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

Abstract

Intranasal chitosan-formulated DNA vaccination promotes IgA secretion in the intestine. However, the mechanism whereby chitosan-DNA skews IgA class switch recombination (CSR) of B cells in the Gut-associated lymph tissue (GALT) is not fully resolved. In this study, we investigated the effects of nasally administered chitosan-DNA (pcDNA3.1-VP1 plasmid encoding VP1 capsid protein of Coxsackievirus B3) on IgA production, DC activation and Tfh/Th17 response in the intestine. Compared to DNA immunization, intranasal chitosan-DNA vaccination induced antigen-specific IgA production in feces, a pronounced switching of antigen-specific IgA⁺ plasmablast B cells in the mesenteric lymph nodes (MLNs) and an enhanced expression of post-recombination Iα-CH transcripts/IgA germline transcript (αGT) as well as activation-induced cytidine deaminase (AID) in MLN B cells. MLN Tfh frequency was markedly enhanced by chitosan-DNA, and was associated with VP1-specific IgA titer. 24 h after immunization, intranasal chitosan-DNA induced a recruitment of CD103⁺DCs into the MLN that paralleled a selective loss of CD103⁺DCs in the lamina propria (LP). In vivo activated MLN-derived CD103⁺DCs produced high levels of IL-6 and BAFF in response to chitosan-DNA, which up-regulated transmembrane activator and CAML interactor (TACI) expression on MLN B cells. Upon co-culture with IgM⁺B in the presence of chitosan-DNA, MLN CD103⁺DCs induced IgA production in a T-dependent manner; and this IgA-promoting effect of CD103⁺DC was blocked by targeting TACI and, to a lower extent, by blocking IL-6. MLN CD103⁺DCs displayed an enhanced capacity to induce an enhanced CD4⁺Th17 response in vivo and in vitro, and IL-17A deficient mice had a pronounced reduction of specific intestinal IgA following immunization. Taken together, mesenteric CD103⁺DCs are indispensable for the adjuvant activity of chitosan in enhancing DNA vaccine-specific IgA switching in gut through activating BAFF-TACI and IL-6-IL-6R signaling, and through inducing Th17/Tfh differentiation in the MLN.

Keywords: CD103+DC; IgA; TACI; chitosan; class switch recombination (CSR).

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 and frequencies of IgA⁺ plasma cells in MLN and LP with mean values ± SEM were shown (n = 6). ^**p < 0.01; ^*P < 0.05. (E) Representative semi-quantitative (left) or quantitative (right) RT-PCR on sorted B cells from wk8-chitosan-DNA-immunized MLNs for expression of indicated transcripts (Aicda, α-GT, Iμ-Cα, and α-CT). Relative expression of Aicda and α-GT mRNA are displayed as the fold change of respective transcript expression over the expression by Gapdh. Data present mean ± SEM of three independent experiments (n = 6). ^**P < 0.01; ^*P < 0.05; ns, not significant. (F) MLN B cells were subdivided into B1a (B220^loCD5^lo), B1b (B220^loCD5^neg), and B2-B (B220^hiCD5^neg) cells according to B220 and CD5 expression. Bar graph represents the absolute number of B2-B cells. Histograms represent expression of mIgA by MLN B1 and B2-B cells after chitosan-DNA immunization. Data present mean ± SEM of three independent experiments (n = 6). ^**P < 0.01; ^*P < 0.05.

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 CXCR5^high PD1^high 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.

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 CD11c^intMHCII^hi MLN migratory DCs (mDCs), CD11c^hiMHCII⁺ MLN resident DCs (rDCs) and CD11c^hiMHCII⁺ LP mDCs, CD11c^medMHCII⁺ 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 after excluding CD64⁺ macrophages from CD11c⁺MHCII^high cells. (E) CD11c⁺MHCII⁺ PP DCs were further evaluated for expression of CD11b. Cell numbers of CD103⁺CD11b⁺ and CD103⁺CD11b⁻ DC and CD11b⁺DC subsets in the MLNs, LPs and PPs of mice were cumulated in bar graph. Data are mean ± SEM (n = 6) from three independent experiments. ^**P < 0.01; ^*P < 0.05; ns, not significant.

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 CD103⁺DCs (1.0 × 10⁵) were incubated with chitosan, DNA, chitosan-DNA (5 μg/ml) or anti-IgM mAb (5 μg/ml) plus anti-CD40 mAb (5 μg/ml). Supernatants were harvested to measure cytokine levels by ELISA at 48 h. Data are representative of three independent experiments. ^***p < 0.001; ^**P < 0.01; ^*p < 0.05; ns, not significant.

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.

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 production in the supernatant were detected by ELISA. Data presented as the mean ± SEM of three independent experiments. ^***P < 0.001; ^**P < 0.01; ^*P < 0.05; ns, not significant. (G) MLN lymphocytes were isolated from wk8 chitosan-DNA-immunized mice and gated on CD45⁺CD3⁺ T cells before IL-17A intracellular staining. IL-17A expression by CD4⁺Th and γδT cells was compared among various groups. Representative flow cytometry plots of IL-17⁺cells and cumulative data quantifying of the number of Th17 or γδT17 cells were shown. Data are representative of three independent experiments and presented as the mean ± SEM (n = 6). ^***P < 0.001; ^*P < 0.05; ns, not significant. (H) chitosan-DNA vaccine was i.n. administrated to WT and IL-17A KO mice biweekly for 4 times and the fecal SIgA was detected by ELISA at wk0 and wk8. Data are representative of three independent experiments and presented as the mean ± SEM (n = 6). ^***P < 0.001; ns, not significant.