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Sustained intratumoral delivery of IL-12 and GM-CSF induces tumor regression via restoration of tumor-resident CD8+ T-effector/memory cell cytotoxicity and subsequent re-priming of a secondary CD8+ T-effector cell response in tumor-draining lymph nodes (TDLN). However, treatment-induced T-effector activity is transient and is accompanied with a CD4+ CD25+ Foxp3+ T-suppressor cell rebound. Molecular and cellular changes in post-therapy tumor microenvironment and TDLN were monitored to elucidate the mechanism of counter-regulation. Real-time PCR analysis revealed a 5-fold enhancement of indoleamine 2, 3 dioxygenase (IDO) expression in the tumor and the TDLN after treatment. IDO induction required IFNγ and persisted for up to 7 days. Administration of the IDO inhibitor D-1-methyl tryptophan (D-1MT) concurrent with treatment resulted in a dramatic enhancement of tumor regression. Enhanced efficacy was associated with a diminished T-suppressor cell rebound, revealing a link between IDO activity and post-therapy regulation. Further analysis established that abrogation of the regulatory counter-response resulted in a 10-fold increase in the intratumoral CD8+ T-cell to CD4+ Foxp3+ T-cell ratio. The ratio of proliferating CD8+ T-effector to CD4+ Foxp3+ T-suppressor cells was prognostic for efficacy of tumor suppression in individual mice. IFNγ-dependent IDO induction and T-suppressor cell expansion were primarily driven by IL-12. These findings demonstrate a critical role for IDO in the regulation of IL-12-mediated antitumor immune responses.
Cancer vaccines can break tolerance to tumor antigens and induce potent anti-tumor T-cell activity (1, 2). However, in the great majority of cases anti-tumor T-cells fail to induce effective tumor regression, independent of the intensity of the response (1–3). The immune suppressive nature of the tumor microenvironment has been identified as a major factor contributing to the inability of T-cells to mediate effective tumor regression (4, 5). Blocking of suppressive mechanisms during therapy results in improved tumor eradication however, durable regressions are rarely achieved (6, 7). Whether the lack of long-term efficacy is due to the inherent transient nature of effector T-cell responses (8) and the associated T-cell intrinsic regulatory pathways (9), to resurgence of tumor-mediated immune dysfunction (4) or a combination of these factors is not established.
Previous studies demonstrated that sustained intratumoral delivery of IL-12 and GM-CSF restored tumor-resident CD8+ T-effector/memory cell (Tem) cytotoxicity and induced de novo priming of antitumor CD8+ T-effector cells in the TDLN (10, 11). Treatment-induced immune activation however, subsided within a week and was followed by a T-suppressor cell rebound (12). Since IL-12 directly restored full effector function to pre-existing quiescent CD8+ Tem in the presence of T-suppressor cells (10), we hypothesized that repeated stimulation could revive and extend the window of CD8+ Tem cytotoxicity, and enhance long-term tumor suppression. Chronic treatment however, resulted in progressive loss of therapeutic efficacy culminating in tumor resurgence (12). Long-term monitoring of intratumoral T-cell populations demonstrated that multiple treatments led to the intensification of the T-suppressor cell rebound after each cycle of therapy and ultimately resulted in the loss of anti-tumor Tem (12). These findings suggested that feedback inhibition may represent an important tumor-independent mechanism that can limit long-term therapeutic efficacy.
Whereas the majority of tumor immune therapy protocols focus on the induction phase of the anti-tumor immune response, few studies have addressed the mechanism of post-activation regulatory rebound. Consistent with our findings, these studies demonstrated that repeated vaccination or immune stimulation can promote the expansion of the CD4+ CD25+ Foxp3+ T-suppressor cell subset, in murine models of autoimmunity and cancer (13–15). On the other hand, the molecular basis of therapy-induced T-suppressor cell expansion has not been delineated. To this end, quantitative and qualitative monitoring of the tumor microenvironment and the TDLN was undertaken to investigate the mechanism of post-IL-12/GM-CSF T-suppressor cell amplification. The results demonstrate a central role for the IL-12-IFNγ-IDO axis in treatment-induced CD4+ Foxp3+ T-cell expansion and the associated loss of therapeutic efficacy in our model.
