Indoleamine 2,3-dioxygenase controls conversion of Foxp3+ Tregs to TH17-like cells in tumor-draining lymph nodes

Abstract

The immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) is expressed by a subset of murine plasmacytoid DCs (pDCs) in tumor-draining lymph nodes (TDLNs), where it can potently activate Foxp3+ regulatory T cells (Tregs). We now show that IDO functions as a molecular switch in TDLNs, maintaining Tregs in their normal suppressive phenotype when IDO was active, but allowing inflammation-induced conversion of Tregs to a polyfunctional T-helper phenotype similar to proinflammatory T-helper-17 (TH17) cells when IDO was blocked. In vitro, conversion of Tregs to the TH17-like phenotype was driven by antigen-activated effector T cells and required interleukin-6 (IL-6) produced by activated pDCs. IDO regulated this conversion by dominantly suppressing production of IL-6 in pDCs, in a GCN2-kinase dependent fashion. In vivo, using a model of established B16 melanoma, the combination of an IDO-inhibitor drug plus antitumor vaccine caused up-regulation of IL-6 in pDCs and in situ conversion of a majority of Tregs to the TH17 phenotype, with marked enhancement of CD8+ T-cell activation and antitumor efficacy. Thus, Tregs in TDLNs can be actively reprogrammed in situ into T-helper cells, without the need for physical depletion, and IDO serves as a key regulator of this critical conversion.

Figure 1

Activation of Treg suppressor activity by IDO and effector T cells. (A) Resting splenic Tregs (FACS-sorted CD4⁺CD25⁺) were cocultured with pDCs from TDLNs (CD11c⁺B220⁺) plus OT-I T cells, OVA peptide antigen, and feeder layer (all on the B6 background). After 2 days, the Tregs were harvested, resorted, and added to readout assays to measure suppressor activity (A1 T cells + congenic spleen DCs, CBA background). Graph represents proliferation in the readout assay by tritiated-thymidine incorporation, using either IDO-activated Tregs or the same Tregs activated with αCD3 cross-linking plus recombinant IL-2 (with IDO blocked using 1MT). Bars represent SD of replicate wells. (B) Tregs were activated with IDO⁺ pDCs as in panel A, with or without the cognate OVA peptide antigen for OT-I. (C) Tregs in panel A were activated in the presence of OT-I and OVA, with (○) or without (□) D-1MT to block IDO. Experiments in each panel were repeated 3 to 10 times with similar results.

Figure 2

In the absence of IDO, activated T cells drive conversion of Tregs to a TH17-like phenotype. (A) Sorted CD4⁺GFP⁺ Tregs from Foxp3^GFP knockin mice were activated in cocultures with pDCs and OT-I as in Figure 1, with or without OVA and 1MT as indicated. After 2 days, cocultures were stained for intracellular IL-17 after a 4-hour stimulation with phorbol myristate acetate/ionomycin plus brefeldin A. The top dot plot represents an example of a representative gate for the Foxp3^GFP-positive CD4⁺ Tregs. The bottom plots represent the gated Treg population in each treatment group. (B) Tregs (CD4⁺CD25⁺) were sorted from homozygous-null Rorγt ^gfp/gfp mice lacking functional RORγt, or from WT controls, and activated in cocultures with and without 1MT as shown. After 2 days, cultures were stained for CD4 to identify Tregs versus IL-17. (The only CD4⁺ cells in cocultures were the original sorted Tregs.) (C) Tregs from Rorγt-null mice, or control wild-type Tregs, were activated in cocultures for 2 days, with (□) or without (◊) 1MT. Tregs were sorted and functional suppressor activity measured against A1 T-cell readout as in Figure 1. (D) Foxp3^GFP Tregs were sorted and activated in cocultures with 1MT and OVA. Plots represent 4-color staining from a representative sample, gated on GFP⁺CD4⁺. (E) Foxp3^GFP Tregs were activated as in panel A and stained for IL-17 versus the cytokines shown. Plots represent the gated CD4⁺GFP⁺ cells. Experiments were repeated 3 to 12 times with similar results.

Figure 3

Up-regulation of IL-17 is driven by IL-6. (A) Cocultures were performed as in Figure 2, with and without 1MT. After 2 days, cultures were stained for IL-6 versus CD11c (to mark the sorted pDCs). The right graph represents IL-6 measurement by enzyme-linked immunosorbent assay on supernatants of cocultures (error bars show SD of quadruplicate wells). (B) Tregs were sorted from Foxp3^GFP mice and activated in cocultures, with or without 1MT plus neutralizing polyclonal antibody against IL-6. After 2 days, cocultures were stained for CD4 versus IL-17. Plots represent the gated CD4⁺ (Treg) population. (C) Sorted Foxp3^GFP Tregs were activated in cocultures with or without 1MT. Recombinant IL-6 (100 ng/mL) was added as shown. Plots represent the gated CD4⁺ (Treg) population. Plot at right indicates a representative example of coexpression of IL-22 and IL-17 on gated Tregs in IL-6–treated cocultures. (D) IL-6 up-regulation in pDCs requires OVA antigen or CD28→B7 engagement. Cocultures were performed in the presence of 1MT, with and without OVA or recombinant CD28-Ig fusion protein (20 μg/mL), as indicated. After 2 days, cultures were stained for IL-6 versus CD11c (top plots), and for CD4 versus IL-17 versus IL-22 (bottom plots, gated on the CD4⁺ Treg population). All experiments were repeated 3 to 10 times with similar results.

