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. 2010 Jul 1;16(13):3409-19.
doi: 10.1158/1078-0432.CCR-10-0644. Epub 2010 May 14.

Role of type 1 IFNs in antiglioma immunosurveillance--using mouse studies to guide examination of novel prognostic markers in humans

Mitsugu Fujita  1 , Michael E Scheurer, Stacy A Decker, Heather A McDonald, Gary Kohanbash, Edward R Kastenhuber, Hisashi Kato, Melissa L Bondy, John R Ohlfest, Hideho Okada
Affiliations

Role of type 1 IFNs in antiglioma immunosurveillance--using mouse studies to guide examination of novel prognostic markers in humans

Mitsugu Fujita et al. Clin Cancer Res. .

Abstract

Purpose: We hypothesized that the type 1 IFNs would play a pivotal role in antiglioma immunosurveillance through promotion of type 1 adaptive immunity and suppression of immunoregulatory cells.

Experimental design: We induced de novo gliomas in Ifnar1(-/-) (deficient for type 1 IFN receptors) or wild-type mice by intracerebroventricuar transfection of NRas and a short hairpin RNA against P53 using the Sleeping Beauty transposon system. We analyzed the survival of 587 glioma patients for single nucleotide polymorphisms (SNP) in type 1 IFN-related genes.

Results: Ifnar1(-/-) mice exhibited accelerated tumor growth and death. Analyses of brain tumor-infiltrating lymphocytes in Ifnar1(-/-) mice revealed an increase of cells positive for CD11b(+)Ly6G(+) and CD4(+)FoxP3(+), which represent myeloid-derived suppressor cells and regulatory T cells, respectively, but a decrease of CD8(+) cytotoxic T lymphocytes (CTLs) compared with wild-type mice. Ifnar1(-/-) mouse-derived glioma tissues exhibited a decrease in mRNA for the CTL-attracting chemokine Cxcl10, but an increase of Ccl2 and Ccl22, both of which are known to attract immunoregulatory cell populations. Dendritic cells generated from the bone marrow of Ifnar1(-/-) mice failed to function as effective antigen-presenting cells. Moreover, depletion of Ly6G(+) cells prolonged the survival of mice with developing gliomas. Human epidemiologic studies revealed that SNPs in IFNAR1 and IFNA8 are associated with significantly altered overall survival of patients with WHO grade 2 to 3 gliomas.

Conclusions: The novel Sleeping Beauty-induced murine glioma model led us to discover a pivotal role for the type 1 IFN pathway in antiglioma immunosurveillance and relevant human SNPs that may represent novel prognostic markers.

