Platinum-based drugs disrupt STAT6-mediated suppression of immune responses against cancer in humans and mice

Tumor microenvironments feature immune inhibitory mechanisms that prevent T cells from generating effective antitumor immune responses. Therapeutic interventions aimed at disrupting these inhibitory mechanisms have been shown to enhance antitumor immunity, but they lack direct cytotoxic effects. Here, we investigated the effect of cytotoxic cancer chemotherapeutics on immune inhibitory pathways. We observed that exposure to platinum-based chemotherapeutics markedly reduced expression of the T cell inhibitory molecule programmed death receptor-ligand 2 (PD-L2) on both human DCs and human tumor cells. Downregulation of PD-L2 resulted in enhanced antigen-specific proliferation and Th1 cytokine secretion as well as enhanced recognition of tumor cells by T cells. Further analysis revealed that STAT6 controlled downregulation of PD-L2. Consistent with these data, patients with STAT6-expressing head and neck cancer displayed enhanced recurrence-free survival upon treatment with cisplatin-based chemoradiation compared with patients with STAT6-negative tumors, demonstrating the clinical relevance of platinum-induced STAT6 modulation. We therefore conclude that platinum-based anticancer drugs can enhance the immunostimulatory potential of DCs and decrease the immunosuppressive capability of tumor cells. This dual action of platinum compounds may extend their therapeutic application in cancer patients and provides a rationale for their use in combination with immunostimulatory compounds.

Recently, it has become evident that chemotherapeutic drugs not only have a direct cytotoxic effect on tumor cells but may also potentiate the immune system via so-called off-target effects (1, 2). For example, direct immune-activating effects of cancer chemotherapy have been observed: treatment with low concentrations of several chemotherapeutics resulted in increased antigen cross-presentation, T lymphocyte expansion, and T cell infiltration of tumors by to-date-unknown molecular mechanisms (3, 4). In addition, the cytotoxic drug oxaliplatin was shown to induce an immunogenic type of cell death in a TLR4/high-mobility group box 1–dependent manner (5, 6). However, the effect of treatment with cancer chemotherapeutics on immune inhibitory pathways that play a crucial role in tumor immune escape is unknown (7).

We hypothesized that anticancer chemotherapeutics may influence the type of T cell–mediated immune responses induced by DCs by acting on immune inhibitory pathways. Here we demonstrate that platinum-based chemotherapeutics downregulate the inhibitory molecule programmed death receptor-ligand 2 (PD-L2) in a STAT6-dependent manner, both on DCs and on tumor cells, resulting in enhanced T cell activation and increased tumor cell recognition. The clinical relevance of these observations is underpinned by our finding in head and neck cancer patients that tumor STAT6 expression is correlated with an improved clinical outcome when these patients received platinum-based radiochemotherapy, but not when these patients were treated with radiotherapy only. These findings provide a rationale for the use of platinum-based chemotherapy in combination with other immunostimulatory compounds to increase the clinical efficacy of cancer treatment.

The T cell–stimulating potential of DCs is enhanced by platinum-based chemotherapy. We first determined the effect of a large number of clinically relevant concentrations of chemotherapeutic agents on the allogeneic T cell stimulatory potential of monocyte-derived DCs (Supplemental Table 1; supplemental material available online with this article; doi: 10.1172/JCI43656DS1). Surprisingly, DCs exposed to carboplatin during their maturation by cytokines induced a significantly higher T cell proliferation compared with all other chemotherapeutics tested (Figure 1A). We also observed this similar enhanced immune-stimulating activity in a dose-dependent manner for the platinum compounds oxaliplatin and cisplatin, which are frequently used in the clinic (Figure 1, B and C, and Supplemental Table 1). T cells that were activated by DCs exposed to platinum compounds produced significantly higher levels of IFN-γ and IL-2 compared with untreated DCs (Figure 1, D and E).

