Role of innate lymphoid cells and dendritic cells in intradermal immunization of the enterovirus antigen

Figures

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  Fig. 1. EV71 and CA16 antigen distribution in various tissues and pathological features. Immunohistochemical observation of the EV71 and CA16 antigens after administration via a intradermal (ID) and b intramuscular (IM) routes at 12 h and 24 h post inoculation. c EV71 viral RNA and d CA16 viral RNA in local tissues of three groups. Pathological observation of e intradermal (ID) tissues and f intramuscular (IM) tissues. Scale bar is 100 µm. Data are representatives of three independent experiments (error bars represent SD).; Statistical significance were assessed by unpaired t tests (*p < 0.01, **p < 0.001)

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  Fig. 2. Expression profiles of signaling molecules in peripheral tissues. a IKKα, IKKβ, TAK, and NIK; b IFNα, IFNβ, and TNFα, and c TLIA, OX40L, CD160, and BTLA were detected in tissues from mice inoculated with EV71, CA16, or EV71 combined with CA16 via the intradermal (ID) or intramuscular (IM) route. All data for each control group (IM, ID) were treated as reference values to calculate the relative fold change. Data are representatives of three independent experiments (error bars represent SD)

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  Fig. 3. Relationships between the EV71 or CA16 antigen and ILC1 cells in local tissues post inoculation. a Representative confocal fluorescence images of EV71 expression (purple), GATA3 (green) and NKp46 (red) after intradermal (ID) or intramuscular (IM) administration of EV71 antigen at 12 and 24 h post inoculation. c Representative confocal fluorescence images of CA16 expression (purple), GATA3 (green) and NKp46 (red) after intradermal (ID) or intramuscular (IM) administration of the CA16 antigen at 12 and 24 h post inoculation. e Representative confocal fluorescence images of EV71+CA16 expression (purple), GATA3 (green) and NKp46 (red) at 12 and 24 h post inoculation of the EV71+CA16 antigens via the intradermal (ID) or intramuscular (IM) route. Statistical analysis of colocalization with b EV71, d CA16, and f EV71+CA16 cells. g Colocalization rates of ILC1, ILC2, and ILC3 cells after intradermal (ID) or intramuscular (IM) inoculation. Representative fluorescent cells in the white rectangle are shown at ×20 magnification after confocal microscopy. Red scale bar is 100 µm, white scale bar is 5 µm. Data are representatives of three independent experiments (error bars represent SD).…

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  Fig. 4. Relationship between the EV71 and/or CA16 antigen and DCs in tissues. a Representative confocal fluorescence images of EV71 expression (green) and CD11c (red) at 12 h and 24 h post inoculation of the EV71 antigen via the intradermal (ID) or intramuscular (IM) route. c Representative confocal fluorescence images of CA16 expression (green) and CD11c (red) at 12 and 24 h post inoculation of the CA16 antigen via the intradermal (ID) or intramuscular (IM) route. e Representative confocal fluorescence images of EV71+CA16 expression (green) and CD11c (red) at 12 and 24 h post inoculation of the EV71+CA16 antigen via the intradermal (ID) or intramuscular (IM) route. Statistical analysis of colocalization with b EV71, d CA16, f EV71+CA16 cells. g Colocalization rates of DCs after intradermal (ID) or intramuscular (IM) inoculation. Representative fluorescent cells in the white rectangle are shown at ×20 magnification after confocal microscopy. Red scale bar is 100 µm, white scale bar is 5 µm. Data are representatives of three independent experiments (error bars represent SD). Statistical significance were assessed by unpaired t tests (*p < 0.01, **p < 0.001, ***p < 0.0001)

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  Fig. 5. ILC and DC coordination is capable of enhancing T cell proliferation. a Flow cytometric analysis and comparison of CD94−CD45+CD127+Lin− (ILCs) from skin and muscle at 12 h post inoculation. Data are representative of 3 separate experiments for each subset. b Specific IFN-γ proliferation of cocultured T cells and ILCs, T cells and DCs, and T cells, ILCs and DCs stimulated with EV71 antigen (left panel) and CA16 antigen (right panel) at 12 h post inoculation. c Specific IFN-γproliferation of cocultured T cells and ILCs, T cells and DCs, and T cells, ILCs and DCs stimulated with EV71 antigen (left panel) and CA16 antigen (right panel) at 24 h post inoculation. d mRNA expression profiles of skin from each group at 12 h post inoculation. All data from each control group (IM, ID) were treated as reference values to calculate the relative fold change. Data are representatives of three independent experiments (error bars represent SD). Statistical significance were assessed by unpaired t tests (*p < 0.01, **p < 0.001, ***p < 0.0001)

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  Fig. 6. Immunological evaluation of mice immunized with the EV71 and/or CA16 antigen. Serum samples were tested for neutralization of a EV71 and b CA16 in each group. Each symbol represents one mouse, and the line indicates the mean titer value of each group. Specific IFN-γ proliferation abilities of spleen cells stimulated with the c EV71 or d CA16 antigen. Each group of 1-day-old BALB/c mice was challenged with EV71/FY23 or CA16/G20 to test the protective efficacy of adaptive immunity. The neonatal mice were monitored daily for survival for two weeks. Data are representatives of three independent experiments (error bars represent SD). Statistical significance were assessed by unpaired t tests (*p < 0.01, **p < 0.001)