Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8+ T cells directly within the tumor microenvironment
© Spranger et al.; licensee BioMed Central Ltd. 2014
Received: 29 October 2013
Accepted: 22 January 2014
Published: 18 February 2014
Blockade of immune inhibitory pathways is emerging as an important therapeutic modality for the treatment of cancer. Single agent treatments have partial anti-tumor activity in preclinical models and in human cancer patients. Inasmuch as the tumor microenvironment shows evidence of multiple immune inhibitory mechanisms present concurrently, it has been reasoned that combination therapies may be required for optimal therapeutic effect.
To test this notion, we utilized permutations of anti-CTLA-4 mAb, anti-PD-L1 mAb, and/or the IDO inhibitor INCB23843 in the murine B16.SIY melanoma model.
All three combinations showed markedly improved tumor control over single treatments, with many mice achieving complete tumor rejection. This effect was seen in the absence of vaccination or adoptive T cell therapy. The mechanism of synergy was investigated to examine the priming versus effector phase of the anti-tumor immune response. Only a minimal increase in priming of anti-tumor T cells was observed at early time points in the tumor-draining lymph nodes (TdLN). In contrast, as early as three days after therapy initiation, a marked increase in the capacity of tumor-infiltrating CD8+ T cells to produce IL-2 and to proliferate was found in all groups treated with the effective combinations. Treatment of mice with FTY720 to block new T cell trafficking from secondary lymphoid structures still enabled restoration of IL-2 production and proliferation by intratumoral T cells, and also retained most of the tumor growth control.
Our data suggest that the therapeutic effect of these immunotherapies was mainly mediated through direct reactivation of T cells in situ. These three combinations are attractive to pursue clinically, and the ability of intratumoral CD8+ T cells to produce IL-2 and to proliferate could be an important biomarker to integrate into clinical studies.
KeywordsAnti-CLTA-4 PD-1/PD-L1 IDO inhibitor Combinatorial immunotherapy Tumor-infiltrating lymphocytes T cell anergy/exhaustion Tumor microenvironment Immune inhibitory pathways
Despite expression of numerous antigens, tumor evasion from host immunity still occurs. Over the past several years, a working model has emerged suggesting the existence of at least two major categories of immune resistance based on biologic features of the tumor microenvironment . One major subset shows infiltration with CD8+ T cells at baseline, along with a specific chemokine profile and a type I interferon (IFN) transcriptional signature, all indicative of active Th1-type inflammation. These tumors appear to resist the ongoing immune response through the dominant inhibitory effect of immune suppressive mechanisms. In contrast, the other major subset lacks these chemokines and T cell markers and appears to escape immune effects through immunologic ignorance or exclusion. With this as a working model, one might envision distinct immunotherapies being necessary for optimal therapeutic effects in these two patient groups. This conceptual framework is being integrated into biomarker development as novel immunotherapies are being explored in patients . While most of these analyses have been carried out on biopsy material from patients with advanced melanoma, similar results are emerging in other solid tumors, including lung cancer, ovarian cancer, colorectal cancer, breast cancer, and head and neck cancer [3–6].
Focusing in on the T cell “inflamed” tumor subset, at least four immune inhibitory mechanisms have been identified to be involved in human specimens and validated mechanistically in preclinical models. These are expression of the ligand PD-L1 (programmed death-ligand 1), which can engage the inhibitory receptor PD-1 (programmed death-receptor 1) on activated T cells; presence of the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO), which exploits the exquisite sensitivity of T cells to tryptophan depletion; infiltration with FoxP3+ regulatory T cells (Tregs), which can mediate extrinsic suppression of effector T cell function; and T cell-intrinsic anergy, characterized by defective IL-2 (interleukin-2) production and proliferation and driven in part through the transcription factor Egr2 (Early growth response protein 2) [6–11]. We recently have observed that increased expression of PD-L1, IDO, and FoxP3+ Tregs in the melanoma tumor microenvironment is driven by infiltrating CD8+ T cells, arguing that these mechanisms are part of an immune-intrinsic negative feedback loop . Thus, immunotherapies aiming to uncouple these pathways may be most effective in tumors showing the T cell-infiltrated phenotype, during the negative feedback phase of a chronic, smoldering immune response.
