Maki Ikeura

STING Contributes to Antiglioma Immunity via Triggering Type I IFN Signals in the Tumor Microenvironment

Ohkuri, Ghosh, Kosaka, and colleagues show that a STING-mediated DNA-sensor signaling is involved in IFN induction in the sterile microenvironment of brain tumor that enhances antitumor immunity, providing a proof-of-concept for developing STING agonists for cancer immunotherapy. Although type I 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 affect tumor-infiltrating immune cells. Mouse de novo gliomas show increased expression of type I IFN messages, and in mice, CD11b+ brain-infiltrating leukocytes (BIL) are the main source of type I IFNs that are induced partially in a STING (stimulator of IFN genes)-dependent manner. Consequently, glioma-bearing StingGt/Gt mice showed shorter survival and lower expression levels of Ifns compared with wild-type mice. Furthermore, BILs of StingGt/Gt mice showed increased CD11b+ Gr-1+ immature myeloid suppressor and CD25+ Foxp3+ regulatory T cells (Treg) and decreased IFNγ-producing CD8+ T cells. CD4+ and CD8+ T cells that received direct type I IFN signals showed lesser degrees of regulatory activity and increased levels of antitumor activity, respectively. Finally, intratumoral administration of a STING agonist (cyclic diguanylate monophosphate; c-di-GMP) improved the survival of glioma-bearing mice associated with enhanced type I IFN signaling, Cxcl10 and Ccl5, and T-cell migration into the brain. In combination with subcutaneous OVA peptide vaccination, c-di-GMP increased OVA-specific cytotoxicity of BILs and prolonged their survival. These data demonstrate significant contributions of STING to antitumor immunity via enhancement of type I IFN signaling in the tumor microenvironment and suggest a potential use of STING agonists for the development of effective immunotherapy, such as the combination with antigen-specific vaccinations. Cancer Immunol Res; 2(12); 1199–208. ©2014 AACR.

LOH in the HLA Class I region at 6p21 is Associated with Shorter Survival in Newly Diagnosed Adult Glioblastoma

Purpose: Glioblastoma (GBM) shows downregulated expression of human leukocyte antigen (HLA) class I, thereby escaping from cytotoxic T cells and limiting the efficacy of immunotherapy. Loss of heterozygosity (LOH) of HLA class I (6p21) and/or β-2 microglobulin (B2m) (15q21) regions represents irreversible downregulation. In this study, we examined the prevalence of these LOH events and their relations with overall survival in GBM. Experimental Design: In a cross-sectional analysis on 60 adult patients with GBM, DNA from formalin-fixed, paraffin-embedded specimens were evaluated for 10 microsatellite regions of HLA class I, B2m, HLA class II, HLA class III, and 6q by PCR as well as immunohistochemical evaluation of HLA class I expression and CD8+ T-cell infiltration. Results: LOH in HLA class I, B2m, HLA class II, HLA class III, and 6q regions was present in 41.4%, 18.2%, 9.4%, 77.8%, and 36.0% of informative cases, respectively. LOH of HLA class I was associated with shorter overall survival (HR = 4.89, P = 0.0078). HLA class I was downregulated in 22% to 43% of cases based on immunohistochemistry. Cases that displayed negative staining were significantly younger. HLA class I expression correlated with intratumoral CD8+ T-cell infiltration. Conclusion: LOH in the HLA class I region is frequent in adult GBMs. The association of shorter survival with LOH in this region suggests a crucial role for these genes in immunosurveillance. Clin Cancer Res; 19(7); 1816–26. ©2013 AACR.

Expression of antigen processing and presenting molecules in brain metastasis of breast cancer.

Defects in human leukocyte antigen class I antigen processing machinery (APM) component expression can have a negative impact on the clinical course of tumors and the response to T cell-based immunotherapy. Since brain metastases of breast cancer are of increasing clinical significance, the APM component expression levels and CD8+ T cell infiltration patterns were analyzed in primary breast and metastatic brain lesions of breast cancer by immunohistochemistry. Comparison of unpaired 50 primary and 33 brain metastases showed lower expression of β2-microglobulin, transporter associated with antigen processing (TAP) 1, TAP2 and calnexin in the brain lesions. Although no significant differences were found in APM component scores between primary breast and brain lesions in 15 paired cases, primary breast lesions of which patients eventually developed brain metastases showed lower levels of β2-microglobulin, TAP1 and calnexin compared with breast lesions without known brain metastases. The extent of CD8+ T cell infiltration was significantly higher in the lesions without metastasis compared with the ones with brain metastases, and was positively associated with the expression of TAP1 and calnexin. Furthermore, mouse tumor cells stably transfected with silencing hairpin (sh)RNA for TAP1 demonstrated a decreased susceptibility to cytotoxic T lymphocytes in vitro and enhanced spontaneous brain metastasis in vivo. These data support the functional significance of TAP1 expression in tumor cells. Taken together, our data suggest that patients with low or defective TAP1 or calnexin in primary breast cancers may be at higher risks for developing brain metastasis due to the defects in T cell-based immunosurveillance.

