Blocking Indolamine-2,3-Dioxygenase Rebound Immune Suppression Boosts Antitumor Effects of Radio-Immunotherapy in Murine Models and Spontaneous Canine Malignancies

IDO upregulation in the tumor microenvironment after immunostimulatory therapies

To test our hypothesis that IDO-mediated “rebound immune suppression” maintains immune suppression and limits treatment efficacy, we employed the poorly immunogenic and highly metastatic mouse 4T1 orthotopic breast tumor model (27). We first evaluated IDO expression within the tumor microenvironment after immunostimulatory therapies and observed a significant increase in IDO-expressing cells compared with untreated controls in the glandular nests of malignant cells (Fig. 1A and B; P < 0.0001). IDO can be upregulated by tumor-infiltrating immune cells as well as tumor cells. In our model, IDO upregulation appears to be predominately in the neoplastic epithelial cells. IDO upregulation within 4T1 cells was verified in vitro as well (data not shown). We likewise observed a parallel statistically significant 3- to 5-fold increase in IDO1 mRNA expression (Fig. 1C). Importantly, this upregulation of IDO was not limited to RT and CpG but was also observed with other immunotherapy strategies (Fig. 1A–C). These results indicate that immunotherapy can paradoxically upregulate immunosuppressive pathways, such as IDO, which may limit efficacy.

Systemic 1MT significantly decreases IDO activity and augments the antitumor efficacy of local RT + CpG

To investigate if IDO-mediated “rebound immune suppression” after RT + CpG limits treatment efficacy, we next tested the effects of adding systemic IDO blockade. Our treatment schema (Fig. 1D) consisted of two 8 Gy fractions of local RT administered to the primary tumor over a 7-day period. Each fraction of RT was accompanied by intratumoral injection of 20 μg of CpG based on dose response data. CpG was administered locally to mirror human clinical trials and minimize systemic toxicity (6, 7). For IDO blockade, 1MT was administered by daily 2 mg i.p. injections throughout the study period. The triple combination decreased IDO enzymatic activity, as measured by serum kynurenine/tryptophan ratio, below the level of untreated controls (Fig. 1E; P = 0.03). We next evaluated the antitumor effects of 1MT, RT, and CpG, alone or in combination. CpG and 1MT alone or in combination had no significant effect on tumor growth (Fig. 1F). Local RT alone significantly reduced tumor growth, but by day 34, there was accelerated tumor outgrowth, and mean tumor size was no longer statistically different than controls (Fig. 1F and G). Although CpG and 1MT had no antitumor effects alone or in combination, when either one was combined with RT, they significantly inhibited tumor growth (Fig. 1F and G). Notably, the triple combination was significantly better than either RT + CpG or RT + 1MT decreasing tumor growth by an additional 3-fold (day 34 mean tumor size: 377 mm³, 1,060 mm³, 1,057 mm³, respectively; Fig. 1F). The triple therapy also significantly improved the survival of 4T1-bearing mice (Fig. 1H, P < 0.001). To ensure these results were not tumor or mouse strain specific, we tested this regimen in C57BL/6 mice bearing B16 melanoma tumors. Again, mice treated with the triple combination had significantly smaller tumors than mice treated with RT + CpG (Fig. 1I, P = 0.042). Importantly, in both models, there was no evidence of autoimmune or other toxicities induced by the therapy. Thus, as hypothesized, the addition of 1MT significantly improved the antitumor effects of RT + CpG in 4T1 and B16 tumors (Fig. 1G and I).

We next examined the systemic antitumor effects of this triple therapy in 4T1-bearing mice. At day 40, heavy burdens of pulmonary metastatic disease could be grossly identified in control but not treated mice (Fig. 1J). The primary tumors (located in the distal mammary fat pad) were treated with 2-cm radiation portals, and the lungs were well outside of the radiation fields. Similar results were also observed by CT imaging (Fig. 1K) and further corroborated in quantifiable fashion using in-vitro lung tumor colony-forming assays with a 6-fold reduction in lung colony-forming units in the triple combination group compared with controls at day 28 (Fig. 1L, P = 0.001). Crucially, lungs from the majority of the triple combination–treated mice grew no tumor colonies.

Taken together, our findings indicate that RT + CpG resulted in antitumor effects but also upregulated IDO expression. The addition of IDO blockade with 1MT decreased IDO activity and significantly improved the antitumor effects. This triple therapy was well tolerated, increased survival, and markedly reduced systemic metastases.

