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The Journal of Immunology, 1998, 160: 1552-1554.
Copyright © 1998 by The American Association of Immunologists

CUTTING EDGE

Cutting Edge: The Chicken TCR {zeta}-Chain Restores the Function of a Mouse T Cell Hybridoma1

Thomas W. F. Göbel2 and Luca Bolliger

Basel Institute for Immunology, Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The TCR/CD3 complex has been intensively studied in mammals, but it has been difficult to isolate homologues in other vertebrates. Here, we characterize the chicken {zeta}-chain, the first nonmammalian homologue identified. The comparison of mammalian and chicken {zeta} proteins revealed high identity of the transmembrane and the C-terminal cytoplasmic domains. Transfection of a mouse {zeta}-deficient cell line, with the chicken {zeta} gene, restored surface expression of the murine TCR/CD3 complex. The chicken {zeta}-chain was stably associated with the mouse TCR/CD3 components and fully restored its signaling capacity upon stimulation with Ab, superantigen, and peptide Ag. This is the first report of a nonmammalian TCR component that is capable of fully restoring a mammalian TCR in every aspect analyzed, thus demonstrating the enormous selective pressure to maintain the {zeta}-chain as a structural and signaling component over a period of 300 million years.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The mammalian TCR/CD3 complex consists of six proteins that are indispensable for its surface expression and function (1). The clonotypic {alpha}ß or {gamma}{delta} TCR chains are noncovalently associated with three signal transduction units, a CD3{delta}{epsilon} heterodimer, a CD3{gamma}{epsilon} heterodimer, and a {zeta}{zeta} homodimer. The {zeta}-chain has at least two distinct functions in the TCR/CD3 complex. It is the rate-limiting chain during the assembly of the TCR/CD3 complex, and it is an important signaling module (2, 3). Numerous studies employing chimeric receptors, truncations of the immunoreceptor tyrosine-based activation motifs (ITAMs)3, and the generation of {zeta}-chain knockout mice provide evidence for the unique role of the {zeta}-chain during T cell development and activation (4, 5, 6, 7).

Evolutionary comparison of the TCR/CD3 proteins mainly focused on the {alpha}ß and {gamma}{delta} TCR chains. In fact, nonmammalian TCR genes have first been isolated in the chicken (8, 9) and recently in other species, including cartilaginous fish (10). In contrast, the information about the signal transduction units of the TCR/CD3 complex of nonmammalian species is limited to a chicken and Xenopus laevis CD3 protein with equal homology to mammalian CD3{gamma} and CD3{delta} (11, 12) and the chicken CD3{epsilon} chain (13).

To draw analogies, in particular for the signaling pathways, to the mammalian TCR/CD3 complex, the gene encoding the chicken {zeta}-chain was isolated and characterized in mouse {zeta}-chain-deficient cells. Unexpectedly, the chicken {zeta}-chain was able to rescue the surface expression and function of the mouse TCR. These experiments demonstrate that the components involved in the signal transduction of the TCR/CD3 complex are extremely well conserved and that nonmammalian species use similar modular signaling units.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
PCR amplification and DNA sequence analysis

The Marathon cDNA amplification kit (Clontech, Palo Alto, CA) was employed for cDNA synthesis and adaptor ligation from chicken T cell-derived mRNA. The adaptor-ligated cDNA was amplified using an adaptor-specific primer and a degenerate primer encoding the amino acid sequence YDALHMQ (5'-TGCATA/GTGIAA/GIGCA/GTCA/GTA-3'), with the cycling conditions: 95°C/5 s; 50°C/30 s; 72°C/2 min for 35 cycles followed by a 72°C extension for 10 min. 3' rapid amplification of cDNA ends was employed to amplify a full length cDNA. The PCR products were subcloned and sequenced with the ABI PRISM dye terminator cycle sequencing ready reaction kit (Perkin-Elmer, Foster City, CA) on an ABI 373A STRETCH sequencer.

DNA transfections and analysis of transfectants

The chicken {zeta} cDNA was amplified with specific oligonucleotides and ligated into EcoRI cut LXSP vector. An amount equal to 10 µg of purified LXSP DNA was used to transiently transfect 2.5 x 106 Bosc23 cells by a standard calcium-phosphate procedure. Following an overnight transfection, cells were provided with 6 ml of fresh 10% FCS Iscove’s modified Dulbecco’s medium medium, and 1 ml of the supernatant harvested 24h later was subsequently used to infect 106 MA5.8 cells (14) in the presence of 40 µg/ml DEAE-dextran. After 12 h incubation, 3 mg/ml puromycin was added to select for infected cells. Quantification of the TCR surface expression was conducted by flow cytometry using mAbs H57-597 (anti-mouse TCRCß), 2C11 (anti-mouse CD3{epsilon}), and KJ25 (anti-mouse TCRVß3; all PharMingen, San Diego, CA). Surface biotinylation and immunoprecipitations were performed according to standard procedures.