Figure 4

Evidence that IDO acts via the GCN2-kinase pathway in pDCs to block IL-6 up-regulation. (A) Hypothesized pathway for IDO-induced translational regulation of NF-IL-6 transcription factor. (B) pDCs were isolated from TDLNs of tumors grown in genetically defined hosts (IDO1-KO, GCN2-KO, or WT). pDCs were then used in activation cocultures with OT-I and Tregs, as Figure 2. After 2 days, cocultures were stained for CD11c versus IL-6. (C) Foxp3^GFP Tregs were cocultured as in Figure 2A, using pDCs from TDLNs of WT, IDO1-KO, or GCN2-KO hosts. All cultures were without 1MT. After 2 days, cultures were harvested and stained for intracellular IL-17 versus CD4. (D) Analysis of the inhibitory LIP isoform of NF-IL-6 in T-REX cells stably transfected with inducible IDO cDNA. IDO was either uninduced or induced by treatment with doxycycline (20 ng/mL) as indicated. Induced cells were treated with 50, 25, and 10 μM of the IDO inhibitors L-1MT or methyl-thiohydantoin-tryptophan (MTHT), as indicated. Graph documents production of kynurenine by functional IDO (error bars show SD of triplicate wells). The top Western blot represents expression of IDO after induction; the bottom Western blot represents induction of the 21-kDa LIP isoform of NF-IL-6, and the higher molecular weight LAP isoforms. All experiments were repeated 3 to 4 times with similar results.

Figure 5

Generation of TH17-like cells in TDLNs in vivo. (A) Foxp3^GFP mice with B16-OVA tumors were treated with OVA-Lv vaccine, oral D-1MT, and adoptive transfer–sorted OT-I cells, as shown. On day 11, TDLNs were harvested and stained for IL-17. Percentages in the right-upper quadrants of each plot give the fraction of the Foxp3^GFP-positive cells that coexpressed IL-17. Percentages below give total Foxp3^GFP-positive cells in each LN. (B) Mice were treated as in panel A and TDLN cells stained for CD11c versus B220 versus intracellular IL-6. Plots represent total LN cells; inset represents gated CD11c⁺ population from the (+)1MT group. (C) Wild-type B6 mice with B16-OVA tumors were treated with control (vehicle only) or oral D-1MT plus OVA-Lv vaccine. All mice received coadoptive transfer of 10⁶ CD8⁺ OT-I cells mixed with 10⁶ sorted Foxp3⁺ Tregs (CD4⁺GFP⁺Thy1.1⁺) from OT-II^{Foxp3-GFP Thy1.1} mice. On day 11, TDLNs were harvested and stained for CD4/Thy1.1/IL-17 versus Foxp3^GFP by 4-color FACS. Each left plot represents the population of transferred Tregs (CD4⁺Thy1.1⁺) as a percentage of total TDLN cells; right plots represent GFP versus IL-17 expression in the gated GFP⁺ Tregs (the percentage gives the fraction of Foxp3^GFP-positive cells that coexpress IL-17). (D) Bone marrow chimeras (RORγt-null marrow into wt B6 hosts, or control wtB6→wtB6) received B16-OVA tumors, and mice were treated as in panel A with either control (vehicle only) or oral D-1MT plus OVA-Lv vaccine. All mice received OT-I adoptive transfer on day 7. Plots indicate representative IL-17 up-regulation in gated CD4⁺CD25⁺ population in TDLNs from each treatment group on day 11. Experiments were repeated 3 to 8 times with similar results.

Figure 6

Replacement of Tregs by TH17-like cells is associated with enhanced antitumor efficacy. (A) Foxp3^GFP mice with established B16-OVA tumors were treated using the protocol shown in Figure 5, with or without resting OT-I cells, OVA-Lv vaccine, and oral D-1MT, as indicated below the axis. On day 11, tumors were dissected and the tumor area measured as the product of orthogonal diameters. Values reflect the mean of pooled data from 7 independent experiments (error bars show SD); the total number of tumors analyzed in each treatment (n) is shown. *P < .01 by analysis of variance versus all other groups; bars represent SD. (B) B6 mice with established B16-OVA tumors were treated with adoptive transfer of resting OT-I cells (control) or OT-I cells plus OVA-Lv vaccine plus oral D-1MT, as in the previous panel. Data points represent average of 5 mice; bars represent SD. One of 2 experiments. (C) Foxp3^GFP mice were injected with 10⁶ B16F10 tumor cells. On day 5, mice received muTRP1-Lv vaccine (or control). D-1MT in drinking water (or control) was started on day 6. On day 11, TDLNs were harvested and stained for intracellular IL-17 as in Figure 5. Percentages give the fraction of Foxp3^GFP-positive cells coexpressing IL-17. Representative of4 independent experiments. (D) Foxp3^GFP mice with established B16F10 tumors were treated with or without muTRP1-Lv vaccine and D-1MT in drinking water, as indicated. Tumor size was measured at necropsy on day 11. Each data point is a mean of 6 tumors from 3 independent experiments (error bars show SD). *P < .01 by analysis of variance.

Figure 7

Enhancement of CpG-based vaccine by 1MT. (A) Wild-type B6 mice with established B16-OVA tumors were treated with adoptive transfer of OT-I cells, with or without 1MT, and with or without vaccine (OVA protein in incomplete Freund adjuvant plus CpG-1826). In parallel experiments, wild-type hosts were compared with RORγt-null bone marrow chimeric hosts (as in Figure 5D) or with MHC class II-deficient (IA^b-KO) hosts. Mean data are indicated, pooled from a total of 8 experiments; bars represent SD. *P < .05 by analysis of variance. (B) B6 mice with B16-OVA tumors received CFSE-labeled OT-I cells plus OVA/CpG/IFA vaccine, with or without 1MT as shown. After 4 days, tumors were disaggregated and stained for CD8 versus granzyme B or CXCR3.