RT + CpG + 1MT induces systemic antitumor effects in spontaneous canine malignancies

Based on our murine studies, we initiated a pilot clinical trial for outbred companion canines with spontaneous metastatic melanomas and sarcomas. Due to the aggressive nature and poor prognosis of these cancers, the standard of care for these canine patients is palliative RT to the primary tumor consisting of 4 weekly fractions of 8 Gy. Our treatment protocol (Supplementary Fig. S1A) built on this palliative regimen but mirrored our mouse studies with an intratumoral injection of CpG at the time of RT and daily administration of 1MT for the treatment course. CpG was administered at 2 mg/dose to mirror human clinical studies. The activity of CpG ODN 2006 used in human clinical trials was verified in canines with an in-vitro peripheral blood mononuclear cell proliferation assay (Supplementary Fig. S1B). 1MT was administered orally at 1,200 mg per day based on pharmacokinetic studies in canines (28). We verified the ability of 1MT to reduce IDO enzymatic activity in canines by measuring serum kynurenine/tryptophan ratio in samples collected from trial canines (Supplementary Fig. S1C; P = 0.02). We enrolled 5 canines with rapidly progressing metastatic disease to this pilot trial (Supplementary Table S1). Four had mucosal melanomas with pulmonary metastases, and one had soft tissues sarcoma with nodal and pulmonary metastases (Supplementary Table S1). Canines with metastatic melanomas have an extremely poor prognosis (29). The survival of canines in this study (5.8 months, 95% confidence interval: 3.2–9.2 months) exceeded published historic controls (29). All 5 canines responded at the primary site of disease which was within the radiation portals (Supplementary Table S1; Fig. 2A–D). The local response to therapy was robust with a 50% to 100% reduction in tumor mass (Fig. 2A–D). Photographic and CT imaging examples documenting local responses are shown in Fig. 2B–D. Crucially, when examining best systemic responses by immune-related response criteria (25) at distant metastatic sites outside of the radiation portals, we observed one subject with a complete response, two with partial responses, one with disease stabilization, and one with progressive disease (Supplementary Table S1; Fig. 2E–H). CT imaging examples of partial (Fig. 2F–G) and complete responses (Fig. 2H) of index lesions are depicted. Mirroring human cancer immunotherapy trials (1, 3), responses were rapid and robust with >80 to 100% reduction in the volume of index lesions (Fig. 2E) and regression of even large bulky tumors (Fig. 2F and H). Some of the partial responses consisted of a mixed response with regression of some lesions and growth or stability of others (Fig. 2G). This type of mixed response has also been observed in human immunotherapy trials and is associated with a good prognosis (25). Thus, in canines with previously rapidly progressing disease, the local response rate was 100% and the systemic response rate was 60%, with another 20% disease stabilization. Upon cessation of treatment in this trial, all dogs eventually had CT documented or clinical/symptomatic progression. No further treatment was provided beyond the brief treatment course outlined in the schema, and assessment of longer administration, as is used in human trials, is needed.

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Figure 2.

Efficacy of radiation + CpG + 1MT in a canine clinical trial. Therapeutic response of local irradiated tumors (A–D) and untreated metastatic lesions (E–H) in a canine clinical trial are depicted. Waterfall plot of best response at the primary-treated tumor (A). Photographs (B) and CT (C) depicting response of a melanoma of the buccal mucosa. CT depicting response of an abdominal wall sarcoma (D). Waterfall plot of best response at untreated metastatic index lesions (E). CT demonstrating a partial response (F), mixed response (G), and complete response (H) of metastatic pulmonary index lesions.

Canines were monitored closely for toxicity with regular physical examinations and lab work. Importantly, the only adverse effects observed were mild mucositis and skin toxicity within the radiation portals that did not exceed what would be expected from palliative RT alone (Supplementary Table S1). These results demonstrate that this triple combination therapy can be safely administered resulting in significant antitumor effects in metastatic disease and validate the findings of our mouse studies in a model more representative of human cancer.