The first successful immunotherapy aiming to block a negative regulatory pathway on T cells is the anti-CTLA-4 (Cytotoxic T-Lymphocyte Antigen-4) mAb ipilimumab, which was approved by the FDA (Food and Drug Administration) in 2011 for treatment of patients with advanced melanoma . CTLA-4 is an inhibitory receptor also expressed by tumor-infiltrating T cells. This success has catalyzed extensive investigation into the potential for inhibitors of other immune suppressive pathways to have clinical activity in advanced cancer patients. Early clinical results have been reported with anti-PD-1 and anti-PD-L1 mAbs , IDO inhibitors , and Treg depletion targeting surface CD25 [16, 17]. Numerous additional immunotherapeutic agents are being developed that target a plethora of positive or negative immunoregulatory molecules. These include agonistic Abs against 4-1BB, Ox40, ICOS (Inducible T-cell COStimulator), and CD40; blocking Abs against LAG3 (Lymphocyte-activation gene 3), B7-H3, B7-H4, Tim3, and KIRs (killer inhibitory receptors); and the cytokines IL-7, IL-15, and IL-21 [18–26], among others. Based on the presumption that even the best of these agents will have modest single agent activity, and because of the complexity of immune regulation in vivo that involves networks of regulatory pathways, it is envisioned that combination immunotherapies ultimately will be required for maximal therapeutic benefit.
The prospect of investigating combination immunotherapy doublets in early phase clinical trials in an empiric fashion without solid mechanistic data seems daunting and perhaps impossible to pursue based on available patient resources. We recently have assembled a framework for logical combination of immunotherapeutic agents based on presumed modes of action . This framework provides a motivation for testing such logical combinations in suitable mouse preclinical models to aid in the prioritization of regimens for clinical development. Mouse models also facilitate elucidation of the mechanism by which two interventions may be synergistic.
In the current report, we have started with αCTLA-4 mAb as a successful backbone, along with αPD-L1 and IDO inhibition. As a tumor model, we utilized B16 melanoma, which does develop an “inflamed” tumor microenvironment at early time points that leads to accumulation of PD-L1, IDO, and Tregs over time . We found that doublets of αCTLA-4 +/− αPD-L1 +/− an IDO inhibitor each showed improved tumor control in vivo. Mechanistic studies revealed that the major biologic effect of the successful doublets was restoration of IL-2 production and proliferation by CD8+ T cells within the tumor microenvironment. Our results provide support for continued development of these combinations in patients, and also suggest a new predictive biomarker that should be integrated into clinical development.
Combinatorial blockade of CTLA-4, PD-L1 or IDO pathways results in improved tumor control in vivo
To assess if the anti-tumor effect was restricted to the presence of the model-antigen SIY we also evaluated all three double therapies using the parental cell line B16F10 (Additional file 2: Figure S2). Using the same dose of tumor cells as for B16.SIY we detected a significant delay in tumor outgrowth for all three double therapies compared to the no treatment control, although the effect was less dramatic with this less immunogenic variant. To investigate whether the doublet therapy would also mediated tumor control in larger more established tumors, we delayed the initiation of treatment until day 7. As depicted in Additional file 2: Figure S3, delayed treatment with αCTLA-4 + αPD-L1 also resulted in significant tumor control compared to the no treatment, although therapeutic efficacy was less potent than with earlier treatments. Together, these results suggest that combinatorial targeting of CTLA-4 +/− PD-L1 +/− IDO could translate into a therapeutic advantage in vivo.
Effective combination therapies do not substantially increase the frequency of anti-tumor CD8+ T cells in the tumor-draining lymph node at early time points
Recent studies have focused on the potential Treg-depleting effect of αCTLA-4 antibody therapy . To address this possibility in our model, we evaluated Treg frequencies using FoxP3 intracellular flow cytometry analysis in the lymphoid organs and within the tumor. However, neither on day 7 nor on day 14 were we able to detect a decrease in Treg frequency in any organ (Additional file 2: Figure S4, Additional file 1: Table S2). This difference might be explained by different αCTLA-4 mAb being used in various studies, and exclude a requirement for Treg depletion in the efficacy of these combination therapies in our model system.
