IDO-orchestrated crosstalk between pDCs and Tregs inhibits autoimmunity

Abstract

Plasmacytoid dendritic cells (pDCs) have been shown to both mediate and prevent autoimmunity, and the regulation of their immunogenic versus tolerogenic functions remains incompletely understood. Here we demonstrate that, compared to other cells, pDCs are the major expressors of Indoleamine-2,3-dioxygenase (IDO) in steady-state lymph nodes (LNs). IDO expression by LN pDCs was closely dependent on MHCII-mediated, antigen-dependent, interactions with Treg. We further established that IDO production by pDCs was necessary to confer suppressive function to Tregs. During EAE development, IDO expression by pDCs was required for the generation of Tregs capable of dampening the priming of encephalitogenic T cell and disease severity. Thus, we describe a novel crosstalk between pDCs and Tregs: Tregs shape tolerogenic functions of pDCs prior to inflammation, such that pDCs in turn, promote Treg suppressive functions during autoimmunity.

Keywords: Antigen presentation; Experimental autoimmune encephalomyelitis; Indoleamine 2,3-dyoxygenase; Plasmacytoid dendritic cells; Regulatory T cells; Tolerance.

Graphical abstract

Fig. 1

IDO expression by pDCs is induced after Ag-specific interactions with Tregs. (A) IDO mRNA expression in B cells, cDCs, pDCs and macrophages (MØ) sorted from total skin LNs of naïve WT. (B) IDO mRNA in pDCs isolated from total skin LNs of naive WT, Rag2^−/−, OTII Rag2^−/− and IDO^−/− mice. (C) CD5 expression levels by AND Rag2^−/−, Smarta Rag1^−/− (SMA) and Marilyn Rag2^−/− (MAR) CD4⁺ TCR tg T cells (left), and IDO mRNA levels in pDCs isolated from skin LN of AND, SMA, MAR and WT mice (right). (C) Foxp3⁺CD4⁺ Treg frequencies in LNs of OTII Rag2^−/− and female Marilyn Rag2^−/− mice. (E) Foxp3⁺CD4⁺ Treg frequencies in LN cells of 2D2 TCR tg mice (left), and IDO mRNA levels in pDCs isolated from skin LN of Rag2^−/− mice and co-cultured in vitro with 2D2 TCR tg CD4⁺ T cells and MOG_35–55 peptide for 16 h (right). (F) IDO mRNA levels in ex-vivo pDCs sorted from skin LN of 3wk-old WT, pIII + IV^−/− and Scurfy mice. (G) IDO mRNA levels of ex vivo WT pDCs and WT BM derived pDCs loaded or not with MOG_35–55 and co-cultured in vitro with 2D2 CD4⁺ T cells for 16 h. (H) IDO mRNA levels of ex vivo WT pDCs and WT BM derived pDCs loaded with MOG_35–55 and co-cultured in vitro for 16 h with 2D2 CD4⁺ CD25⁻ cells, 2D2 CD4⁺ T cells or 2D2 CD4⁺ CD25⁺ Treg cells. (A–H) Results are representative of at least 2 independent experiments. Error bars depict mean ± SEM. (A, B, C, G, and H) One-way ANOVA with Bonferroni post Hoc test or (E and F) two-tailed Mann-Whitney test was used. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS = Non significant.

Fig. 2

IDO expression by pDCs is dependent of MHCII-TCR interactions. (A-C) Mixed BM chimeric mice were generated by co-transferring (1:1) CD45.2 GFP⁺ WT and CD45.2 pIII + IV^−/− BM cells in lethally irradiated CD45.1 WT mice. (A) Experimental design. (B) WT GFP⁺ cDCs and pDCs, and pIII + IV^−/− cDCs and pDCs were sorted from total skin LN of naïve mice based on CD45.2, CD11c, PDCA-1 and GFP markers. (C) IDO mRNA expression level in indicated cells is represented. Results are representative of at least 3 independent experiments. Error bars depict mean ± SEM. One-way ANOVA with Bonferroni post Hoc test was used. **P < 0.01, NS = Non significant.

