טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentDikla Berko
SubjectMembrane-Anchored Beta2 Microglobulin as a Tool to Elevate
Presentation of Tumor Associated Antigens:
Expression and Biochemical and
Immunological Characterization
DepartmentDepartment of Biology
Supervisors Full Professor Admon Arie
Professor Gidi Gross


Abstract

The magnitude of response elicited by CTL vaccines correlates with the density of immunogenic MHC-I-peptide complexes formed on the surface of antigen presenting cells. We have explored b2 microglobulin as a potential core component of novel CTL vaccines by genetically converting it into an integral membrane protein and studying its effect on the resulting MHC-I proteins. We have constructed two different membrane-anchored derivatives of β2m. One harbors an antigenic peptide fused to its amino terminus, and the second carries no antigenic peptide. In this work, we showed that expression of β2m with covalently linked peptides leads to high level of presentation of the specific peptide. In addition, we demonstrated that specific CTLs and complex-specific antibodies could recognize the chimeric peptide/β2m-heavy chain complexes. Expression of membranal human b2m in RMA-S cells elevates thermal stability of MHC-I molecules, in addition, RMA-S transfectants bind exogenous peptide at concentrations more than 1000-fold lower than parental RMA-S. Moreover, saturation of transfectants MHC-I by exogenous peptide occurs in less than one minute, as compared to approximately one hour required for parental cells. At saturation, however, level of peptide bound by modified cells is only 3-5-fold higher. Antibody inhibition analyses suggest that both prolonged persistence of peptide-receptive heavy chain/b2m heterodimers and fast heterodimer formation via lateral diffusion may contribute to stabilization. Our findings support the role of an allosteric mechanism in governing ternary MHC-I complex stability and propose that membranal anchorage of b2m can be instrumental in the design of CTL vaccines.