BRIEF DEFINITIVE REPORT:
Signaling Efficiency of the T Cell Receptor Controlled by a Single Amino Acid in the beta  Chain Constant Region

By B. Thomas Bäckström, Barbara T. Hausmann, and Ed Palmer

From the Basel Institute for Immunology, CH-4005 Basel, Switzerland

SUMMARY
Materials and Methods
Results
Discussion
FOOTNOTES
ACKNOWLEDGEMENTS
References


Summary

A single amino acid residue, Gln136, located within the connecting peptide domain of Cbeta controls the ability of the alpha /beta TCR to transmit a full signal. TCRs in which this Cbeta residue is mutated to Phe, the residue found in TCR-gamma , are unresponsive to antigenic ligands. Interestingly, this Cbeta residue is either polar or charged in every species studied thus far, including the trout and the skate. In contrast, the analogous residue in Cgamma is always hydrophobic. In spite of their compromised antigen responsiveness, the mutant TCR complex contains the CD3-gamma , -delta , -epsilon , and -zeta chains, and undergoes zeta  chain phosphorylation and ZAP-70 recruitment. However, the biological response of the mutant TCR could be rescued with a calcium ionophore, implying that mutant TCRs are defective in generating a calcium-mediated signal. The implications of the differences between Cbeta and Cgamma are considered.


One of the primary goals of immunologists over the last 10 yr has been to understand how the TCR transduces a signal (1) . The TCR complex consists of an antigen-binding alpha /beta heterodimer supported by noncovalently associated CD3-gamma , -delta , and -epsilon as well as zeta  chains (2). The alpha  and beta  constant regions are presumably required to propagate a signal to the CD3 and zeta  components of the complex.

Considered together, the cytoplasmic tails of the CD3 and zeta  chains contain 10 immunoreceptor tyrosine-based activation motifs (ITAMs), which are rapidly phosphorylated upon TCR engagement (3, 4). Subsequently, the protein tyrosine kinase ZAP-70 is recruited to the TCR complex and activated (5, 6). Thereafter, large multimolecular complexes of proteins participating in the signaling cascades assemble around the cytoplasmic domains of activated TCRs (7), which leads to the initiation of intracellular signaling pathways.

Although homologous to its alpha /beta relative, the gamma /delta TCR is expressed in a separate lineage of T lymphocytes (8). While it has been generally assumed that both classes of TCR use similar mechanisms to generate a signal, this may not be the case, as there are several features that distinguish these two types of T cell receptors. The CD3-epsilon chains contained within human alpha /beta receptors express an epitope which is masked in gamma /delta receptors (9, 10). Furthermore, unlike alpha /beta receptors, gamma /delta TCRs can be expressed in the absence of CD3-delta , can develop in MHC deficient mice, and can recognize unprocessed antigens in the absence of MHC presentation (11). Finally, the TCR-alpha chain contains a motif within its connecting peptide (CP) domain that is required for the transduction of antigenic signals (15). alpha /beta TCRs lacking this alpha  chain-connecting peptide motif (alpha -CPM) do not properly interact with the CD3 complex and fail to generate Ca2+-mediated signals. The alpha -CPM is specific for the alpha /beta TCR and seems to have evolved after the divergence of TCR-alpha and -delta chains. That this motif is found within alpha /beta but not gamma /delta TCRs implies that these two types of TCRs may use subtly different signaling mechanisms.

We wondered whether there exists a region within the TCR-beta chain, analogous to the alpha -CPM, which is required to produce a functional alpha /beta receptor. The experiments reported here identify a single amino acid within the beta  chain- CP domain that controls TCR signaling efficiency. Mutation of this beta  chain amino acid to a residue commonly seen in TCR-gamma chains generates an alpha /beta receptor which is particularly inefficient in transducing signals from antigenic ligands.


Materials and Methods

Generation of DNA Constructs. The Valpha 2.1 and Vbeta 8.1 TCR cDNAs were isolated from the T cell hybridoma, 3BBM74, and confer reactivity to the I-Abm12 alloantigen and the staphylococcal enterotoxin B (SEB) superantigen (16). The wild-type (wt) and mutated TCR constructs were generated using overlapping oligo nucleotides and PCR as previously described (15). All constructs were verified by DNA sequencing using the SequiThermTM cycle sequencing kit (Epicentre Technologies Corp., Madison, WI) and the deduced amino acid sequences of the mutant beta  chains are shown in Fig. 1.