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Figures

Figure 1
Figure 1. Ifnar1−/− mice exhibit accelerated tumor growth and death following SB-based induction of gliomas
(A) CNS gliomas were induced in C57BL/6-background Ifnar1−/− (red lines) or WT (black lines) neonatal mice by intraventricular transfection of the following plasmids: pT2/C-Luc//PGK-SB1.a3 (0.2 μg), pT/CAG-NRas (0.4 μg), and pT/shp53 (0.4 μg). Tumor growth (left) and symptom-free survival (right) were monitored. P-values are based on log-rank test. (B) Glioma cells derived from Ifnar1−/− (solid lines) or WT (dashed lines) mice were injected into the basal ganglia of Ifnar1−/− (red lines) or WT (black lines) host mice (1 × 105/mouse). Tumor growth (left) and symptom-free survival (right) were monitored. (C) 51Cr release assay was performed to evaluate cytolytic ability of cervical LN cells obtained from the mice bearing Ifnar1−/− cells (solid lines in B) against the Ifnar1−/− and WT cell lines. (D) ELISA was performed to evaluate the amount of mIFN-γ secretion by the CTLs used in (C) per the last 24 hrs. (C and D) I: Ifnar1−/−; W: WT. Lines within boxes denote means; box upper and lower bounds indicate SD; whiskers indicate minimum and maximum values. P-values are based on Holm’s post hoc test.
Figure 2
Figure 2. Ifnar1−/− mice demonstrate increased tumor-infiltration of CD11b+Ly6G+ and CD4+FoxP3+ cells but decreased Tc1 effector cells and CD11c+ DCs
(A and B) The mice bearing SB-induced tumors were sacrificed at around days 50–60, and BILs were isolated based on similar tumor size observed by BLI. BILs obtained from 3 mice in a given group were pooled and then evaluated by flow cytometry for the following subpopulations: CD11b+Ly6G+, CD4+FoxP3+, CD11c+CD86+ (A) and CD8+gp100 tetramer+ (B). Numbers in dot plots indicate percentage of gated subpopulations in leukocyte-gated BILs. Absolute numbers are depicted in the rightmost panels. P-values are based on Student’s t-test. (C and D) Total RNA was extracted from each mouse brain (3 mice/group). Quantitative RT-PCR was performed to evaluate the mRNA expression levels of the following molecules: Ccl2, Ccl22, Cxcl10 (C) and Gp100 (D).
Figure 3
Figure 3. Ifnar1−/− DCs demonstrate defects in their APC function and the ability to attract CTLs in the glioma sites
(A) Bone marrow-derived mature DCs were prepared as described in Materials and Methods. (Upper panels) Flow cytometry was performed to evaluate the following surface markers: I-Ab (an MHC class II), CD86, and CCR7. Histograms represent the following DC types: Ifnar1−/− (shaded), WT (open), and WT DCs stained with isotype control IgG (dashed line). (Lower panels) Immature DCs were matured with 250 ng/ml LPS in the presence or absence of 10 ng/ml rmIFN-α. ELISA was performed to evaluate the production levels of the following molecules per the last 24 hours: mIL-12p70, mCXCL10, and mCCL22. (B) WT mice received s.c. immunization with either Ifnar1−/− or WT DCs loaded with 5 μg/ml hgp10025–33 (upper) or Garc177–85 (lower) peptide on Day 0. Control mice received PBS. (Upper panels) On day -1, mice received an i.v. injection of 5 × 106 naive Pmel-I CD8+ T cells labeled with CFSE. On day 6, inguinal LN cells were collected to evaluate the proliferation of CSFE-labeled gp100-reactive cells by flow cytometry. (Lower panels) On day 6, the Garc177–85-specific cytolytic ability of effector T cells was evaluated by in vivo cytolytic assay as described in Materials and Methods. (C) Mice with developing gliomas received intratumoral injections of DCs derived from Ifnar1−/− or WT mice (1 × 105/mouse) on day 45 after birth, and i.v. Tc1 infusions on the same day (3 × 106/mouse). Mice were then sacrificed on day 5 following the DC injection for flow-cytometric evaluation of Tc1 infiltration into the tumor sites. Numbers in dot plots indicate percentage of CD8+gp100-teramer+ cells in lymphocyte-gated BILs. Absolute numbers are depicted in the rightmost panel.
Figure 4
Figure 4. mAb-mediated depletion of Ly6G+ cells, but not CD25+ cells, inhibit the growth of SB-induced gliomas
(A and B) Ifnar1−/− or WT mice with developing gliomas received i.p. injections of anti-Ly6G mAb (RB6-8C5; 0.25 mg/dose) or control IgG on days 21, 23, 25, and 27 after tumor induction. (A) The mice were sacrificed, and BILs were isolated for CD11b+Ly6G+ subpopulations. (B) Symptom-free survival was monitored. (C and D) Ifnar1−/− or WT mice with developing gliomas received i.p. injections of anti-CD25 mAb (PC61; 0.25 mg/dose) or control IgG on days 21 and 24 after tumor induction. (C) The mice were sacrificed, and BILs were isolated for CD4+FoxP3+ subpopulations. (D) Symptom-free survival was monitored.
Figure 5
Figure 5. Treatment with poly-ICLC prolongs the survival of mice bearing SB-induced gliomas in an Ifnar1-dependent manner
Mice with developing gliomas received i.m. injections of either poly-ICLC (2.5 mg/kg/dose) or mock PBS starting on days 21 and 24 after birth, and weekly thereafter. Symptom-free survival was monitored.
Figure 6
Figure 6. Association of SNPs in IFN-related genes and the survival of patients with WHO grade 2–3 gliomas
Overall survival was evaluated among 304 glioma patients with grade 2–3 gliomas by genotype for SNPs in IFN-related genes. (A) Patients with AA genotype (red line) for IFNAR1 rs1041868 exhibited a significantly shorter survival than those with the AG/GG genotypes (black line). (B) Patients with AC genotype (red line) for IFNA8 rs12553612 exhibited a significantly shorter survival than those with the AA genotype (black line).
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