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Signaling efficiency of the T cell receptor controlled by a single amino acid in the beta chain constant region.

Bäckström BT, Hausmann BT, Palmer E - J. Exp. Med. (1997)

Bottom Line: TCRs in which this Cbeta residue is mutated to Phe, the residue found in TCR-gamma, are unresponsive to antigenic ligands.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.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, CH-4005 Basel, Switzerland.

ABSTRACT
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.

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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-Vβ8 (C), and the  culture supernatants were assayed for IL-2. Similar results were obtained  using plate-bound anti–CD3-ε and anti-Vα2 mAbs (data not shown).  Results shown are representative of two or more experiments.
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Figure 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-Vβ8 (C), and the culture supernatants were assayed for IL-2. Similar results were obtained using plate-bound anti–CD3-ε and anti-Vα2 mAbs (data not shown). Results shown are representative of two or more experiments.

Mentions: To test this idea, we constructed two additional chimeras, βV and βVI, which respected the conserved β chain length (13 amino acids) in this region (αwt; Fig. 1 A). Both the βV and the βVI chimeras were expressed at the cell surface in conjunction with a wt α chain (αwt; Fig. 1 B). In fact, the αwt/βV and the αwt/βVI chimeric TCRs were expressed at two- to threefold higher levels on the cell surface than were the αwt/βwt receptor. Hybridomas expressing these chimeric TCRs were stimulated with the superantigen, SEB bound to APCs. As seen in Fig. 2 A, the αwt/βV TCR responded to SEB about as well as the wt TCR, while the αwt/βVI 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 αwt/βVI mutant receptor was compared to that of an αwt/βwt hybridoma sorted for an equivalently high level of TCR expression (Fig. 2 B). Furthermore, transgenic mice expressing the αwt/βVI 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 αwt/βVI TCR were not intrinsically defective. Therefore, TCRs comprised of the chimeric βVI chain seemed to be specifically deficient in transducing signals from antigenic ligands.


Signaling efficiency of the T cell receptor controlled by a single amino acid in the beta chain constant region.

Bäckström BT, Hausmann BT, Palmer E - J. Exp. Med. (1997)

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-Vβ8 (C), and the  culture supernatants were assayed for IL-2. Similar results were obtained  using plate-bound anti–CD3-ε and anti-Vα2 mAbs (data not shown).  Results shown are representative of two or more experiments.
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Related In: Results  -  Collection

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Figure 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-Vβ8 (C), and the culture supernatants were assayed for IL-2. Similar results were obtained using plate-bound anti–CD3-ε and anti-Vα2 mAbs (data not shown). Results shown are representative of two or more experiments.
Mentions: To test this idea, we constructed two additional chimeras, βV and βVI, which respected the conserved β chain length (13 amino acids) in this region (αwt; Fig. 1 A). Both the βV and the βVI chimeras were expressed at the cell surface in conjunction with a wt α chain (αwt; Fig. 1 B). In fact, the αwt/βV and the αwt/βVI chimeric TCRs were expressed at two- to threefold higher levels on the cell surface than were the αwt/βwt receptor. Hybridomas expressing these chimeric TCRs were stimulated with the superantigen, SEB bound to APCs. As seen in Fig. 2 A, the αwt/βV TCR responded to SEB about as well as the wt TCR, while the αwt/βVI 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 αwt/βVI mutant receptor was compared to that of an αwt/βwt hybridoma sorted for an equivalently high level of TCR expression (Fig. 2 B). Furthermore, transgenic mice expressing the αwt/βVI 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 αwt/βVI TCR were not intrinsically defective. Therefore, TCRs comprised of the chimeric βVI chain seemed to be specifically deficient in transducing signals from antigenic ligands.

Bottom Line: TCRs in which this Cbeta residue is mutated to Phe, the residue found in TCR-gamma, are unresponsive to antigenic ligands.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.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, CH-4005 Basel, Switzerland.

ABSTRACT
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.

Show MeSH