Fig. 3

MHC-II sufficient pDC express IDO during EAE. (A) IDO mRNA expression in B cells, cDCs, pDCs and macrophages (MØ) sorted from dLNs of WT EAE mice 10 days after immunization. (B) EAE was induced in WT → WT and pIII + IV^−/− → WT chimeric mice, and IDO mRNA was measured in cDCs and pDCs sorted from dLN 10 days after immunization. (C) Mixed BM chimeric mice were generated by co-transferring (1:1) CD45.2 GFP⁺ WT and CD45.2 pIII + IV^−/− BM cells in lethally irradiated CD45.1 WT mice. WT GFP⁺ cDCs and pDCs, and pIII + IV^−/− cDCs and pDCs were sorted from dLNs 10 days after EAE induction based on CD45.2, CD11c, PDCA1 and GFP markers. IDO mRNA expression level in indicated cells (A–C) Results are representative of at least 3 independent experiments. Error bars depict mean ± SEM. One-way ANOVA with Bonferroni post Hoc test was used. *P < 0.05, ****P < 0.0001, NS = Non significant.

Fig. 4

IDO is required during EAE priming phase (A-C) EAE was induced in WT → WT (■), pIII + IV^−/− → WT (○) and IDO^−/− → WT () BM chimeras. (A) Clinical scores were followed daily (two-way ANOVA with Bonferroni post Hoc test). Frequencies of (B) IFN-γ⁺ (left) and IL-17⁺ (right) CD4⁺ T cells, (C) Ki67⁺ (left), Ki67⁺CD103⁺ICOS⁺ (middle) and IL10⁺ (right) among CD25^hiFoxp3⁺CD4⁺Tregs in dLN at d9 (one-way ANOVA with Bonferroni post Hoc test). (A–C) Results are representative of at least 3 independent experiments with 8 mice per group. Error bars depict mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, NS = Non significant.

Fig. 5

IDO⁺pDCs drive suppressive Tregs during EAE. (A–E) MOG_35–55 loaded BM-derived pDCs from WT (■) and IDO^−/− () were transferred (arrow) or not (○) into BDCA2-DTR → WT BM chimeras and EAE was induced 1 day after. Mice received 5 consecutive DT injections every 3–4 days. (A) Clinical scores were followed daily (two-way ANOVA with Bonferroni post Hoc test). Frequencies of (B) CD4⁺CD25⁺ Foxp3⁺ Treg cells (one-way ANOVA with Bonferroni post Hoc test), (C) CD103⁺ICOS⁺ Tregs, (D) CD25^high Tregs and (E) CD69⁺ Tregs from dLNs are represented at d10 after EAE immunization (two-tailed Mann-Whitney test). (A–E) Results are representative of at least 2 independent experiments with 6–8 mice per group. Error bars depict mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6

Tregs primed by IDO⁺pDCs are suppressive in vivo and control EAE development. (A, B) CD4⁺CD25^hi cells were purified from dLNs of WT → WT, pIII + IV^−/− → WT and IDO^−/− → WT BM chimeras 10 days after EAE induction, and transferred (arrow) into WT recipients further immunized for EAE the day after. (A) Experimental design is represented. Foxp3 expression in sorted cells. (B) Clinical scores were followed daily in control mice (⊡) and in mice transferred with WT Treg (■), IDO^−/− Tregs () or pIII + IV^−/− Tregs (○) (two-way ANOVA with Bonferroni post Hoc test). (A, B) Results are representative of at least 2 independent experiments. Error bars depict mean ± SEM. *P < 0.05, **P < 0.01. See also Table 1. (C, D) CD4⁺CD25^hi cells were purified from total skin LNs of naïve WT → WT and IDO^−/− → WT BM chimeras or from dLNs of WT → WT and IDO^−/− → WT BM chimeras 10 days after EAE induction. (C) CD4⁺CD25^hi cells were with proliferation dye-labeled 2D2 CD4⁺ T cells and LPS activated, MOG_35–55 loaded, cDCs. 2D2 T cell proliferation was assessed after 5 days. Flow cytometry histograms represent 2D2 T cell proliferation for indicated conditions. Histograms represent the percentages of Treg-mediated suppression (two-tailed Mann-Whitney test). Results are representative of 2 independent experiments. Error bars depict mean ± SEM. **P < 0.01, NS = Non significant. (D) CD4⁺CD25^hi cells were transferred into WT recipients further immunized for EAE the day after. Clinical scores and incidence (d15) are depicted. Data are representative of 2 experiments. Error bars represent mean ± SEM. One-way ANOVA with Bonferroni post Hoc test was used. *P < 0.05.