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

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.

Figure 1

Amino acid sequences and surface expression of chimeric TCR-β chains. (A) The sequences of the wt TCR-β chain (Cβ), wt TCR-γ chain (Cγ), and the 3 chimeric TCR-β γ chains (βV-βVII) are shown using the single letter amino acid code. The boxes indicate the TCR-γ chain–derived amino acids. Only the CP, transmembrane, and Cyto domains of the TCR constant regions are shown. The complete α and β chain cDNAs have been previously described (16). The NH₂-terminal amino acid in A represents the interchain Cys₁₂₇ of the TCR-β 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 (α⁻/β⁻)T cell hybridomas expressing wt α chains and wt β or chimeric TCR-β γ chains were stained with the biotinylated anti-Vβ8 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 α/β TCR. Similar results were obtained using anti-Vα2, anti–CD3-ε, or anti–Cβ specific mAbs (data not shown).

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.

Figure 3

ζ 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-ζ 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 ζ chains, p34, unphosphorylated ζ chains, and ZAP-70 are indicated.

Figure 3

ζ 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-ζ 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 ζ chains, p34, unphosphorylated ζ chains, and ZAP-70 are indicated.

Figure 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).

Figure 5

Point mutation of Gln₁₃₆ affects antigen responsiveness. Hybridomas expressing the wt TCR-α chain and wt or mutant β 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-β and γ chain CP domains are indicated in boldface.