Intestinal CD103+CD11b+ cDC2 Conventional Dendritic Cells Are Required for Primary CD4+ T and B Cell Responses to Soluble Flagellin

Abstract

Systemic immunization with soluble flagellin (sFliC) from Salmonella Typhimurium induces mucosal responses, offering potential as an adjuvant platform for vaccines. Moreover, this engagement of mucosal immunity is necessary for optimal systemic immunity, demonstrating an interaction between these two semi-autonomous immune systems. Although TLR5 and CD103⁺CD11b⁺ cDC2 contribute to this process, the relationship between these is unclear in the early activation of CD4⁺ T cells and the development of antigen-specific B cell responses. In this work, we use TLR5-deficient mice and CD11c-cre.Irf4^fl/fl mice (which have reduced numbers of cDC2, particularly intestinal CD103⁺CD11b⁺ cDCs), to address these points by studying the responses concurrently in the spleen and the mesenteric lymph nodes (MLN). We show that CD103⁺CD11b⁺ cDC2 respond rapidly and accumulate in the MLN after immunization with sFliC in a TLR5-dependent manner. Furthermore, we identify that whilst CD103⁺CD11b⁺ cDC2 are essential for the induction of primary T and B cell responses in the mucosa, they do not play such a central role for the induction of these responses in the spleen. Additionally, we show the involvement of CD103⁺CD11b⁺ cDC2 in the induction of Th2-associated responses. CD11c-cre.Irf4^fl/fl mice showed a reduced primary FliC-specific Th2-associated IgG1 responses, but enhanced Th1-associated IgG2c responses. These data expand our current understanding of the mucosal immune responses promoted by sFliC and highlights the potential of this adjuvant for vaccine usage by taking advantage of the functionality of mucosal CD103⁺CD11b⁺ cDC2.

Keywords: cDC2; dendritic cells; flagellin; immune response; mucosa.

Figure 1

Mucosal CD103⁺cDC2 respond to sFliC immunization in a TLR5-dependent manner. Wild-type (WT) or TLR5^−/− mice were immunized i.p. with sFliC and cDCs (Lin⁻MHC-II^hiCD11c^hi) were evaluated 24 h later, alongside non-immunized (N.I.) mice. (A) MLN representative flow cytometry plots (including percentages) of cDC1 (Lin⁻MHC-II^hiCD11c^hiCD11b⁻CD103⁺), CD103⁺cDC2s (Lin⁻MHC-II^hiCD11c^hiCD11b⁺CD103⁺) and CD103⁻cDC2 (Lin⁻MHC-II^hiCD11c^hiCD103⁻) are shown with adjacent graphs of absolute numbers. Representative photomicrographs of MLN sections stained for CD11c; blue, CD103; red, CD11b; green, and IgM; white (scale bar = 200 μm) are shown (top right). Zoom-in insets (white boxes) show single staining and a merge of CD11c, CD103, and CD11b (scale bar = 20 μm). T, T zone; B, B zone. (B) WT or NAIP5^−/− mice were immunized i.p., with sFliC and absolute numbers of MLN CD103⁺cDC2s were evaluated 24 h later, alongside non-immunized (N.I.) mice. (C) Wild-type (WT) or TLR5^−/− mice were immunized i.p. with sFliC and splenic cDCs (Lin⁻MHC-II^hiCD11c^hi) were evaluated 24 h later, alongside non-immunized (N.I.) mice. Representative flow cytometry plots (including percentages) of cDC2s (Lin⁻MHC-II^hiCD11c^hiCD11b⁺) are shown with adjacent graphs of absolute numbers. Representative photomicrographs of spleen sections stained for CD11c; blue, Dec205; green, DCIR2; red, and IgM; white (scale bar = 100 μm). Zoom-in insets (white boxes) show the differential location of cDC1s (Dec205⁺) in the T zone and cDC2s (DCIR2⁺) in the bridging channels. Data shown as mean+s.d. of 4 mice and are representative of 3 independent experiments. **P < 0.001, by two-way analysis of variance (ANOVA) N.S., not significant.

Figure 2

The primary T cell response to sFliC is dependent upon CD103⁺cDC2 in the mucosa but not in the spleen. cDC populations in Irf4^fl/fl or Cd11c-cre.Irf4^fl/fl mice were evaluated in (A) MLN and (B) spleen. Irf4^fl/fl or Cd11c-cre.Irf4^fl/fl mice were either non-immunized (N.I.) or immunized with sFliC and T cell responses were evaluated 7 days later. Representative flow cytometry plots (percentages) and absolute numbers (graphs) of activated CD4⁺ T cells (CD3⁺CD4⁺CD44⁺CD62L⁻) in the (C) MLN and (D) spleen. Ex vivo sFliC-specific restimulations, single cell suspensions were restimulated with 5 μg/ml of sFliC in the presence of anti-CD40 (2 μg/ml), biotinylated anti-CD154 (5 μg/ml) for 48 h. Antigen-specific CD4⁺ T cells were identified by detection of the anti-CD154 by streptavidin. (E) MLN and (F) spleen representative flow cytometry plots (percentages) and absolute numbers (graphs) of CD154⁺ CD4⁺ T cells. Data shown as mean + s.d. (n = 4 mice/group) representative experiment of 3 performed. **P < 0.001; *P < 0.05, by two-way analysis of variance (ANOVA), N.S., not significant.

Figure 3

The generation of primary B cell responses to sFliC in the MLN, but not the spleen, are dependent upon CD103⁺cDC2. Irf4^fl/fl or Cd11c-cre.Irf4^fl/fl mice were either non-immunized (N.I.) sFliC-immunized and GC B cells (TCRβ⁻CD19⁺GL7⁺CD95⁺) were evaluated 7 days later. (A) MLN and (B) spleen representative flow cytometry plots (percentages) and absolute numbers (graphs) of GC B cells. Data are mean+s.d. (n = 4 mice/group) representative experiment of 3 performed. **P < 0.001; *P < 0.05, by two-way analysis of variance (ANOVA), NS, not significant. (C) Serum anti-sFliC IgG, IgG1, and IgG2c evaluated by enzyme-linked immunosorbent assay (ELISA). Data shown as mean + s.d. (n = 12 mice/group) and shows three independent experiments pooled together. *P < 0.05, two-way analysis of variance (ANOVA), N.S., not significant.

Figure 4

Switching to IgG1 is abrogated in the CD11c-cre.Irf4^fl/fl mice. Irf4^fl/fl or Cd11c-cre.Irf4^fl/fl mice were sFliC-immunized for 7 days. (A) Representative photomicrographs of serial sections from MLN and spleen stained for: PNA-binding cells (blue) and IgD-expressing cells (brown) (first column) or sFliC-binding cells (blue) and IgD-expressing cells (brown) (second column), scale bar = 200 μm. The third and fourth columns show zoom-in insets (black-boxed areas) stained to detect sFliC-binding cells and IgG1 and IgG2c respectively (scale bar = 50 μm). T, T zone; B, B zone. (B) Quantification of sFliC⁺IgG1⁺ cells and sFliC⁺IgG2c⁺ cells in the MLN and spleen. A total of 10 random fields were evaluated per slide. Data shown as mean + s.d. (n = 8 mice pooled from two independent experiments). ***P < 0.0001, by Mann-Whitney. N.D. non-detected.