Photoaffinity labeling of the T cell receptor on cloned cytotoxic T lymphocytes by covalent photoreactive ligand.
The interaction of the T cell antigen receptor with a photoreactive antigenic peptide derivative bound covalently to the H-2Kd (Kd) molecule was studied by photoaffinity labeling on cloned, CD8 positive cytotoxic T lymphocytes. The Kd-restricted Plasmodium berghei circumsporozoite peptide 253-260 (YIPS-AEKI) was conjugated with iodo-4-azidosalicylic acid at the N terminus and with 4-azidobenzoic acid at the T cell receptor residue Lys-259. Cell-associated or soluble Kd molecules were photoaffinity-labeled with the peptide derivative by selective photoactivation of the N-terminal photoreactive group. Incubation of cell-associated or soluble covalent Kd-peptide derivative complexes (ligands) with cytotoxic T lymphocytes that recognized this peptide derivative and activation of the orthogonal photoreactive group resulted in specific photoaffinity labeling of the T cell receptor. The labeling was inhibitable by an anti-Kd antibody and was absent on Kd-restricted cytotoxic T lymphocytes of different specificity. The binding of the soluble ligand reached a maximum after 2-4 min at 37 degrees C, after 30 min at 18 degrees C, and after 3 h at 4 degrees C. In contrast, binding of the cell-associated ligand reached a transient maxima after 50 and 110 min at 37 and 18 degrees C, respectively. The degree of binding at 37 degrees C was approximately 30% lower than that at 18 degrees C. No binding took place at 4 degrees C. Inhibition studies with antibodies and drugs indicated that the binding of the cell-associated, but not the soluble ligand, was highly dependent on T cell-target cell conjugate formation, whereas the binding of the soluble ligand was greatly dependent on CD8.
Luescher
I F
IF
Ludwig Institute for Cancer Research, Epalinges, Switzerland.
Cerottini
J C
JC
Romero
P
P
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000953:Antigens, Protozoan / Q000378:metabolism; D002999:Clone Cells; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010777:Photochemistry; D010962:Plasmodium berghei; D015800:Protozoan Proteins / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013602:T-Lymphocytes, Cytotoxic / Q000737:chemistry* / Q000276:immunology
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan; D008024:Ligands; D015800:Protozoan Proteins; D011948:Receptors, Antigen, T-Cell; C041595:circumsporozoite protein, Protozoan; D004533:Egtazic Acid; D004492:Edetic Acid