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

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tel-00827710, version 1 - 29 May 2013 A B Fig.5. Gating strategy and Enrichment. (A) Example of the gating strategy applied to an Influenza A-Matrix 158-66 enriched human sample. SSC-A low /SSC-W low initial gating permit to exclude doublets, then Dump vs. CD3 contour plot permits to isolate viable pure CD3 for further analysis. Upper line shows evaluation of background on a Multimer-PE vs CD8 contour plot still gated on the total CD3 population. Bottom line illustrates CD3 + CD8 + selection, then finally evaluation of Multimer-PE + cells percentages within CD8 + T cells. (B) Left plots: PBMCs from a healthy donor have been incubated with CMV, Flu or HCV MHCI multimers, then enriched as described in the protocol. Right plots : C57BL/6 mice were immunized intradermally, either with male (HY model) or K bm1 mOva (Ova model) splenocytes. On day 11, the spleen and lymph nodes were harvested and enrichment was performed as described in the protocol, using D b -UTY (HY model) or K b -SIINFEKL (Ova model) multimers. 232

tel-00827710, version 1 - 29 May 2013 Fig.6. Multi-enrichment. A. PBMCs from a healthy donor are incubated with a cocktail of MHCI multimers. Each specificity (CMV, Flu, MART1) is labeled with PE and with another color (PE-Cy7, APC, APC-Cy7 respectively). Enrichment is performed with anti-PE microbeads as described in Section 3.4. B. After applying the gating strategy described in Section 3.5 and Figure 5A, CD8 + PE + cells are gated (left plot). Each specificity is then identified within CD8 + PE + population using the second color readout (middle and right plots). Page 233 of 256

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