Influence of the NH2-terminal amino acid of the T cell receptor alpha chain on major histocompatibility complex (MHC) class II + peptide recognition

Abstract

The alpha/beta T cell receptor (TCR) recognizes peptide fragments bound in the groove of major histocompatibility complex (MHC) molecules. We modified the TCR alpha chain from a mouse T cell hybridoma and tested its ability to reconstitute TCR expression and function in an alpha chain-deficient variant of the hybridoma. The modified alpha chain differed from wild type only in its leader peptide and mature NH2-terminal amino acid. Reconstituted cell surface TCR complexes reacted normally with anti-TCR and anti-CD3 antibodies. Although cross-linking of this TCR with an antibody to the TCR idiotype elicited vigorous T cell hybridoma activation, stimulation with its natural MHC + peptide ligand did not. We demonstrated that this phenotype could be reproduced simply by substituting the glutamic acid (E) at the mature NH2 terminus of the wild type TCR alpha chain with aspartic acid (D). The substitution also dramatically reduced the affinity of soluble alpha/beta-TCR heterodimers for soluble MHC + peptide molecules in a cell-free system, suggesting that it did not exert its effect simply by disrupting TCR interactions with accessory molecules on the hybridoma. These results demonstrate for the first time that amino acids which are not in the canonical TCR complementarity determining regions can be critical in determining how the TCR engages MHC + peptide.

Figure 1

Complete nucleotide sequence of the Vα13.1 leader and its 5′ untranslated region. The sequence of the Vα13.1 leader was assembled from six independently cloned PCR products of different lengths, which were obtained by anchored reverse transcriptase PCR from the B10.D2derived T cell hybridoma, 22.3. Deduced amino acid residues are shown. Underlined triplets in the 5′ untranslated region indicate stop codons, which are found in all three reading frames. The Vα11.2 leader is shown for comparison. Assignment of the boundary between the leaders and the mature variable domains is based on standard mouse TCR α chain sequence alignments (54).

Figure 2

DOα chain variant constructs. Circled letters indicate the predicted NH₂-terminal amino acid of the mature α chain.

Figure 3

Infectants bearing the variant TCR α chain DOαL11terD respond to immobilized antiidiotype mAb as well as the parental hybridoma, 22.3, does. 22.3.111 and 22.3.145 are low TCR expressing subclones of 22.3. Data are representative of multiple experiments. TCR level indicates the mean linear fluorescence of antiidiotype mAb staining of the hybridomas.

Figure 4

Infectants bearing the variant TCR α chain DOαL11terD respond poorly to IA^d/OVA stimulation. cOVA 327–339 peptide was presented to the hybridomas by the IA^d-expressing B cell lymphoma, A20. Data points indicate the means of duplicate wells in one representative experiment.

Figure 5

Infectants bearing any one of four DO TCR α chain variants respond equally well to immobilized antiidiotype mAb. Data points represent the means of two independent experiments with duplicate wells. Error bars indicate standard errors of the mean. Four infectants of each variant type were tested. TCR levels of all infectants varied by less than twofold.

Figure 6

Infectants bearing variant DOα chains with D, but not E, at their predicted NH₂ termini respond poorly to IA^d/ OVA. Data points represent the means of three independent experiments with duplicate wells, except for those at 250 and 500 μg/ml OVA which represent only two such experiments. Error bars indicate standard errors of the mean. Four infectants of each type were tested. TCR levels of all infectants varied by less than twofold.

Figure 7

Substitution of D for E at the DOα chain NH₂ terminus lowers the affinity of cell-free α/β-TCR for IA^d-OVA. Various concentrations of IA^d-OVA were injected in flow cells with immobilized DOα/β TCRs bearing DOα chains with either D or E at the NH₂ terminus as described in the Materials and Methods. A flow cell with immobilized free β chain from this receptor was used as a control for signal from protein in solution and buffer differences.

Figure 8

Position of the Vα NH₂ terminus in three TCR crystal structures. The program Molscript was used to create a ribbon representation of three Vα elements based on their crystal structures. In each case, a wire frame representation of the NH₂-terminal amino acid is shown. The mouse Vα4 structure (12) was of a Vα dimer in the absence of Vβ. The mouse Vα3 (13) and human Vα2 (14) structures were of complete α/βTCRs. The views are of the Vαs with their solvent exposed faces toward and their Vβ interaction surfaces facing away from the reader.