BALB/c mice were purchased from Taconic laboratories (Germantown, NY) or bred in our facility. IFNγ knockout mice in BALB/c background were purchased from Jackson Laboratories (Bar Harbor, ME). The BALB/c syngeneic mammary carcinoma cell line 4T1 (16) was used in all experiments.
Recombinant murine IL-12 was a gift from Wyeth Pharmaceuticals (Wyeth, Andover, MA). Recombinant murine GM-CSF was purchased from Peprotech Inc. (Rocky Hill, NJ). Cytokines were encapsulated into polylactic acid microspheres via phase inversion nanoencapsulation (10). Mice were treated with a single intratumoral injection of 2 mg of each preparation (1 μg of cytokine) as described previously (10).
Mice were injected subcutaneously with 3 × 105 tumor cells in 0.1 ml of PBS in the mammary tissue and tumors were allowed to grow to 6–7 mm in diameter (14 days) to establish metastases. Primary tumors were treated with a single intratumoral injection of microspheres and tumors were surgically resected one week after treatment (17). Mice were euthanized 3 weeks after surgery and lung tumor burden was quantified using a clonogenic metastasis assay (17).
qRT-PCR was performed essentially as described (17). Transcript levels were calculated by the comparative threshold cycle (Ct) method (17). The relative target quantity, normalized to an endogenous control (GADPH) and relative to the day zero calibrator, was expressed as 2−ΔΔCt (fold), where ΔCt = Ct of the target gene – Ct of endogenous control gene, and ΔΔCt = ΔCt of samples for the target gene – ΔCt of zero day calibrator for the target gene. CD8 and IFNγ primers were published previously (17). The primer sequences for Foxp3, IDO-1 and IDO-2 were: Foxp3, 5'-TCC CAC GCT CGG GTA CAC-3' (forward), 5'-TTG CCA GCA GTG GGT AGG AT-3' (reverse); IDO-1, 5'-CAG GCC AGA GCA GCA TCT TC-3' (forward), 5'-GCC AGC CTC GTG TTT TAT TCC-3' (reverse); IDO-2, 5'-TGTCCTGGTGCTTAGCAGTCATGT-3' (forward), 5'-TGCAGGATGTGAACCTCTAACGCT-3'(reverse).
CD8+ T-cells were depleted via in vivo antibody administration (17).
BrdU labeling was performed as described by us previously (11).
Single cell suspensions from tumors and TDLN were prepared essentially as described (10).
Flow cytometric analysis of single cell suspensions prepared from tumors and TDLN was performed on a 4-color FACSCalibur Flow Cytometer (BD Pharmingen) using established protocols (10). All antibodies and staining protocols were as described previously (10, 17).
D- and L-1MT (Sigma-Aldrich) were prepared and administered to tumor-bearing mice in drinking water starting 2 days prior to microsphere treatments until day 14 post-microsphere injection as described (18).
Student's t-test was used to determine the significance of the differences between control and experimental groups. In experiments with multiple groups homogeneity of inter-group variance was analyzed by ANOVA. Log-Rank analysis was employed to determine significance in survival studies. In all analyses p ≤ 0.05 was considered significant.