Fig. 1. Amino acid sequences and surface expression of chimeric TCR-beta chains. (A) The sequences of the wt TCR-beta chain (Cbeta ), wt TCR-gamma chain (Cgamma ), and the 3 chimeric TCR-beta gamma  chains (beta V-beta VII) are shown using the single letter amino acid code. The boxes indicate the TCR-gamma chain-derived amino acids. Only the CP, transmembrane, and Cyto domains of the TCR constant regions are shown. The complete alpha  and beta  chain cDNAs have been previously described (16). The NH2-terminal amino acid in A represents the interchain Cys127 of the TCR-beta constant region. The dotted lines indicate the approximate boundary of the TM domain, defined using the Lasergene Navigator Protean Software program (DNASTAR, Inc., Madison, WI). (B) 58hCD4 (alpha -/beta -)T cell hybridomas expressing wt alpha  chains and wt beta  or chimeric TCR-beta gamma  chains were stained with the biotinylated anti-Vbeta 8 mAb, F23.1, and SAPE, and then analyzed by flow cytometry. Dashed lines represent fluorescence of the same cells stained with streptavidin-phycoerythrin alone. The solid vertical lines indicate the mean fluorescence intensity of 58hCD4 cells expressing the wt alpha /beta TCR. Similar results were obtained using anti-Valpha 2, anti-CD3-epsilon , or anti-Cbeta specific mAbs (data not shown).
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Production of Retroviral Supernatants and Infection of T Cell Hybridomas. Retroviruses encoding wt or chimeric TCR chains were generated and used to infect the alpha -/beta - T cell hybridoma, 58hCD4, as previously described (15).

Cell Lines. The 58hCD4 cell line has been previously described (15, 17) and was provided by O. Acuto (Pasteur Institute, Paris, France). The murine fibroblast cell line DAP.3 transfected with human HLA class II DR1 molecules (18) or the human, DR1-expressing, B lymphoblastoid cell line .221 (19) was used to present SEB. The use of the cytokine indicator cell lines HT-2 and FDC-P1 have been previously described (15).

Antibodies. The use of anti-Valpha 2 (B20.1), anti-Vbeta 8.1 (F23.1), and anti-CD3-epsilon (145-2c11) mAbs, as well as anti-CD3-gamma , anti- CD3-delta , and anti-CD3-epsilon rabbit antisera have been previously described (20). The anti-zeta chain mAb, H146-968 (21), was purified from culture supernatants using protein G-Sepharose beads (Pharmacia Biotech AB, Uppsala, Sweden). The rabbit anti- ZAP-70 antiserum was provided by Dr. L. Samelson (National Institutes of Health, Bethesda, MD). The antiphosphotyrosine mAb 4G10 was purchased from Upstate Biotechnology Inc. (Lake Placid, NY).

Flow Cytometry, T Cell Stimulation, and IL-2 and IL-3 Assays. The levels of cell-surface antigens were detected by immunofluorescence and analyzed on a FACScan® using the CELLQuest analysis software (Becton Dickinson, Rutherford, NJ). T cell stimulation and IL-2 and IL-3 assays were performed as previously described (15).

Immunoprecipitation and Western Blotting. T hybridoma cells (2 × 107/sample) were lysed in buffers containing 1% digitonin and the TCR complex immunoprecipitated with 2 µg of B20.1 (anti Valpha 2). Immunoprecipitations, Western blotting, and detection of CD3 chains were carried out as previously described (20).

zeta Chain Tyrosine Phosphorylation Analysis. For superantigen stimulation, 106 .221 APCs were incubated with or without SEB (10 µg/ml) for 2 h in 1 well of a 24-well plate. 107 T hybridoma cells were added to the APCs, centrifuged at 1,000 rpm for 30 s, and then incubated at 37°C for 20 min. Cells were washed twice with ice cold PBS containing 0.4 mM Na3VO4 and 0.4 mM EDTA, and were then lysed in 0.5 ml lysis buffer (10 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM PMSF, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) containing 2 mM Na3VO4, 25 mM NaF, and 1% Triton X-100. Lysates were centrifuged at 12,000 g for 10 min, and the relevant proteins in the supernatant were immunoprecipitated with 1.5 µg of H146-968 (anti-zeta ) mAb or 3 µl of a rabbit polyclonal anti-ZAP-70 antiserum. Immunoprecipitates were recovered using protein G-Sepharose beads. The tyrosine phosphorylated proteins present in these immunoprecipitates were analyzed by Western blotting and detected using the antiphosphotyrosine mAb 4G10.


