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

tel-00827710, version 1 - 29 May 2013
were pooled for our analysis. CD11c + cells were enriched using CD11c magnetic beads
(Miltenyi Biotech), and the enriched fraction was analyzed by flow cytometry to identify the
different subsets of DCs present. In parallel, the flow-through fraction was stained to identify
the different populations of cells stained with the PKH26 dye.
IV. CHARACTERIZATION OF ANTIGEN PERSISTENCE
A. Persistence of antigen cross-presentation
CD45.1 OT-I splenocytes were isolated and stained using 5µM carboxyfluorescein diacetate
succinimidyl ester (CSFE) in PBS. After washing with ice-cold PBS, 5x10 6 OT-I splenocytes
were injected i.v. into immunized mice. Three days later the draining and non-draining lymph
nodes, and the spleen were harvested. Organs were processed independently and cells were
labeled with CD8b and CD45.1 antibodies allowing for the identification of the transferred
CD8 OT-I T cells and the determination of CFSE intensity.
B. Persistence of antigen
1) Bioluminescence
FVB/N female mice were immunized with 5x10 6 FVB/N-luciferase + male splenocytes. For in
vivo imaging, mice were injected at a given time point with 3mg of D-luciferin (Synchem),
followed by isoflurane inhalation to keep animals sedated during acquisition. Images from
mice were acquired over 10 minutes and the bioluminescent signal was expressed in photon/
s/cm 2 /steradian. For ex vivo imaging, organs were harvested and placed in wells containing
PBS and D-luciferin to determine the total bioluminescent signal from each organ; the
bioluminescence is expressed as the total flux/organ in photons/s. Bioluminescence imaging
was performed by using an IVIS Lumina II system (Caliper Life Sciences). Quantification of
the light emission was analyzed using Living Image Software version 3.1 (Xenogen
Corporation).
2) PCR
To investigate the presence of remaining DNA from injected splenocytes, mice were tattooed
around the site of injection prior to immunization. At defined time points, the skin from the
site of immunization was harvested and DNA was extracted using the QIAamp DNA Mini Kit
from Qiagen. PCR was performed on DNA using the primers specific for the ovalbumin gene:
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