Previous studies demonstrated that intratumoral IL-12 and GM-CSF promoted rapid activation of tumor-resident CD8+ T-effector/memory cells followed by priming of an anti-tumor CD8+ T-effector cell response in the TDLN (10, 11). Although the secondary CD8+ T-effectors effectively homed to and infiltrated the tumors, whether they mediated effective long-term tumor killing was not determined (11). To this end, we monitored the cytotoxicity window for the secondary CD8+ T-effectors in a murine surgical tumor metastasis model that allows quantitative analysis of systemic tumor eradication (19, 20). In this model, subcutaneous primary tumors are established and are allowed to metastasize to the lungs (Figure 1A). Primary tumors are then treated with a single intratumoral injection of IL-12 and GM-CSF microspheres followed by resection of the primary tumors one week after treatment. Lung tumor burden in post-surgical mice is then quantified 3 weeks after surgery using a clonogenic metastasis assay (17). In the current study mice were depleted of CD8+ T-cells in a time-dependent manner to identify the interval of CD8+ T-effector cell activity required for eradication of systemic disease (Figure 1A). The results are shown in Figure 1B. The data establish that a single treatment resulted in effective elimination of established lung metastases, reducing tumor burden from an average of 560 ± 89 to 65 ± 48 colonies/lung. Importantly, 60% of the mice in the treatment group were found to be lung tumor-free whereas all mice in the control group had advanced lesions. Depletion of CD8+ T-cells starting one day prior to IL-12/GM-CSF microsphere injection resulted in the complete loss of anti-tumor efficacy demonstrating the central role of CD8+ T-cells in the eradication of systemic disease. Similar results were obtained when CD8+ T-cell depletion was initiated starting on day 4 post-treatment, revealing that CD8+ T-cell activity was required beyond 4 days to achieve effective tumor kill. In contrast, depletion of CD8+ T-cells starting on day 10 still resulted in significant tumor elimination, suggesting that peak effector activity occurred during the first 10 days.
The above findings suggested that treatment-induced effector and/or memory CD8+ T-cells were not effective against tumors that persisted beyond 10 days. Previous studies had demonstrated that treatment was followed by a rebound in intratumoral CD4+ CD25+ Foxp3+ T-suppressor cells (12). We therefore hypothesized that CD8+ T-cell priming could be paralleled by CD4+ Foxp3+ T-cell expansion, potentially curbing long-term T-effector cell activity in the tumor. To this end, CD8 and Foxp3 mRNA kinetics were monitored in the TDLN and tumors of treated mice via qRT-PCR. The results are shown in Figure 2A. Consistent with previous findings (11), treatment was followed by a 4-fold enhancement of CD8 mRNA in the TDLN on day 3 and an 8-fold increase in the tumor on day 7. Importantly, a similar increase (3-fold) in Foxp3 mRNA in the TDLN on day 3 was followed by a 2-fold increase in intratumoral Foxp3 on day 7. The increase in Foxp3 expression in the TDLN was slightly delayed when compared to CD8 during the first 24 hours suggesting that CD8+ T-cell-priming preceded T-suppressor cell expansion. Collectively, these data demonstrate that treatment-induced CD8+ T-cell priming and proliferation was accompanied by a similar expansion of T-suppressor cells in the TDLN and that both populations eventually homed to tumors.
The overlapping nature of post-therapy CD8 and Foxp3 mRNA expression patterns in the TDLN and tumors raised the possibility that treatment itself was responsible for the T-suppressor cell rebound. IFNγ is the primary downstream cytokine induced by IL-12 and is central to its antitumor effects (21). Among more than 200 immunologically-relevant genes induced by IFNγ is IDO (22–24), a tryptophan-catabolizing enzyme with immune suppressive function (25). Importantly, recent studies demonstrated a role for IDO in CD4+ Foxp3+ T-suppressor cell activation and generation (26, 27). These findings provided the rationale for the next series of experiments in which the putative link between the IL-12-IFNγ-IDO axis and post-therapy immune suppression was investigated. Initially, expression of IFNγ and IDO were monitored in the TDLN and the tumors of treated mice by qRT-PCR. Treatment induced a rapid increase in IFNγ mRNA both in the TDLN and the tumor (Figure 2B). This enhancement was more dramatic in the TDLN (5- and 34-fold increases at 6 and 24 hours over background, respectively) compared to the tumor (2- and 3-fold enhancement at 6 and 24 hours, respectively). Upregulation of IFNγ expression in tumors was transient and declined rapidly after day 1 returning to background levels on day 7. IFNγ mRNA levels also declined in the TDLN but remained above pre-therapy levels for at least 7 days. Importantly, treatment promoted a similar increase in the expression of IDO (referred to as IDO-1 from this point on) as well as the more recently described IDO-2 (28) in the TDLN except that induction of these enzymes was delayed by 24 hours in comparison to IFNγ. In contrast, treatment resulted in the upregulation of IDO-1 but not IDO-2 in tumors (Figure 2B). Whereas IDO-1 mRNA returned to background levels in the tumor on day 3, both IDO-1 and 2 remained high in the TDLN for at least 7 days.