Results

To better understand the functional relationships between the alpha /beta heterodimer and the CD3 and zeta  components, we made a number of chimeric TCR-beta chains by replacing domains from the beta  chain constant region with homologous domains from the TCR-gamma chain. We first generated a chimeric beta  chain, beta VII, which contained TCR-beta -derived V, D, J, and C region sequences up to and including the interchain Cys, followed by Cgamma sequences encoding the COOH-terminal part of the CP domain, the transmembrane domain, and the cytoplasmic tail (Fig. 1 A). As seen in Fig. 1 B, this chimeric beta VII chain was not expressed at the cell surface when paired with a wt alpha chain. However, Cbeta and Cgamma differ in the length of their CP domains. There are 13 amino acids between the interchains Cys127 and Ala141 in all beta  chains and 16 amino acids between Cys117 and Ala134 in all mammalian gamma  chains (Fig. 1 A and reference 22). Thus, the fact that the beta VII chimera could not be expressed at the cell surface might have been due to the possibility that the Cgamma length in this region (16 amino acids) was incompatible with surface expression in the context of an alpha /beta TCR.

To test this idea, we constructed two additional chimeras, beta V and beta VI, which respected the conserved beta  chain length (13 amino acids) in this region (alpha wt; Fig. 1 A). Both the beta V and the beta VI chimeras were expressed at the cell surface in conjunction with a wt alpha  chain (alpha wt; Fig. 1 B). In fact, the alpha wt/beta V and the alpha wt/beta VI chimeric TCRs were expressed at two- to threefold higher levels on the cell surface than were the alpha wt/beta wt receptor. Hybridomas expressing these chimeric TCRs were stimulated with the superantigen, SEB bound to APCs. As seen in Fig. 2 A, the alpha wt/beta V TCR responded to SEB about as well as the wt TCR, while the alpha wt/beta VI TCR was clearly less sensitive (~100-fold) to this superantigen. This signaling deficit was seen even more clearly when the SEB response of a hybridoma expressing the alpha wt/beta VI mutant receptor was compared to that of an alpha wt/beta wt hybridoma sorted for an equivalently high level of TCR expression (Fig. 2 B). Furthermore, transgenic mice expressing the alpha wt/beta VI TCR were defective in responding to SEB and the I-Abm12 alloantigen (data not shown). Thus, the signaling defect of this mutant TCR was not limited to superantigens. On the other hand, these chimeric TCRs could be activated by plate-bound anti-TCR mAbs (Fig. 2 C and data not shown), indicating that the hybridomas expressing the alpha wt/beta VI TCR were not intrinsically defective. Therefore, TCRs comprised of the chimeric beta VI chain seemed to be specifically deficient in transducing signals from antigenic ligands.


Fig. 2. Response of chimeric TCRs to SEB and anti-TCR mAbs. In each experiment, 5 × 104 58hCD4 cells/well expressing wt or chimeric TCRs were stimulated with 2 × 104 DAP.3-DR1 cells and increasing doses of SEB (A and B) or plate-bound anti-Vbeta 8 (C), and the culture supernatants were assayed for IL-2. Similar results were obtained using plate-bound anti-CD3-epsilon and anti-Valpha 2 mAbs (data not shown). Results shown are representative of two or more experiments.
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TCRs from these hybridomas were immunoprecipitated, and the associated polypeptides were analyzed by Western blotting (data not shown). The CD3-gamma , -delta , and -epsilon chains as well as the zeta  polypeptides were coprecipitated from hybridomas expressing either the wt TCR or the signaling-defective, alpha wt/beta VI TCR (data not shown). In spite of a pronounced signaling defect (Fig. 2), the interactions with the CD3 and zeta  chains were preserved in complexes containing the functionally defective, alpha wt/beta VI heterodimer.

The tyrosine phosphorylation of the zeta  chain was examined in these hybridomas as well. From the data in Fig. 3 A, it was apparent that the zeta  chains in the alpha wt/beta wt and alpha wt/beta VI TCRs could be tyrosine phosphorylated and that both forms (p21 and p23) of the phosphorylated zeta  chain could be generated. Although the tyrosine phosphorylation of ZAP-70 could not be demonstrated even in hybridomas expressing the alpha wt/beta wt receptor (data not shown), an anti-ZAP-70 antiserum was used to evaluate ZAP-70 recruitment (Fig. 3 B). In superantigen-stimulated cells expressing either wt or mutant TCRs, the p21 and p23 forms of the zeta  chain as well as the p34 phosphoprotein were coprecipitated with ZAP-70. These experiments indicated that even in hybridomas expressing the signaling-defective alpha wt/beta VI TCR, ZAP-70 was nevertheless recruited to the phosphorylated zeta  chains. Thus, the signaling defect in hybridomas expressing this mutant TCR was likely downstream from ZAP-70 recruitment.



