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The Journal of Immunology, Vol 143, Issue 11 3423-3429, Copyright © 1989 by American Association of Immunologists

ARTICLES

Probing the structure of processed antigen by using biotin and avidin. MHC-dependent inhibition of responses to selected biotinyl-insulin derivatives

PE Jensen and KD Wilkinson
Department of Pathology, Emory University School of Medicine, Atlanta, GA 30322.

Current models suggest that Ag undergoes proteolytic cleavage in APC and that resultant peptide fragments associate with class II histocompatibility glycoproteins before recognition by helper T cells. Little direct information is available concerning the physical structure and membrane association of Ag processed under physiologic conditions. A model system, employing a series of biotinylated insulin derivatives, was used to examine the domains of Ag that are presented by APC. We reasoned that avidin should block the response of T cells to a given derivative only if biotin is retained on the functionally relevant form of Ag after processing. By utilizing derivatives modified at selected sites one should be able to determine whether specific sites remain after processing. By using F1 APC pulsed with biotinyl- insulin derivatives modified through the free amino groups of the A1, B1, or B29 amino acids, and T cell hybridomas restricted to I-Ad or I- Ab, we found that avidin inhibited the I-Ad-restricted response to A1, but not B1 or B29 derivatives. By contrast, specific inhibition of the I-Ab-restricted response was observed by using all three derivatives. These results suggest that the processed form of insulin recognized in association with I-Ab is largely intact and includes residues from both chains (A1, B1, and B29). The differential inhibition observed by using T cells restricted to different class II alleles demonstrates that processed Ag associated with I-Ab differs in conformation or structure from that associated with I-Ad. This experimental approach should prove valuable in characterizing the actual structure of processed Ag recognized by T cells.


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W. P. Thayer, J. R. Kraft, S. M. Tompkins, J. C. Moore IV, and P. E. Jensen
Assessment of the Role of Determinant Selection in Genetic Control of the Immune Response to Insulin in H-2b Mice
J. Immunol., September 1, 1999; 163(5): 2549 - 2554.
[Abstract] [Full Text] [PDF]


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