The sequential nature of IFNγ and IDO-1/IDO-2 expression kinetics in the TDLN between days 0 and 2 suggested that their transcription were linked. To test this hypothesis, tumors were induced in either wild-type or IFNγ-knockout (GKO) mice, and IDO-1 mRNA was quantified in pre- and post-therapy tumors and TDLN. The results are shown in Figure 2C. In wild-type mice treatment promoted 5 and 2-fold increases in IDO-1 mRNA in the tumor and the TDLN on day 1, respectively (Figure 2C). In contrast, treatment failed to induce detectable IDO-1 mRNA in the GKO mice (Figure 2C). Analysis of day 3 TDLN revealed a similar loss of IDO-2 induction in GKO mice demonstrating that IDO-2 upregulation was also IFNγ-dependent (data not shown).
The above findings were consistent with the notion that the IL-12-IFNγ-IDO axis represented a critical feedback inhibitory mechanism in our model. D-1MT, an inhibitor of IDO-2 activity, enhances tumor suppression in immune-competent but not in immune-deficient mice (18). To this end, we tested whether co-administration of D-1MT with IL-12 and GM-CSF to mice could enhance tumor suppression by blocking post-therapy IDO activity. Tumor-bearing mice were treated either with IL-12/GM-CSF microspheres, D-1MT alone or a combination of IL-12/GM-CSF + D-1MT and tumor growth was monitored. The results are shown in Figure 3. In the control group (blank microspheres) all tumors grew rapidly and mice had to be sacrificed by day 21 or earlier. Administration of D-1MT alone resulted in delayed tumor growth during the first week but tumors grew rapidly thereafter. Administration of IL-12/GM-CSF microspheres suppressed tumor growth during the first 2 weeks but all tumors eventually resumed growth. In contrast, complete tumor regression was achieved in 5 of 11 mice in the combination group resulting in long-term cure (see Supplemental Figure 1 for survival analysis). Since both IDO-1 and 2 were upregulated in the TDLN in response to treatment, we also tested the antitumor efficacy of L1-MT, which specifically targets IDO-1. L1-MT also delayed tumor growth when administered in combination with IL-12/GM-CSF therapy however complete regressions were achieved only with D1-MT suggesting that IDO-2 was the primary mediator of post-therapy feedback inhibition in this model (Supplemental Figure 2).
To determine whether IDO-2 activity and CD4+ Foxp3+ T-cell expansion were linked, post-therapy T-suppressor cell numbers were monitored in the presence or absence of D-1MT. The results shown in Figure 4A reveal that IL-12/GM-CSF treatment resulted in a dramatic 6-fold expansion of T-suppressor cells in tumors and a more modest 1.4-fold increase in the TDLN between days 3 and 10. Importantly, co-administration of D-1MT resulted in a significant reduction (1.8 to 6-fold) in the numbers of CD4+ Foxp3+ T-cells in the TDLN and the tumors on days 3 and 7. However, this inhibition was transient and the CD4+ Foxp3+ T-cell numbers in the experimental mice rebounded to levels observed in control mice on day 10. These findings establish that D-1MT transiently inhibited post-therapy T-suppressor cell expansion.
Since inhibition of T-suppressor cell expansion is expected to result in enhanced CD8+ T-cell activity, the effect of IDO-2 inhibition on CD8+ T-cell proliferation was monitored. To this end, CD8+ T-cell proliferation was quantified in both the TDLN and tumors of control and D-1MT-treated mice. The results are shown in Figure 4B. These data demonstrate that initially TDLN CD8+ T-cells proliferated equally well in both control and D-1MT-treated mice independent of Foxp3+ T-cell levels. On day 7 however, CD8+ T-cell proliferation declined in the TDLN of control mice but remained high in D-1MT-treated animals consistent with extended priming activity. On day 10, proliferation returned to background levels in both groups. In tumors CD8+ T-cell proliferation on day 3 was minimal in both groups. On both days 7 and 10 however, CD8+ T-cells incorporated BrdU more vigorously (>3-fold higher) in the D-1MT group demonstrating that CD8+ T-effectors continued to proliferate in the tumors for at least until day 10. These data establish that the diminished T-suppressor cell expansion correlated with extended CD8+ T-cell activity.
