dextramer teachology slides (pdf)dextramer技术的全面介绍 (20页 ppt).docVIP

MHC DextramerTM technology Improving immune monitoring Liselotte Brix, PhD COO, Immudex Agenda Dextramer structure and function Detection of low-affinity interactions Stability of MHC Dextramers Detection of CD4 + antigen-specific T cells Detecting antigen-specific T cells in situ Dextramers – a new generation of MHC multimers with more MHCs Dextramer structure Flow cytometer – principle Fl Fl Fl Fl Fl Fl Fl Dextramer Tetramer Pentamer Dextramers bind and label Cells pass by a laser beam, antigen-specific T-cells one at a time, allowing detection of fluorescence- labelled cells Traditional MHC multimers Detection of antigen-specific T cells Shortcomings ?Isolated MHC Multimer specific population ? Detection of low-affinity TCR-MHCp interactions: – Cancer-specific CD8+ T cells ?Easy to enumerate – Antigen-specific CD4+ T cells ? Low reproducibility ?No clear MHC Multimer specific population ? Reagent-stability ?Difficult to enumerate MHC multimer More MHCs, stronger cooperative binding Benefits ? Dextramers have 10-100 fold higher avidity than Tetramers ? The Dextramer is a flexible ”ball of yarn” that can adapt (unfold-refold) around its target, to position the MHC monomers for optimal interaction with TCRs, and lock the structure Dextramers have higher resolution and less background than conventional multimers Comparison Experiment and conclusion Dextramers ? High ? CMV-specific detected in HPBMC resolution ? Low background ? Each multimer used according to its recommended protocol Tetramers ? Low ? Gating: Live lymphocytes, CD3+, CD4-, CD14- resolution ? Medium background ? Dextramers have less background and better resolution Pentamers ? Medium resolution ? High Dextramers allow more accurate enumeration of antigen-specific T cells. background Detection of Tumor Infiltrating Lymphocytes Staining Mart-1 -specific T cells Dextramer Resolution: 373 Median fl.: 1104 Tetramer Resolution: 131 Median fl.: 301 Resolution using Dextramers is 3 times as high as for