A type I IFN-Flt3 ligand axis augments plasmacytoid dendritic cell development from common lymphoid progenitors

Abstract

During infections and inflammation, plasmacytoid dendritic cells (pDCs) are the most potent type I interferon (IFN-I)-producing cells. However, the developmental origin of pDCs and the signals dictating pDC generation remain incompletely understood. Here, we report a synergistic role for IFN-I and Flt3 ligand (FL) in pDC development from common lymphoid progenitors (CLPs). Both conventional DCs (cDCs) and pDCs were generated from CLPs in response to FL, whereas pDC generation required higher concentrations of FL and concurrent IFN-I signaling. An absence of IFN-I receptor, impairment of IFN-I signaling, or neutralization of IFN-I significantly impeded pDC development from CLPs. Furthermore, FL induced IFN-I expression in CLPs, which in turn induced Flt3 up-regulation that facilitated survival and proliferation of CLPs, as well as their differentiation into pDCs. Collectively, these results define a critical role for the FL/IFN-I/Flt3 axis in pDC differentiation from CLPs.

Figure 1.

Impaired pDC development from Ifnar1^−/− CLPs is cell autonomous. (A) BM or splenocytes from WT or Ifnar1^−/− mice were stained, gated on CD11c^intCD11b⁻, and analyzed for pDCs (B220⁺Siglec-H⁺). Mean percentages (B) and numbers (C) of pDCs are shown (n = 4). Results represent four experiments. (D) CLPs from the BM of WT or Ifnar1^−/− mice were co-cultured with AC-6.21, supplied with 100 ng/ml FL, and gated on CD11c⁺. cDC (CD11b⁺B220⁻) and pDCs (CD11b⁻B220⁺) populations were enumerated. Mean percentages (E) and numbers (F) of DCs are shown (n = 4–8). Results represent three experiments. (G) Competitive adoptive transfer was performed by co-transferring 10⁴ WT (CD45.1) and 10⁴ Ifnar1^−/− (CD45.2) CLPs with 2 × 10⁵ BM cells (CD45.1xCD45.2) into lethally irradiated donors (CD45.1xCD45.2). After 12 d, BM was analyzed for CLP-derived DCs. Mean percentages of pDCs (H) and cDCs (I) are shown (n = 5). Results represent two experiments.

Figure 2.

Ifnar1^−/− CLPs display reduced Flt3 expression and impaired survival, proliferation, and differentiation in response to FL. (A) CLPs were isolated from WT or Ifnar1^−/− mice and Flt3 expression was analyzed by flow cytometry (n = 7). Results represent five experiments. (B) BM was stimulated with or without 100 ng/ml of FL for the indicated times, cells were gated on lin⁻Flt3⁺ cells, and expression of pAkt was analyzed by flow cytometry (n = 3). One experiment out of three is shown. (C) CLPs were stimulated with 100 ng/ml of FL for the indicated times and stained for Annexin V (n = 4). Results represent four experiments. (D) Same as in C, except CLPs were CFSE labeled before FL stimulation for the indicated times. (E) Mean percentages of CFSE^lo population are shown (n = 4). Results represent two experiments. (F) CLPs that had been transduced with MiGR1 or MiGR1-Flt3 were co-cultured with AC-6.21 supplied with 100 ng/ml of FL, gated on CD11c⁺GFP⁺, and analyzed for DC populations. Mean percentages of GFP⁺ cDCs (G) and pDCs (H) are shown (n = 4). Results represent four experiments. (I) CLPs were co-cultured with AC-6.21 supplied with the indicated doses of FL, gated on CD11c⁺, and analyzed for DC populations. Mean percentages (J and L) and numbers (K and M) of DCs are shown (n = 3–9). Results represent five experiments. Same as in I, except 400 ng/ml of FL was supplied. CLP-derived cDCs or pDCs were sorted, and expression of Tcf4 (N), Rag1 (O), or Id2 (P; n = 3) was evaluated by RT-qPCR. One experiment out of three is shown.

Figure 3.

FL induces IFN-α production in CLPs, which promotes FL-dependent pDC development. (A) WT CLPs were stimulated with the indicated doses of FL plus anti-Flt3 or isotype antibody (1 µg/ml each) for 4 h, and expression of Ifna4 was evaluated by RT-qPCR. (B) Same as in A, except CLPs were stimulated with 100 ng/ml of FL for the indicated times (n = 3). One experiment out of three is shown. (C) WT CLPs were stimulated with or without 100 ng/ml of FL plus anti-Flt3 or isotype antibody (1 µg/ml each) for 24 h, and IFN-α expression was evaluated by intracellular staining (n = 3). One experiment out of three is shown. (D) EV or FL-expressing vector was delivered in vivo using the HGT method, followed by sorting out CLPs and intracellular staining for IFN-α (n = 3). One experiment out of three is shown. (E) Same as in C, except anti-IFNAR1 or isotype antibody (1 µg/ml each) was used. The treated cells were stained for BST2. CLPs stimulated with IFN-α4 (125 and 250 U/ml) served as positive controls (n = 4). Results represent three experiments. (F and G) WT CLPs were stimulated with 100 ng/ml of FL for the indicated times, and Ifit3 (F) and Oas2 (G) expression was examined by RT-qPCR (n = 3). One experiment out of three is shown. (H) WT CLPs were co-cultured with AC-6.21 cells and supplied with 50 ng/ml of FL in the presence or absence of IFN-α (100 U/ml). Cells were gated on CD11c⁺ and analyzed for DC populations. Mean percentages (I) and numbers (J) of DCs are shown (n = 3). Results represent two experiments. (K) Same as in H, except 100 ng/ml of FL and anti-IFNAR1 or isotype antibody were used (1 µg/ml each). Mean percentages (L) and numbers (M) of DCs are shown (n = 5). Results represent three experiments.

Figure 4.

IFN-I is required for FL-dependent up-regulation of Flt3 on CLPs. (A) BM cells stimulated with or without IFN-α4 (1,000 U/ml) for 24 h and Flt3⁺ CLPs were enumerated by flow cytometry. Mean percentages of Flt3^hi CLPs (B) and MFI of Flt3 on CLPs (C) are shown (n = 3). Results represent three experiments. (D) Same as in A, except sorted CLPs were stimulated for the indicated times. (E) EV or IFN-α4–expressing vector (1 µg each) was delivered in vivo using the HGT method for 24 h, and Flt3 expression on CLPs was evaluated by flow cytometry. Mean percentages of Flt3^hi CLPs (F) and MFI of Flt3 on CLPs (G) are shown (n = 3–4). Results represent two experiments. (H and I) Same as in E, except percentages (H) and numbers (I) of CLPs were evaluated by flow cytometry. (J) Same as in A, except BM was stimulated with or without 100 ng/ml of FL. Mean percentages of Flt3^hi CLPs (K) and MFI of Flt3 on CLPs (L) are shown (n = 4). Results represent two experiments. (M) Same as in E, except EV or FL-expressing vector (5 µg each) was delivered into mice. Mean percentages of Flt3^hi CLPs (N) and MFI of Flt3 on CLPs (O) are shown (n = 3). (Results represent three experiments.) (P) A model for FL/IFN–I/Flt3 axis in promoting pDC development from CLPs. High-dose FL triggers the production of IFN-I (1), which facilitates FL-stimulated up-regulation of Flt3 (2), resulting in increased survival, proliferation, and differentiation into of CLPs. It remains to be determined whether IFN-I is directly involved in these processes.