IL-18 Induces PD-1–Dependent Immunosuppression in Cancer Free

Cancer progression constitutes one of the best-characterized pathological conditions in which tolerance is actively induced. Tumors employ several mechanisms to avoid or actively suppress anticancer immune responses (1, 2). Tumor progression subverts the adaptive arm of antitumor immune responses, either directly by compromising T-cell functions or indirectly by inhibiting antigen-presenting cells (3). The secretion by tumor cells of soluble factors (such as IL-6, transforming growth factor-β, vascular endothelium growth factor, macrophage-colony stimulating factor, and indoleamine 2, 3-dioxygenase) can directly block T-cell proliferation, promote T-cell apoptosis, or render tumor cells resistant against T-cell attack (4). The tumor microenvironment can also release IL-23 which is a key NK cell immunosuppressant promoting metastases dissemination of otherwise NK cell-controlled cancers (5).

Driven by clinical observations linking elevated serum levels of IL-18 with defective NK cell functions (6, 7) and the potential role of tumor derived-IL-18 in cell autonomous tumor progression (8, 9), we investigated the potential immunosuppressive effects of IL-18 in NK cell-controlled tumors (10, 11). We found that IL-18 could upregulate PD-1 expression on NK cells and facilitated metastases dissemination of NK cell-dependent tumors in a PD-1–dependent manner. Depletion or neutralization of IL-18 produced by tumor cells markedly stimulated NK cell–mediated immunosurveillance against cancer.

Female C57Bl/6 or BALB/c mice were obtained from C.River Laboratories. C57Bl/6 Nude mice were purchased from Taconic. MyD88^−/− and IL-18Rα^−/− mice backcrossed on a C57Bl/6 background were kindly provided by B. Ryffel. RET^+/− mice were obtained from A. Prévost-Blondel (Institut Cochin, Paris, France) with the permission of M. Kato (Chubu University, Japan). All animals were maintained in IGR animal facilities according to the Animal Experimental Ethics Committee Guidelines. The tumors used were B16F10 melanomas and CT26 colon carcinomas (obtained from ATCC). To establish pulmonary metastases, 3 × 10⁵ B16F10 or CT26 were inoculated i.v. into C57Bl/6 or BALB/c mice, respectively. Mice were sacrificed between day 9 and 12 and pulmonary metastases were enumerated by binocular microscopy.

rmIL-18 (endotoxin-and BSA-free) was purchased from R&D Systems. Neutralizing anti-PD-1 antibody (RPM1-14) was kindly provided by Dr. Hideo Yagita (12). Monoclonal antibodies (purchased from Becton Dickinson Pharmingen, eBioscience, and R&D Systems) to the following mouse antigens were conjugated to FITC, PE, PerCP, APC, Pacific Blue or biotin: NK1.1 (PK136), NKp46 (29A1.4), CD3 (145-2C11), CD117 (ACK2), PD-1 (J43). Cells were pretreated with anti-CD16/CD32 Ab before staining and were analyzed with LSRII cytofluorometer using FACS Diva Software (Becton Dickinson), and Flow-JO Software (TreeStar, Ashland, OR).

Serum concentrations were evaluated by mouse IL-18 ELISA Kit.

IL-18BP was kindly provided by C. Dinarello (13). 3 × 10⁵ B16F10 or CT26 tumor cells were injected i.v. into C57Bl/6 or BALC/c mice, respectively. Mice received 20 μg of IL-18BP (or saline buffer) i.p. daily from day 0 to day 8 with or without depleting anti-NK1.1 Ab (300 μg injected i.p. on day 0, 3, 6, 9). Anti-PD-1 antibody was injected i.p. (250 μg/mice) at day 0, 1, 3, 6, 9.

B16F10 tumor cells were transfected with mouse IL-18 siRNA [IL-18 Stealth Select RNAi (MSS205424, MSS205426] and control siRNA (Stealth RNAi Negative Control Med GC; Invitrogen) using HiPerfect reagent.

