TGF secreted by B16 melanoma antagonizes cancer ... - Springer Link

Cancer Immunol Immunother (2008) 57:1197–1206 DOI 10.1007/s00262-008-0453-1

O R I G I N A L A R T I CL E

TGF secreted by B16 melanoma antagonizes cancer gene immunotherapy bystander eVect Claudia Penafuerte · Jacques Galipeau

Received: 18 October 2007 / Accepted: 9 January 2008 / Published online: 24 January 2008 © Springer-Verlag 2008

Abstract Tumor-targeted delivery of immune stimulatory genes, such as pro-inXammatory cytokines and suicide genes, has shown to cure mouse models of cancer. Total tumor eradication was also found to occur despite subtotal tumor engineering; a phenomenon coined the “bystander eVect”. The bystander eVect in immune competent animals arises mostly from recruitment of a cancer lytic cell-mediated immune response to local and distant tumor cells which escaped gene modiWcation. We have previously described a Granulocyte–Macrophage Colony Stimulating Factor (GM-CSF) and Interleukin 2 (IL2) fusokine (aka GIFT2) which serves as a potent anticancer cytokine and it here served as a means to understand the mechanistic underpinnings to the immune bystander eVect in an immune competent model of B16 melanoma. As expected,

This work was supported by a Canadian Institute for Health Research operating grant MOP-15017. C. P. is recipient of Montreal Centre for Experimental Therapeutics in Cancer Scholarship and US Army Graduate study Scholarship and J. G. is a Fonds de recherché en santé du Québec chercheur-boursier senior. Electronic supplementary material The online version of this article (doi:10.1007/s00262-008-0453-1) contains supplementary material, which is available to authorized users. C. Penafuerte Division of Experimental Medicine, Department of Medicine, McGill University, Montreal, QC, Canada e-mail: [email protected] J. Galipeau (&) Department of Medicine and Oncology, Lady Davis Institute for Medical Research, 3999 Cote Ste-Catherine Road, Montreal, QC, Canada H3T1E2 e-mail: [email protected]

we observed that GIFT2 secreted by genetically engineered B16 tumor cells induces a bystander eVect on non modiWed B16 cells, when admixed in a 1:1 ratio. However, despite keeping the 1:1 ratio constant, the immune bystander eVect was completely lost as the total B16 cell number was increased from 104 to 106 which correlated with a sharp reduction in the number of tumor-inWltrating NK cells. We found that B16 secrete biologically active TGF which in turn inhibited GIFT2 dependent immune cell proliferation in vitro and downregulated IL-2R expression and IFN secretion by NK cells. In vivo blockade of B16 originating TGF signiWcantly improved the immune bystander eVect arising from GIFT2. We propose that cancer gene immunotherapy of pre-established tumors will be enhanced by blockade of tumor-derived TGF. Keywords B16 melanoma · TGF · Immunology · Bystander eVect · Gene therapy Abbreviations TGF Transforming growth factor beta IL-2 Interleukin 2 GM-CSF Granulocyte–macrophage colony-stimulating factor GIFT2 GM-CSF and IL-2 fusion transgene

Introduction Many immunogene therapy strategies have been developed for the treatment of malignant melanoma. These approaches include the introduction of “suicide genes”, the expression of tumor suppressor genes by tumor cells or the inactivation of oncogene expression, as well as the introduction of genes encoding pro-inXammatory proteins such as co-stimulatory

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molecules and cytokines. In particular, the genetic modiWcation of melanoma cells to secrete pro-inXammatory cytokines enhances the immunogenicity of these cells by providing signals required to trigger an eVective cell mediate immune response [16]. Since it is not possible to modify all pre-existing tumor cells with suicide or proinXammatory genes in situ by any contemporary gene transfer technology, an important feature to consider for cancer gene immunotherapy is the bystander eVect [20]. Whereas a small fraction of tumor is gene modiWed to initiate an immune response in vivo against a vastly bulkier pre-established native cancer. Indeed, a cytokine-secreting live cell cancer vaccine approach is also wholly dependent upon a robust immune “bystander” eVect for clinical eVectiveness [5]. A vast array of pro-inXammatory cytokines and derivatives has been studied as a means to initiate an anticancer immune response in animal models of melanoma and in clinical trials. However, despite tightly controlled conditions, we––as others [13]- have observed treatment failures in mouse models of melanoma where the immune “bystander” eVect was lost as the experimental tumor burden was increased at onset of treatment. As a possible explanation for this observation, we may invoke the balance between inhibitory and stimulatory signals essential in the maintenance of homeostasis and in the regulation of the immune response as possibly antagonistic to the immune bystander eVect. During cancer progression this balance is disrupted and inhibitory signals may prevail, leading to an immunosuppression in the tumor-microenvironment and resulting in tumor growth. Various mechanisms have been proposed for tumor cell evasion from physiological immunosurveillance and these include the dysregulation of MHC class I and tumor antigen expression as well as adhesion/ accessory molecules expression, induction of anergy or clonal deletion of eVector cells, and the secretion of suppressive soluble factors [16]. Previous studies from our laboratory have shown that the fusion protein between GM-CSF and IL-2, aka GIFT2–– has novel immunological properties compared to both cytokines in combination, such as greater melanoma site recruitment of macrophages and functional NK cells, and circumvents the limitations of each individual cytokine [21]. With the use of GIFT2 fusokine as means to initiate an anticancer immune response, we here analyzed the immune bystander eVect of GIFT2-secreting melanoma cells on wild type B16 present in the tumor site in vivo. We observed that the bystander eVect is lost as tumor burden increases and that B16-derived TGF was responsible in good part of this acquired refractoriness by its direct eVect on innate eVector cells despite local production of a potent pro-inXammatory fusokine. These data strongly support the need to target tumor-derived suppressor cytokines––such as TGF, for an optimal immune bystander response to a cancer immunogene platform.

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Materials and methods Animals, cell lines, and reagents All experimental mice were females 6–8 weeks old (Jackson Laboratory, Bar Harbor, ME). The C57Bl/6-derived B16F0 (B16) mouse melanoma cells (American Type Culture Collection [ATCC], Manassas, VA) as well as a polyclonal population of B16 derivative (B16GIFT2 [21]) were maintained in Dulbecco’s modiWed Eagle’s medium (Wisent Technologies, Rocklin, CA), supplemented with 10% fetal bovine serum (Wisent Technologies) and 50 U/ml Pen/Strep (Wisent Technologies). The cell lines CTLL-2 and mouse embryonic Wbroblasts (MEF) (American Type Culture Collection [ATCC], Manassas, VA) were grown according to ATCC’s recommendations. Recombinant mouse TGF and IL-2, as well as TGF neutralizing antibody (anti-TGF1, 2, 3 isoforms) and soluble TGF receptor II (TRII) were obtained from R&D Systems, Minneapolis, MN; antiphosphorylated SMAD2 and SMAD3 antibodies were obtained from Cell Signalling Technology, Danvers, MA; -tubulin antibody was obtained from Santa Cruz Biotechnology, Santa Cruz, CA. Anti-mouse FcR III/II, CD3, CD8, CD4, CD25, NK1.1, CD80, CD86, CD105, MHC class I, MHC class II, CD122 (IL-2R chain) and the isotype control antibodies for Xow cytometry were obtained from BD Biosciences, San Diego, CA. The enzyme-linked immunosorbent assay (ELISA) kit for mouse IFN- was obtained from BD Biosciences. Murine B16F0 tumor implantation in immunocompetent C57Bl/6 mice and immune inWltrate analysis Wild type and genetically modiWed GIFT2 fusokine-secreting B16 cells (0.7 § 0.2 pmol per 106 cells per 24 h) were injected subcutaneously in C57Bl/6 mice, and tumor growth was monitored over time by performing external measurements in two dimensions and calculating using the equation volume = lengthxwidth2 £ 0.5. For immune inWltrate analysis, 104 or 106 genetically modiWed cytokine secreting and/or non modiWed B16 cells were mixed with 500 l Matrigel (BD Biosciences) at 4°C and injected subcutaneously in C57Bl/6 mice. Implants were surgically removed 6 days after transplantation and enzymatically dissociated as reported previously [21]. After incubation with anti-FcR III/II mAb for 1 h, inWltrated cells were incubated for 1 h at 4°C with appropriate antibodies and analyzed by Xow cytometry using a FACS Calibur cytometer (BD). Flow cytometry analysis of non modiWed and genetically modiWed cytokine expressing B16 cells Flow cytometry analysis was performed in phosphatebuVered saline (PBS) with 2% FBS with the following


antibodies: R-phycoerythrin (PE)-conjugated anti-mouse H-2 Kb (MHC class I, clone AF6-88.5), I-Ab (MHC class II, clone AF6-120.1), CD80 (clone 16-10A1), CD86 (clone GL1) and CD105. Isotype control analysis was performed in parallel. Non modiWed and genetically modiWed B16 cells expressing mouse GIFT2 were incubated with the appropriate antibodies for 1 h at 4°C and the expression of these cell surface markers was determined by using FACS Calibur cytometer (BD) and analyzed using Cellquest software (BD). Cytokine-dependent CTLL-2 proliferation assay CTLL-2 cells were pre-incubated in medium conditioned by non-modiWed B16 cells and pretreated with TGF neutralizing antibody or isotype control, as well as in medium conditioned by MEF. The cells were plated at 104 cells/well of a 96-well plate with increasing concentration of mouse recombinant IL-2. The cells were incubated for 48 h and 20 l of 5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution were incorporated for the last 4 h of incubation. The reaction was stopped by adding 200 l of dimethyl sulfoxide and absorbance read at 570 nm. The expression of CD8 and CD122 (IL-2R chain) on CTLL-2 cells pre-incubated as previously described were determined by Xow cytometry analysis using FACS Calibur Cytometer (BD). Murine NK cell isolation Mouse NK cell population was obtained by resuspending 107 splenocytes/ml in PBS containing 0.5% bovine serum albumin and treated with biotin-antibody cocktail containing anti-CD5 (Ly-1), CD8a (Ly-2), CD4 (L3T4), Gr-1 (Ly-6G/C), CD19 and Ter-119 MicroBeads (Miltenyi Biotec, Gladbach, Germany). After incubation for 15 min at 4°C, cells were washed and the cell populations were depleted by magnetic cell sorting (MACS) system with an autoMACSTM column (Miltenyi Biotec) according to the manufacturer’s instructions. NK cell population purity assessed by Xow cytometry was 90% (data not shown). NK cells were incubated in the conditioned media from non-modiWed or genetically modiWed B16 cells expressing GIFT2 in the presence or absence of TGF neutralizing antibody for 72 h and IL-2R (CD122) expression was determined by Xow cytometry. After 72 h, the supernatant was collected and IFN production was determined by ELISA. NK cell extracts were immunoblotted using anti-phosphorylated SMAD2, SMAD3, total SMAD2/3 antibodies and anti -tubulin antibody as loading control. In vivo blockade of TGF 104 genetically modiWed cytokine secreting and/or non modiWed B16 cells were mixed with 500 l Matrigel (BD Biosciences) at 4°C plus 40 g

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TGF neutralizing antibody or isotype control, and injected subcutaneously in C57Bl/6 mice. Tumor volume was monitored over time and statistic analysis was performed. Similar experiments were carried out using 10 g of soluble TGF receptor II as a TGF blocking agent.

Results MHC class I and II expression in B16 melanoma cells B16 melanoma derived from C57Bl/6 mice [6, 7] is known as “poorly immunogenic” [15] yet under certain circumstances, it is possible to upregulate MHC I and MHC II expression in these cells in vitro [4]. To verify the immune phenotype of B16 and B16GIFT2 cells here utilized as model systems, we performed Xow cytometric analysis for cell surface expression of MHC I, MHC II and the co-stimulatory molecules CD80 and CD86. As shown in supplementary data 1, wild type B16 cells and their B16GIFT2 derivatives do not express detectable levels of these surface proteins, yet robustly express CD105 (endoglin) a co-receptor for TGF. This phenotype is consistent with low immunogenicity and is unaVected by expressing GIFT2 fusokine. Immune bystander eVect is lost with increased B16 melanoma tumor burden To test the eVect of tumor burden on immune bystander eVect, we admixed B16 melanoma cells with a polyclonal population of GIFT2-secreting B16 cells (hereafter B16GIFT2) at a constant 1:1 ratio. We measured tumor growth in mice having received an initial tumor cell inoculum of either: 1 £ 104, 1 £ 105 or 1 £ 106 of each cell type. As previously reported, Fig. 1a shows that mice implanted with B16GIFT2 melanoma cells remain tumor free long term and mice implanted with B16 cells promptly develop palpable tumors within 20 days. The cohort of immunocompetent mice implanted with 1 £ 104 B16GIFT2 cells mixed with 1 £ 104 B16 cells displayed the highest percentage of survival and cure (40% of mice), indicating that the paracrine secretion of GIFT2 from B16GIFT2 cells induced a local bystander antitumor response against B16 cells present at the tumor site. However, this bystander eVect is lost in a cell dose dependent manner as the number of total B16 cells increases (despite a constant 1:1 mix of B16 and B16GIFT2 cells), with a complete loss of the immune bystander eVect with a 1 £ 106 cell dose. Figure 1b, c details a replicate in vivo experiment where B16:B16GIFT2 at 1:1 ratio either 1 £ 104 (B) or 1 £ 106 (C) per cell type were implanted at day 0 and tumor growth monitored over time. Whereas mice receiving solely B16GIFT2 remained tumor-free independently of the size

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of the day 0 inoculum, virtually all mice receiving 1 £ 106 of each B16:B16GIFT2 at 1:1 grew tumors as quickly as B16 controls. Only in the “low tumor burden” group receiving 1 £ 104 of each B16:B16GIFT2 at 1:1 had a long term 50% cure rate (P < 0.05 log rank).

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Days post tumor implantation Fig. 1 In vivo immune bystander eVect of GIFT2-secreting cells. Kaplan–Meier survival curve of: a cohort of 10 C57Bl/6 mice per each experimental group were injected subcutaneously with (Wlled circle) 1 £ 105 B16GIFT2 cells (positive control), (Wlled triangle) 1 £ 105 B16 cells (negative control), or a mixture of both at 1:1 ratio varying the cell number: (Wlled square) 1 £ 104, (open circle) 1 £ 105 and (open cross) 1 £ 106 of each cell type. b Cohorts of 10 mice per each experimental group were injected subcutaneously with (Wlled circle) 1 £ 104 B16GIFT2 cells, (Wlled triangle) 1 £ 104 B16 cells and (Wlled square) 1 £ 104 of each admixed cells at 1:1 ratio (2 £ 104 total cell number). c A similar experiment was performed using a cohort of 10 mice per group injected with (Wlled square) 1 £ 106 of each admixed cells at 1:1 ratio (2 £ 106 total cell number), (Wlled circle) B16GIFT2 cells and (Wlled triangle) B16 cells. These experiments were repeated three times with similar results and statistic analysis indicated signiWcant diVerences between the test groups (P < 0.05 log rank)

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We observed that the bystander eVect was operative in implants of 1 £ 104 of each B16:B16GIFT2 at 1:1 yet lost in similar implants inoculated at a dose of 1 £ 106 cells as shown in Fig. 1c. We speculate that the host-derived cell mediated immune response must be involved in this discrepancy. To analyze the diVerential response to low (1 £ 104) and high (1 £ 106) dose implants, we performed an in vivo matrigel cell inWltrate analysis as previously described [21]. In brief, melanoma cells were embedded in matrigel, injected in mice subcutaneously, surgically retrieved 6 days later and enzymatically dissociated to produce a cellular suspension amenable to Xow cytometry analysis. We analyzed the recruitment of host-derived CD4+, CD8+, NK, NKT cells, and CD4+CD25+ cells to the tumor site. Immune inWltrate analysis of the matrigel plugs of 1 £ 104 of each B16:B16GIFT2 at 1:1 admixed cells per implant, exhibited a pattern of cell migration similar to that seen in implants where all B16 cells express GIFT2 (Fig. 2a). There was no signiWcant diVerence in the proportion of CD4, CD8 or CD4/CD25 cells between B16GIFT2 and B16:B16GIFT2 admixed cells at low and high tumor cell dose. The cohort of mice injected with wild type B16 shows overall a poor recruitment of immune eVector cells to the tumor site. However, the cohort of mice implanted with 1 £ 106 of each B16:B16GIFT2 at 1:1 admixed cells revealed a signiWcantly reduced NK and NKT cell recruitment to the tumor site (Fig. 2b), suggesting a selective suppression of innate cellular eVectors as B16 tumor burden increases and immune bystander eVect is lost. We also performed a cell inWltrate analysis in implants containing 1 £ 105 of each B16:B16GIFT2 and we observed a pattern of cell migration similar to that seen in 1 £ 106 cell dose, although no statistically signiWcant diVerence was observed (P = 0.08 log rank, supplementary data 2). B16 tumor cells secrete biologically active TGF The loss of the immune bystander eVect with tumor cell dose is associated with a decrease of host-derived NK and NKT cells inWltration at tumor site. This observation suggests that a secreted inhibitory factor is released by B16 cells (and their GIFT2 derivatives) which acts as a dominant negative modulator of the immune bystander eVect driven by the GIFT2 fusokine. B16 have been previously shown to release latent TGF which would be a likely


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Fig. 2 Immune inWltrated analysis of tumor implants: Immunocompetent C57Bl/6 mice were injected subcutaneously with (Wlled bars) B16 cells, (open bars) B16GIFT2 cells, (dotted bar) admixed cells and (chequered bar) mouse embryonic Wbroblasts (MEF) embedded in matrigel. Implants were retrieved 6 days post implantation and digested with collagenase to collect immune inWltrated cells, which were ana-

lyzed by Xow cytometry. a Cohorts of six mice were implanted with 1 £ 104 cells. b Cohorts of six mice were implanted with 1 £ 106 cells. SigniWcant diVerences were observed of NK cells tumor recruitment between implants contained 1 £ 106 of each admixed cells and 1 £ 106 B16GIFT2 cells (P < 0.05). These experiments were performed in triplicate with similar results

candidate suppressor of immunogene-driven bystander eVect [18]. We here determined whether B16 cells produced functionally active TGF. An IL-2 dependent cell line (CTLL-2) was used to analyze this immunosuppressive property and was used as a bioassay for active TGF. CTLL-2 cells cultured in medium conditioned by B16 cells and increasing doses of recombinant IL-2 showed a signiWcant reduced proliferation in MTT assay compared to the control (medium conditioned by mouse embryonic Wbroblasts). The addition of TGF neutralizing antibody rescued the ability of these cells to proliferate in response to IL-2 (Fig. 3a). Active TGF induces de novo expression of CD8 on CTLL-2 cells and on normal immature thymocytes [8]. Based on this property, we observed the expression of CD8 on CTLL-2 cultured in medium conditioned by B16 cells. Similarly, recombinant TGF (1 ng/ml) induced de novo CD8 expression on CTLL-2 cells (Fig. 3b). The expression of CD8 was abrogated with TGF neutralizing antibody, indicating a speciWc property of active TGF secreted by B16 cells (Fig. 3c). We analyzed surface expression of components of the interleukin-2 (IL2) receptor complex by CTLL-2 in response to B16 conditioned media. We found that CTLL-2 cells cultured in medium conditioned by B16 cells downregulated the expression of IL-2R after 72 h of incubation and such expression increased signiWcantly after treatment with TGF neutralizing antibody (Fig. 3d). The expression of the IL-2R on CTLL-2 was not altered by B16 conditioned media (data not shown).

B16 derived TGF blocks NK cell recruitment and function IL-2/IL-2R interaction on NK cells has relevant implications in the expansion and development of NK cells, as well as in the cytotoxicity and cytokine production by these cells. In particular, the common IL-2/IL-15R signaling activates transcription factors involved in the control of perforin expression and secretion of proinXammatory cytokines such as GM-CSF and IFN [24]. Since we observed a reduced recruitment of NK cells to the tumor site as B16 tumor burden increases, we evaluated the responsiveness of mouse NK cells to IL-2 by culturing NK cells in medium conditioned by B16 and B16GIFT2 cells. As shown in Fig. 4a, the expression of IL-2R chain was signiWcantly reduced on NK cells cultured in medium conditioned by B16 cells supplemented with recombinant IL-2 (23.5 pmol/ml). Pre-treating medium conditioned by B16 cells with anti-TGF neutralizing antibody restored the expression of IL-2R on NK cells to similar levels as the positive control. On the other hand, NK cells cultured in medium conditioned by B16GIFT2 cells containing equimolar concentration of GIFT2, display a signiWcantly greater expression of IL-2R despite the presence of a similar concentration of active TGF secreted also by B16GIFT2 cells. This expression was not altered by TGF neutralizing antibody pre-treatment. This increase in the expression of IL-2R demonstrated that GIFT2 fusokine released by B16GIFT2 cells overrides the suppressive eVects of TGF on NK cells. We also evaluated the expression

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