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IL-6 Signaling Blockade During CD40-Mediated Immune Activation Favors Antitumor Factors by Reducing TGF-Β, Collagen Type I

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IL-6 Signaling Blockade During CD40-Mediated Immune Activation Favors Antitumor Factors by Reducing TGF-Β, Collagen Type I IL-6 Signaling Blockade during CD40-Mediated Immune Activation Favors Antitumor Factors by Reducing TGF-β, Collagen Type I, and PD-L1/PD-1 This information is current as of September 26, 2021. Emma Eriksson, Ioanna Milenova, Jessica Wenthe, Rafael Moreno, Ramon Alemany and Angelica Loskog J Immunol 2019; 202:787-798; Prepublished online 7 January 2019; doi: 10.4049/jimmunol.1800717 Downloaded from http://www.jimmunol.org/content/202/3/787 References This article cites 50 articles, 12 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/202/3/787.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 26, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology IL-6 Signaling Blockade during CD40-Mediated Immune Activation Favors Antitumor Factors by Reducing TGF-b, Collagen Type I, and PD-L1/PD-1 Emma Eriksson,* Ioanna Milenova,* Jessica Wenthe,* Rafael Moreno,† Ramon Alemany,† and Angelica Loskog*,‡ IL-6 plays a role in cancer pathogenesis via its connection to proteins involved in the formation of desmoplastic stroma and to immunosuppression by driving differentiation of myeloid suppressor cells together with TGF-b. Inhibition of IL-6 signaling in the tumor microenvironment may, thus, limit desmoplasia and myeloid suppressor cell differentiation. CD40 signaling can further revert myeloid cell differentiation toward antitumor active phenotypes. Hence, the simultaneous use of IL-6 blockade with CD40 stimuli may tilt the tumor microenvironment to promote antitumor immune responses. In this paper, we evaluated the mecha- Downloaded from nisms of LOAd713, an oncolytic adenovirus designed to block IL-6R signaling and to provide myeloid cell activation via a trimerized membrane-bound isoleucine zipper (TMZ) CD40L. LOAd713-infected pancreatic cancer cells were killed by oncolysis, whereas infection of stellate cells reduced factors involved in stroma formation, including TGF-b-1 and collagen type I. Virus infection prevented IL-6/GM-CSF–mediated differentiation of myeloid suppressors, but not CD163 macrophages, whereas infec- tion of dendritic cells led to upregulation of maturation markers, including CD83, CD86, IL-12p70, and IFN-g. Further, IL-6R blockade prevented upregulation of programed death ligand 1 (PD-L1) and PD-1 on the stimulated dendritic cells. These results http://www.jimmunol.org/ suggest that LOAd713 can kill infected tumor cells and has the capacity to affect the tumor microenvironment by stimulating stellate cells and myeloid suppressors with TMZ-CD40L and IL-6R blockade. Gene transfer of murine TMZ-CD40L prolonged survival in an animal model. LOAd713 may be an interesting therapeutic option for cancers connected to IL-6 signaling, such as pancreatic cancer. The Journal of Immunology, 2019, 202: 787–798. he tumor microenvironment is important for the devel- measured by upregulated a-smooth muscle actin (aSMA), is opment and progression of cancer, and it also plays a role correlated with a poor prognosis (3). in the response to treatment or lack thereof. Pancreatic The cytokine IL-6 is upregulated in pancreatic cancer, and a high T by guest on September 26, 2021 cancer is a challenging disease highly resistant to conventional serum level of IL-6 correlates with a more-advanced disease and cancer therapeutics. The tumor microenvironment is characterized a poorer health status (4). In pancreatic cancer, type 2 tumor- by desmoplasia with stroma including fibroblasts, stellate cells, associated macrophages in the tumor stroma are the main pro- myeloid cells, and extracellular matrix (1). Less than 10–20% of ducers of IL-6, but other cell types also contribute, including stellate the cells are the malignant tumor cells. The activated stellate cells cells (5, 6). Signaling through the IL-6 pathway leads to activation produce a high amount of fibronectin and collagen type I and are, of STAT3, which in turn elevates TGF-b1 expression. TGF-b1can hence, the major contributor to the fibrosis seen in the lesions (2). activate production of collagen type I and plays a central role in Fibrosis increases the intralesion pressure, which is believed to fibrosis of the pancreas (7). TGF-b1 is also involved in immuno- reduce