Covalent assembly of a soluble T cell receptor-peptide-major histocompatibility class I complex.
We used stepwise photochemical cross-linking for specifically assembling soluble and covalent complexes made of a T-cell antigen receptor (TCR) and a class I molecule of the major histocompatibility complex (MHC) bound to an antigenic peptide. For that purpose, we have produced in myeloma cells a single-chain Fv construct of a TCR specific for a photoreactive H-2Kd-peptide complex. Photochemical cross-linking of this TCR single-chain Fv with a soluble form of the photoreactive H-2Kd-peptide ligand resulted in the formation of a ternary covalent complex. We have characterized the soluble ternary complex and showed that it reacted with antibodies specific for epitopes located either on the native TCR or on the Kd molecules. By preventing the fast dissociation kinetics observed with most T cell receptors, this approach provides a means of preparing soluble TCR-peptide-MHC complexes on large-scale levels.
Grégoire
C
C
Centre d'Immunologic, Institut National de la Santé et de la Recherche Médicale, Marseille, France.
Lin
S Y
SY
Mazza
G
G
Rebai
N
N
Luescher
I F
IF
Malissen
B
B
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D003432:Cross-Linking Reagents; D017931:DNA Primers; D006183:H-2 Antigens / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D006825:Hybridomas; D007700:Kinetics; D008285:Major Histocompatibility Complex; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011948:Receptors, Antigen, T-Cell / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D017931:DNA Primers; D006183:H-2 Antigens; C024755:H-2K(K) antigen; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins