Robust tumor immunity to melanoma mediated by interleukin 9

Deficiency of ROR-γ is associated with inhibited melanoma growth and increased tumor lymphocytic infiltration

To examine the role of ROR-γ in tumor immunity, we used a B16F10 murine melanoma model. RORc^−/− mice do not develop secondary lymph nodes and have fewer CD4⁺ and CD8⁺T cells as compared to RORc^+/+ mice ¹⁸. We therefore generated ROR-γ deficient chimeric mice (RORc^−/−ch) and ROR-γ normal wild type chimeric mice (RORc^+/+ch) by administering bone marrow cells from RORc^−/− or RORc^+/+ mice into sublethally irradiated Rag1^−/− C57BL/6 mice^14,16. After full reconstitution of T cells and restoration of the intact immune system (8–10 weeks), mice were used for tumor growth experiments (Supplementary Fig. 1). B16F10 melanoma cells were injected subcutaneously into RORc^+/+ch and RORc^−/−ch mice, and tumor growth was monitored over time. Melanoma growth was strongly inhibited in RORc^−/−ch mice, and survival of the RORc^−/−ch mice was significantly increased compared to control mice (Fig. 1a–b). We examined the infiltration of lymphocytes in tumors removed from these mice. Melanomas from RORc^−/−ch mice contained 3–fold higher numbers of CD4⁺T cells and CD8⁺T cells as compared to RORc^+/+ch controls (Fig. 1c). T cells from draining lymph nodes of RORc^−/−ch melanoma bearing mice secreted negligible IL-17A, and increased amounts of IFN-γ as well as TNF-α (Fig. 1d). There was no difference in the number of melanoma tumor-infiltrating CD4⁺CD25⁺FoxP3⁺T cells (T-regulatory cells) in RORc^+/+ch (7.2% ± 0.4) and RORc^−/−ch (5.9% ± 0.7).

Increased IL-9 expression in ROR-γ⁻ T cells

To further explore the mechanism of melanoma growth inhibition in RORc^−/−ch mice, we performed transcriptional profiling analysis using CD4⁺T cells from RORc^−/− and RORc^+/+ mice differentiated under T_H17 polarizing conditions. As expected, expression of IL-17A, IL-17F and IL-23R in ROR-γ⁻CD4⁺T cells was much lower compared to ROR-γ⁺CD4⁺T cells (Fig. 2). However, expression of IL-9 in ROR-γ⁻CD4⁺T cells was dramatically increased as compared to ROR-γ⁺CD4⁺T cells (Fig. 2, Supplementary Fig. 2). Granzyme-B and Granzyme-C expression were also significantly enhanced. Because of the striking upregulation of IL-9 under these conditions, we focused our subsequent experiments on the potential role of IL-9 in tumor immunity.

To be certain that the T cells from RORc^−/−ch mice did not have an intrinsic property that predisposed them to IL-9 production; we examined CD4⁺T cells from these mice. There was increased expression of IL-9 in ROR-γ⁻CD4⁺T cells (Supplementary Fig. 3a–b) and memory CD4⁺T cells (Supplementary Fig. 3 c–e). However, negligible IL-9 expression was observed in naïve CD4⁺T cells, and importantly there was no difference in IL-9 expression between ROR-γ⁺ and ROR-γ⁻naïve CD4⁺T cells (Supplementary Fig. 3f). T_H2 and T_H9 cells both secrete IL-9^19,20; there was increased IL-9 expression in ROR-γ⁻T_H2 cells. However, ROR-γ⁻CD4⁺T cells polarized under T_H9 conditions showed similar IL-9 production when compared to RORγ⁺T_H9 cells (Supplementary Fig. 3g–h).

To determine if the melanoma growth inhibitory response in RORc^−/− ch mice could be attributed to IL-9, melanoma cells were injected subcutaneously into RORc^−/−ch mice. Tumor growth in these mice lagged significantly behind that of RORc^+/+ch mice. Mice were next treated with neutralizing antibodies to IL-9. As shown in Figure 3a, the impaired melanoma growth in RORc^−/−ch mice was significantly, but not completely, reversed by neutralization of IL-9. Therefore, we conclude that the melanoma growth inhibition observed in RORc^−/−ch mice was partially dependent on IL-9.

The role of IL-9 in melanoma immunity in IL-23R^−/− mice

The most strongly downregulated gene in the transcriptional profiling experiments on ROR-γ⁻CD4⁺T cells was IL23R. We therefore explored melanoma tumor growth in IL23R^−/− mice as well. We differentiated naïve CD4⁺T cells from IL23R^+/+ and IL23R^−/− mice under T_H17 polarizing conditions and re-stimulated (anti-CD3/CD28 mAbs) these cells. After 48h, supernatant was collected and IL-9 and IL-17 were measured. As expected, IL-23R⁻CD4⁺T cells produced significantly less IL-17A (Fig. 3b). However, there was increased IL-9 expression compared to IL-23R⁺CD4⁺T cells (Fig. 3b).

Next, melanoma cells were injected into IL23R^+/+ and IL23R^−/− mice. Melanoma growth was greatly impaired in IL23R^−/− mice. We next asked what role IL-9 played in this tumor growth inhibition. Neutralization of IL-9 also led to enhanced melanoma tumor growth in IL23R−/− and IL23R^+/+ mice (Fig. 3c), suggesting that the production of IL-9 by TIL’s (Fig. 3d) was partially effective in tumor growth suppression. In addition, tumor site-draining lymph node cells (LNCs) from IL23R^+/+ and IL23R^−/− mice were isolated and stimulated (anti-CD3/CD28 mAbs) for 48 h. There was increased IL-9 secretion by IL-23R⁻T cells compared to IL-23R⁺T cells (Fig. 3e).

T_H9 cells inhibit melanoma growth

Since IL-9 is mainly produced by CD4⁺T cells, we examined the role of effector subsets of CD4⁺T cells in melanoma immunity. T_H1, T_H2, T_H9 and T_H17 cells were generated from naïve CD4⁺T cells of OT-II mice. CD4⁺T cells from OT-II mice express a transgenic TCR that recognizes residues 323-339 from ovalbumin in the context of I-A^b. B16F10-ova melanoma cells express the full-length ovalbumin molecule. Before adoptive transfer into mice, in vitro cytokine polarized CD4⁺T cells were analyzed for their cytokine expression (Supplementary Fig. 5a). Differentiated T_H cells were transferred into syngeneic immunocompetent mice, and, B16F10-ova melanoma cells were injected subcutaneously. Mice treated with T_H9 cells showed the greatest resistance to melanoma growth (Fig. 4a).

To determine if Th9 cells could inhibit the melanoma development independent of endogenous T cells, we transferred T_H0 and T_H9 cells generated from OT-II mice into immunocompromised hosts. In this setting, Th9 cells inhibited B16F10-ova induced melanoma growth (Fig. 4b), and survival of T_H9-treated mice was significantly increased (Fig. 4c). This data suggest that T_H9 cells are capable of inhibiting melanoma development, even in the absence of CD8⁺T cells. Finally, IL-9 blockade accelerated melanoma growth in T_H9 treated mice, suggesting partial dependence of this effect directly on IL-9 (Fig. 4b).

To determine whether T_H9 cells could directly kill melanoma cells, we co-cultured B16F10-ova cells with OT-II T_H9 cells for 24 h. At that point, B16F10-ova cells were stained with 7-AAD. T_H9 cells alone were capable of inducing apoptosis in melanoma cells, whereas T_H0 and T_H17 cells were much less effective in this regard (Supplementary Fig. 6a). Next, the mechanism of T_H9 cell direct cytotoxicity was assessed. We measured a limited profile of effector molecules in T_H9 cells (Supplementary Fig. 5b). We observed increased expression of granzyme-B in T_H9 cells. Inhibition of granzyme-B significantly attenuated the T_H9 cell cytotoxic activity (Supplementary Fig. 6b). To further explore the direct cytotoxic effects of Th9 cells, we used another cytotoxic assay⁵. We incubated OT-II T_H9 cells with CFSE-labeled B16F10-ova cells and CFSE-labeled EL-4 cells for 36 h. There were dose dependent effects of T_H9 cells on tumor cell lysis (Supplementary Fig. 6c). More importantly, OT-II T_H9 cells killed B16F10-ova cell but not a tumor cell line that did not express Ova (Supplementary Fig. 6c).

Abrogation of IL-9/IL-9R signaling promotes melanoma development, and treatment with rIL-9 inhibits melanoma development

To analyze the role of IL-9 in tumor growth more directly, melanoma cells were injected into IL9R^−/−mice, and tumor growth was monitored. Melanoma growth was accelerated in IL9R^−/−mice compared to IL9R^+/− mice (Fig. 5a). We next asked if administration of rIL-9 could protect mice against melanoma growth and progression. Treatment of melanoma bearing mice with recombinant IL-9 (rIL-9) both impaired melanoma growth (Fig. 5b) and increased the survival of mice (data not shown). To determine if rIL-9 could potentiate the anti-melanoma response in mice already vaccinated against melanoma, we vaccinated mice with 10⁶ irradiated melanoma cells. Live B16F10 melanoma cells were injected 7 days later, and mice were treated with rIL-9. Again, rIL-9 treated mice showed significantly reduced melanoma growth (Supplementary Fig. 4a). We asked if rIL-9 could inhibit an unrelated tumor—Lewis Lung Carcinoma (LLC). LLC-1 tumor development was significantly suppressed in rIL-9 treated mice compared with a control group (Fig. 5c), However, rIL-9 had no effect on EL-4 lymphoma growth (data not shown).

Mast cells, but not adaptive immune cells, are required for the anti-tumor effect of IL-9

We next investigated the mechanism of IL-9 mediated tumor immunity. Melanoma cells were injected into Rag1^−/−C57BL/6 mice, which lack T and B cells, and mice were treated with rIL-9. Treatment with rIL-9 still significantly inhibited melanoma growth (Fig. 5d), suggesting that the target of the rIL-9 effect was not a T cell or B cell. Because IL-9 is also known to promote mast cell development and function, we injected B16F10 melanoma and LLC-1 cells, respectively, into mast cell deficient mice (Kit W-sh/HNihrJaeBsmJ) and then measured tumor growth in mice treated systemically with rIL-9 (B16F10: Fig 5e, LLC-1: Fig 5f). Strikingly, under these conditions, there was no significant difference in tumor development. These data suggest that tumor growth inhibition mediated by rIL-9 administration depends upon the presence of mast cells, but not on the presence of T cells or B cells.

IL-9 producing T cells (T_H9 cells) can be found in normal human skin and peripheral blood, and in metastatic melanoma lesions

For this work to be relevant to patients with melanoma, we felt it important to attempt to identify T_H9 cells in human tissues. Specifically, we asked if T_H9 cells can be identified in humans, and if so, whether they showed a phenotype comparable to murine T_H9 cells. Murine T_H9 cells are defined as a distinct population of CD4⁺T cells that produce IL-9 but not IFN-γ, IL-4 or IL-17. We were able to demonstrate that human memory T cells isolated from peripheral blood contained a distinct population of IL-9 producing T cells that do not produce IFN-γ, IL-4 and IL-17. T cells with an identical phenotype, and in even greater abundance, were also found in resident T cells (T_RM) isolated from healthy skin (Fig. 6a–b). Finally, we looked for the presence of IL-9 producing T cells in TIL’s extracted from patients with Stage IV metastatic melanoma (metastasis to lung (n=4), skin (n=2), adrenal gland (n=1), and bone (n=1)). We observed detectable IL-9 producing T cells in 6 out of 8 biopsies; however, T_H9 cells were less abundant in TIL’s from melanoma lesions, and the level IL-9 production was significantly lower in these T cells melanomas compared to memory T cell populations from healthy donors (Fig. 6c).

Mice

WT C57BL/6, Rag1^−/− C57BL/6 and IFNG^−/− mice were obtained from Jackson Laboratories. IL9R^{−/− 39}, IL23R^−/− and RORc^{−/− 17} and their control mice (RORc^+/+, and IL9R^−/+) were used, Mice were housed in conventional, pathogen-free facilities at the animal facility of Harvard Medical School.

