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

tel-00827710, version 1 - 29 May 2013
C. Injection strategies
1) Immunization
Splenocytes isolated from K bm1 mOva mice were used for immunization. The spleen was
harvested, mashed and red blood cells were lyzed by adding 2.5mL NH4Cl 1.66% and
incubating at 37°C for 5 minutes. Cells were washed with PBS and counted. 5x10 5 (or 5x10 6
depending on the experiment) cells in a volume of 100µl were injected intradermally (i.d.) or
intravenously (i.v.). The intradermal injection was performed in the right flank with the
inguinal lymph node being the draining lymph node. Retro-orbital injection was used to
deliver antigen intravenously.
2) TCR-transgenic T cell transfer
For OT-I transfer, bulk splenocytes were isolated from OT-I mice. After red blood cell lysis,
10 3 , 10 6 or 5x10 6 splenocytes were transferred i.v. in a volume of 100µl.
3) Adjuvant delivery
100µg of Poly I:C (high molecular weight product from Invivogen) was injected i.v. in a final
volume of 100µl.
4) Antibody injection
The antibody binding the MHC-I-peptide complexe H-2K b -SIINFEKL was isolated from the
hybridoma 25-D1.16 and obtained from National Cell Culture Center. An isotype control
(MOPC-21) was injected into control mice.
II. CHARACTERIZATION OF THE T CELL RESPONSE
A. Tetramer-based enrichment
See manuscript 2 for further information
1) Tetramer preparation
Monomers were prepared by Fabrice Lemaître using a modified version of the methods
previously described (Altman et al., 1996), and tetramerization was performed prior to use,
using PE-Streptavidin (Invitrogen), added for 1 hour at 25°C.
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