Protein lysates of tumor cells transfected or not with control or IL-18 siRNA were examined by immunoblotting. Aliquots of 20 μg of total protein extracts were solubilized in Laemmli loading buffer, separated by 10% SDS-PAGE and blotted onto nitrocellulose membranes (Bio-Rad Laboratories). After blocking for 1 h, blots were incubated overnight at 4°C with antibodies against IL-18 (rabbit anti-mouse IL-18, 2 μg/mL; BioVision, Inc.) and GAPDH (mouse anti-glyceraldehyde-3-phosphate dehydrogenase, 1/10,000; Chemicon International). Protein detection was carried out with HRP-conjugated secondary antibodies (Southern Biotech) and SuperSignal West Pico Chemiluminescent Substrate.

The cell-autonomous protumorigenic effects of endogenous IL-18 have been described in B16F10 melanoma where IL-18 induced adhesiveness of melanoma cells to sinusoidal endothelium, thereby facilitating metastatic dissemination (14). Since B16F10 metastatic spreading is controlled by NK cells (10), we investigated the mechanisms by which rIL-18 could facilitate tumor progression. We compared different schedules of recombinant mouse endotoxin-free IL-18 (rIL-18) administration (Supplementary Fig. S1A) for their ability to modulate the establishment of B16F10 lung metastases. rIL-18 enhanced the development of B16F10 metastases when it was injected twice a week (2×; Fig. 1A), whereas daily administration (5×) of rIL-18 reduced tumorigenesis consistent with published results (15; Fig. 1A). Accordingly, the (2×) rIL-18–induced expansion of lung metastases was inversely correlated with intratumoral mature NK cells, defined as NK1.1⁺CD3⁻Kit (CD117)⁻ NK cells (16; Fig. 1B). Circulating serum levels of IL-18 accumulating by day 7 were significantly higher with the 5× versus 2× regimen (Fig. 1C). These circulating levels of rIL-18 were associated with distinct NK cell phenotypes (Supplementary Fig. S1B and C) and cytokine patterns in spleens. The quantitative real-time PCR (qRT-PCR) analyses of 96 immune gene products using a microfluidic card designed for qRT-PCR on splenocytes harvested from mice treated with once (1×) versus twice a week (2×) versus 5 times (5×) daily administrations of rIL-18 revealed different gene signatures (Fig. 1D and E). We observed a significant upregulation of the IL-12p40 and inflammatory gene products (TNF, CCL5, IL-1β, IL-15, and CCR4) with 5 daily injections of rIL-18, whereas 1 (1×) or 2 shots (2×) of rIL-18 promoted an immunosuppressive microenvironment (significant downregulation of CCR7, Fas, upregulation of IL-10, trend toward a reduction in CXCR3, CD3ϵ, CD4, CD8, Pfr, CD28, CD40, STAT1; Fig. 1D and E). These data underscore the capacity of low doses of rIL-18 to favor tumor progression of B16F10 metastases.

Next, we determined whether tumor cells may spontaneously secrete bioactive IL-18. Mature (18 kDa) bioactive IL-18 was detected in B16F10 and various tumor cell lysates (CT26, P815, RMA, and RMAS) by immunoblotting (Fig. 2A, top) and was released into the supernatant, as determined by an IL-18 bioassay (17) that measures IFNγ induction in splenocytes (Fig. 2A, bottom). Transfection of B16F10 tumor cells with 2 distinct IL-18 siRNAs reduced the capacity of tumor cells to produce bioactive IL-18 (Fig. 2B, top) and compromised B16F10 tumorigenesis (Fig. 2B, bottom). In IL-18R^−/− or MyD88^−/− mice, the dissemination of B16F10 metastases was markedly reduced, suggesting that B16F10 progression was dependent on the IL-18R/MyD88 signaling pathway of the host (Fig. 2C). It is noteworthy that upon inoculation of transplantable tumors (B16F10) and in spontaneous melanomas arising in RET transgenic mice (expressing the RET proto-oncogene driven by the metallothionein promoter; ref. 18), the serum levels of IL-18 were markedly increased by 10 days (premortem) and at 12 months, respectively (Supplementary Fig. 1D and E), in the same ranges as those obtained with 2× rIL-18 scheduling (Fig. 1C). Next, we used saturating amounts of IL-18 binding protein (IL-18BP), a naturally occurring IL-18 antagonist (13), that successfully compromised tumor progression in 2 NK cell–controlled lung metastases models (Refs. 10, 11; CT26 and B16F10, Fig. 2D, top and bottom). IL-18 neutralization by IL-18BP did not cause tumor regression in the absence of NK1.1⁺ cells, suggesting that IL-18 modulated NK cell functions (Fig. 2E). Therefore, IL-18 produced by tumor cells could subvert the NK-mediated antitumor host defense.