Figure 1

DCs exposed to platinum-based chemotherapy display enhanced allogeneic T cell stimulatory capacity. (A) DCs were cultured in clinically relevant concentrations of chemotherapeutics during 48 hours of cytokine maturation and subsequently used in a mixed lymphocyte reaction with allogeneic PBMCs. T cell proliferation was measured by ³H-thymidine incorporation after 5 days. Ctrl, control DCs (nontreated); MTX, methotrexate; Cyta, cytarabine; Vinc, vincristine; Irino, irinotecan; Eto, etoposide; Bleo, bleomycin; DTIC, dacarbazine; Gem, gemcitabine; 5-FU, 5-fluorouracil; Doxo, doxorubicin; Lena, lenalidomide; Carb, carboplatin. For all proliferation experiments, the statistical analyses were performed against the control unless otherwise specified in the figure; the mean of 6 replicates per experiment and the SEM are depicted. A representative experiment is shown. n = 3. **P < 0.01; ***P < 0.001. (B) Same experiment with different platinum-based chemotherapeutics. Oxali, oxaliplatin; cis, cisplatin. A representative experiment is shown. n = 3. (C) Concentration-dependent effect of platinum exposure on T cell stimulatory potential of DCs. This effect was observed irrespective of whether the platinum drugs were added during or after DC maturation (data not shown). Oxaliplatin concentrations 0.5, 2.0, 3.0, and 4.0 μg/ml. A representative experiment is shown. n = 5. (D and E) IFN-γ and IL-2 secretion by T cells in MLR upon exposure to DCs cultured with or without platinum. A representative experiment is shown. n = 3. In the IL-2 experiment, the P value was 0.07, but the difference may be important in a microenvironment of marked T cell proliferation and hence increased IL-2 consumption. Data are depicted as mean + SEM.

Notably, this increased T cell–stimulating activity of platinum-treated DCs was also observed in antigen-specific T cells using keyhole limpet hemocyanin (KLH) as a model antigen (Figure 2A). Moreover, we observed that the T cell stimulatory capacity of DCs matured in vitro through TLRs instead of cytokines was also further enhanced by platinum exposure (Figure 2A). The observed effect on TLR-DCs was not only limited to enhanced T cell proliferation but also supported by abundant IFN-γ production by these antigen-specific T cells (Figure 2B). Although the platinum-induced immunogenic effect is more pronounced in TLR ligand–matured DCs, the background T cell proliferation and IFN-γ production is also more elevated when DCs were activated with TLR ligands. Therefore, we conclude that the observed effect of platinum compounds is independent from stimuli used to activate and/or mature DCs.

Figure 2

Enhanced T cell stimulatory potential of DCs matured with cytokines or TLR ligands in the presence of platinum chemotherapy in an antigen-specific model. (A) KLH-specific T cells were cocultured with KLH-loaded DCs, matured with cytokines or R848 and poly(I:C), in the presence or absence of platinum chemotherapy. cDC, cytokine-matured DCs; TLR-DC, TLR ligand–matured DCs; Ox, oxaliplatin. A representative experiment is shown. n = 3. (B) Enhanced IFN-γ secretion by T cells in MLR upon exposure to TLR ligand–matured DCs cultured with platinum as compared with platinum-untreated DCs. A representative experiment is shown. n = 3. *P < 0.05; **P < 0.01; ***P < 0.001. Data are depicted as mean + SEM.

The enhanced immunogenicity of platinum-treated DCs is dependent on PD-L2/STAT6. To investigate why platinum-treated DCs display enhanced immunogenicity, we determined their phenotype (Figure 3A, shows effects of oxaliplatin; similar results were obtained with carboplatin and cisplatin, not shown). No significant differences were observed in expression of MHC class I and II or the costimulatory molecules CD80 and CD86 on platinum-treated versus nontreated DCs. Although we detected fluctuations in pro- and antiinflammatory cytokine production of DCs matured in the presence of platinum compounds (Figure 3, B and C, and Supplemental Figure 1), these differences were very small and not consistent for all platinum compounds, and they did not correlate with enhanced T cell activation. We therefore concluded that the enhanced immunogenicity is not caused by increased expression of MHC or costimulatory molecules or by increased proinflammatory cytokine secretion upon platinum treatment.

Figure 3

Platinum compounds do not alter DC costimulatory molecule expression or cytokine secretion. (A) Expression of MHC class I and II, costimulatory molecules CD80, CD83, and CD86 by DCs exposed to 2.0 μg/ml oxaliplatin during maturation as compared with untreated mature DCs.The MFIs of positive cells are shown as compared with platinum-untreated cells with SEMs, for 3 independent experiments for HLA class I and II and for 5 independent experiments for CD80, CD83, and CD86. (B and C) Production of proinflammatory cytokines TNF-α and IL-8 by DCs exposed to platinum chemotherapy during maturation. Supernatants were harvested 24 hours after stimulation. Means with SEMs of 3 separate experiments are shown. *P < 0.05; **P < 0.01. Data are depicted as mean + SEM.