Cell stimulation and IL-2 quantitation

For Ab stimulation a 96-well plate was coated with the H57 mAb (anti-mouse TCRCß region) and blocked with IMDM/5% FCS before addition of cells. An amount equal to 5 x 104 cells was added to each well for 20 h. Anti-Thy-1 stimulation was performed similarly with serial dilutions of soluble G7 mAb supernatant (15). Superantigen stimulation was conducted with DAP3 cells incubated with the indicated amounts of Staphylococcus aureus A toxin (Sigma, Brunschwig, Switzerland) at 5 x 105 cells/ml. An amount equal to 50 µl DAP3 cells was incubated with 5 x 104 transfected cells for 20 h. The Ag stimulation was performed similarly using apocytochrome c peptide-pulsed LK35.2 cells. The culture supernatants were assayed in triplicates for IL-2 production using the standard HT-2 bioassay (16).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Commonly evolved features of {zeta}-chains

The gene encoding the chicken {zeta}-chain was isolated with a degenerate PCR-RACE approach utilizing a primer specific for the highly conserved YAQLHMQ motif. The 1598-bp-long chicken {zeta} cDNA clone is 50% identical to the human homologue and contains a 42-bp 5' untranslated region, a 501-bp open reading frame, and a 1055-bp 3' untranslated region4 . The 166-amino acid-long protein has a Mr of 16,235, an isoelectric point (pI) of 9.4, and lacks N-glycosylation sites. The comparison to the mammalian {zeta} sequences allows the assignment of a 22-amino acid signal peptide, a 9-amino acid extracellular (EC) domain, a 21-amino acid transmembrane (TM) region, and a 114-amino acid cytoplasmic (CY) domain (Fig. 1Go).



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  		FIGURE 1. Comparison of chicken and mammalian {zeta} peptide sequences. Predicted peptide sequences inferred from respective cDNA sequences were aligned using the CLUSTAL program (DNASTAR Inc., Madison, WI). The signal peptide is not shown. The location of the TM domain is indicated with a bar above the sequence.

 
In mammals the entire EC domain is identical, whereas only 4 of 9 amino acids and a positively charged residue have been conserved in the chicken {zeta} EC. The highest identity (81%) between chicken and mammalian {zeta}-chain sequences is found in the TM domain, including the cysteine residue involved in the covalent dimerization of the {zeta}-chain and the negatively charged residue, a putative site of interaction with respective positive charges of the TCR chains (Fig. 1Go).

An increasing gradient of homology is found within the CY domain toward the C terminus. This is reflected by 56% overall amino acid identity of the human and chicken CY domains, whereas a comparison of the three ITAMs yields 67%, 81%, and 87% identity, respectively. Interestingly, the 8-amino acid spacing of the second {zeta}-chain ITAM has also been conserved. The chicken {zeta}-chain also harbors a putative GTP/GDP binding site located between the second and third ITAM (17). In summary, areas of high amino acid identity in the chicken {zeta}-chain support the functional evolution of commonly used motifs.

The chicken {zeta}-chain associates with the mouse TCR/CD3 complex

Due to the high identity of mouse and chicken {zeta}-chains, the {zeta}-deficient and TCR surface-negative MA5.8 cell line (14) was transfected with either the chicken {zeta} gene (MA5.8-c{zeta}) or the wild-type mouse {zeta} gene (MA5.8-m{zeta}). Both the mouse and the chicken {zeta}-chain similarly restored TCR expression (Fig. 2Go). The variation in TCR/CD3 levels in both MA5.8-m{zeta} and MA5.8-c{zeta} cells was due to the bulk infection procedure and was not observed in stable clones (data not shown). Potential epitope alterations as tested with different TCR/CD3-specific mAb (anti-CD3{epsilon}, anti-Cß, anti-Vß3) could not be detected (Fig. 2Go, and data not shown). Following surface biotinylation of MA5.8-c{zeta} cells, the chicken {zeta}-chain homodimer (34- and 36-kDa NR; 17-kDa R) was coimmunoprecipitated along with the mouse TCR-{alpha}ß (90-kDa NR; 40- and 50-kDa R), CD3{epsilon} (23-kDa NR; 25-kDa R), CD3{delta} (27-kDa NR; 28-kDa R) and CD3{gamma} (23-kDa NR; 21-kDa R; Fig. 3Go). Thus the chicken {zeta}-chain is able to restore mouse TCR surface expression by stably assembling with all mouse TCR/CD3 components.



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  		FIGURE 2. The mouse TCR surface expression is restored by the chicken {zeta}-chain. Flow cytometric analysis with a mouse CD3{epsilon}-specific FITC-conjugated mAb (2C11) of MA5.8 cells, MA5.8-m{zeta}, and MA5.8-c{zeta} cells. The mean fluorescence intensity is indicated in the plots.

 


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  		FIGURE 3. The chicken {zeta}-chain associates with the mouse TCR/CD3 complex. Biotinylated MA5.8-c{zeta} cell lysates were immunoprecipitated with a mouse CD3{epsilon}-specific mAb, separated on a 4 to 20% gradient PAGE under nonreducing (NR) or reducing (R) conditions, blotted, and detected using a streptavidin-horseradish peroxidase conjugate and enhanced chemiluminescence.

