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. 2003 Jan 6;197(1):101-9.
doi: 10.1084/jem.20021908.

Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation

André Boonstra  1 , Carine Asselin-Paturel, Michel Gilliet, Chad Crain, Giorgio Trinchieri, Yong-Jun Liu, Anne O'Garra
Affiliations

Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation

André Boonstra et al. J Exp Med. .

Abstract

Distinct dendritic cell (DC) subsets have been suggested to be preprogrammed to direct either T helper cell (Th) type 1 or Th2 development, although more recently different pathogen products or stimuli have been shown to render these DCs more flexible. It is still unclear how distinct mouse DC subsets cultured from bone marrow precursors, blood, or their lymphoid tissue counterparts direct Th differentiation. We show that mouse myeloid and plasmacytoid precursor DCs (pDCs) cultured from bone marrow precursors and ex vivo splenic DC subsets can induce the development of both Th1 and Th2 effector cells depending on the dose of antigen. In general, high antigen doses induced Th1 cell development whereas low antigen doses induced Th2 cell development. Both cultured and ex vivo splenic plasmacytoid-derived DCs enhanced CD4(+) T cell proliferation and induced strong Th1 cell development when activated with the Toll-like receptor (TLR)9 ligand CpG, and not with the TLR4 ligand lipopolysaccharide (LPS). The responsiveness of plasmacytoid pDCs to CpG correlated with high TLR9 expression similarly to human plasmacytoid pDCs. Conversely, myeloid DCs generated with granulocyte/macrophage colony-stimulating factor enhanced Th1 cell development when stimulated with LPS as a result of their high level of TLR4 expression. Polarized Th1 responses resulting from high antigen dose were not additionally enhanced by stimulation of DCs by TLR ligands. Thus, the net effect of antigen dose, the state of maturation of the DCs together with the stimulation of DCs by pathogen-derived products, will determine whether a Th1 or Th2 response develops.

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Figures

Figure 1.
Figure 1.
The differential development of naive CD4+ T cells by bone marrow–derived DC subsets into a Th1 or Th2 phenotype depends on the antigen dose. Bone marrow cells were cultured for 7 d with GM-CSF to generate CD11c+ CD11b+ B220− myeloid DCs or for 10 d with Flt3 ligand to generate CD11c+ CD11b− B220+ plasmacytoid pDCs. (A) Proliferation of CD4+ CD62L+ T cells obtained from DO11.10 mice stimulated by FACS®-purified CD11c+ myeloid DCs or CD11c+ B220+ plasmacytoid pDCs was assessed after 60 h in the presence of 1 μM OVA peptide and varying DC numbers (left) or different OVA peptide doses (right). (B) To assess Th cell development, GM-CSF and Flt3 ligand–generated bone marrow–derived DC subsets were cocultured with naive DO11.10 CD4+ T cells in the presence of 10, 1, 0.1, or 0.01 μM OVA peptide. After 9 d, cells were washed, counted, and restimulated with PMA and ionomycin. The cytokine profile was determined by intracellular cytokine staining for IL-4 and IFN-γ using flow cytometry.
Figure 2.
Figure 2.
Myeloid and plasmacytoid bone marrow–derived DCs induce Th1 development with CpG whereas only myeloid DCs are responsive to LPS. FACS®-purified bone marrow–derived DCs were cultured with 1 μM OVA and CD4+ Mel14+ T cells for 9 d under neutral conditions with LPS or CpG. To some cultures, anti–IL-12p40 mAbs were added. At day 9, the percentage of IL-4 and IFN-γ+ T cells was determined by flow cytometry after restimulation with PMA and ionomycin.
Figure 3.
Figure 3.
Splenic DC subsets direct Th1 or Th2 cell development of naive CD4+ T cells depending on the antigen dose. (A) Proliferation of CD4+ CD62L+ T cells from DO11.10 mice stimulated by FACS®-purified CD11c+ CD8α+, CD11c+ CD8α− DCs, or splenic B220+ plasmacytoid pDCs was assessed after 60 h in the presence of 1 μM OVA peptide and varying numbers of DCs (left) or titration of the dose of OVA peptide (right). (B) To assess Th cell development, splenic DC subsets were cocultured with naive CD4+ T cells from DO11.10 mice in the presence of the 10, 1, 0.1, or 0.01 μM OVA peptide. After 9 d, cells were washed, counted, and restimulated with PMA and ionomycin. The cytokine profile was determined by intracellular cytokine staining for IL-4 and IFN-γ.
Figure 4.
Figure 4.
Plasmacytoid pDCs and splenic CD8α+, but not CD8α−, DCs direct Th1 cell development with CpG. Classical splenic CD11c+ CD8α+ and CD11c+ CD8α− DCs were cultured with 0.01 μM OVA peptide (A and B) or CD11cdull B220+ plasmacytoid pDCs with 1 μM OVA peptide (C) in cocultures with CD4+ Mel14+ T cells for 9 d under neutral conditions with LPS or CpG DNA. To neutralize endogenous IL-12, anti–IL-12p40 mAb was added to some cultures. After 9 d, the cytokine profile was determined by flow cytometry.
Figure 5.
Figure 5.
Quantitative real time PCR analysis of TLR4 and TLR9 expression on bone marrow–derived and splenic DC subsets. (A) Expression is relative to 18s. (B) 7 × 104 sorted DCs were stimulated in a volume of 200 μl with medium, LPS, or CpG for 24 h. IL-12p70 levels were determined by immunoassay. The sensitivity of the ELISA was 25 pg/ml.
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