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STING contributes to anti-glioma immunity via triggering type-I IFN signals in the tumor microenvironment

  • Takayuki Ohkuri1, 6,
  • Arundhati Ghosh4, 8,
  • Akemi Kosaka1, 6,
  • Jianzhong Zhu4, 8,
  • Maki Ikeura6,
  • Michael David9,
  • Simon C Watkins5,
  • Saumendra N Sarkar3, 4, 8 and
  • Hideho Okada1, 2, 3, 6, 7
Journal for ImmunoTherapy of Cancer20142(Suppl 3):P228

https://doi.org/10.1186/2051-1426-2-S3-P228

Published: 6 November 2014

Keywords

Green Fluorescence ProteinTumor MicroenvironmentIntratumoral AdministrationMouse GliomaGlioma Microenvironment
While type-I interferons (IFNs) play critical roles in antiviral and antitumor activity, it remains to be elucidated how type-I IFNs are produced in sterile conditions of the tumor microenvironment and directly impacts tumor-infiltrating immune cells. We report that both human and de novo mouse gliomas show increased expression of type-I IFN messages, and in mice, CD11b+ brain-infiltrating leukocytes (BILs) are the main source of type-I IFNs that is induced partially in a STING (stimulator of IFN genes)-dependent manner. Consequently, glioma-bearing Sting Gt/Gt mice showed shorter survival, and lower expression levels of Ifns compared with wild-type mice. Furthermore, BILs of Sting Gt/Gt mice show increased CD11b+ Gr-1+ immature myeloid suppressor and CD25+ Foxp3+ regulatory T (Treg) cells, while decreased IFN-γ-producing CD8+ T cells. To determine the effects of type-I IFN expression in the glioma microenvironment, we utilized a novel reporter mouse model, in which the type-I IFN signaling induces the Mx1 (IFN-induced GTP-binding protein) promoter-driven Cre recombinase, which turns the expression of loxp-flanked tdTomato off, and turns green fluorescence protein (GFP) expression on, thereby enabling us to monitor the induction and effects of IFN signaling in the glioma microenvironment. CD4+ T cells that received direct type-I IFN signals (i.e., GFP+ cells) demonstrate lesser degrees of regulatory activity based on lower Foxp3 and Tgfb1 expression levels (Figure 1) as well as lesser suppression of CD8+ T cell proliferation (Figure B). IFN-sensed CD8+ T cells exhibit enhanced levels of Th1 markers, Tbx21 and Igfng (Figure C), as well as cytotoxic T-cell activity based on reverse antibody-dependent T-cell-mediated cytotoxicity assay (Figure D). Finally, intratumoral administration of a STING agonist (cyclic diguanylate monophosphate; c-di-GMP) improves the survival of glioma-bearing mice associated with enhanced type-I IFN signaling, Cxcl10 and Ccl5 and T cell migration into the brain. In a combination with subcutaneous OVA peptide-vaccination, c-di-GMP increased OVA-specific cytotoxicity of BILs and prolonged the survival. These data demonstrate significant contributions of STING to antitumor immunity via enhancement of the type-I IFN signaling in the tumor microenvironment, and imply a potential use of STING agonists for development of effective immunotherapy, such as the combination with antigen-specific vaccinations.
Figure 1
Figure 1

Type-I IFNs directly impact on T-cell functions in glioma-developing mice. (A) CD4+ cells from draining LN derived from glioma-developing tdTomato mice were sorted into GFP- or GFP+ cells and incubated with (black bars) or without (grey bars) anti-CD3mAb. After 4 h, total RNA was extracted for evaluation of Foxp3 and Tgfb1 mRNA levels by qRT-PCR. (B) CFSE-labeled WT CD8+ T-cells were co-cultured with GFP- or GFP+ CD4+ T-cells in the presence of CD3 beads. After 60 h, division of CFSE-labeled CD8+ T-cells gated by reactivity to PE-Cy7-condjugated anti-CD8mAb was evaluated by CFSE intensity. As a negative control, CFSE-labeled WT CD8+ T-cells were cultured without any stimulation (left panel). Histograms are representative of two independent experiments. The bar graph shows the percentage of CD8+ cells that have divided at least twice in each of two stimulation conditions (N = 4/group; *p < 0.05). (C) GFP- or GFP+ CD8+ T-cells were incubated with (black bar) or without (grey bar) anti-CD3mAb. After 4 h, total RNA was extracted for evaluation of Tbx21 and Ifng mRNA expression levels by qRT-PCR (U: undetected). (D) Cytotoxic activity of GFP- and GFP+ CD8+ T-cells was evaluated by 51Cr-release assay. RMA-S cells untreated (left panel) or pretreated (right panel) with anti-CD3mAb (10 g/mL) were used as target cells. *p < 0.05 compared at the same E/T ratio.

Authors’ Affiliations

(1)
Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, USA
(2)
Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, USA
(3)
Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, USA
(4)
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, USA
(5)
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, USA
(6)
Department of Brain Tumor, University of Pittsburgh School of Medicine, Pittsburgh, USA
(7)
Cancer Immunology, University of Pittsburgh Cancer Institute, Pittsburgh, USA
(8)
Cancer Virology Programs, University of Pittsburgh Cancer Institute, Pittsburgh, USA
(9)
Division of Biology, UCSD, La Jolla, USA

Copyright

© Ohkuri et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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