subunits from the TCR–CD3 complex by binding to the TCR{alpha}ß chain, thereby compromising signal transduction via the TCR. So far we have not detected any high-affinity binding of CD3–Ig fusion proteins to intact normal PBMC and T cell lines (data not shown). Likewise, we also have not detected any significant effects of CD3–Ig fusion proteins on the proliferation of normal PBMC induced by either superantigens or anti-CD2 + anti-CD28mAb (data not shown). A couple of explanations can be offered for the failure of the CD3–Ig fusion proteins to displace CD3 subunits from the TCR–CD3 complex. First, the overall binding avidity among components of the TCR–CD3 complex may be too high to be disrupted by soluble CD3–Ig fusion proteins. Second, the Fc tails of the CD3–Ig proteins may impose strong steric hindrance to prevent them from interacting with components of the TCR–CD3 complexes expressed on T cells. Additional studies to examine the ability of CD3–Ig to bind targets in detergent-solubilized cell lysates or purified TCR{alpha}ß/{delta}{gamma} chains should provide more insight into this question.

The availability of soluble recombinant human CD3{epsilon}{delta} fusion proteins resembling the native proteins in the TCR–CD3 complex provides us with the starting materials for three-dimensional structural determination of the CD3{epsilon}{delta} subunits. This could be the first step to elucidate how the EC domains of CD3 subunits interact with each other and the TCR{alpha}ß/{delta}{gamma} chains. Such information would facilitate the screening and identification of high potency molecules that can disrupt the native TCR–CD3 complexes expressed on T cells. Such agents could be small molecules or biologics like new anti-CD3 mAb directed against specific regions of the CD3 chains. Not only can these molecules be applied to further our understanding in the signaling function of the TCR–CD3 complex during T cell development, they can also be tested as immunosuppressive agents in the clinic.