chemotherapy exposure. Indeed, high stromal activity, as suppression, another characteristic of pancreatic cancer, by inhibi- tion of Th1 cells and expansion of T regulatory cells (8, 9). IL-6 is used in some dendritic cell (DC) activation protocols, but it can also interfere with the differentiation and maturation of DCs because it *Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, 751 85 Uppsala, Sweden; †L’Institut d’Investigacio´ Biome`dica promotes STAT3 activation (10, 11), and it is therefore one of the de Bellvitge–Institut Catala` d’Oncologia, L’Hospitalet de Llobregat, 08908 Barcelona, factors to promote differentiation of myeloid-derived suppressor ‡ Spain; and Lokon Pharma AB, 751 83 Uppsala, Sweden cells (MDSCs) (6). The complete role of IL-6 in tumor immunology ORCIDs: 0000-0003-1677-4230 (I.M.); 0000-0003-4385-7568 (J.W.); 0000-0001- is, therefore, obscure. Nevertheless, the presence of macrophages in 9957-3105 (R.M.); 0000-0001-8583-6138 (A.L.). the tumor and/or an increased level of circulating MDSCs correlate Received for publication May 22, 2018. Accepted for publication November 22, to a poorer prognosis (12, 13). 2018. We previously demonstrated that immunostimulatory gene This work was supported by grants to A.L. from the Swedish Cancer Society and Swedish State Support and by a contract research agreement with Lokon Pharma AB. therapy using viruses transferring CD40L (AdCD40L) is a potent Address correspondence and reprint requests to Prof. Angelica Loskog, Department stimulator of the tumor microenvironment because of its capacity to of Immunology, Genetics, and Pathology, Uppsala University, Rudbeck Laboratory activate DCs and tilt M2 to M1 macrophages. AdCD40L showed C11, Dag Hammarskjo¨lds Va¨g 20, 751 85 Uppsala, Sweden. E-mail address: efficacy in mouse, dog, and human (14–17). In the current paper, [email protected] we explored the possibility of combining CD40L-based immune Abbreviations used in this article: BV421, Brilliant Violet 421; DC, dendritic cell; ffu, fluorescent forming unit; MDSC, myeloid-derived suppressor cell; PD-1, pro- activation with IL-6 pathway blockade using virus-mediated gene gramed death-1; PD-L1, programed death ligand-1; sIL, soluble IL; scFv, single therapy. We constructed LOAd713, an oncolytic adenovirus se- chain fragment; scFv-aIL-6R, scFv against the IL-6R; aSMA, a-smooth muscle rotype 5/35 chimera that carries a gene encoding a single chain actin; TMZ, trimerized membrane-bound isoleucine zipper. fragment (scFv) against the IL-6R in combination with a gene Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 encoding a trimerized membrane-bound isoleucine zipper (TMZ) www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800717 788 CD40 STIMULI DURING IL-6 BLOCKADE IN THE TUMOR MILIEU human CD40L. In the current paper, the effect of LOAd713 in- a total volume of 100 ml. Postinfection, the infected cell suspensions were fection of pancreatic cancer cells as well as the stellate cells and diluted to 100,000 cells/ml, and 100 ml in quadruplicates was added to a myeloid immune cells that constitute most of the tumor micro- 96-well plate. After 48 and 72 h, the viability of the cells were measured by MTS CellTiter AQueous One Solution Cell Proliferation Assay kit environment were evaluated. (Promega, Madison, WI) according to the manufacturer’s instructions. The relative cell viability in percent was calculated as the absorbance for infected Materials and Methods cells divided by the absorbance for uninfected cells multiplied by 100. Primary cells and cell lines Phosphorylation of STAT3 The pancreatic cell lines BxPc3, MiaPaCa2, PaCa3, and Panc01 were a Panc01 cells were infected with 25 ffu/cell of LOAd(2), LOAd700, or kind gift from Dr. R. Heuchel (Karolinska Institute, Stockholm, Sweden). LOAd713 or left uninfected and cultured for 48 h. Supernatants were The cell origin was confirmed by short tandem repeat analysis by the harvested and added to fresh Panc01 cells. rIL-6 (20 ng/ml; BioLegend) Uppsala Genome Center, Uppsala, Sweden. BxPc3 were cultured in was then added to the cells followed by 20 min incubation at 37˚C. The RPMI 1640 supplemented with 10% FBS and 1% penicillin–strepto- level of STAT3-Y705 phosphorylation in the different groups was deter- mycin. MiaPaCa2, PaCa3, and Panc01 were cultured in DMEM sup- mined with flow cytometry according to the manufacturer’s protocol (BD plemented with 10% FBS and 1% penicillin–streptomycin.
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