PD-1 is one of the major checkpoints downregulating T-cell functions in tumor beds. Recently, myeloma associated-human NK cells were reported to express PD-1 that may cause their exhaustion (19). Therefore, we examined PD-1 expression on T and NK cells in metastases and lymph nodes (LN) of tumor bearers. Tumor sites contained significant levels of tumor infiltrating PD-1⁺ T lymphocytes but no PD-1⁺ NK cells (Fig. 3A, top). Interestingly, NK cells (but not T cells) exhibited a significant upregulation of PD-1 expression in LN (Fig. 3A, bottom). As determined by intracellular staining, PD-1 molecules were present in freshly isolated NK cells (Supplementary Fig. S2A), which, however, lacked surface expression of PD-1. Stimulation of NK cells with rIL-2 or other NK cell stimulatory compounds (such as IFNγ, CpG ODN, PolyI:C) induced surface exposure of PD-1 (Supplementary Fig. S2B), indicating that NK cell activation results in surface expression of PD-1. Importantly, rIL-18 could induce PD-1 expression on splenic NK cells, both in vitro and in vivo (Fig. 3B and C, respectively). The prometastatic effects of rIL-18 in Nude mice (lacking T lymphocytes) was mostly attributable to PD-1 triggering because neutralizing anti-PD-1 Ab administration abrogated the IL-18–mediated flare up of B16F10 metastases (Fig. 3D).

IL-18 plays a pivotal role in inflammation and immune responses. Evidence in favor of the IL-18–mediated anticancer effects involve NK, T cells, or IFNγ and are usually obtained with high doses of IL-18 often combined with IL-2 or IL-12 (8, 20, 21). Cytokine doses and schedules have been discussed in the context of pathogenesis and cancer therapy and molecules such as IL-2 or IL-10 have led to opposite biological outcomes depending on their cellular targets and/or their mode of delivery [reviewed in (22, 23)]. In patients with cancer, increased IL-18 serum levels accompany tumor progression and have a negative prognostic impact (9). In the absence of Th1-like cytokines, IL-18 alone accelerates tumor progression (24), in part through cell autonomous effects on cancer and endothelial cells (24, 25). In addition, as here and to our knowledge for the first time, low dosing of IL-18 could mediate immunosuppression on the NK cell arm of immunity (as shown using a schedule of IL-18 administration twice a week or a natural tumor outgrowth leading to low-circulating levels of IL-18). Importantly, IL-18 could drive the expression of PD-1 on mature NK cells, whereas the prometastatic effects of recombinant or tumor-derived IL-18 on NK-controlled tumors involved PD-1 receptors. However, we cannot exclude that other players of innate immunity such as DC could also express PD-1 (26) in the context of IL-18 (data not shown) that might inhibit the DC/NK cell cross-talk. Further studies will be needed to clarify this question. These data could potentially impact the clinical development of anti-PD1 and PDL-1 antibodies in IL-18–secreting human malignancies.

No potential conflicts of interest were disclosed.

We thank C. Dinarello (Denver, USA) for providing IL-18BP, and A. Mackensen (Erlangen, Germany) for critically reading the manuscript.

This work was supported by INCa, ANR, Ligue contre le cancer (équipes labellisées de LZ and GK), and INFLACARE EU grant. M. Terme, E. Ullrich, K. Meinhardt, and L. Aymeric were supported by Cancéropole IDF and the Ligue Nationale Contre le Cancer, the German Research Foundation (DFG), the Fondation pour la Recherche Medicale (FRM), the German Cancer Aid, the Deutscher Akademischer Austauschdienst (DAAD), and Institut National du Cancer (INCa), respectively.

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