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

tel-00827710, version 1 - 29 May 2013
(b) Barcoding technology
An elegant strategy of cellular barcoding has recently been developed to analyze the kinship
between different T cell populations (Schumacher et al., 2010). A retroviral plasmid library
was generated in which each individual virus carried a unique molecular “barcode”. T cells
were infected and, thus, labeled by retroviral transduction and then reintroduced into mice.
After immunization and the resulting differentiation of transferred T cells, different cell
subsets were sorted, DNA was isolated and the overlap of barcodes between different
functional subsets was analyzed. Using this technique, lineage relationships could be analyzed
between T cell subsets. The power of cellular barcoding has already been harnessed to answer
two long-standing questions. First, it was used to determine whether populations of T cells
found in a specific location or those that share a common functional activity, come from a
common progenitor (Schepers et al., 2008). Following this study, van Heijst and colleagues
made use of this approach to determine the number of precursors that are recruited to form a
given effector T cell population depending on the conditions of immunization (van Heijst et
al., 2009).
(c) Combicolor approach
The consistent improvement of flow cytometry technology has also provided new tools by
which to study T cell responses. It is now possible to combine many fluorophores in the same
experiment. While one tetramer labeled by one colour was used before to characterize a T cell
response, it is now possible to simultaneously detect multiple different antigen-specific T
cells with several tetramers within the same sample. In particular, Hadrup and colleagues
developed a novel combinatorial method, in which each specific T cell is labeled with a mix
of identical tetramers conjugated to different fluorophores. Each antigen-specificity is labeled
and identified by a unique fluorophore combination. In this way, 15 specificities can be
detected concurrently by using 4 different fluorophores (Hadrup et al., 2009). Depending on
the frequency of the cells of interest, cells can be stained directly or upon tetramer
enrichment.
Although previous studies that characterized the T cell response were performed by
examining cells at the population level, the trend is now to study rare populations and
characterize the response at the single cell level by using the multiple new techniques
described here, either alone or in combination, including tetramer-based enrichment. Indeed,
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