In vitro T cell differentiation

CD4⁺CD25⁻CD62Lhigh cells from RORc^−/− mice or RORc^+/+ controls were sorted by CD4⁺CD62L⁺ isolation kit II from Miltenyi Biotech (USA) according to manufacturer’s protocol. Purity of CD4⁺CD25⁻CD62Lhigh was >95%.

Sorted CD4⁺CD25⁻CD62Lhigh cells were differentiated into T_H1 (IL-12: 10 ng ml⁻¹), T_H2 (IL-4: 10 ng ml⁻¹), T_H9 (TGF-β plus IL-4: 1 ng ml⁻¹ and 10 ng ml⁻¹), and T_H17 (TGF-β plus IL-6: 1 ng ml⁻¹ and 10 ng ml⁻¹) in presence with plate bound anti-CD3 (1 μg ml⁻¹) and irradiated splenocytes (1:5 ratio). After 48 h cells were fed with IL-2 (10 ng ml⁻¹) containing fresh media and split into two parts, if needed. On day 5, cells were harvested and processed for cytokine analysis at RNA or protein level by real-time RT-PCR, flow cytometry and ELISA.

For adoptive transfer experiments, CD4⁺T_H cells differentiation was carried out using above mentioned protocol with few modifications. Plate bound antibodies to CD3 (2 μg ml⁻¹) and CD28 (1 μg ml⁻¹) was used in place of irradiated splenocytes. In addition to above mention polarization condition, anti-IFN-γ mAb (10 μg ml⁻¹) was added into bv9 cultures and anti-IFN-γ mAb (10 μg ml⁻¹) plus anti-IL-4 mAb (10 μg ml⁻¹) was added into T_H17 cultures.

Tumor (Melanoma, Lewis lung carcinoma and thymic lymphoma) induction, In vivo T cell transfer and IL-9 neutralization

Melanoma cell lines (B16F10 cells or B16F10-ova cells), T cell lymphoma (EL-4) and Lewis lung carcinoma (LLC-1) were grown in RPMI1640 supplemented with 10% FBS, and penicillin/streptomycin. B16F10 cells (2–4 × 10⁵ cells 150 μl⁻¹), EL-4 (2×10⁵ cells 150 μl⁻¹), or LLC1 (5×10⁵ cells 150 μl⁻¹) were injected subcutaneously into the right or left flank of the mice and tumor development was monitored over time. Tumor volume was calculated by following formula: (major circumference X minor circumference²)/2. Mice were sacrificed when there was external necrosis or/and tumor volume reached no greater than 2 cm in any direction.

To investigate the role of effector subsets of T_H cells on melanoma and thymic lymphoma growth, 2-million differentiated cells (T_H1, T_H2, T_H9 and T_H17) from CD45.1⁺CD45.2⁻OT2 TCR transgenic mice were injected (i.v.) into WT-C57BL6 mice or Rag1^−/− (C57BL6 background) mice and, on the same day tumor cells (B16F10-ova cells: 3×10⁵ cells 150 μl⁻¹) were injected subcutaneously. Tumor growth was monitored over time.

IL-9 activity in vivo was neutralized by injecting (i.p.) 100 μg neutrilizing antibody to IL-9 mAb (clone: MM9C1, a generous gift by Jacques van Snick from Ludwig Institute, Belgium) for 4 times on day 0, day 3, day 6 and day 10. Melanoma cells were injected on day 0 and melanoma growth was monitored over time.

rIL-9 (5 μg: from Cell Sciences, USA) was injected (i.p.) from day 0 and every third day till the termination of the experiment. Unlike Cell Science rIL-9 (source: E.Coli), rIL-9 from RnD systems is glycosylated and therefore has stronger biological activity. Thus, we used 100 times less rIL-9 from RnD systems (i.e. 50 ng on every alternate day till the termination of experiment) in LLC-1 tumor model experiments compared to melanoma model experiments. In addition, we also treated melanoma bearing mice with rIL-9 from RnD systems, USA which produced similar results as were observed with rIL-9 from Cell Sciences (data not shown).

