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

tel-00827710, version 1 - 29 May 2013
population observed in the pre-enrichment fraction (Obar et al., 2008). By contrast, the
tetramer-positive cell population represents a substantial number of cells following
enrichment (Figure 19B).
B. Combination with intracellular staining
The tetramer-based enrichment strategy allowed us to detect antigen-specific T cells,
enumerate them and characterize them phenotypically by looking at surface marker
expression. However, we were not able to obtain any information about the functionality of
these cells. To address this, we developed a protocol that combined the tetramer-based
enrichment with intracellular staining (Figure 20). Unfortunately, several technical hurdles
became immediately apparent, most importantly that these cells needed restimulation in order
to secrete cytokines; however, this restimulation also appeared to induce TCR
downregulation, inhibiting enrichment with tetramers.
In order to find a solution to this problem, we initially developed a protocol using in vivo
restimulation of the cells. Three hours prior to organ harvest, mice were injected with CpG
formulated with DOTAP combined with SIINFEKL peptide. After the restimulation period
and harvest, we performed tetramer-based enrichment as previously described, followed by an
intracellular staining (Figure 20A, Protocol 1). In vivo, the restimulation effect was strong
enough to allow for the observation of
IFNγ production in, and tetramer-positive cells at the same time (Figure 20B).
Nonetheless, when we attempted to examine the production of other cytokines, we were not
able to detect a signal, perhaps due to the lower extent of restimulation in vivo. We tried again
to optimize a protocol with ex vivo restimulation with peptide-pulsed splenocytes. This
resulted in a robust restimulation that led to a loss of tetramer staining at this step. Our final
protocol involved first performing the enrichment step, followed by the restimulation with
peptide-pulsed splenocytes for several hours before applying the established staining panels
(Figure 20A, Protocol 2). Although we lost the positive tetramer staining, we knew from the
initial trial (Protocol 1) that IFNγ-positive cells are also tetramer-positive cells and, in this
way, we were able to investigate additional cytokines that are secreted by these cells.
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