Several studies showed a correlation between intratumoral CD8+ T-cell to CD4+ Foxp3+ T-cell ratio and tumor progression (29, 30). Since D-1MT administration resulted in reduced T-suppressor cell numbers and enhanced CD8+ T-cell proliferation, the BrdU+ CD8+ T-cell to CD4+ Foxp3+ T-cell ratio was evaluated as a potential prognostic marker for tumor regression. To this end, the ratios were determined between days 0 and 10 in the TDLN and the tumors of control (IL-12/GM-CSF alone) and D-1MT (IL-12/GM-CSF + D-1MT) groups. These data are presented in Figure 5A. The results show that treatment did not significantly change the ratio in control mice between days 0 and 7 in the TDLN or tumors. On day 10 however, the ratio declined by 2-fold suggestive of the development of a more immune suppressive microenvironment. In contrast, the ratio of actively-dividing CD8+ T-cells to T-suppressor cells increased dramatically in both the TDLN and the tumors on days 3 and 7 (2 to 3-fold in the TDLN and 6 to 15-fold in the tumor compared to day 0) in the D-1MT-treated group. Although the ratio declined in both microenvironments on day 10 it still remained above pre-therapy levels.
The absolute values for total or proliferating CD8+ T-cell to CD4+ Foxp3+ T-suppressor cell ratios in tumors were plotted against tumor weight to determine whether the ratio correlated with tumor size. More specifically, the correlation coefficients (r-values) were determined in control and D-1MT groups on days 3, 7 and 10 (Supplemental Figure 3). Of these, the r-values for proliferating CD8+ T-cell to CD4+ Foxp3+ T-cell ratio on days 3 and 7 were most predictive. Data for day 7 tumors with or without D-1MT are shown in Figure 5B. The correlation between BrdU+ CD8+ T-cell to CD4+ Foxp3+ ratio and tumor suppression was significant in both groups with the D-1MT group displaying stronger linkage. In contrast, total CD8+ T-cell to CD4+ Foxp3+ T-cell ratio was found to be less predictive (Table 1).
Since IL-12 and GM-CSF were found to be synergistic in mediating tumor regression in previous studies (19) we wanted to determine their respective roles in the observed feedback inhibition. Combined administration of IL-12 + GM-CSF promoted 5.1 ± 0.45 and 5.2 ± 1.01-fold increases in intratumoral IFNγ and IDO-1 mRNA on day 1, respectively. IL-12 alone resulted in similar increases in both IFNγ (4.5 ± 1.27-fold) and IDO-1 (3.1 ± 0.2-fold) expression. In contrast, GM-CSF alone did not promote any significant increases in IFNγ (1.3 ± 0.04-fold) or IDO-1 (0.7 ± 0.17) expression (n = 3–4 mice per group). With regard to the T-suppressor cell rebound between days 3 and 7, combination, IL-12-alone, GM-CSF-alone and control (blank microsphere) groups demonstrated 13.9 ± 4.7, 6.0 ± 0.2, 2.5 ± 0.6 and 2.3 ± 0.4-fold increases in T-suppressor cell numbers, respectively. These data demonstrate that T-suppresssor cell expansion was driven by IL-12. GM-CSF appeared to contribute to the T-suppressor cell rebound when administered with IL-12, but had no effect when injected alone.
The above studies demonstrate that the pro-inflammatory activity of IL-12 is regulated via a feedback inhibitory mechanism involving the IFNγ-IDO-T-suppressor cell axis. Our results also establish that blocking of IDO activity with D-1MT can be effectively employed to overcome feedback inhibition and enhance IL-12-mediated antitumor responses. Finally, the above data identify the proliferating CD8+ T-cell to CD4+ Foxp3+ T-cell ratio in tumors as a highly accurate prognostic marker of therapeutic efficacy.