Effective doublets result in increased frequency of IL-2-producing and proliferating polyfunctional T cells within the tumor
Increased frequency of polyfunctional T cells within the tumor does not require new T cell migration
Combinatorial treatments lead to prolonged persistence and higher frequency of tumor-reactive lymphocytes in the periphery at later time points
Our current work has indicated that doublet therapies using either αCTLA-4, αPD-L1 and/or IDOi show a synergistic retardation of tumor outgrowth in vivo. The major biologic correlate to this improved efficacy was restored IL-2 production and proliferation of tumor-infiltrating CD8+ T cells. In addition, this functional restoration did not require new T cell migration as assessed using FTY720 administration. Together, these data suggest that successful combination immunotherapies function, at least in part, by correcting functional defects of T cells directly within the tumor microenvironment.
It is noteworthy that CD8+ TILs (tumor-infiltrating lymphocytes) without any therapy showed significant production of IFN-γ production when analyzed ex vivo. Consistent with this observation, human melanoma metastases showing a T cell-infiltrated phenotype usually show expression of IFN-γ-induced target genes and in many cases IFN-γ itself . Recently, we have observed that IFN-γ produced by CD8+ T cells is necessary for the induction of the negative regulatory factors PD-L1 and IDO within the tumor microenvironment . Our current data indicate that IDO1 is further upregulated upon blockade of PD-L1/PD-1 interactions. Thus, the retained ability of TIL to produce at least some IFN-γ may in fact contribute to the negative regulatory network within the tumor site that enable tumor immune evasion.
Although several previous studies have been conducted to investigate if combining blockade of two inhibitory pathways could act synergistically in retarding the tumor growth in vivo, only limited studies have addressed the mechanism leading to this effect [30, 33, 42, 43]. In particular the recently published studies by Duraiswamy and colleagues (αCTLA-4 + αPD-L1) and Holmgaard et al. (αCTLA-4 + IDOi) indicated that proliferation of TIL in combination with increased functional capacity (IFN-γ, granzyme B) were increased with effective combinations [44, 45]. Taken together, these collective results converge on the notion that restored function of existing TIL may be a critical mechanism of action of these interventions. Presence of polyfunctional T cells has been described to be associated with improved anti-tumor immunity in preclinical mouse models and also in patients [37, 38, 46]. It is not yet known if other immunotherapy doublets will similarly involve reactivation of T cells only within the tumor microenvironment. Nonetheless, the data presented here show a striking importance of TIL reactivation mediated by the doublet therapies we have tested. The mechanism leading to this could be compensatory immune regulation by a second pathway when only one pathway is inhibited.
Recent studies have suggested that αCTLA-4 antibody could lead to depletion of regulatory T cells selectively within the tumor microenvironment [36, 47]. In our own hands, we were not able to detect diminished Tregs after αCTLA4 mAb either alone or in combination, either measuring percentage or absolute number in the tumor site. The reason for this discrepancy is not clear, but could be related to the use of distinct mAbs against CTLA-4. While studies by Allison et al. were performed with the 9D9 mAb (mouse IgG2b), our study was conducted with the 4 F10 mAb (hamster IgG1). Further work will be required to determine under what circumstances Treg depletion may be functionally relevant with αCTLA-4 agents. Our own data suggest that it is not mandatory for αCTLA-4 to deplete intratumoral Tregs in order to have therapeutic benefit in vivo.
Our experiments with FTY720 suggest that new T cell migration is not required in order to improve CD8+ T cell function in response to immunotherapy combinations in vivo. However, it is clear that successful immunotherapies trigger tumor cell death, renewed inflammation in the tumor site, antigen cross-presentation, and epitope spreading , ultimately leading to increased T cell infiltration in the tumor microenvironment. This entire cascade of events may be required for optimal tumor elimination. Indeed, our experiments of FTY720 administration at later time points showed a modest effect on tumor growth control. However, our results demonstrate that new T cell migration is not required for the early improved TIL function observed as well as for maintaining a steady-state control of the tumor outgrowth. These data have limitations, in that approximately 10% of peripheral blood T cells were still present after FTY720 treatment, which could potentially contribute to tumor infiltration. However, an important contribution of new migration in the presence of FTY720 would likely have been associated with a similar 90% reduction of T cell numbers in the tumor which is indicated by the blunted tumor control when FTY720 is administered on day −1. In addition, our direct in vivo analysis of TIL proliferation using BrdU administration support the notion of rapid restoration of TIL proliferation in situ.
