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

tel-00827710, version 1 - 29 May 2013
conditions of immunization. This lower dose of antigen will lead to a less robust response
and, therefore, an enrichment step is required to be able to study the antigen-specific T cells.
Figure 19. Tetramer-based enrichment. (A) The gating strategy used is detailed. Single cells were
selected using SSC-W. Then cells were stained with a mixture of antibodies for lineage markers and
DAPI to exclude cells that are not of interest (DUMP gate). CD3 + cells were selected and CD8 and K b -
SIINFEKL tetramer labeling were used to detect antigen-specific CD8 + T cells. (B) C57BL/6 mice
were immunized i.d., either with male splenocytes (HY model) or K bm1 mOva splenocytes (Ova
model). On day 11, the spleen and lymph nodes were harvested and enrichment was performed using
D b -UTY (HY model) or K b -SIINFEKL (Ova model) tetramers. The tetramer staining on cells prior to
enrichment and on the enriched fraction is shown. The numbers indicated represent the percentages of
tetramer-positive cells among CD8 + T cells for each sample. Of note, approximately 350 000 cells
were acquired for both samples: this corresponds to the total enriched fraction, but it reflects only a
part of the pre-enrichment cell suspension.
To validate this technique in our hands, we tested it in two different models, HY and
Ovalbumin, using the respective peptide-MHC-I tetramers. We compared what was obtained
with and without enrichment, both in the enriched and in the flow-through fraction. Without
enrichment, it is extremely difficult to detect tetramer-positive cells for mainly two reasons:
first, it was not possible to collect the total pre-enrichment cell suspension for flow cytometry
analysis as it is comprised of approximately 200 million cells. Additionally K b -SIINFEKL
tetramers are known for their high background staining, seen in the intermediately stained
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