Human type 1 and type 2 conventional dendritic cells express indoleamine 2,3-dioxygenase 1 with functional effects on T cell priming

Abstract

Dendritic cells (DCs) are key regulators of the immune system that shape T cell responses. Regulation of T cell induction by DCs may occur via the intracellular enzyme indoleamine 2,3-dioxygenase 1 (IDO), which catalyzes conversion of the essential amino acid tryptophan into kynurenine. Here, we examined the role of IDO in human peripheral blood plasmacytoid DCs (pDCs), and type 1 and type 2 conventional DCs (cDC1s and cDC2s). Our data demonstrate that under homeostatic conditions, IDO is selectively expressed by cDC1s. IFN-γ or TLR ligation further increases IDO expression in cDC1s and induces modest expression of the enzyme in cDC2s, but not pDCs. IDO expressed by conventional DCs is functionally active as measured by kynurenine production. Furthermore, IDO activity in TLR-stimulated cDC1s and cDC2s inhibits T cell proliferation in settings were DC-T cell cell-cell contact does not play a role. Selective inhibition of IDO1 with epacadostat, an inhibitor currently tested in clinical trials, rescued T cell proliferation without affecting DC maturation status or their ability to cross-present soluble antigen. Our findings provide new insights into the functional specialization of human blood DC subsets and suggest a possible synergistic enhancement of therapeutic efficacy by combining DC-based cancer vaccines with IDO inhibition.

Keywords: IDO; cDC1; cDC2; epacadostat; plasmacytoid dendritic cells.

Figure 1

IDO1 is among the genes that best defines peripheral blood cDC1s under homeostatic conditions and its expression is increased upon stimulation with TLR agonists. Gene expression was analyzed from RNA‐seq datasets. Z scores were calculated using normalized and log transformed counts per million values. Means are depicted. (A) Expression of selected genes was compared between immature cDC1s, immature cDC2s, and immature (unstimulated or IL‐3‐treated) pDCs, of 4 (cDC1) or 3 (cDC2 and pDC) different donors. (B) IDO1 gene expression among cDC1s of three or four different donors cultured overnight in absence or presence of a clinical grade TLR3 agonist (Hiltonol) and/or TLR7/8 agonist (protamine‐RNA).

Figure 2

Immature cDC1s, but not cDC2s and pDCs, express functionally active IDO. (A) IDO1 protein expression among steady‐state human blood DCs of three different donors was analyzed from a proteomics dataset. Means are depicted. (B) IDO intracellular protein expression was analyzed by flow cytometry in DCs cultured overnight. The graph shows the geometric mean fluorescent intensity (MFI) of IDO expression subtracted by the MFI of the isotype control, with at least seven different donors per condition from 10 independent experiments. Means are depicted. Significance was determined by one‐way ANOVA with Bonferroni correction (^*** P < 0.001). (C) IDO activity was analyzed by measuring l‐kynurenine in supernatants of 24‐h DC cultures by HPLC. Results are from six different donors from four independent experiments; in one experiment (squares), pooled supernatants from 3 different donors were used. Means are depicted and symbols correspond to measurements belonging to the same donors. Significance was determined by repeated measures one‐way ANOVA with Bonferroni correction (^* P < 0.05).

Figure 3

IDO protein expression and activity among stimulated blood DC subsets. (A) Flow cytometry histograms from a representative donor showing isotype control (solid grey line), IDO expression in absence of stimulus (dashed black line), and IDO expression for either IFN‐γ or CpG‐C stimulation (solid black line) for cDCs or pDCs, respectively. (B) The figure shows the geometric mean fluorescence intensity (MFI) of IDO expression subtracted by the MFI of the isotype control, with at least 3 different donors per condition from 10 independent experiments. Bars represent means. Significance was determined by one‐way ANOVA with Bonferroni correction, comparing stimulated DCs with immature control (^* P < 0.05; ^** P < 0.01). (C) IDO activity was analyzed by measuring l‐kynurenine in supernatants of 24‐h DC cultures by HPLC. The results are from six different donors from four independent experiments; in one experiment (squares), pooled supernatants from 3 different donors were used. Bars represent means and symbols correspond to measurements belonging to the same donors. Significance was determined by repeated measures one‐way ANOVA with Bonferroni correction, comparing stimulated DCs with their immature control (^* P < 0.05).

Figure 4

IDO expressed by cDCs inhibits T cell proliferation. Blood DCs were stimulated with indicated stimuli and/or epacadostat in medium containing 10 μM tryptophan. (A) PBLs were stimulated with anti‐CD3/CD28‐coated beads in supernatants of 48‐hour DC cultures and proliferation was measured after three days by tritiated thymidine incorporation. Mean proliferation in counts per minute (cpm) of three different donors from 3 independent experiments with technical triplicates (cDC2, pDC) or duplicates (cDC1) is shown. Symbols correspond to measurements belonging to the same donors. Significance was determined by two‐tailed paired t‐test comparing absence vs presence of IDO inhibitor (^* P < 0.05; ^** P < 0.01). (B) Allogeneic PBLs were cultured with overnight‐stimulated DCs and proliferation was measured after another three days by tritiated thymidine incorporation. Mean proliferation of at least three different donors from four independent experiments with technical triplicates (cDC2, pDC) or duplicates (cDC1) are shown. Symbols correspond to measurements belonging to the same donors. Significance was determined by two‐tailed paired t‐test, comparing absence versus presence of IDO inhibitor.

Figure 5

IDO inhibition by epacadostat does not affect the ability of DCs to cross‐present soluble antigen to T cells. Blood DCs were stimulated overnight with R848 (pDCs) or R848 and poly(I:C) (cDCs) in medium containing 1 μM tryptophan, with or without epacadostat, and loaded with gp100_(154‐162) (irrelevant), gp100_(280‐288) (short), or gp100_(272‐300) (long) peptide. After overnight culture, T cells expressing the T cell receptor recognizing gp100_(280‐288) were added at a 1:5 DC:T cell ratio for 24 h. CD69 expression by T cells was used as a readout for DC‐induced T cell stimulation and was measured by flow cytometry. Mean frequency of CD69^high cells among live CD3⁺ T cells is shown, with at least seven different donors per condition from 11 independent experiments, with technical duplicates for cDC2 and pDC conditions. Significance was determined by two‐tailed paired t‐test, comparing absence versus presence of IDO inhibitor (ns, non‐significant).