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

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tel-00827710, version 1 - 29 May 2013 and received poly I:C 7 hours post-immunization. This time point was known from previous work to induce an inhibition of priming (Figure 39). On day 3 post-immunization, CFSE- labeled antigen-specific OT-I T cells were transferred into the mice and 3 days later, OT-I division was assessed by looking at CFSE dilution. If DCs presenting antigen were present, OT-I would proliferate. As shown in Figure 41A, OT-I transferred into WT recipients did not divide in the non-immunized animals whereas a robust proliferation can be observed in immunized mice. As expected, inhibition of OT-I division in immunized mice was conferred by poly I:C treatment 7 hours post-immunization. In contrast, the same level of OT-I proliferation was observed in IFNAR -/- recipients that had been immunized, irregardless of their treatment with poly I:C (Figure 41B). These results demonstrated that in WT recipients there are not DCs available to cross-present antigen after poly I:C injection and this phenomena is type I IFN-dependent. Figure 41. Inhibition of cross-presentation by poly I:C is type I IFN-dependent. WT and IFNAR -/- mice were immunized i.d. with 5x10 5 K bm1 mOva splenocytes and received 100µg of poly I:C 7 hours later. On day 3, 5x10 6 CD45.1 + CFSE-labeled OT-I were transferred and CFSE dilution was evaluated 3 days later. Representative results of CFSE dilution of CD8 + OT-I cells obtained from WT mice are shown (A). The percentage of undivided OT-I was determined and plotted (B) for WT and IFNAR -/- mice. p-values were calculated using a Mann-Whitney test. NI, non-immunized mice. 120

tel-00827710, version 1 - 29 May 2013 2) Type I IFN act on cDCs but cell-intrinsic production is not required As type I IFN are produced by several cell types following poly I:C treatment, particularly the cDCs themselves, we asked whether the cell-intrinsic production of these cytokines by cDCs was required to inhibit cross-presentation. Bone marrow chimeras were generated by transferring IRF3/7 DKO bone marrow into WT recipients, allowing for type I IFN production from only the stromal cells in these mice. While IRF3/7 DKO mice do not produce any type I IFN upon poly I:C stimulation, a significant level of these cytokines can be detected in these chimeras, albeit at lower levels than in their WT counterparts (Figure 42A). As previously described, the transfer of CFSE-labeled OT-I was performed to assess the presence of antigen-presenting DCs following adjuvant treatment. As expected, cross- presentation occured in IRF3/7 DKO mice after poly I:C injection. Interestingly, cross- presentation was completely abrogated in the BM-chimeras suggesting that the indirect, stromal production of type I IFN was sufficient to inhibit cross-presentation (Figure 42B). Page 121 of 256

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