Influence of the NH2-terminal Amino Acid of the T Cell Receptor α Chain on Major Histocompatibility Complex (MHC) Class II + Peptide Recognition

Abstract

The α/β T cell receptor (TCR) recognizes peptide fragments bound in the groove of major histocompatibility complex (MHC) molecules. We modified the TCR α chain from a mouse T cell hybridoma and tested its ability to reconstitute TCR expression and function in an α chain–deficient variant of the hybridoma. The modified α 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 α chain with aspartic acid (D). The substitution also dramatically reduced the affinity of soluble α/β-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.

T cell positive selection by a high density, low affinity ligand

“…1, the 5Ј-oligonucleotides used in the PCRs introduced restriction enzyme sites (XhoI, EcoRI or SalI) just 5Ј to the V region start ATGs. The 3Ј-oligonucleotides introduced restriction enzyme sites described (18)(19)(20)(21) at the 5Ј end of either C␣ (BspEI) or C␤ (BglII). The genes were cloned in a baculovirus transfer vector (18)(19)(20)(21) in frame with sequences encoding C␣ and C␤.…”

“…The 3Ј-oligonucleotides introduced restriction enzyme sites described (18)(19)(20)(21) at the 5Ј end of either C␣ (BspEI) or C␤ (BglII). The genes were cloned in a baculovirus transfer vector (18)(19)(20)(21) in frame with sequences encoding C␣ and C␤. These C region sequences were truncated just past the codons for the cysteines forming the interchain disulfide, thus removing sequence encoding the transmembrane and cytoplasmic tail regions.…”

“…The eluted TCRs were further purified by size exclusion chromatography. A control soluble TCR (20,21) from the T cell hybridoma DO-11.10, specific for an ovalbumin peptide plus IA d , was also prepared. Surface Plasmon Resonance Studies.…”

Multiepitope Trojan Antigen Peptide Vaccines for the Induction of Antitumor CTL and Th Immune Responses

“…The amino acid sequences of minimal T cell epitopes (12)(13)(14)(15)(16)(17)(18) and multiepitope TAs used in this study are presented in Table I. All synthetic peptides were synthesized according to standard solid-phase synthesis methods using an Applied Biosystems (Foster City, CA) apparatus and were purified by reverse-phase HPLC.…”

Identification of T Cell Ligands in a Library of Peptides Covalently Attached to HLA-DR4

“…TCR expression vectors. pSFFMC␣ was constructed by destroying an XhoI site upstream of the SFFV-LTR in pSFFpJS␣1, which itself consists of the EcoRI-flanked DO-11.10 TCR V␣-chain (27) cloned into pSFFVSVneo (28). pH␤AcPrMC␤2 was constructed by destroying the BglII and EcoRI sites in pH␤AcPr-1-neo (29) and inserting a PCR-amplified mouse C␤2 fragment (30) between the BamHI and SalI cloning sites.…”