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

tel-00827710, version 1 - 29 May 2013
4) Early poly I:C treatment affects antigen uptake
In the previously described experiment, only poly I:C was injected without antigen. To mimic
physiologic vaccination conditions, it made sense to add the antigen back to the experimental
protocol to examine how poly I:C affects antigen uptake and presentation by the DCs. To
study the behaviour of injected antigenic splenocytes, the cells were stained with PKH26 dye
prior to injection, allowing for their detection in the draining lymph node and even inside DCs
upon phagocytosis by flow cytometry. Due to technical limitations, we needed to inject 10
times more splenocytes to ensure detection. WT CD45.1 recipients were immunized with
5x10 6 PKH-26-labeled CD45.2 K bm1 mOva splenocytes and received poly I:C 7 hours later.
On day 2, the draining lymph node was collected, collagenase digested and analyzed for
labeled splenocytes as well as for the detection of splenocytes engulfment by different DC
subsets. Many more PKH-26-labeled DCs were detected in immunized mice as compared to
mice that received the combination of poly I:C and splenocytes (Figure 44A). As previously
described, the CD8α + DC subset appeared to be reduced in the latter condition and
furthermore, no CD8α + DCs labeled for PKH26 (indicating antigen engulfment) were
detected. We also examined the draining lymph node for injected splenocytes. Lymphocytes
are the main cell population present in the splenocyte injected preparation and furthermore,
these cells are able to migrate and thus, reach the draining lymph node on their own.
Interestingly, we found PKH26-labeled B and T cells in all immunized animals, irregardless
of poly I:C treatment (Figure 44B). These data demonstrated that while early poly I:C
treatment inhibits antigen uptake by DCs, injected splenocytes can still migrate to the draining
lymph node even upon poly I:C injection.
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