Fig. 3. zeta chain phosphorylation of SEB-stimulated hybridomas expressing chimeric TCRs. Hybridomas (58hCD4) expressing wt or chimeric TCRs were stimulated using .221 cells with (10 µg/ml) or without SEB. Cells were lysed in 1% Triton X-100, and the relevant chains were immunoprecipitated with an anti-zeta mAb (A) or with an anti-ZAP-70 antiserum (B). After Western blotting, the presence of tyrosine phosphorylated proteins was detected with the antiphosphotyrosine mAb 4G10 as described in Materials and Methods. The positions of phosphorylated zeta  chains, p34, unphosphorylated zeta  chains, and ZAP-70 are indicated.
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Binding of an appropriate ligand to the TCR eventually leads to the activation of the protein kinase C/ras and the calcium intracellular signaling pathways (1, 7). Deficient activation of the PKC/ras pathway can be compensated for by the addition of the phorbol ester, PMA, whereas deficient activation of the calcium pathway can be compensated for by the addition of a calcium ionophore, such as ionomycin. In Fig. 4, hybridomas expressing the wt or the defective alpha wt/beta VI TCR were stimulated with SEB in the presence of PMA or ionomycin. The impaired response of the alpha wt/beta VI TCR was only marginally affected by several different concentrations of PMA, but was rescued in the presence of ionomycin used at several different concentrations (Fig. 4 and data not shown). These findings suggested that the mutant TCR was defective or inefficient in activating the calcium pathway.


Fig. 4. Effects of calcium ionophore and PMA on superantigen stimulation. The 58hCD4 hybridoma (5 × 104 cells/well) expressing wt or chimeric TCRs was stimulated with 2 × 104 DAP.3-DR1 cells and the indicated combinations of 3 µg/ml SEB, 100 ng/ml ionomycin, or 30 ng/ml PMA. Culture supernatants were assayed for IL-2 (A) or IL-3 (B).
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The functional chimera, beta V, contains the beta  chain sequence ASYQQ, which has been replaced with the gamma  chain sequence, LQFQF, in the nonfunctional chimeric chain, beta VI (Fig. 1 A). The most striking amino acids in this region are Tyr134 and Gln136, both of which are encoded by Phe in murine Cgamma 1. Point mutations were introduced into the wt beta  chain cDNA, and the resulting mutant beta  chains were paired with an alpha wt chain and were tested for their reactivity to SEB. As seen in Fig. 5, A and B, the Tyr134 right-arrow Phe mutation had a negligible effect on the response to SEB, whereas TCRs carrying the Gln136 right-arrow Phe mutation had a marked and reproducible effect on SEB responsiveness. Considering the pronounced effect of the Gln136 right-arrow Phe exchange on superantigen responsiveness, we examined the amino acid residues present at this position in all Cbeta and Cgamma sequences, some of which are shown in Fig. 5 C. Strikingly, the amino acids at this position in the beta  chain are always polar or charged. In contrast, gamma  chains always contain hydrophobic amino acids at this position (Fig. 5 C and reference 23).


Fig. 5. Point mutation of Gln136 affects antigen responsiveness. Hybridomas expressing the wt TCR-alpha chain and wt or mutant beta  chains were stimulated with DAP.3-DR1-presenting cells and SEB as described in Fig. 2. IL-2 and IL-3 responses are shown in A and B, respectively. Sequences of the CP domains from different species (22, 23) are shown in C, using the single letter amino acid codes. Sequences were aligned using the Lasergene Navigator MegAlign Software program (Clustal alignment method with the PAM250 residue weight table). The conserved amino acids present in the TCR-beta and gamma  chain CP domains are indicated in boldface.
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Discussion

