Date of Award


Degree Name

Master of Science


Molecular Biology Microbiology and Biochemistry

First Advisor

Martinko, John


AN ABSTRACT OF THE THESIS OF DJAMILATOU SAIDOU HANGADOUMBO, for the Master's of Science, presented on July 31st, 2009 at Southern Illinois Carbondale. Title: Epitope Stability in Single Chain Trimers of a Major Histocompatibility Complex Class I Molecule. Major Professor: Dr. John Martinko The major histocompatibility complex class I molecule (MHC I) binds antigenic peptides of 8-10 amino acids and present them to CD8+ cytotoxic T cells. Peptide epitopes displayed on the MHC I are targeted by the T cells. Better presentation of peptides was accomplished with a MHC single chain trimer (SCT). A single chain trimer is an engineered version of the MHC class I molecule with the β2m leader peptide, the antigen peptide, the β2m and the heavy chain covalently linked through linker peptides. An engineered disulfide trap improved presentation and recognition of peptides by T cells and B cells. In this project, we investigated changes in epitope recognition of the disulfide-trapped single chain trimers. We hypothesized that the disulfide trap in single chain trimers will display a T-cell recognizable epitope even with low affinity peptides. In order to demonstrate and confirm the hypothesis, the SIINFEKL/H-2Kb single chain trimer was mutated to obtain different peptide epitope analogs. The constructs were tested for expression with specific monoclonal antibodies 25-D1.16 (specific for Kb/Ova complex), B8-24-3 (specific for folded Kb), and the 64-3-7, specific for unfolded MHC I. The single chain trimer constructs were tested for T cell recognition with the B3Z assay, T cell clones that recognize SIINFEKL/Kb. A peptide competition assay was used to test displacement of peptides in single chain trimers by exogenous OVA, and a heterologous competitor, SIYRYYGL, both Kb high affinity binding peptides. We showed that the disulfide trap stabilizes interaction with strong binding peptides SIINFEKL and SIINYEKL, excluding competitors. However, weak binding peptides, even when bound to the site via disulfide trap are still displaced. We also found that the disulfide trap does not allow monoclonal antibody or T cell recognition in the absence of conserved anchor residues. We demonstrated that the B3Z assay is more sensitive in our hands the flow cytometry, showing expression of Kb on the surface.




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