In total, the results presented herein suggest that doublet combinations of anti-CTLA-4, anti-PD-1/PD-L1, or IDO inhibition are attractive for clinical translation. Phase I clinical trial development of the anti-CTLA-4 + anti-PD-1 combination has already been pursued in advanced melanoma, with profound clinical response rates observed that appear superior to single agent activity based on historical controls [47, 49]. Development of the other combinations described herein may have a similarly strong rationale for clinical testing. Our data also suggest that proliferation and/or IL-2 production by CD8+ TIL should be considered as a pharmacodynamic biomarker for clinical response to these combinations.
Mice and tumor inoculation
C57BL/6 mice were purchased from Taconic Farms and were maintained according to National Institute of Health Animal Care guidelines. Ethical approval was obtained by the Council for Animal Research at The University of Chicago and followed international guidelines. B16F10 and B16-dsRed-SIY tumor cells  were maintained as previously described and B16-dsRed-SIY will be specified as B16.SIY throughout the paper. On day 0 of the experiments, 2×106 B16-dsRed-SIY cells were inoculated subcutaneously into the flank of the mice. Mice with same age and gender were used as controls.
Treatment was initiated on day 4 post tumor inoculation with the following regimens for each drug (illustrated in Figure 1A). αCTLA-4 antibody (clone UC10-4 F10-11, Bio-X-Cell) was given i.p. on day 4, 7 and 10 at a dose of 100 μg/mouse. αPD-L1 antibody (clone 10 F.9G2, Bio-X-Cell) was given i.p. (100 μg/mouse) every other day starting on day 4 ending on day 16 post tumor inoculation. IDOi (INCB23843, Incyte Corporation) was dissolved in 0.5% methylcellulose and administered at 300 mg/kg po QD on a 5 days on/2 days off schedule starting on day 4 [35, 51]. In the case of functional experiments with an earlier endpoint, treatment regimens were carried out as described until the day of T cell analysis. For delayed therapy the same treatment scheme was applied starting on day 7 post-tumor inoculation, when tumors were palpable.
Flow cytometry and antibodies
For flow cytometric analysis, spleen, TdLN, and tumor tissues were harvested at the indicated time point or when tumors reached a volume of 200 mm2. Single cell suspensions were prepared and a Ficoll-Hypaque purification step was performed for the tumor-derived cell suspension. Following a washing step, approximately 2×106 cells were used for antibody staining. Antibodies against the following molecules were used throughout the paper if not otherwise indicated: CD3 (AX700, 17A2, eBioscience), CD4 (PerCP-Cy5.5, RM4-5, Biolegend), CD8 (APCCy7, 53–6.7, Biolegend), FoxP3 (APC, FJK-16a, eBioscience), IL-2 (PerCP, JES6-5H4, eBioscience), IFN-γ (APC, XMG1.2, eBioscience), and TNF-α (FITC, MP6-XT22, eBioscience). Fixable life/dead cell discrimination was performed using Fixable Viability Dye 450 or 506 (eBioscience). Staining was carried out at RT for 30 min if not indicated differently and intracellular staining was performed using the FoxP3-staining kit according to manufacturer’s instructions (BD).
Staining of SIY-specific cells was performed using the SIYRYYGL-pentamer (Proimmune), conjugated with Phycoerythrin (PE), or as a non-specific control with the SIINFEKL-pentamer. For staining, pentamers were diluted 1:50 in PBS + 10% FCS and incubated for 20 minutes at room temperature (RT). Following a washing step, cells were stained with specific antibodies for 30 minutes on ice prior to fixation in 4% PFA. All flow cytometric analyses were done using an LSR II blue instrument (BD) and analyzed using FlowJo software (Tree Star).
