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

Abstract

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.

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.

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.

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.

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.

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.

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.

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.