Cytotoxicity Assay

For the cytotoxicity assay, CFSE labeled B16F10-ova cells (5×10⁵ cells 0.5ml⁻¹) were cultured with differentiated T_H cells (OT2-T_H0, OT2-T_H9 and OT2-T_H17) in several different ratios. After 24 h of co-culture, cells (gate on CFSE labeled B16F10-ova cells) were analyzed for 7-AAD staining, a sensitive indicator of apoptotic cell death by flow cytometry. In addition, we used another cytotoxicity assay as described before ⁵.

Growth curve assay

Effect of rIL-9 on growth of B16F10 cells were studied by growth curve assay. B16F10 cells were seeded with rIL-9. After each incubation period cells were fixed (10% acetic acid in 10% ethanol). Cells were subsequently stained with 0.4% crystal violet in 10% ethanol for 30 min. Subsequently, 200 μl 10% acetic acid was added. After 30 min, 100 μl solution was transferred into 96-well plate and OD was measured at 595-wavelength.

Measurement of cytokines by intracellular cytokine staining, CBA, ELISA and quantitative RT-PCR

Intracellular cytokines by Lymph node (LNs) cells, splenocytes, TILs or in vitro differentiated T_H cells were quantified after restimulation with PMA plus ionomycin in presence of GolgiStop for 6 h as described previously⁴⁰.

Cytokines were quantified in cell free culture supernatants by cytometric bead array (CBA by BD Biosciences) or by ELISA (eBioscience) according to the manufacturer’s instructions.

RNA was extracted with High pure RNA isolation kit (Roche), cDNA was made by First strand cDNA synthesis kit (BioRad) and quantitative RT-PCR was done using multiplex kit (BioRad) on iCycler (BioRad) according to the manufacturer’s instructions. IL-9R PCR was carried out by using IL-9R specific taqman probes and AB Biosystem PCR machine.

Cell purification, sorting, Intracellular cytokine staining and cytokine quantification in supernatants (Human study)

PBMCs were isolated from buffy coats of healthy donors by density centrifugation. Memory CD4⁺T cells were purified from freshly isolated PBMCs by negative selection using a Memory CD4⁺T cells Isolation Kit (Miltenyi Biotech, Germany) and stimulated with anti-CD3/CD2/CD28 beads (Milyenyi) in presence of TGF-β (3 ng ml⁻¹).

Normal human skin samples were obtained as discarded material after cosmetic surgery according to Institutional Review Board of Partners Human Research Committee. Total skin T cells from healthy donors and T cells of melanoma metastasis were isolated from explant cultures grown with IL-2 and IL-15 as previously⁴¹ described.

Memory T cells (CD4⁺CD45RO⁺) from peripheral blood after stimulation with anti-CD3/CD28 mAbs plus TGF-β and skin-resident T cells isolated by skin-explant culture of healthy donors were restimulated for 5 h in the presence of PMA plus ionomycin (Sigma-Aldrich) in presence of golgistop. After incubation, CD4⁺T cells were stained for IFN-γ (anti-IFN-γ: B27), IL-4 (anti-IL-4: MP4-25D2), IL-9 (antiIL-9: MH9A4) and IL-17 (anti-IL-17: eBio64DEC17,) using intracellular staining and analyzed by flow cytometry⁴⁰.

Memory T cells from blood, skin T cells from healthy donors and tumor-infiltrating lymphocytes of subjects with melanoma metastasis were stimulated at 10⁶ cells ml⁻¹ with beads coated with antibodies to CD3/CD2/CD28 (bead: T cell ratio: 1:2 from Miltenyi Biotech) in the presence of IL-2 (50 IU ml⁻¹) and TGF-β (3 ng ml⁻¹) for 2 days. IL-9 in culture supernatants was measured by Luminex bead-based multiplex assays using a custom-made Luminex bead assay as described previously⁴².

Statistical analysis

Student t-test (two tailed) was performed for the data analysis using GraphPad Prism software program. A paired t-test was used in Supplementary Fig 2h, and 2J. The p value <.005, .025 and .05 are represented as ***, ** and * respectively.

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