Fig. 3: DC uptake and CD4+ T-cell priming ability in vivo following i.n immunizations with the combined LNP vector. | Mucosal Immunology

Fig. 3: DC uptake and CD4+ T-cell priming ability in vivo following i.n immunizations with the combined LNP vector.

From: A vaccine combination of lipid nanoparticles and a cholera toxin adjuvant derivative greatly improves lung protection against influenza virus infection

Fig. 3

a Gating strategy used for identifying migratory and resident DCs in the mediastinal lymph node (mLN) using labeled antibodies against CD11c and MHC II (upper, left panel). Increase in migratory DC after administration of LNPs relative to that seen after use of FPEa alone (upper, middle panel). Uptake of i.n administered FPEα:LNP and FPEα in migratory, but not resident, DCs in the mLN (upper right panel). Representative FACS dot plots of Y-Ae MFI (middle panels) or bars showing the mean expression of co-stimulatory molecules (lower panels) on migratory (MHC IIhigh, CD11c+) DCs at 48 h after a single i.n immunization with 10 μg FPEα or FPEα:LNP. The mean percentage (%) of Y-Ae+ cells or MFI of CD40, CD80 and CD86-expression on migratory DCs was calculated from 3 independent experiments and given as mean ± SD. b Schematic representation of the experimental protocol used for analyzing CD4+ T-cell priming in vivo after adoptive transfer of 2 × 106 CFSE-labeled Eα TCR Tg CD4+T cells into C57BL/6 mice and immunization with 5 μg of FPEα or FPEα:LNP (left panel). Gating strategy used to identify proliferating CFSE-labeled Eα TCR Tg CD4+T cells following i.n immunizations (middle panel). Representative FACS histograms of proliferating Eα TCR Tg CD4+T cells in the mLN on day 6 after a single i.n. immunization (right panel). The percentage of proliferating (>2 cell divisions) CFSE-labeled Eα TCR Tg CD4+T cells was calculated and given as means ± SD of 3 mice in each group and 3 independent experiments (lower panel). c The same model as described in B was used, but the T-cell priming ability in mLN was assessed at different days after a single i.n. immunization with FPEα or FPEα:LNP (left panel). Gating strategy (middle panel) and representative histograms are given of proliferating CFSE-labeled Eα TCR Tg CD4+T cells following injection of cells at 2, 4, 6, and 8 days after a single i.n. immunization with 5 μg of FPEα or FPEα:LNP (right panel). The percentage of proliferating (>2 cell divisions) CFSE-labeled Eα TCR Tg CD4+ T cells was calculated and given as means ± SD of 3 mice in each group and 3 independent experiments (lower panel). Statistical significance was calculated by unpaired t test and p values are given as *p < 0.05 and **p < 0.01.

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