Notch2 receptor signaling controls functional differentiation of dendritic cells in the spleen and intestine

Abstract

Dendritic cells (DCs) in tissues and lymphoid organs comprise distinct functional subsets that differentiate in situ from circulating progenitors. Tissue-specific signals that regulate DC subset differentiation are poorly understood. We report that DC-specific deletion of the Notch2 receptor caused a reduction of DC populations in the spleen. Within the splenic CD11b(+) DC subset, Notch signaling blockade ablated a distinct population marked by high expression of the adhesion molecule Esam. The Notch-dependent Esam(hi) DC subset required lymphotoxin beta receptor signaling, proliferated in situ, and facilitated CD4(+) T cell priming. The Notch-independent Esam(lo) DCs expressed monocyte-related genes and showed superior cytokine responses. In addition, Notch2 deletion led to the loss of CD11b(+)CD103(+) DCs in the intestinal lamina propria and to a corresponding decrease of IL-17-producing CD4(+) T cells in the intestine. Thus, Notch2 is a common differentiation signal for T cell-priming CD11b(+) DC subsets in the spleen and intestine.

Figure 1. Notch2 signaling regulates splenic DC development

Mice with Itgax-cre -mediated deletion of Notch1 or Notch2 or DC-specific overexpression of DNMAML1 were analyzed along with the respective cre-negative littermate controls. Statistically significant differences are indicated as follows: ***, P<0.001; **, P<0.01; *, P<0.05. (A) Representative staining profiles of total splenocytes with the fraction of CD11c^hi MHC II⁺ DCs indicated. (B) The fraction (top) and absolute number (bottom) of splenic DCs (mean ± S.D. of 3–6 animals per group). (C) Staining profiles of gated CD11c^hi MHC II⁺ splenic DCs, with CD11b⁺ (blue), CD8⁺ (green), and double-negative (purple) subsets highlighted. The percentages represent mean ± S.D., n=3–6 for DC-Notch1^Δ and DC-DNMAML1 and 11–12 for DC-Notch2^Δ. (D) The percentage among total splenocytes (top) and absolute number (bottom) of CD11b⁺ and CD8⁺ DC subsets (mean ± S.D. of 8–10 animals per group). (E) The expression of SIRPα and CD24 in gated CD11c^hi MHC II⁺ CD11b⁻ CD8⁻ double-negative DCs from conditional Notch2 (DC-Notch2^Δ) and littermate control (Ctrl) spleens. The SIRPα⁺ and CD24⁺ populations indicative of the differentiation towards CD11b⁺ and CD8⁺ DC subsets, respectively, are indicated (representative of 2 animals). (F) Expression of surface markers in CD11b⁺ DCs from DC-Notch2^Δ and control spleens.

Figure 2. Notch2-RBPJ signaling specifies a distinct population of CD11b⁺ cDC

(A) The expression of Cx3cr1-GFP reporter in CD11b⁺ splenic DCs from conditional RBPJ-deficient (DC-Rbpj^Δ) mice. Shown is the histogram of GFP expression in gated CD11c^hi MHC II⁺ CD11b⁺ DCs from Cx3cr1-GFP⁺ control (Ctrl) or DC-Rbpj^Δ mice (representative of 4 animals per group). (B) The expression of Esam vs GFP in gated CD11b⁺ splenic DCs from Cx3cr1-GFP⁺ control or DC-Rbpj^Δ mice. (C) The expression of Esam vs Clec12a in gated CD11b⁺ splenic DCs from DC-Rbpj^Δ or control mice. (D) The percentage among total splenocytes (top) and absolute number (bottom) of the Esam^hi and Esam^lo CD11b⁺ DC subsets in mice with DC-specific targeting of the indicated genes (mean ± S.D. of 3–5 animals). Significance is indicated as in Fig. 1. (E) Splenic DC populations in mice with DC-specific expression of Notch1 intracellular domain (DC-NICD). Shown are staining profiles of gated splenic CD11c^hi MHC II⁺ DCs and of their CD11b⁺ subset from DC-NICD mice or Cre-negative littermate controls (Ctrl). Representative of 8 animals per genotype. (F) The expression of Esam in splenic CD11b⁺ and CD8⁺ DCs from DC-NICD and control mice.

Figure 3. The development of Esam^hi CD11b⁺ cDCs requires LTβR signaling

(A) Splenic CD11b⁺ Esam^hi DC subset in experimental (Expt) Flt3^−/− or Ltbr^−/− animals or respective wild-type controls (Ctrl). The DC-Rbpj^Δ splenocytes that lack Esam^hi DCs (Fig. 2D) are included for comparison. Shown are staining profiles of gated B220⁻ CD11b⁺ CD8⁻ splenocytes, with the CD11c^hi Esam^hi DC population highlighted. (B) The fraction of Esam^hi and Esam^lo DC subsets among total splenocytes from Flt3^−/−, Ltbr^−/− or DC-Irf4^Δ experimental (Expt) animals or controls (Ctrl). Significance is indicated as in Fig. 1. (C) Splenic CD11b⁺ Esam^hi DC population among gated wild-type competitor (WT) or Ltbr^−/− donor populations in competitively reconstituted BM chimeras, shown as in panel A (mean ± S.D. of 3 animals). (D) The fraction of WT competitor- or Ltbr^−/− donor-derived CD11b⁺ DC subsets among total splenocytes in the BM chimeras (mean ± S.D. of 3 animals).