Addition of IDO blockade to RT + CpG transforms the immune-suppressive tumor microenvironment

The primary rationale for adding 1MT to RT + CpG was our hypothesis that IDO-mediated rebound immune suppression limited efficacy by maintaining Tregs and an immunosuppressive tumor microenvironment despite inflammatory signals. Clinical efficacy has been observed with RT + CpG, but patients whose tumors induce Tregs are unlikely to respond (6). We examined the fate of immunosuppressive Tregs after the completion of therapy in mice and canines. In mice, Treg levels were maintained within the tumor after RT + CpG (Fig. 3). The addition of 1MT significantly and substantially reduced the Treg subset of CD4⁺ T cells within the tumor microenvironment but not in the periphery (Fig. 3; Supplementary Figs. S2 and S3). The addition of 1MT resulted in a greater than 4-fold decrease in intratumoral Tregs compared with RT + CpG alone 14 days after therapy (Fig. 3A and B; P = 0.02). This triple combination, but not immunotherapy alone (CpG + 1MT), RT alone, or RT + CpG, significantly reduced Tregs compared with control mice (Fig. 3A and B; control: 24.4% ± 2.1% vs. RT + CpG + 1MT 6.2% ± 2.2%; P < 0.01). Similar results were seen if Tregs were analyzed as a percentage of total cells in the tumor (control: 0.093% ± 0.072% vs. RT + CpG + 1MT 0.018% ± 0.003%; P = 0.07, data not shown). IL6 induction after immunostimulatory therapies contributes to the reduction of Tregs. IDO can maintain Tregs in tumors by downregulating IL6 (8). We observed a 9-fold increase in IL6 expression within the tumor microenvironment after triple combination therapy (Supplementary Fig. S4; P = 0.03). We also observed a significant 3- to 4-fold increase in the CD4⁺/Treg ratio, which is a known indicator of immune status and prognosis (11, 30, 31), in RT + CpG + 1MT–treated mice compared with tumor-bearing control mice (P < 0.01), CpG + 1MT–treated mice (P < 0.05), RT-treated mice (P < 0.01), and RT + CpG–treated mice (P < 0.01; Fig. 3C). The addition of 1MT provided no added benefit in terms of Treg reduction and improvement in the conventional CD4/Treg ratio in the dLN (Supplementary Fig. S2A–S2C), spleens (Supplementary Fig. S2D–S2F), or nondraining lymph nodes (data not shown). This is in contrast with the tumor, where the greatest Treg reductions were seen in the 1MT-containing groups (CpG + 1MT and RT + CpG + 1MT; Fig. 3A–C). Similar results were also seen at earlier time points (Supplementary Fig. S3). Thus, although 1MT was administered systemically, its primary action is within the tumor microenvironment where IDO is also upregulated.

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Figure 3.

Radiation + CpG + 1MT reduces intratumoral regulatory CD4⁺ T cells in mice and canines. Day-28 levels of tumor-infiltrating regulatory CD4⁺ T cells as assessed by flow cytometry and immunofluorescence in 4T1-bearing mice (A–C) or canine patients (D–G) treated with RT + CpG + 1MT. Representative flow cytometry contour plots demonstrating staining of intratumoral CD4⁺ cells for FoxP3 and CD25 (A). Flow cytometry data represented as a bar graph expressed as %Treg (CD4⁺, CD25⁺, FoxP3⁺) of CD4⁺ cells (B). Bar graph representation of CD4⁺ to Treg ratio as measured by flow cytometry (C). Representative flow cytometry plots demonstrating staining of canine intratumoral CD4⁺ cells for FoxP3 pre- and post-RT + CpG + 1MT therapy (D). Bar graph representation of intratumoral Tregs pre- and posttherapy expressed as a percentage of CD3⁺ cells in 4 canine patients as assessed by flow cytometry (E). Line graph demonstrates changes in Treg levels in individual patients as assessed by flow cytometry (E). Immunofluorescent staining of canine tumor samples for FoxP3 (F). FoxP3⁺ cells stain bright pink, and nuclei are counterstained by DAPI; white arrows point out examples of positive staining cells. Bar graph quantification of intratumoral FoxP3-positive cells (G). n = 3 to 4 mice per group and 4 canine patients. Bar graphs represent mean ± SEM. Results analyzed by one-way ANOVA or Student t test between the indicated groups (*, P < 0.05; **, P < 0.01).

