טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentSegal Nimrod
SubjectHigh-Throughput Method for Analyzing T Cell Interactions
with their Cognate MHC-Peptide Ligands
DepartmentDepartment of Biology
Supervisor Professor Arie Admon
Full Thesis textFull thesis text - English Version


Abstract

The specific binding of T cells to their cognate ligand HLA-peptide complexes has been widely studied by the use of HLA tetramers technology. The use of HLA-peptide tetramers opened the way for higher screening throughput and for detection of HLA-peptide interactions with their target T cells without having to clone and grow the T cells ex-vivo. It allows for sorting the T cells directly from people’s blood, simply by decorating them with the premade tetramers, followed by isolation and enumeration of the T cells by flow cytometry.

Nevertheless, the production of tetramers is tedious, expensive and time consuming. It requires large amounts of the synthetic peptides and a considerable loss of the peptide molecules during the process. Evaluation of the numbers of specific T cells, directed against assortment of peptides, in the peripheral blood of patients inflicted with cancer, autoimmunity or viral diseases, requires a simple and high throughput approach for the production of large repertoires of HLA-peptides multimers. Only a small amount of the multimers are usually needed for every FACS reaction and therefore a simple procedure is called for loading different peptides on HLA molecules in an effective way and small amounts, and most importantly without having to utilize a complex preparation procedures, which make the process too slow and tedious.

Our new, high throughput approach, for preparation of soluble HLA peptide monomers and multimers, is based on the exchange of the bound peptides on the surface of tagged soluble HLA molecules with studied peptides, using heat treatment, and all in a single fast step and in small volume, ready for use for decoration of the T cells. Specificity and efficiency of both monomers and dimers for direct decoration of T cell clones and T cells from donor's blood were tested using FACS analyses.

Further improvements of these new methodological approaches are of great potential for development of new and interesting opportunities in the field of T cells analysis.