Figure 4. Phenotypic analysis of the splenic CD11b⁺ DC subsets

Unless indicated otherwise, Cx3cr1-GFP reporter mice were used to separate the Esam^hi GFP^lo and Esam^lo GFP^hi subsets of CD11c^hi CD11b⁺ CD8⁻ splenic DCs. (A) Expression of the indicated surface markers in the gated Esam^hi GFP^lo and Esam^lo GFP^hi subsets. Dashed lines indicate positive staining threshold. (B) Expression of LTβR on the indicated DC subsets from wild-type mice. (C) Microphotographs of the sorted DC subsets stained by Giemsa stain on cytospin preparations. The CD11c⁻ CD11b⁺ F4/80⁺ Cx3cr1-GFP⁺ macrophages (MΦ) are shown for comparison (magnification, 400x). (D) Histograms of BrdU staining in the gated DC subsets after 2 days of in vivo BrdU pulse. The percentages of BrdU⁺ cells are indicated; representative of 4 animals. (E) Cell cycle profiles of DC subsets after 2 hr of in vivo BrdU pulse. Shown are gated DC subsets stained for DNA content (DAPI) and BrdU incorporation; the fractions of cells in S and G2/M phases are indicated (representative of 2 animals).

Figure 5. Gene expression profiles of splenic CD11b⁺ DC subsets

(A) The comparison of control and RBPJ-deficient CD11b⁺ DCs. Splenic CD11b⁺ DCs from Cx3cr1-GFP⁺ DC-Rbpj^Δ mice were sorted along with the Esam^hi GFP^lo and Esam^lo GFP^hi subsets of CD11b⁺ splenic DCs from control Cx3cr1-GFP⁺ mice. Shown is unsupervised clustering analysis of the resulting microarray expression profiles. (B) Pairwise comparison of the Esam^hi and Esam^lo subsets. The probes overexpressed >2 fold in the respective populations are shown in green and red, and their percentage among the total probes is indicated. Probes for select relevant genes are highlighted in blue. (C) The expression of signature probe sets in the Esam^hi and Esam^lo subsets. The signature sets of total DCs, CD11b⁺ DCs and monocytes were derived from Immgen database and their distribution among the Esam^hi and Esam^lo subsets is depicted as in panel B. (D) The expression of Ccr2-RFP reporter in DC subsets. Shown are histograms of RFP expression in the indicated splenic populations including CD11c⁻ CD11b⁺ monocytes and macrophages (Mo-MΦ), and Clec12a⁻ and Clec12a⁺ CD11b⁺ DCs corresponding to Esam^hi and Esam^lo subsets, respectively. (E) Efficiency of Lyz2-cre recombination in DC subsets. Shown are histograms of YFP expression in the indicated splenic populations of Lyz2-cre⁺ mice with cre-inducible YFP reporter allele.

Figure 6. Functional properties of splenic CD11b⁺ DC subsets

(A) The expression of indicated genes in GFP^lo Esam^hi and GFP^hi Esam^lo subsets of splenic CD11b⁺ DCs sorted from Cx3cr1-GFP mice. Data represent normalized expression values relative to the Esam^hi sample as determined by qPCR (mean ± S.D. of triplicate reactions). (B) The expression of indicated genes in the same DC subsets as well as in CD11c⁻ CD11b⁺ F4/80⁺ GFP⁺ macrophages (MΦ), determined by qPCR as in (A) and shown on a log scale. (C) Cytokine production by CD11b⁺ DCs in response to TLR9 ligand CpG. Lymphocyte-depleted splenocytes from Cx3cr1-GFP mice were incubated for 6 hr with CpG and stained for surface markers and intracellular TNF-α or IL-12. Shown are cytokine expression profiles in GFP^lo Esam^hi or GFP^hi Esam^lo subsets of CD11c^hi CD11b⁺ DCs, or in CD11c⁻ CD11b⁺ GFP^hi monocytes and macrophages (Mo-MΦ). Data are representative of three independent experiments. (D) TNF-α production by CD11b⁺ DCs in response to TLR ligands. Splenocytes from Cx3cr1-GFP mice were activated with CpG or TLR2 ligand heat-killed L.monocytogenes (HKLM) and stained as above. (E) Phenotypic maturation of CD11b⁺ DCs in vitro. Splenocytes from Cx3cr1-GFP mice were activated with CpG or HKLM and stained for DC markers as above. Shown are staining profiles of CD11b⁺ DC subsets for CD40, with mean fluorescence intensities indicated. (F) In vivo CD4⁺ T cell priming in the absence of Esam^hi DCs. DC-Rbpj^Δ or littermate control (Ctrl) mice were administered CFSE-labeled OVA-specific OT-II T cells and immunized with OVA. Shown are CFSE staining profiles of gated OT-II T cells from the recipient spleens 3 days after immunization. The fractions of T cells that did not divide or divided 1–3 and >3 times are indicated (mean of two recipients).

Figure 7. Notch2 controls the development of intestinal CD11b⁺ CD103⁺ DCs

(A) DC populations in the intestinal lamina propria from DC-Notch2^Δ mice and littermate controls. Shown are staining profiles of CD45⁺ CD11c^hi MHC II⁺ DCs and of the gated CD11b⁺ DCs, highlighting the CD11b⁺ CD103⁺ DC subset. Fractions represent average ± S.D. of 4 animals per group. Statistical significance is indicated as in Fig. 1. (B) Absolute numbers of the indicated DC subsets (mean ± S.D. of 4 animals). (C) Staining profiles of CD11c^hi MHC II⁺ DCs from the mesenteric lymph nodes. Fractions represent average ± S.D. of 7 animals per group. (D) Effector T cell populations in the intestine. Lymphocytes were isolated from the LP of small intestine and stained ex vivo for intracellular FoxP3 or activated in vitro with PMA plus ionomycin and stained for intracellular IL-17. Shown are profiles of CD11c^hi MHC II⁺ LP DCs and of gated TCRβ⁺ CD4⁺ T cells isolated ex vivo or activated in vitro (two pairs of littermates shown; representative of 4 DC-Notch2^Δ animals).