Since DCs can also express several T cell inhibitory molecules, including PD-L1 and PD-L2, the enhanced T cell stimulatory potential upon platinum exposure could result from decreased PD-L expression. Indeed, treatment with oxaliplatin, cisplatin, or carboplatin during DC maturation moderately reduced the expression of PD-L1 and profoundly reduced the expression of PD-L2 (Figure 4A). We did not observe these effects with other tested chemotherapeutic agents. Expression of other known inhibitory receptors remained unaltered upon platinum treatment (Figure 4B). Blocking of the PD-1/PD-L1/PD-L2 axis by anti–PD-L1 and –PD-L2 antibodies enhanced the capacity of mature DCs to stimulate T cells to a similar level when compared with platinum-treated DCs. However, the increased allostimulatory potential of DCs upon platinum treatment was abolished in the presence of PD-L2 antibodies (Figure 4C). This strongly supports the notion that downregulation of PD-L1, and particularly PD-L2, is a major cause of the enhanced T cell stimulatory capacity of platinum-treated DCs.

Figure 4

Platinum-based chemotherapeutics downregulate PD-L2 expression on DCs, resulting in enhanced T cell activation. (A and B) Expression of T cell inhibitory molecules PD-L1 and PD-L2 (light gray, isotype; white, untreated DC; dark gray, platinum-treated DC) and B7-H2/H3/H4 and IDO by DCs exposed to platinum chemotherapy during maturation. A representative experiment is shown. n > 5 for PD-Ls; n = 2 for other inhibitory molecules. (C) MLR with DCs matured in the presence (black bars) or absence (white bars) of 5 μg/ml oxaliplatin in the presence of blocking antibodies against PD-L1, PD-L2, or control immunoglobulin. ^#P < 0.05, compared with IgG control DCs; **P < 0.01; ***P < 0.001. A representative experiment is shown. n = 3. Data are depicted as mean + SEM.

The most important regulator of PD-L2 is the molecule STAT6. Macrophages from Stat6^–/– mice are virtually unable to express PD-L2 (8). DCs from NF-κB p50^–/–p65^–/+ mice also have defective PD-L2 upregulation upon activation (9). Therefore, we measured STAT6 and phosphorylated (active) STAT6 expression in DCs treated with or without platinum chemotherapy. We observed that treatment of DCs with platinum compounds resulted in a strong decrease in phosphorylated STAT6 (Figure 5A). To investigate whether the STAT6 dephosphorylation was the cause of the observed enhanced immunogenicity of DCs upon platinum exposure, we tested the T cell stimulatory activity of platinum-treated DCs exploiting siRNA against STAT6 (Figure 5B and Supplemental Figure 2). Despite the fact that inhibition was not complete, Stat6 knockdown DCs now lacked the previously observed enhanced T cell response upon platinum treatment, whereas DCs transfected with control siRNA still exhibited that response. To confirm that platinum-induced Stat6 dephosphorylation indeed mediated the observed PD-L2 downregulation, we cultured bone marrow–derived DCs from Stat6^–/– and wild-type BALB/C mice in the absence or presence of platinum chemotherapeutics (Figure 5C). We found that PD-L2 was significantly more downregulated upon platinum exposure in wild-type DCs when compared with Stat6^–/– DCs. Together, these data provide evidence that the enhanced immunogenicity of platinum-treated DCs depends on inactivation of STAT6 resulting in downregulation of PD-L2.

Figure 5

Enhanced T cell activation by DCs upon platinum exposure is mediated through STAT6 dephosphorylation. (A) Western blot analysis of cytokine-matured DCs treated for 24 hours with 5 or 7.5 μg/ml cisplatin or oxaliplatin or medium during maturation. Blot lanes were run on the same gel but were noncontiguous (white line). (B) MLR with DCs that were transfected with STAT6 siRNA or control siRNA and subsequently matured with or without 5 μg/ml oxaliplatin. A representative experiment is shown. n = 3. (C) PD-L2 expression of BM-derived CD11c⁺ IL-4/LPS–stimulated DCs from wild-type and Stat6^–/– BALB/c mice, cultured in the presence or absence of cisplatin. Depicted are the normalized MFI and SEM of 1 of 2 independent experiments performed in triplicate. *P < 0.05; ***P < 0.001.