We took advantage of the differences between alpha /beta and gamma /delta TCRs to generate signaling-defective receptor mutants. Since the TCR-beta and -gamma constant regions are structurally homologous, we could replace domains of Cbeta with similar domains of Cgamma . Most of the resulting chimeric chains paired with a wt alpha  chain and were expressed on the cell surface (Fig. 1 B). Hybridomas expressing alpha wt/beta VI TCRs are 100-fold less sensitive to antigenic signals than hybridomas expressing wt receptors (Fig. 2, A and B). The signaling defect seems specific for antigenic ligands, since alpha wt/ beta VI TCRs respond fully to mAbs capable of receptor cross-linking (Fig. 2 C and data not shown). What distinguishes the functional beta V chimera from the defective beta VI chimera are the amino acids present from position 132 to 136 that are derived from Cbeta and Cgamma , respectively (Fig. 1 A). Site-directed mutagenesis defined Gln136 as a critical residue in this region. This position has been conserved in the beta  chains of all known species from the skate to humans (23) and is either polar or charged (Fig. 5 C). In contrast, the analogous residue in the gamma  chain has been conserved as a hydrophobic amino acid (Fig. 5 C). This conservation is functionally significant since changing this residue from a hydrophilic to a hydrophobic amino acid is accompanied by a significant loss in signaling efficiency (Figs. 2 and 5).

There is evidence that the alpha /beta and gamma /delta heterodimers do not interact with the CD3 complex in the same way. For example, gamma /delta TCRs can be expressed in the absence of CD3-delta (11). Furthermore, a CD3-epsilon epitope, contained within human alpha /beta receptors and recognized by the mAb WT31 is masked in gamma /delta receptors (9, 10, 24). Many of these differences might be attributed to the CP (membrane proximal) domains of the different TCR chains. We have previously identified a motif within the alpha  CP domain which is strikingly absent from TCR-delta chains. This motif controls the proper association of CD3-delta and -zeta to the TCR complex and is required for antigen-driven signal transduction (15).

Examining the CP domain of the beta  chain, we have found a region within Cbeta which controls the efficacy of antigen-driven signal transduction. Although alteration of this region does not lead to changes in the subunit composition of the TCR complex (data not shown), a clear biological defect was observed (Fig. 2). Mutation of this region had no observable effect on the amount of zeta  chain phosphorylation nor on the recruitment of ZAP-70 to the zeta  homodimer (Fig. 3). Thus, the signaling defect in this mutant is likely downstream from ZAP-70 recruitment. The signaling defect can be corrected or at least compensated in the presence of a calcium ionophore (Fig. 4), suggesting that the mutation within the beta chain may lead to the inefficient induction of Ca2+ mobilization.

There is little structural information available for the CP domains since this region has been truncated from the TCR heterodimers used in crystallographic analyses (25, 26). Nevertheless, the CP domains of the alpha  and beta  chains may function as a unit since amino acids within these domains have been conserved in alpha /beta TCRs during the last 500 million yr (15, 22, 23). The length of the CP domains has been conserved as well. Considering the region between the interchain Cys and the transmembrane domain (defined by the beginning of the CART motif [22]), all known beta chains contain 13 amino acids (22, 23). On the other hand, alpha  chains contain from 17 to 20 amino acids in this region. Thus, in alpha /beta TCRs, a length disparity between the alpha  and beta  chain CP domains has been conserved throughout vertebrate evolution. In contrast, the lengths of the TCR-gamma and -delta chain CP domains (between the interchain Cys and the transmembrane domain) are comparable in each species examined thus far (22, 23). Therefore, a length similarity between the delta  and gamma  chains has been conserved in this region in gamma /delta receptors during a similar period of vertebrate evolution.

Functionally, the CP domains may mediate an interaction with the zeta  homodimer. In this regard, alpha /beta TCRs compete more avidly for the zeta  chain compared to a gamma /delta TCR (27). Furthermore, mutation of the alpha -CPM leads to a TCR complex that is unstably associated with the zeta  chains (15). Nevertheless, why the CP domains have diverged between alpha /beta and gamma /delta TCRs is puzzling. It is conceivable that the CP domains in the alpha /beta TCR have a specialized function such as interacting with the CD4 and CD8 coreceptors or that they have evolved to optimize the efficiency of signal transduction from MHC encoded ligands.


Footnotes

Address correspondence to Dr. Ed Palmer, Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland. Phone: 41-61-605-1277; FAX: 41-61-605-1364; E-mail: Palmer{at}bii.ch  B. Thomas Bäckström's current address is Malaghan Institute of Medical Research, PO Box 7060, Wellington South, New Zealand.

Received for publication 16 July 1997 and in revised form 26 September 1997.

   The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche LTD, Basel, Switzerland.

The authors thank Drs. Luca Bolliger, Thomas Göbel, Wayne Hein, Michel Record, and Sabine Stotz for careful reading of the manuscript, Beatrice Pfeiffer for photography, Dr. L. Samelson for the rabbit anti- ZAP-70 antiserum, and O. Acuto for the 58hCD4 cell line.


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