Mesenteric CD103+DCs Initiate Switched Coxsackievirus B3 VP1-Specific IgA Response to Intranasal Chitosan-DNA Vaccine Through Secreting BAFF/IL-6 and Promoting Th17/Tfh Differentiation

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  Figure 1. Intranasal immunization with chitosan-formulated DNA enhances specific IgA induction in the gut and B cell IgA CSR in the MLN. (A) Mice were immunized intranasally with chitosan, DNA (pcDNA3-VP1, 50 μg) or chitosan-DNA biweekly for 4 times. VP1-specific IgA in fecal extracts was measured by ELISA at indicated time points. (B) Representative flow cytometry plots (left) and frequency (right) of B220+IgA+ plasmablasts from spleens, MLNs, PPs, and LPs of wk8-chitosan-DNA-immunized mice. Data represent mean ± SEM for six individual mice. Data in A and B is representative of three independent experiments. ***P < 0.001; **p < 0.01; *P < 0.05; ns, not significant. (C) Representative examples of ELISPOT results from wells coated with VP1 peptides for enumeration of VP1-specific VP1-specific IgA antibody-forming cells (AFCs, plasmablasts) in spleen, MLN, PP, and LP of wk8-immunized mice. Experiments were independently repeated three times (n = 6). Numbers of AFCs with mean values ±SEM are shown. **p < 0.01; ns, not significant. (D) IgA+ plasma cells, defined as B220−CD138+IgA+, within the MLN and LP were ensured by flow cytometry, Representative plots were previously gated on B220− cells…

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  Figure 2. Intranasal chitosan-DNA increased Tfh differentiation in MLN, which correlated with IgA+ plasmablast frequency and IgA titer. (A) GALT MNC were stained with anti-mouse CD3, CD4, CXCR5, and PD-1 antibodies followed by flow cytometry. Within CD3+CD4+T cells, Tfh were identified as CXCR5high PD1high T cells. Absolute numbers of Tfh in MLNs and LPs of mice after chitosan-DNA immunization. Data present mean ± SEM of three independent experiments (n = 6). **P < 0.01. (B,C) Correlation of frequencies of MLN Tfh as determined by flow cytometry and MLN IgA+ Plasma cells (% of B cells) (B, r = 0.86, n = 6, P < 0.0001) or fecal IgA titer (C, r = 0.92, n = 6, p < 0.0001) is shown.

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  Figure 3. Intranasal chitosan-DNA immunization recruits CD103+DCs into the MLNs. (A) Representative flow cytometry plots of SI-LP, PP, MLN DC, and spleen DC in BALB/c mice 24 h after DNA or chitosan-DNA immunization. Cells are pre-gated on live, CD45+ cells. Absolute cell numbers of CD11c+MHCII+ cells from various lymphoid organs were summarized in the bar graph. Data are mean ± SEM (n = 6) from three independent experiments. **P < 0.01. (B) Representative histogram depicting the increase in mean fluorescence intensity of CD80 and CD86 on CD11c+MHCII+ DCs in various GALTs. Cumulative MFI units data for CD80 and CD86 expression were shown. Data are mean ± SEM (n = 6) from three independent experiments. ***P < 0.001; **P < 0.01. (C) Intestinal DC subset composition. Representative flow cytometry plots (left panel) and cell numbers (right panel) of CD11cintMHCIIhi MLN migratory DCs (mDCs), CD11chiMHCII+ MLN resident DCs (rDCs) and CD11chiMHCII+ LP mDCs, CD11cmedMHCII+ LP macrophages from DNA or chitosan-DNA-immunized mice were shown. Data are mean ± SEM (n = 6) from three independent experiments. **P < 0.01. (D) Representative flow cytometry plots of CD103+CD11b−DC and CD103−CD11b+DC subsets…

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  Figure 4. Freshly isolated MLN CD103+DCs induce specific IgA production. (A) CD103+DCs were sorted from 24 h chitosan-DNA-immunized mice. Naïve MLN IgM+B cells (5 × 105) were co-cultured with naïve CD4+Th0 cells (5 × 105) with FACS-sorted MLN CD103+ DCs (2.5 × 104) for 7 days in the presence of DNA or chitosan-DNA (5 ug/ml). VP1-specific IgA secreted in the culture was quantified by ELISA. (B) Representative flow cytometry plots of IgA+B cells after co-culture and cumulative data quantifying of the number of IgA+B cells were shown. Data in (A,B) are mean ± SEM representative of three independent experiments. ***p < 0.001; **P < 0.01; ns, not significant. (C) Quantitative PCR analysis for expression of Aicda and α-GT in co-cultured B cells. mRNA expression is relative to Gapdh. Data are representative of three independent experiments. **P < 0.01; *P < 0.05. (D,E) IL-6, BAFF, IL-17A, IL-12p40, IL-12p70, and IFNγ production in the CD103+DC-Th0-B co-culture (D) or CD103+DCs after stimulation (E). MACS-purified naive CD4+Th0 cells (5 × 105) were co-cultured with MLN CD103+DCs (2.5 × 104) and naïve MLN IgM+B cells (5 × 105) for 48 h in the presence of DNA or chitosan-DNA (5 μg/ml). Sorted…

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  Figure 5. CD103+DCs induce specific IgA production in an IL-6 and BAFF-dependent manner. (A–C) BAFF receptors expression by immunized MLN B cells. Flow sorted CD19+B cells from immunized mice were incubated with chitosan, DNA and chitosan-DNA (5 ug/ml). The mRNA expression of BCMA (A), BAFF-R (B) and TACI (C) were determined by real-time PCR at indicated time point. Data presented as the mean ± SEM of three independent experiments (n = 6). ***p < 0.001; **P < 0.01; *P < 0.05; ns, not significant. (D) FACS-sorted MLN CD103+DCs (2.5 × 104) were primed with DNA or chitosan-DNA (10 ug/ml) and cultured with MLN CD19+IgM+B cells (5 × 105) and CD4+Th0 cells (5 × 105) for 7 days. Either isotype control Ab (10 μg/ml), IL-6-neutralizing Ab (10 μg/ml), or TACI-neutralizing Ab (10 μg/ml) was added to some cultures as indicated. VP1-specific IgA secreted in the culture was quantified by ELISA. Data are mean±SEM from three independent experiments. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.

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  Figure 6. MLN CD103+DCs induce T cell proliferation and Th17 differentiation in vitro and in vivo which are required for IgA induction. (A) T cell division after 4-days culture of CD103+DCs and CD4+Th0. FACS-sorted CD103+DCs (1 × 104) from 24-h chitosan-DNA-immunized mice were co-cultured with CFSE-labeled naïve CD4+Th0 cells (5 × 104) in the presence of DNA or chitosan-DNA (5 ug/ml), and representative FACS plots of CFSE dilution were shown. (B–D) Representative dot plots show Foxp3 (B), RORγt (C) and IL-17A (D) induction in primed T cells at the start of culture and after 4 d of culture with CD103+DCs. T cells were stained for FoxP3, RORγT, IL-17A, and CD4 and analyzed by FACS. The graphs show percentages of Foxp3+, RORγt+ and IL-17+ cells among CD4+T cells in the presence of various antigen-pulsed CD103+DCs. Data from three independent experiments are summarized as mean ± SEM. **P < 0.01; *P < 0.05; ns, not significant. (E) T cell activation was assessed by evaluation expression of CD69 and downregulation of CD62L. Cells were gated as CD3+CD4+. Representative histograms show the MFI for CD69 and CD62L. Experiments were independently repeated three times (n = 3). (F) IL-17A and IL-23p19…