Platinum dephosphorylates STAT6 in tumor cells, resulting in decreased PD-L2 and subsequent increased T cell recognition. Since many cancer cells are capable of inducing an immunosuppressive microenvironment and upregulating PD-Ls in order to evade antitumor immunity (10), we next tested to determine whether the same platinum compounds also affected PD-L expression on tumor cells. To mimic an immunosuppressive microenvironment in vitro, we exposed BLM melanoma cells to cytokine combinations known to induce PD-L1/2 expression on endothelial cells, i.e., IL-4 together with IFN-γ, LPS, or TNF-α (11). Subsequent exposure to platinum compounds clearly reversed PD-L2 expression, but not PD-L1 expression (Figure 6A and Supplemental Figure 3).

Figure 6

Platinum-based chemotherapeutics downregulate PD-L2 on tumor cells via STAT6 dephosphorylation, resulting in enhanced tumor cell recognition by tumor antigen–specific T cells. (A) PD-L1 and PD-L2 expression by BLM melanoma cells cultured in IL-4 and IFN-γ with or without platinum chemotherapy for 24 hours. cis, 10 μg/ml cisplatin. A representative experiment is shown. n = 5. (B) Western blot of BLM melanoma cells treated with or without IL-4, IFN-γ, LPS, TNF-α, and 20 μg/ml cisplatin for 8 hours, as indicated. A representative experiment is shown. n = 5. (C) IFN-γ production of gp100-specific T cells upon coculture with gp100-expressing BLM melanoma cells, preincubated with or without 10 μg/ml cisplatin for 24 hours. A representative experiment is shown. n = 3. **P < 0.01. Data are depicted as mean + SEM.

Because PD-L2 is regulated by STAT6 and NF-κB (8, 9, 12), we measured protein levels of STAT6 and phosphorylated (active) STAT6 in BLM tumor cells upon coculture with IL-4 and several NF-κB–activating stimuli (Figure 6B and Supplemental Figure 4). We found that cisplatin caused a strong dephosphorylation of STAT6. Although several STAT molecules are constitutively expressed in multiple types of cancer and play an important role in the proliferative capacity and antiapoptotic and immune-evasive potential of cancer cells (13), a direct STAT-inactivating mechanism in cancer cells induced by clinically used cytotoxic chemotherapy has not been reported before.

To further explore whether chemotherapy-induced reversal of an immunosuppressive tumor microenvironment not only reduces PD-L2, but also effectively enhances susceptibility of tumor cells to cytotoxic T cells, we compared the recognition of platinum-treated and nontreated gp100 antigen expressing melanoma cells by gp100-specific T cells (14). As expected, we observed that platinum-induced reduction of PD-L2 coincides with enhanced T cell recognition as measured by antigen-specific IFN-γ production (Figure 6C). Together, these findings indicate that platinum compounds not only directly affect immune cells, but also modulate an immunosuppressive microenvironment by dephosphorylating STAT6 in tumors, resulting in downregulation of PD-L2 and subsequent enhanced tumor cell recognition by cytotoxic T lymphocytes.

Tumor STAT6 dictates response to platinum-based cancer treatment. Finally, to verify the clinical relevance of platinum-induced STAT6 modulation in tumor cells in cancer patients, we analyzed tumor STAT6 expression in a cohort of patients with squamous cell carcinoma of the head and neck that had been treated in our institute with cisplatin in combination with radiotherapy (Figure 7, A and B, and Supplemental Table 2). We could exclude a possible effect of radiotherapy on STAT6 activation by in vitro analysis of IL-4–induced STAT6 phosphorylation in tumor cells, either irradiated or not (Supplemental Figure 5). After a median follow-up of 68 months, we observed a clinically highly relevant and statistically significant difference between patients with STAT6-negative tumors and STAT6-positive tumors with a 3-year recurrence-free survival of 48% and 80%, respectively (Figure 7C). In a matched cohort of patients that had been treated during that same period with radiotherapy only, tumor STAT6 expression was not correlated with an improved clinical outcome (Figure 7D). Rather, there was a clear trend toward a shorter progression-free survival. This could be explained by the immune-evasive potential of STAT6-expressing tumor cells, if not attacked by platinum-based chemotherapy. These data demonstrate the significance of platinum-induced STAT6 modulation in the tumor microenvironment in cancer patients.