    Acknowledgements
 
This study was supported by Xcyte Therapies, Inc. and NIH grant CA90143


    Abbreviations
 
EC—extracellular

ITAM—immunoreceptor tyrosine-based activation motif

ER—endoplasmic reticulum

PBMC—peripheral blood mononuclear cell

sc—single chain

SPR—surface plasmon resonance

TM—transmembrane


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Clevers, H., Alarcon, B., Wileman, T. and Terhorst, C. 1988. The T cell receptor/CD3 complex: a dynamic protein ensemble. Annu. Rev. Immunol. 6:629.[ISI][Medline]
  2. Weiss, A. and Littman, D. R. 1994. Signal transduction by lymphocyte antigen receptors. Cell 76:263.[ISI][Medline]
  3. Wange, R. L. and Samelson, L. E. 1996. Complex complexes: signaling at the TCR. Immunity 5:197.[ISI][Medline]
  4. Cantrell, D. 1996. T cell antigen receptor signal transduction pathways. Annu. Rev. Immunol. 14:259.[ISI][Medline]
  5. Minami, Y., Weissman, A. M., Samelson, L. E. and Klausner, R. D. 1987. Building a multichain receptor: synthesis, degradation, and assembly of the T-cell antigen receptor. Proc. Natl Acad. Sci. USA 84:2688.[Abstract]
  6. Alarcon, B., Berkhout, B., Breitmeyer, J. and Terhorst, C. 1988. Assembly of the human T cell receptor–CD3 complex takes place in the endoplasmic reticulum and involves intermediary complexes between the CD3-gamma.delta.epsilon core and single T cell receptor alpha or beta chains. J. Biol. Chem. 263:2953.[Abstract/Free Full Text]
  7. Chen, C., Bonifacino, J. S., Yuan, L. C. and Klausner, R. D. 1988. Selective degradation of T cell antigen receptor chains retained in a pre-Golgi compartment. J. Cell Biol. 107:2149.[Abstract]
  8. Bonifacino, J. S., Cosson, P. and Klausner, R. D. 1990. Colocalized transmembrane determinants for ER degradation and subunit assembly explain the intracellular fate of TCR chains. Cell 63:503.
  9. Wileman, T., Carson, G. R., Concino, M., Ahmed, A. and Terhorst, C. 1990. The gamma and epsilon subunits of the CD3 complex inhibit pre-Golgi degradation of newly synthesized T cell antigen receptors. J. Cell Biol. 110:973.[Abstract]
  10. Wileman, T., Pettey, C. and Terhorst, C. 1990. Recognition for degradation in the endoplasmic reticulum and lysosomes prevents the transport of single TCRß and CD3{delta} subunits of the T-cell antigen receptor to the surface of cells. Int. Immunol. 2:743.[ISI][Medline]
  11. Wileman, T., Kane, L. P., Young, J., Carson, G. R. and Terhorst, C. 1993. Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER. J. Cell Biol. 122:67.[Abstract]
  12. Manolios, N., Letourneur, F., Bonifacino, J. S. and Klausner, R. D. 1991. Pairwise, cooperative and inhibitory interactions describe the assembly and probable structure of the T-cell antigen receptor. EMBO J. 10:1643.[Abstract]
  13. Exley, M., Wileman, T., Mueller, B. and Terhorst, C. 1995. Evidence for multivalent structure of T-cell antigen receptor complex. Mol. Immunol. 32:829.[ISI][Medline]
  14. San Jose, E., Sahuquillo, A. G., Bragado, R. and Alarcon, B. 1998. Assembly of the TCR–CD3 complex: CD3 epsilon/delta and CD3 epsilon/gamma dimers associate indistinctly with both TCR alpha and TCR beta chains. Evidence for a double TCR heterodimer model. Eur. J. Immunol. 28:12.[ISI][Medline]
  15. Fernandez-Miguel, G., Alarcon, B., Iglesias, A., Bluethmann, H., Alvarez-Mon, M., Sanz, E. and de la Hera, A. 1999. Multivalent structure of an alphabetaT cell receptor. Proc. Natl Acad. Sci. USA 96:1547.[Abstract/Free Full Text]
  16. Manolios, N., Kemp, O. and Li, Z. G. 1994. The T cell antigen receptor alpha and beta chains interact via distinct regions with CD3 chains. Eur. J. Immunol. 24:84.[ISI][Medline]
  17. Alarcon, B., Ley, S. C., Sanchez-Madrid, F., Blumberg, R. S., Ju, S. T., Fresno, M. and Terhorst, C. 1991. The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR–CD3 complexes. EMBO J. 10:903.[Abstract]
  18. Weissman, A. M., Frank, S. J., Orloff, D. G., Mercep, M., Ashwell, J. D. and Klausner, R. D. 1989. Role of the zeta chain in the expression of the T cell antigen receptor: genetic reconstitution studies. EMBO J. 8:3651.[Abstract]
