Direct binding of a synthetic multichain polypeptide to class II major histocompatibility complex molecules on antigen-presenting cells and stimulation of a specific T-cell line require processing of the polypeptide.
T-cell activation involves the recognition of foreign antigens as a complex with self-major histocompatibility complex (MHC) proteins on the surface of antigen-presenting cells (APC). Protein antigens usually require uptake by the APC and processing that results in the generation of peptide fragments. The branched synthetic polypeptide (Tyr, Glu)-Ala--Lys was chosen as a model antigen to follow the processing requirements, leading to T-cell activation. It has been demonstrated, by using fixed APC and various inhibitors of proteases, that (Tyr, Glu)-Ala--Lys has to be processed to stimulate a (Tyr, Glu)-Ala--Lys-specific T-cell line of C3H.SW (H-2b) origin to proliferate. To determine whether processing of (Tyr,Glu)-Ala--Lys is required to allow its association with the MHC class II molecules, biotin was covalently attached to it. Binding of the biotinylated (Tyr,Glu)-Ala--Lys to MHC class II gene products on the surface of intact normal APC was directly detected by phycoerythrin-streptavidin. The specificity of the binding was confirmed by its inhibition with anti-I-Ab antibodies as well as with excess of nonlabeled (Tyr,Glu)-Ala--Lys. Furthermore, introducing several inhibitors of proteases to the binding assay, we could substantiate that the proteolysis of (Tyr,Glu)-Ala--Lys is required to allow association of the resulting peptidyl T-cell epitopes with the MHC class II molecules themselves. The presence of the biotin moiety in the resulting peptides suggests that the T-cell epitopes of (Tyr,Glu)-Ala--Lys contain the N-terminal portion of the side chains of the branched polypeptide. An apparent Kd of 8.05 x 10(-8) M was determined, and optimal binding was detected after 10 hr of incubation with the antigen. The latter phenomenon is not due to slow uptake, since uptake of (Tyr,Glu)-Ala--Lys occurs mainly during the first 30 min of incubation, but rather reflects the events of processing that precede MHC interaction.
Zisman
E
E
Weizmann Institute of Science, Rehovot, Israel.
Sela
M
M
Mozes
E
E
D000818:Animals; D000938:Antigen-Presenting Cells / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000378:metabolism*; D008213:Lymphocyte Activation; D051379:Mice; D008815:Mice, Inbred Strains; D010455:Peptides / Q000276:immunology* / Q000378:metabolism; D011480:Protease Inhibitors / Q000378:metabolism; D013154:Spleen / Q000166:cytology; D013601:T-Lymphocytes / Q000276:immunology*
D000949:Histocompatibility Antigens Class II; D010455:Peptides; D011480:Protease Inhibitors; C064216:poly(glutamyl-alanyl-tyrosyl-glycine)