Informed by our murine studies, we examined the fate of Tregs in the tumors, dLN, and systemic circulation of canines receiving RT + CpG + 1MT (Fig. 3; Supplementary Fig. S2). When possible, tumor biopsy, tumor-draining lymph node biopsy, and peripheral blood samples were obtained before and after therapy. Peripheral blood was obtained from all dogs. A posttherapy tumor biopsy could not be obtained from one dog due to a complete response. In addition, some dogs displayed massive tissue death in the posttreatment tumor biopsy samples limiting analyses. Lymph node biopsies could not be obtained from two dogs in which dLNs were not accessible with a minimally invasive procedure. Mirroring the results of our mouse studies, the percentage of tumor-infiltrating Tregs was significantly reduced in all dogs after therapy with virtually no Tregs remaining in the tumor microenvironment after therapy (Fig. 3D and E; P = 0.014). Conversely, in the three dLN samples, we did not observe a statistically significant change in Tregs across the cohort with Tregs dropping substantially (3–4-fold) after therapy in some dogs but increasing in others (Supplementary Fig. S2G). Also mirroring our mouse results, no significant change in peripheral Tregs was observed (Supplementary Fig. S2H). To confirm our results from flow cytometric analysis, immunofluorescence staining for FoxP3 expression in canines that had sufficient remaining biopsy samples was performed. Representative fields and quantification from a canine with Treg decreases in the tumor are depicted in Fig. 3F and G confirming marked reduction in Foxp3⁺ cells following triple combination therapy.

In the mouse tumor models, we also observed significant reductions in other immune-suppressive factors in the tumor microenvironment. Tumor associated macrophages (TAM) are known to play a critical suppressive role in the microenvironment of 4T1 tumors (32). Mirroring the reduction seen in Tregs only, the RT + CpG + 1MT triple combination resulted in a statistically significant reduction of TAMs by nearly 5-fold compared with control (Fig. 4A and B; 18.4% ± 10.3% vs. 3.8% ± 1.5%; P < 0.05). At baseline, intratumoral TAMs were M2 polarized as assessed by arginase to inducible nitric oxide ratio (data not shown). We next examined the expression of the TGF-beta gene (TGFB) which both induces and is produced by Tregs and TAMs (reviewed in 33, 34), is produced by IDO-expressing DCs (35), and is also known to block lymphocyte activation (36). All of the therapies reduced TGFB expression relative to control, but this was most pronounced and most significant in the triple combination (Fig. 4C; P < 0.0001). Taken together, these studies indicate that this therapy reduces multiple immune-suppressive factors and restores IL6 signaling within the tumor microenvironment.

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Figure 4.

Radiation + CpG + 1MT reduces tumor-associated macrophages. Levels of intratumoral CD45⁺CD11b⁺F4/80⁺ macrophages as assessed by flow cytometry in 4T1-bearing mice (A and B). Representative flow cytometry contour plots demonstrating staining of intratumoral CD45⁺ cells for F4/80 and CD11b (A). Flow cytometry data represented as a bar graph expressed as % tumor-associated macrophages of all CD45⁺ cells (B). Bar graph representation of intratumoral TGF-beta mRNA as assessed by qPCR (C). n = 3 to 4 mice per group. Bar graphs represent mean ± SEM. Results analyzed by one-way ANOVA between the indicated groups (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

RT + CpG + 1-MT triple therapy antitumor effects are CD8⁺ T-cell dependent

We next examined the effects of this triple therapy on CD8⁺ T cells and DCs. The percentage of tumor-infiltrating CD8⁺ T cells (expressed as a percentage of total viable cells within the tumor) significantly increased from 0.68% in control mice to 2.71% 7 days after RT + CpG + 1MT (Fig. 5A and B; P = 0.02). The CD8⁺ T-cell/Treg ratio in the tumor was also increased by therapy from 6.3 to 21.7 (Fig. 5C; P = 0.0006). There were no changes in activation/functional/exhaustion markers such as PD-1, CD69, or IFN-gamma in the CD8⁺ T cells after therapy, but the majority of intratumoral CD8⁺ T cells expressed these markers at baseline (data not shown). We also evaluated changes in tumor-infiltrating CD8⁺ T cells in treated canines. By flow cytometric analysis, there was a posttherapy increase in intratumoral CD8⁺ T cells (as a percentage of all CD3⁺ intratumoral cells) in all three evaluable canines (Fig. 5D and E). Overall, the frequency of intratumoral CD8⁺ T cells doubled after therapy, which trended toward statistical significance (Fig. 5D and E; 24% ± 8.5% vs. 47.6% ± 16.4%; P = 0.06). IHC staining likewise demonstrated an increase in intratumoral CD8⁺ T cells after therapy (30 ± 33 vs. 50 ± 39 cells/HPF; Fig. 5F and G; P = 0.09). Mirroring our mouse studies therapy induced a significant increase in the CD8⁺/Treg ratio (Fig. 5H; P = 0.014).

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Figure 5.