Figure 7

Tumor STAT6 dictates clinical response to platinum-based therapy in cancer patients. (A) STAT6 expression by squamous cell carcinoma of the head and neck. (B) STAT6-negative tumor with immune infiltrates as internal positive control. Original magnification, ×250. (C) Kaplan-Meier estimates of recurrence-free survival of patients with (n = 35) or without (n = 21) tumor STAT6 expression (P = 0.038). The time to recurrence was analyzed in a cohort of head and neck cancer patients that had been treated with cisplatin and radiotherapy in our institute in the period of 2003–2007. (D) Kaplan-Meier estimates of recurrence-free survival of patients with (n = 24) or without (n = 24) tumor STAT6 expression (P = 0.065). The time to recurrence was analyzed in a cohort of head and neck cancer patients that had been treated with radiotherapy alone in our institute in the period of 2000–2009.

Platinum-based chemotherapy represents a cornerstone in the systemic treatment of many types of cancer (15). Recent observations suggest that, beside their direct cytotoxic effects, platinum anticancer drugs may also exert their clinical effect through indirect activation of the immune system via induction of an immunogenic type of tumor cell death (5, 6, 16). Furthermore, it has been reported that tumor sensitivity to T cell killing can be increased by platinum due to upregulation of tumor mannose-6-phosphatase receptors (17). Other examples of immunogenic effects of cancer chemotherapy include enhanced antigen presentation by gemcitabine, paclitaxel, and anthracyclines (3, 4); and enhanced homeostatic lymphocyte proliferation and specific tumor infiltration by cyclophosphamide (18). Depletion of myeloid-derived suppressor cells has been described for fluoropyrimidines (19), and depletion of regulatory T cells has been reported for cyclophosphamide (20). So far, the effect of cytotoxic anticancer drugs on inhibitory immunological pathways has not been described before.

The PD-1/PD-L pathway is of pivotal importance in regulating the immune balance between T cell activation and inhibition (10). High PD-L expression by antigen-presenting DCs can result in the induction of tolerant or anergic T cells (10, 21, 22). A wide variety of human cancers express PD-Ls, which are known to correlate with poor prognosis (23–26). Antibody blockade of the PD-1/PD-L pathway results in enhanced tumor-specific T cell expansion and activation (27, 28). Our findings show that treatment with platinum compounds downregulates these PD-L inhibitory molecules, which not only results in enhanced T cell stimulation by DCs, but at the same time enhances the sensitivity of the tumor for lysis by cytotoxic T cells. The importance of our findings is underscored by recent evidence that not only proinflammatory cytokines such as TNF-α (29), but also IL-4 and IL-13, are abundantly present in the tumor microenvironment and induce phosphorylation of STAT6 in tumor cells, resulting in an incompetent Th2 type of immune milieu (30). We propose that platinum-based chemotherapy may revert this incompetent Th2 into effective Th1 antitumor immunity.

While the results of this initial clinical study indicate that STAT6 modulation is of major importance for the antitumor effect of platinum chemotherapy, a prospective trial is needed to truly assess the predictive value of STAT6 expression for tumor platinum sensitivity. In addition, we cannot exclude the possibility that besides PD-L2, other unknown factors in the tumor environment are affected by platinum-induced STAT6 dephosphorylation and may contribute to the observed effect. More research on the effects of platinum-induced STAT6 dephosphorylation is therefore warranted.

In conclusion, our findings may contribute to the design of innovative treatment schedules for cancer patients by the combination of platinum compounds with known immunotherapeutic approaches.

View Supplemental data

Mandy van de Rakt, Tjitske Duiveman, Nicole Meeusen-Scharenborg, Marieke Kerkhoff, Jurjen Tel, Klaartje de Kanter, David Burger, Annechien Lambeck, Monique Link, Hans Jacobs, Jolien Tol, Martijn den Brok, and Erik Aarntzen are acknowledged for their assistance. We thank Niels Schaft (Erlangen, Germany) for providing us with the gp100 TCR construct. This work was supported by grants from the Netherlands Organization for Scientific Research (grant 920-03-250), the Sascha Swarttouw-Hijmans Foundation, The Vanderes Foundation, and the Dutch Cancer Society.

Address correspondence to: I. Jolanda M. de Vries, Department of Tumor Immunology (278), Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB, Nijmegen, Netherlands. Phone: 31.24.361.76.00; Fax: 31.24.354.03.39; E-mail: j.devries@ncmls.ru.nl. Or to: W. Joost Lesterhuis, Department of Medical Oncology (452), Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB, Nijmegen, Netherlands. Phone: 31.24.361.03.53; Fax: 31.24.354.07.88; E-mail: w.lesterhuis@onco.umcn.nl.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2011;121(8):3100–3108. doi:10.1172/JCI43656.

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