  19. Hall, C., Berkhout, B., Alarcon, B., Sancho, J., Wileman, T. and Terhorst, C. 1991. Requirements for cell surface expression of the human TCR–CD3 complex in non-T cells. Int. Immunol. 3:359.[Abstract]
  20. Cosson, P., Lankford, S. P., Bonifacino, J. S. and Klausner, R. D. 1991. Membrane protein association by potential intramembrane charge pairs. Nature 351:414.[ISI][Medline]
  21. Wang, J., Lim, K., Smolyar, A., Teng, M., Liu, J., Tse, A. G., 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. Atomic structure of an alphabeta T cell receptor (TCR) heterodimer in complex with an anti-TCR fab fragment derived from a mitogenic antibody. EMBO J. 17:10.[Abstract/Free Full Text]
  22. Ghendler, Y., Smolyar, A., Chang, H. C. and Reinherz, E. L. 1998. One of the CD3epsilon subunits within a T cell receptor complex lies in close proximity to the Cbeta FG loop. J. Exp. Med. 187:1529.[Abstract/Free Full Text]
  23. Borroto, A., Mallabiabarrena, A., Albar, J. P., Martinez, A. C. and Alarcon, B. 1998. Characterization of the region involved in CD3 pairwise interactions within the T cell receptor complex. J. Biol. Chem. 273:12807.[Abstract/Free Full Text]
  24. Ledbetter, J. A., June, C. H., Martin, P. J., Spooner, C. E., Hansen, J. A. and Meier, K. E. 1986. Valency of CD3 binding and internalization of the CD3 cell-surface complex control T cell responses to second signals: distinction between effects on protein kinase C, cytoplasmic free calcium, and proliferation. J. Immunol. 136:3945.[Abstract/Free Full Text]
  25. Law, C. L., Aruffo, A., Chandran, K. A., Doty, R. T. and Clark, E. A. 1995. Ig domains 1 and 2 of murine CD22 constitute the ligand-binding domain and bind multiple sialylated ligands expressed on B and T cells. J. Immunol. 155:3368.[Abstract]
  26. Salmeron, A., Sanchez-Madrid, F., Ursa, M. A., Fresno, M. and Alarcon, B. 1991. A conformational epitope expressed upon association of CD3-epsilon with either CD3-delta or CD3-gamma is the main target for recognition by anti-CD3 monoclonal antibodies. J. Immunol. 147:3047.[Abstract/Free Full Text]
  27. Dave, V. P., Cao, Z., Browne, C., Alarcon, B., Fernandez-Miguel, G., Lafaille, J., de la Hera, A., Tonegawa, S. and Kappes, D. J. 1997. CD3 delta deficiency arrests development of the alpha beta but not the gamma delta T cell lineage. EMBO J. 16:1360.[Abstract/Free Full Text]
  28. Wang, B., Wang, N., Salio, M., Sharpe, A., Allen, D., She, J. and Terhorst, C. 1998. Essential and partially overlapping role of CD3gamma and CD3delta for development of alphabeta and gammadelta T lymphocytes. J. Exp. Med. 188:1375.[Abstract/Free Full Text]
  29. Malissen, M., Gillet, A., Ardouin, L., Bouvier, G., Trucy, J., Ferrier, P., Vivier, E. and Malissen, B. 1995. Altered T cell development in mice with a targeted mutation of the CD3-epsilon gene. EMBO J. 14:4641.[Abstract]
  30. Haks, M. C., Krimpenfort, P., Borst, J. and Kruisbeek, A. M. 1998. The CD3gamma chain is essential for development of both the TCRalphabeta and TCRgammadelta lineages. EMBO J. 17:1871.[Free Full Text]
  31. Liu, C. P., Ueda, R., She, J., Sancho, J., Wang, B., Weddell, G., Loring, J., Kurahara, C., Dudley, E. C., Hayday, A., et al. 1993. Abnormal T cell development in CD3-zeta–/– mutant mice and identification of a novel T cell population in the intestine. EMBO J. 12:4863.[Abstract]
  32. Ohno, H., Aoe, T., Taki, S., Kitamura, D., Ishida, Y., Rajewsky, K. and Saito, T. 1993. Developmental and functional impairment of T cells in mice lacking CD3 zeta chains. EMBO J. 12:4357.[Abstract]
  33. Koyasu, S., Hussey, R. E., Clayton, L. K., Lerner, A., Pedersen, R., Delany-Heiken, P., Chau, F. and Reinherz, E. L. 1994. Targeted disruption within the CD3 zeta/eta/phi/Oct-1 locus in mouse. EMBO J. 13:784.[Abstract]
  34. Sun, Z. J., Kim, K. S., Wagner, G. and Reinherz, E. L. 2001. Mechanisms contributing to T cell receptor signaling and assembly revealed by the solution structure of an ectodomain fragment of the CD3 epsilon gamma heterodimer. Cell 105:913.
  35. Berkhout, B., Alarcon, B. and Terhorst, C. 1988. Transfection of genes encoding the T cell receptor-associated CD3 complex into COS cells results in assembly of the macromolecular structure. J. Biol. Chem. 263:8528.[Abstract/Free Full Text]