Radiation + CpG + 1MT increases tumor-infiltrating CD8⁺ T cells in mice and canines. Representative flow cytometry contour plots demonstrating staining of tumor-infiltrating CD45⁺CD3⁺ cells for CD8 and CD4 (A). Flow cytometry data of tumor-infiltrating CD8⁺ T cells represented as a bar graph expressed as % CD8⁺ cells of all CD45⁺ cells (B). CD8⁺ T cell to Treg ratio as determined by flow cytometry (C). Levels of tumor-infiltrating CD8⁺ T cells as assessed by flow cytometry in canine tumors (D and E). Representative flow cytometry contour plots demonstrating staining of canine intratumoral CD8⁺ and CD4⁺ T cells pre- and post-RT + CpG + 1MT therapy (D). Bar graph representation of intratumoral CD8⁺ T cells expressed as a percentage of CD3⁺ cells in 3 canine patients as assessed by flow cytometry (E). Line graph demonstrates changes in Treg levels in individual patients as assessed by flow cytometry (E). Immunohistochemical staining with tumor-infiltrating CD8⁺ T cells stained in red, white arrows point out examples of positive staining cells (F). Bar graph quantification of intratumoral CD8-positive cells (G). CD8⁺ T cell to Treg ratio as determined by flow cytometry (H). Line graph demonstrates changes in CD8⁺ T cell to Treg ratio in individual patients before to after treatment (H). n = 3 to 4 mice per group and 3 canine patients. Bar graphs represent mean ± SEM. Results analyzed by Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

We tested the CD8 dependence of this therapy in mouse models. Depletion of CD8⁺ T cells by intraperitoneal administration of anti-CD8 reduced the number of circulating CD8⁺ T cells by >99% (Supplementary Fig. S5A and S5B). In mice depleted of CD8⁺ T cells, we observed that the antitumor effects of RT + CpG + 1MT were significantly diminished. After treatment with the triple combination, tumors were more than tripled in size in CD8-depleted mice compared with those treated with control IgG (Supplementary Fig. S5C; 2,066 mm³ vs. 684 mm³; P < 0.0001). The survival benefit of the triple therapy was also significantly diminished in CD8-depleted mice (Supplementary Fig. S5D; P = 0.03), indicating the critical role of CD8⁺ T cells in the antitumor effects.

In addition to the effects of CD8⁺ T cells, we also evaluated DC activation and phenotypes following triple therapy. IDO expression has been linked to a tolerogenic DC phenotype, associated with decreased expression of CD80 and MHC II and increased TGF-beta production (35). No change in DC numbers or activation within dLNs was observed (data not shown). Recent data demonstrate that intratumoral DCs, although a minor population, play a critical role in antitumor T-cell responses (37, 38). Given that the major effects of this therapy occur in the tumor microenvironment, we also examined DCs in the tumor microenvironment. There was an increased number of activated DCs within treated tumors, with a 4-fold increase in CD80-expressing DCs (Supplementary Fig. S6A–S6C; P = 0.02) and a near doubling of DC MHCII mean fluorescence intensity (MFI) compared with control mice (Supplementary Fig. S6D and S6E; P = 0.01).

Canine immunologic biomarkers

Finally, in three of the canine patients for which sufficient pre- and posttreatment tumor tissue was available, we evaluated gene signatures by qPCR (Fig. 6). Based on our mouse data, we focused on expression of three immunosuppressive markers (IDO1, TGFB, and FOXP3). Two of these patients demonstrated systemic partial responses, and one had systemic progressive disease. Interestingly, in the two responders, expression of all three immunosuppressive genes was markedly decreased after therapy, whereas all three were increased in the setting of progressive disease. These data suggest that this gene signature in the local tumor may represent a predictive biomarker for systemic response to this therapy and requires further evaluation in future studies. Overall, although hypothesis-generating in nature, these canine correlative immune analyses corroborate our mechanistic mouse data and provide rationale for further exploration of these immunologic endpoints in future studies.

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Figure 6.

Intratumoral expression of immunosuppressive molecules as a gene signature of systemic antitumor immune response in canines treated with radiation + CpG + 1MT. Sufficient pre- and posttherapy tumor tissue was available for further analysis in 3 canines: two responders and one with progressive disease. Expression of the immunosuppressive molecules IDO, TGF beta, and FoxP3 was assessed by qPCR, and results are expressed relative mRNA expression of technical triplicates. Bar graphs represent mean ± SEM.