Physiologic control of IDO competence in splenic dendritic cells

Abstract

Dendritic cells (DCs) competent to express the regulatory enzyme IDO in mice are a small but distinctive subset of DCs. Previously, we reported that a high-dose systemic CpG treatment to ligate TLR9 in vivo induced functional IDO exclusively in splenic CD19(+) DCs, which stimulated resting Foxp3-lineage regulatory T cells (Tregs) to rapidly acquire potent suppressor activity. In this paper, we show that IDO was induced in spleen and peripheral lymph nodes after CpG treatment in a dose-dependent manner. Induced IDO suppressed local T cell responses to exogenous Ags and inhibited proinflammatory cytokine expression in response to TLR9 ligation. IDO induction did not occur in T cell-deficient mice or in mice with defective B7 or programmed death (PD)-1 costimulatory pathways. Consistent with these findings, CTLA4 or PD-1/PD-ligand costimulatory blockade abrogated IDO induction and prevented Treg activation via IDO following high-dose CpG treatment. Consequently, CD4(+)CD25(+) T cells uniformly expressed IL-17 shortly after TLR9 ligation. These data support the hypothesis that constitutive interactions from activated T cells or Tregs and IDO-competent DCs via concomitant CTLA4→B7 and PD-1→PD-ligand signals maintain the default potential to regulate T cell responsiveness via IDO. Acute disruption of these nonredundant interactions abrogated regulation via IDO, providing novel perspectives on the proinflammatory effects of costimulatory blockade therapies. Moreover, interactions between IDO-competent DCs and activated T cells in lymphoid tissues may attenuate proinflammatory responses to adjuvants such as TLR ligands.

Figure 1. IDO co-induced by high dose CpG treatment blocks in vivo T cell responses

B6 mice were injected (i/v) with CpGs and 24 hours later spleen and peripheral LNs were analyzed to detect IDO (A) and kynurenine (B). A. Spleen sections were stained to detect IDO. Original magnifications, x200 (top right, x1000). f, follicles. B. HPLC analyses to detect kynurenine in tissue lysates from spleen and pooled inguinal/brachial (ing/bra) LNs of untreated mice, and mice treated with 100μg or 25μg CpG. C. Numbers of OVA-specific (Thy1.1⁺CFSE⁺) OT-2 T cells in inguinal LNs (dLNs) of B6 mice 4 days after CFA/OVA immunization and pre-treatment with CpGs (100μg, i/v) and oral IDO inhibitor (D-1MT). The proportions of dLN OT-2 T cells that expressed intracellular IFNγ after re-stimulation are indicated (open bars). Data are representative of experiments performed two or more times.

Figure 2. IDO-competence in DCs is T cell dependent

B6 and gene-deficient mice were treated with CpGs (100μg, i/v) and 24hrs later MACS-enriched splenic DCs (6–50 ×10³/well) were cultured with responder T cells (5x10⁴/well) from BM3 (A, B) or OT-1 +OVA peptide (C–E) TCR transgenic mice +/−IDO inhibitor (D-1MT). In some cases B6 mice were pre-treated with 500μg anti-TGFβ (D), 100μg anti-CTLA4 (E) mAbs or isotype-matched mAbs 6 hours before CpG treatment, and DCs were isolated 42hrs. later. Thymidine incorporation was assessed after 72hrs (A, B) or 96 (C–E) hrs. Data shown (25 x10³ DCs/well) are the means of triplicate cultures and are representative of experiments performed two or more times with graded numbers of DCs in each experiment. Dotted arrows and percentages highlight significant IDO-dependent suppression mediated by DCs from B6 mice (A–C), and B6 mice treated with isotype-matched mAbs (D, E) before CpG treatment.

Figure 3. PD-1/PD-L interactions mediate IDO-competence in DCs

A, C. Mice (B6) were pre-treated with 100μg (i/p) anti-PD-1 mAbs (A) and anti-PD-L1/PD-L2 mAbs (C) or isotype-matched mAbs 6hrs before CpG treatment (100μg, i/v). 42hrs after CpG treatment MACS-enriched splenic DCs (2.5x10³/well) were cultured with OT-1 T cells, OVA peptide, +/−D-1MT. B, D. PD-1-KO (B) and PD-[L1+L2]-KO (D) were treated with CpGs and 24hrs later splenic DCs were placed in suppression assays. Thymidine incorporation was evaluated after 72 hours. Dotted arrows with percentages highlight IDO-dependent suppression. Data shown are the means of triplicate cultures and are representative of experiments performed on two or more occasions with graded numbers of DCs (6–50×10³/well) in each experiment.

Figure 4. TLR9-mediated IDO induction is PD-1/PD-L dependent

A, C, D. B6 mice were pre-treated with mAbs (100μg, i/p) 6hrs. before CpG treatment then spleens were removed 42hrs. later to detect IDO. B. RAG-1-KO mice were treated with CpGs and spleens were removed 24hrs. later then stained to detect IDO; original magnifications, x200 (f, follicles).

Figure 5. Treg activation after TLR9 ligation is dependent on co-stimulation

A–C. B6 mice were pre-treated with anti-CTLA4, anti-PD-1 mAbs, or anti-PD-L1 + anti-PD-L2 (50:50) or isotype-matched mAbs (100μg), and treated with CpGs 6 hours later. 42 hours after CpG treatment graded numbers of MACS-enriched (CD4⁺CD25⁺) splenic Tregs were added to cultures containing H-Y specific CD4 responder T cells (5x10⁴), APCs (female CBA) and H-Y peptide. Thymidine incorporation was assessed after 72 hours. D, E. PD-1-KO, PD-[L1+L2]-KO and B6 control mice were treated with CpGs and 24 hours later splenic Tregs were assayed for suppressor activity. Data are the means of triplicate cultures (+/−1sd) and were replicated two or more times.

Figure 6. Co-stimulatory blockade allows cytokine expression by activated CD4 cells and Tregs following high dose CpG treatment

A–D. B6 mice were treated with anti-PD-1 (C), anti-PD-L1+PD-L2 (D) or isotype-matched (A) mAbs (100μg/mAb, i/p) 6hrs before high dose CpG treatment. Control IDO1-KO mice were also treated with CpGs (B). After 42hrs. splenocytes were stained and analyzed to detect intracellular IL-17. Dot plots show gated CD4 splenocytes; percentages are the proportions of CD4⁺CD25⁺ cells expressing IL-17. E, F. Foxp3-GFP mice were treated with anti-CTLA4 or isotype-matched mAbs (100μg, i/p) and treated with CpGs as above. Splenocytes were analyzed to detect intracellular IL-17 and IFNγ. Histograms show cytokine staining profiles for gated GFP⁺CD4⁺ cells. Data are representative of experiments performed on two or more occasions.