  36. Kim, K. S., Sun, Z. Y., Wagner, G. and Reinherz, E. L. 2000. Heterodimeric CD3epsilongamma extracellular domain fragments: production, purification and structural analysis. J. Mol. Biol. 302:899.[ISI][Medline]
  37. Sancho, J., Chatila, T., Wong, R. C., Hall, C., Blumberg, R., Alarcon, B., Geha, R. S. and Terhorst, C. 1989. T-cell antigen receptor (TCR)-alpha/beta heterodimer formation is a prerequisite for association of CD3-zeta 2 into functionally competent TCR.CD3 complexes. J. Biol. Chem. 264:20760.[Abstract/Free Full Text]
  38. Jin, Y. J., Koyasu, S., Moingeon, P., Steinbrich, R., Tarr, G. E. and Reinherz, E. L. 1990. A fraction of CD3 epsilon subunits exists as disulfide-linked dimers in both human and murine T lymphocytes. J. Biol. Chem. 265:15850.[Abstract/Free Full Text]
  39. Kearse, K. P. 1998. Calnexin associates with monomeric and oligomeric (disulfide-linked) CD3delta proteins in murine T lymphocytes. J. Biol. Chem. 273:14152.[Abstract/Free Full Text]
  40. Huppa, J. B. and Ploegh, H. L. 1997. In vitro translation and assembly of a complete T cell receptor–CD3 complex. J. Exp. Med. 186:393.[Abstract/Free Full Text]
  41. Moingeon, P., Alcover, A., Clayton, L. K., Chang, H. C., Transy, C. and Reinherz, E. L. 1988. Expression of a functional CD3–Ti antigen/MHC receptor in the absence of surface CD2. Analysis with clonal Jurkat cell mutants. J. Exp. Med. 168:2077.[Abstract]
  42. Tunnacliffe, A., Olsson, C. and de la Hera, A. 1989. The majority of human CD3 epitopes are conferred by the epsilon chain. Int. Immunol. 1:546.[Medline]
  43. David, V., Hochstenbach, F., Rajagopalan, S. and Brenner, M. B. 1993. Interaction with newly synthesized and retained proteins in the endoplasmic reticulum suggests a chaperone function for human integral membrane protein IP90 (calnexin). J. Biol. Chem. 268:9585.[Abstract/Free Full Text]
  44. van Leeuwen, J. E. and Kearse, K. P. 1996. Calnexin associates exclusively with individual CD3 delta and T cell antigen receptor (TCR) alpha proteins containing incompletely trimmed glycans that are not assembled into multisubunit TCR complexes. J. Biol. Chem. 271:9660.[Abstract/Free Full Text]
  45. van Leeuwen, J. E. and Kearse, K. P. 1996. The related molecular chaperones calnexin and calreticulin differentially associate with nascent T cell antigen receptor proteins within the endoplasmic reticulum. J. Biol. Chem. 271:25345.[Abstract/Free Full Text]
  46. Jolliffe, L. K. 1993. Humanized antibodies: enhancing therapeutic utility through antibody engineering. Int. Rev. Immunol. 10:241.
  47. Adair, J. R., Athwal, D. S., Bodmer, M. W., Bright, S. M., Collins, A. M., Pulito, V. L., Rao, P. E., Reedman, R., Rothermel, A. L., Xu, D., et al. 1994. Humanization of the murine anti-human CD3 monoclonal antibody OKT3. Hum. Antibodies Hybridomas 5:41.
  48. Dietrich, J., Neisig, A., Hou, X., Wegener, A. M., Gajhede, M. and Geisler, C. 1996. Role of CD3 gamma in T cell receptor assembly. J. Cell Biol. 132:299.[Abstract]
  49. Geisler, C. 1992. Failure to synthesize the CD3-gamma chain. Consequences for T cell antigen receptor assembly, processing, and expression. J. Immunol. 148:2437.[Abstract/Free Full Text]
  50. Pogue, S. L. and Goodnow, C. C. 1994. Ig heavy chain extracellular spacer confers unique glycosylation of the Mb-1 component of the B cell antigen receptor complex. J. Immunol. 152:3925.[Abstract/Free Full Text]
  51. Li, Q., Santini, R. and Rosenspire, A. R. 1998. Glycosylated extracellular domains of membrane immunoglobulin M contribute to its association with mb-1/B29 gene products and the B cell receptor complex. Immunol. Invest. 27:57.[ISI][Medline]
  52. Portoles, P., Rojo, J., Golby, A., Bonneville, M., Gromkowski, S., Greenbaum, L., Janeway, C. A., Jr, Murphy, D. B. and Bottomly, K. 1989. Monoclonal antibodies to murine CD3 epsilon define distinct epitopes, one of which may interact with CD4 during T cell activation. J. Immunol. 142:4169.[Abstract/Free Full Text]
  53. Muller, B., Cooper, L. and Terhorst, C. 1995. Interplay between the human TCR–CD3 epsilon and the B-cell antigen receptor associated Ig-beta (B29). Immunol. Lett. 44:97.[ISI][Medline]




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