Splenocytes from naïve, tumor-challenged non-treated or treated mice were harvested on day 7 or day 14 after tumor inoculation. Single cell suspensions were prepared and 1×106 splenocytes were assayed per well. Cells were either left un-stimulated or stimulated with 160nM SIY-peptide (SIYRYYGL) or PMA 100 ng/ml and Ionomycin 1 μg/ml as positive control. After a 24 h culture period, detection of INF-γ production was performed according to manufacturer’s instructions.
Quantitative RT-PCR analysis
Tumor was harvested on day 16 and a single cell suspension was prepared. Approximately 107 cells were used for RNA isolation using Qiagen RNAeasy extraction kit according to manufactured instruction. Following cDNA was prepared using Reverse Transcriptase kit (Manufactures instructions; Applied Biosciences). Expression levels of transcripts were analyzed using primer-probe sets specific for IDO1 and PD-L1 and values were normalized against the expression level of 18S (Roche).
Ex vivo T cell functional assays
Single cell suspensions from tumor, spleen, and TdLN were prepared as described above. Cell numbers were determined and cells were labeled with Cell Trace (BD) according to manufacturer’s instructions. A maximum of 1×106 cells was plated per well on either non-treated or anti-CD3 mAb-coated plates. Anti-CD3 mAb coating was performed with a solution of 10 μg/ml αCD3 antibody (145-2C11, Biolegend) in PBS, incubated overnight at 4°C. Following 48 h of incubation, cells were harvested and transferred onto newly anti-CD3-coated or non-treated plates, along with anti-CD28 mAb (2 μg/ml) (EL-4, Biolegend). Medium for all wells included 5 μg/ml BrefeldinA (Sigma). Following a 6 h-incubation at 37°C, cells were harvested and stained for surface markers and intracellular cytokines using the technique described above.
Treatment with FTY720
Prior to the initiation of the therapy regimens (2.5 h pre-treatment), fingolimod (FTY720, Enzo Life Sciences) was given to mice to inhibit lymphocyte migration out of lymphoid organs . FTY720 stock solution (10mg/ml in DMSO) was diluted to a 125μg/ml concentration in PBS directly before administration. Mice received a dose of 25μg FTY720 or PBS containing DMSO as control via oral gavage. Therapy was initiated the same day (2.5h delayed) and mice were analyzed on day 7 to perform the ex vivo functional assay as described above. For long-term FTY720 administration, the dose was reduced to 5μg per mouse per day and was given daily throughout the experiment (day-1 FTY720 was given 24h prior to tumor inoculation; day 4 FTY720 was given 2.5h prior to therapy; day 10 FTY720 was given 2.5h after last dose of αCTLA-4 mAb). Depletion of peripheral lymphocytes was assessed on the endpoint of each group and was always detected to be greater than 90% depletion.
In vivo proliferation assay
Assessment of in vivo proliferation was performed by BrdU pulse in vivo, 24 h prior to flow cytometric analysis. Each mouse received 0.8 mg BrdU in 100 μl injected i.p. either on day 6 or day 13 of the treatment protocol. Mice were analyzed on day 7 or day 14, respectively, and cells were prepared for flow cytometry as described above. Following surface staining, cells were fixed using the FoxP3 staining kit (BD). After the 30 minute fixation period, cells were incubated in 100 μl of PBS/DNase solution (300 μg/ml) for 30 minutes at 37°C. Cells were then washed and incubated for 30 minutes at RT with antibodies for FoxP3 and BrdU (FITC, Bu20a, eBioscience) followed by flow cytometric analysis.
Cytotoxic T-Lymphocyte Antigen-4
Programmed death-ligand 1
Programmed death-receptor 1
Peridinin chlorophyll protein complex
Tumor-draining lymph node
Tumor necrosis factor alpha
Regulatory T cells.
The authors thank Michael Leung for technical support, and Dr. Marisa Alegre for helpful comments. This work was supported by P01 CA97296 and funding from Incyte Cooperation. SS is a postdoctoral fellow of the German Research Association (DFG).
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