טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentRamy Fishler
SubjectT-Cell Activation Studied by Patterned Alkanethiol
Monolayers
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisors Full Professor Sivan Uri
Full Professor Reiter Yoram
Full Thesis textFull thesis text - English Version


Abstract

T-cell activation depends on molecular recognition between the T-Cell Receptor (TCR), present on the membrane of T-cells, and a short peptide of foreign origin presented by a Major Histocompatability Complex (MHC) on the membrane of antigen presenting cells (APCs).  Activated T-cells expand rapidly and eliminate undesirable cells by either killing them or signalling the recognition of a foreign antigen to other cells of the immune system.  The exact mechanism by which the interaction of T-cells and APCs leads to the initiation of T-cell activation pathways inside the T-cell is still not completely clear.  Several experiments have shown that the proximity of adjacent antigens has a determinative effect on T-cell activation.  These experiments support the conjecture that TCR crosslinking is important for T-cell activation.  However, the individual effects of antigen density and antigen amount per T-cell have not been studied systematically.  To better understand the role of antigen density in T-cell activation we constructed a model system that allows independent control over antigen surface density and antigen amount per T-cell.  Arrays of submicrometeric gold islands were prepared by electron beam lithography (EBL) and functionalized with mixed self-assembled alkanethiol monolayers (SAMs).  The SAMs contained amine terminated molecules that were subsequently modified with 2,4,6-trinitrophenyl (TNP), which served as the antigen in this study.  Antigen density was determined by the percentage of TNP terminated molecules in the SAM, while the area of the SAM interacting with each T-cell was controlled by patterning the gold substrate.  We characterized the SAMs by X-ray photoelectron spectroscopy (XPS) to assess their cleanliness, close packing, adjustability of molecular composition, and extent of modification with TNP.  Genetically engineered T-cell hybridomas expressing TNP specific chimeric TCRs were grown on the SAMs and their activation was assessed by IL-2 secretion and CD69 expression.  We found that for this model system: 1. At a constant antigen density the probability for T-cell activation increases monotonically with the amount of antigen per T-cell.  2. When antigen density is varied at a constant amount of antigen per T-cell, an optimal density is observed at an intermediate value, this value is independent of antigen amount per T-cell.  Implications of these results to the mechanism of T-cell activation are discussed.