 
Chicken {zeta} restores signal transduction of a mouse TCR

The functional capabilities of this "xenogenic" TCR complex were tested with mAb, superantigen, or Ag stimulation. Both the MA5.8-m{zeta} cells and MA5.8-c{zeta} cells responded equally well to cross-linking of their TCR/CD3 complex with plate-bound mAb, whereas the nontransfected MA5.8 cells showed only residual stimulation with high amounts of mAb (Fig. 4GoA). Using the superantigen staphylococcal enterotoxin A (SEA), the MA5.8-m{zeta} cells were more efficiently stimulated than the MA5.8-c{zeta} cells, whereas the MA5.8 control cells were not stimulated (Fig. 4GoB). Most importantly, when stimulated with the nominal peptide Ag and APCs, the MA5.8-m{zeta} cells and the MA5.8-c{zeta} cells responded equivalently across the entire dose-response curve (Fig. 4GoC). Previous experiments have suggested a {zeta}-chain-independent T cell activation pathway mediated through the CD3{gamma}{delta}{epsilon} ITAMs (18). To prove the true signaling capacity of the chicken {zeta}-chain in the context of the murine TCR, anti-Thy-1 stimulation as a strictly {zeta}-chain ITAM-dependent T cell activation (18) was compared between MA5.8-m{zeta} cells and MA5.8-c{zeta} cells. Both cell lines responded equally well to increasing concentrations of anti-Thy-1 mAb, thus clearly demonstrating that the chicken {zeta}-chain ITAMs are important to functionally restore the mouse TCR (Fig. 4GoD). Moreover, the early intracellular signaling events as detected by increased TCR associated tyrosine phosphorylation appeared to be identical in MA5.8-m{zeta} cells and MA5.8-c{zeta} cells (data not shown). These results indicate that the chicken {zeta}-chain can fully replace the wild-type mouse {zeta}-chain.



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  		FIGURE 4. The chicken {zeta}-chain restores the function of the mouse TCR. IL-2 secretion of untransfected MA5.8 cells, MA5.8-m{zeta} cells, and MA5.8-c{zeta} cells upon triggering with increasing concentrations of a plate-bound mouse TCRß-specific mAb (A), the superantigen staphylococcal enterotoxin A (SEA) (B), apocytochrome c peptide Ag (C), and soluble Thy-1-specific mAb supernantant (D). One representative experiment of four is shown using three independent bulk infections.

 
This is the first report where a nonmammalian TCR component fully restores the assembly and function of a mouse TCR. The extraordinarily high conservation of the {zeta}-chain over a period of 250 to 300 million years, when the avian and mammalian lineages diverged (19), illustrates the importance of the {zeta}{zeta} homodimer for the assembly, transport, and function of the TCR/CD3 complex. A gradient of conservation with low EC but relatively high CY identity is evident when the chicken and mammalian {zeta}, TCR and CD3 proteins are compared. The low EC conservation of the TCR and the CD3 proteins throughout evolution most likely reflects the adaptation to different selective pressures in various organisms determined by the structure of self MHC molecules and the antigenic environment. In contrast, due to the lack of intrinsic enzymatic activity, the CY regions have been highly conserved to bind downstream signaling modules, like SH2 and SH3 domains. In chickens and Xenopus laevis, there is good biochemical and genetic evidence that only two CD3 genes, CD3{delta} and CD3{epsilon}, exist (11, 12, 20). The nonmammalian TCR/CD3 complexes contain only the CD3{delta}{epsilon} heterodimer and {zeta}{zeta} homodimer as signaling modules, indicating that these two independent dimers are sufficient for TCR function. Despite these differences, our results demonstrate that the chicken {zeta}{zeta} homodimer is not altering the composition of the mouse TCR/CD3 complex. In conclusion, the chicken is the only nonmammalian species where all components of the TCR/CD3 complex have now been characterized, thus providing an excellent model for functional and structural phylogenetic analyses.


   Acknowledgments
 
The authors thank Dr. H. Jacobs and Dr. E. Palmer for critically reading the manuscript, Dr. J. Ashwell for providing the MA5.8 cells, Dr. J. Bluestone for providing the G7 mAb, B. Johansson for excellent technical assistance, H. Stahlberger for artwork, H. Spalinger and B. Pfeiffer for photography. L.B. thanks Dr. E. Palmer for continuous encouragement.


   Footnotes
 
1 The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche & Co. Ltd., Basel, Switzerland Back

2 Address correspondence and reprint requests to Dr. Thomas Göbel, Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland. E-mail address: Back

3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation motif; CY, cytoplasmic; EC, extracellular; TM transmembrane; NR, nonreducing; R, reducing. Back

4 The sequence data presented in this article have been submitted to GenBank under the accession number AJ002317. Back

Received for publication October 24, 1997. Accepted for publication December 15, 1997.


   References
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 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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