Voie d'immunisation et séquence d'administration de l ... - TEL

tel-00827710, version 1 - 29 May 2013
I. DEVELOPMENT OF TETRAMER-BASED ENRICHMENT
STRATEGY
A. Tetramer-based enrichment
The tetramer-based enrichment is a technique that was first developed by Moon and
colleagues to study the naïve antigen-specific CD4 + T cell repertoire in mice (Moon et al.,
2007). It has since been adapted for CD8 + T cells characterization (Obar et al., 2008).
Specifically, the spleen and 14 lymph nodes from a mouse are harvested, representing
approximately 200 million cells. These cells are stained with peptide-MHC-I tetramers
labeled with Phycoerythrin (PE) and then incubated with anti-PE magnetic beads (Figure 18).
This cell-antibody-bead suspension is passed over a magnetic column, which allows for a
100-fold enrichment of tetramer-positive cells.
Figure 18. Tetramer-based enrichment strategy.
The main aim of this technique is to enrich for the totality of antigen-specific T cells and,
consequently there remained many cells in the enriched fraction that are not of interest. To
eclude these cells, a specific gating strategy is used for flow cytometry analysis to focus on
tetramer-positive cells. First, using SSC-W and a dead cell marker, the doublets and the dead
cells are eliminated. Then a dump channel is used to remove all the cells that are not of
interest based on their unique surface marker profile (DC, Macrophages, B cells, NK cells,
CD4 + T cells…). Finally CD3 and CD8 antibodies are used to detect CD8 + T cells and
observe the tetramer-positive cells (Figure 19A). This technique was initially developed to
investigate and characterize naïve T cells. In the context of our studies, we chose to apply it in
a model using a modest dose of antigen and then investigating the immune response in the
first few days post-immunization. These parameters permitted us to work within reasonably
“physiologic” conditions in terms of initial T cell precursor frequency, but also in terms of
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