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

tel-00827710, version 1 - 29 May 2013
4) Combination of tetramer-based enrichment with intracellular staining
For in vivo restimulation, mice were injected with 5µg of CpG ODN2216/ DOTAP
formulated as a mixture with 1µg SIINFEKL peptide 3 hours prior to leukocyte harvest. CpG
was purchased from Invivogen, DOTAP from Roche, and the SIINFEKL peptide from
Polypeptide group. Next, the tetramer-based enrichment protocol was performed with the
addition of Brefedin-A during each incubation step. After the elution step, enriched cells were
stained with Aqua as a dead cell marker, incubated with surface staining antibodies and fixed.
Next, cells were permeabilized and stained with anti-IFNγ as per the manufacturer’s
instructions. Intracellular cytokine staining was performed using the Cytofix/
Cytoperm/Brefeldin-A kit (BD Biosciences). For ex vivo restimulation, the tetramer-based
enrichment was performed first, and the eluted fraction was incubated 4h with SIINFEKL-
pulsed splenocytes at 37°C. Then cells were stained intracellularly for IFNγ, IL-2 and TNFα
(Table 8) as per the manufacturer’s instructions (BD Biosciences).
B. Antibody-based enrichment
Of note, a similar enrichment strategy was used to enrich TCR-transgenic CD45.1/2 WT OT-I
and CD45.2/2 IFNAR -/- OT-I that were transferred into CD45.1/1 WT recipient mice using a
anti-CD45.2-PE staining of leukocytes rather than PE-labeled K b -SIINFEKL tetramers.
C. IFNγ ELISPOT
At different time points following immunization, the spleen and the draining lymph node
were harvested and CD8 + T cells were purified using anti-CD8 microbeads and MS columns
(Miltenyi Biotech). IFNγ ELISPOT was performed as previously described (Blachere et al.,
2006). The Elispot plate evaluation was performed in a blinded fashion by an independent
evaluation service (Zellnet Consulting) using an automated ELISPOT reader (Carl Zeiss).
D. Cytotoxicity in vivo
CD45.1 splenocytes were prepared. Half of them were stained with 0.5µM
carboxyfluorescein diacetate succinimidyl ester (CSFE) using the Vybrant cell tracer kit from
Invitrogen, and pulsed with SIINFEKL peptide. The other half were stained with 5µM CFSE
and left unpulsed. At different time points following immunization, mice received i.v. 5x10 6
of cells from each of these pools. 15 hours later, spleen was harvested and cells were stained
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