Numerous clinical studies have demonstrated the tachyphylactic nature of IL-12 therapy (31–34). However, the mechanism underlying the progressive loss of therapeutic efficacy that accompanies repeated IL-12 administration has not been defined. Our findings, for the first time, demonstrate that IDO activation is, at least in part, responsible for this effect. Whether suppression occurred primarily via T-suppressor cell activity (26, 27) or also involved direct inhibition by tryptophan catabolites (35, 36) was not investigated. In our model, induction of IDO-1 in the tumor microenvironment was rapid and transient with no change in intratumoral IDO-2. These data, combined with our previous finding that tumor-resident CD8+ Tem display full-effector function during this interval (10), are inconsistent with a direct role for tryptophan catabolites in T-effector inhibition. To this end, it was recently reported that IDO-1 overexpression does not inhibit the effector function of pre-existing Tem (36). In contrast, the kinetics of intratumoral T-suppressor cell expansion between days 3 and 10 closely paralleled the loss of post-therapy CD8+ T-effector cytotoxicity in tumors (10). This finding, combined with the well-known ability of T-suppressor cells to inhibit CD8+ T-cell cytotoxicity in vivo (37), suggest that the inhibitory activity of IDO was mediated indirectly via T-suppressor cells.
The sources of post-treatment IDO-1 and IDO-2 were not investigated in this study. Numerous reports have established that plasmacytoid Dendritic Cells (DC), conventional tolerogenic DC and Macrophages are the primary producers of IDO-1 (38–40). IDO-2, on the other hand, is expressed in the kidney, testis and liver, but has also been detected in dendritic cells (28). Our preliminary findings are consistent with the hypothesis that DC are the primary source of IDO-1 and IDO-2 in the TDLN of post-IL-12 mice (Harden and Egilmez, unpublished data). The mechanistic basis of the differential IDO-1 and IDO-2 expression patterns in post-treatment tumors and TDLN, on the other hand, remain undefined. In the case of IDO-1, treatment-induced migration of DC from tumors to TDLN (41) can potentially account for the initial intratumoral spike and the subsequent upregulation and persistence of IDO-1 in the TDLN. The restriction of IDO-2 expression to the TDLN on the other hand, is more difficult to explain. One potential mechanism would involve selective infiltration of tumors and the TDLN by DC subsets with differential IFNγ responsiveness and/or IDO-1 or 2 expression profiles (42).
The finding that D-1MT was more efficient than L-1MT in achieving complete tumor eradication in IL-12/GM-CSF-treated mice is consistent with others' findings in different tumor models (18). The mechanistic basis of the in vivo superiority of D-1MT to L-1MT in promoting tumor suppression is controversial (18, 43, 44). In our study, treatment promoted both IDO-1 and IDO-2 expression in the TDLN demonstrating that the differential antitumor efficacy of the 1-MT isomers was not due to selective expression of the target enzymes in post-treatment TDLN. On the other hand, whether IDO-1 and IDO-2 differ in their abilities to induce T-suppressor cell activation/expansion, and whether they are expressed by different tolerogenic DC subsets is not known. Analysis of post-therapy TDLN DC subsets with regard to IDO-1 and 2 expression and defining their relative abilities to activate/expand T-suppressor cells may shed further light on the respective roles of each enzyme in the development of post-therapy regulatory rebound.
The above results suggest that selective blocking of IFNγ-mediated regulatory pathways can overcome IL-12 tachyphylaxis. Whereas repeated activation of tumor-resident T-effector/memory cells via this strategy represents a potentially effective therapeutic approach, its utility may still be limited by T-cell intrinsic regulatory mechanisms (9) and/or the finite clonal proliferative potential of cytotoxic CD8+ T-cells (45). We are currently investigating the limits of this strategy in a model that allows long-term quantitative monitoring of tumor-specific CD8+ T-effector cell activity.
We thank Dr. Stanley Wolf of Wyeth, Inc. for providing the murine IL-12 and his continued support of our studies.
This work was supported by the NIH grant R01-CA100656-01A1 and the NY State Office of Science Technology and Academic Research (NYSTAR) faculty recruitment award C040070 to NKE