Differential activity of interferon-α8 promoter is regulated by Oct-1 and a SNP that dictates prognosis of glioma

We have previously reported that the single nucleotide polymorphism (SNP) rs12553612 in IFNA8 is associated with better overall survival of glioma patients with the AA-genotype compared with patients with the AC-genotype. As rs12553612 is located in the IFNA8 promoter, we hypothesized that the A-allele allows for an enhanced IFNA8 promoter activity compared with the C-allele. Reporter assays in the human monocyte derived THP-1 cell line demonstrated a superior promoter activity of the A-allele compared with the C-allele. Electrophoretic mobility shift assays (EMSA) further demonstrated that the A-genotype specifically binds to more nuclear proteins than the C-genotype, including the transcription factor Oct-1. Further, co-transfection of plasmids encoding Oct-1 and the reporter constructs revealed that Oct-1 enhanced the promoter activity with the A- but not the C-allele. Taken together, our data demonstrate that the A-allele in the rs12553612 SNP, which is associated with better glioma patient survival, allows for IFNA8 transcription by allowing for Oct-1 binding, which is absent in patients with C allele, and suggests a molecular mechanism of IFNA8 mediated immune-surveillance of glioma progression.

Transgene-derived overexpression of miR-17-92 in CD8+ T-cells confers enhanced cytotoxic activity

MicroRNAs (miRs) play important roles in regulation of a variety of cell functions, including immune responses. We have previously demonstrated that miR-17-92 expression in T-cells enhances Th1 phenotype and provides a long-term protection against glioblastoma when co-expressed as a transgene in T-cells along with a chimeric antigen receptor. To further elucidate the function of miR-17-92 in tumor antigen-specific CD8(+) T-cells, we generated transgenic (Tg) mice in which CD8(+) T-cells overexpress transgene-derived miR-17-92 under the lck promoter as well as T-cell receptor specific for human gp10025-33 (Pmel-1) (miR-17-92/Pmel-Tg). CD8(+) T-cells from miR-17-92/Pmel-Tg mice demonstrated enhanced interferon (IFN)-γ production and cytotoxicity in response to the cognate antigen compared with those from control Pmel-Tg mice without the transgene for miR-17-92. In addition, miR-17-92/Pmel-Tg mouse-derived CD8(+)CD44(+) T-cells demonstrated increased frequencies of cells with memory phenotypes and IFN-γ production. We also found that miR-17-92/Pmel-Tg-derived CD8(+) T-cells expressed decreased levels of transforming growth factor (TGF)-β type II receptor (TGFBR2) on their surface, thereby resisting against suppressive effects of TGF-β1. Our findings suggest that engineering of tumor antigen-specific CD8(+) T-cells to express miR-17-92 may improve the potency of cancer immunotherapy.

Histamine deficiency promotes accumulation of immunosuppressive immature myeloid cells and growth of murine gliomas

To elucidate mechanisms underlying epidemiological findings of decreased risk of glioma development in patients with allergies and asthma, gliomas were induced in mice deficient for histidine decarboxylase (HDC), the enzyme responsible for histamine production. These mice exhibited shortened survival and enhanced tumor growth compared to wild-type (WT) mice. Previous studies have shown a pivotal role of HDC in maturation of bone marrow (BM)-derived myeloid cells. In our glioma models, brain-infiltrating leukocytes (BIL) demonstrated an increased frequency of CD11b+Gr1+ immature myeloid cells (IMC; both CD11b+Ly6G+ and CD11b+Ly6C+ subpopulations) as well as diminished CD8+ T cell infiltration and their effector functions in HDC−/− mice compared with WT mice. Furthermore, HDC−/− IMC demonstrated a more profound immune suppression of CD8+ T cell proliferation and functions associated with increased prostaglandin E2 (PGE2) expression levels. Celecoxib, a cyclooxygenase-2 inhibitor, which is vital for PGE2 production, abrogated suppressive capabilities of HDC−/− IMC. In addition, glioma-bearing HDC-eGFP mice, in which HDC promoter drives green fluorescence protein (GFP) expression, exhibited decreased HDC promoter activities in CD11b+Gr1+ cells in the BM, spleen, and intracranial tumor site compared with non-tumor bearing HDC-eGFP mice. Additionally, in vitro culture with glioma supernatants decreased GFP expression in CD11b+Gr1+, CD11b+Ly6G+, and CD11b+Ly6C+ IMC. HDC expression levels inversely correlated with suppressive functions of CD11b+Gr1+ IMC, as GFP−CD11b+Gr1+ more profoundly inhibited CD8+ T cell proliferation compared with CD11b+Gr1+GFP+ cells. Taken together, these data show a significant role of HDC in the glioma microenvironment via maturation of myeloid cells and resulting activation of CD8+ T cells.

Abstract 3507: Type 17-1-CD8+ T cells (Tc17-1) as potent effector cells for immunotherapy of brain tumors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Malignant gliomas, such as glioblastoma multiforme (GBM), are the most common and dismal primary brain tumors, and there is a strong need for developing novel and effective therapeutic modalities. Although the central nervous system (CNS) and CNS tumors are often considered immunologically privileged, as demonstrated by studies on CNS autoimmunity, activated autoreactive T-cells, especially T helper (Th) 17 cells, infiltrate through the blood brain barrier (BBB) and mediate antigen-specific responses in the CNS. Especially, Th17 express high levels of CCR6, which has a crucial role in Th17 infiltration into the CNS. In addition to Th17 cells, a new putative subtype of IL-17-producing CD4+ T cells with CD4+IL-17+IFN-γ+ (Th17-1 cells) double-positive phenotype has also been identified. Based on our previous studies demonstrating a critical role of Th1-chemokine CXCL10 and its receptor CXCR3 for effective T-cell trafficking in CNS tumors, we hypothesized that Th17-1 cells and CD8+ T cells producing IL-17 and IFN-γ (hereby Type-17-1 CD8+ T-cells) will demonstrate superior CNS tumor infiltration compared with Th1 cells due to the activation of two CNS-relevant chemokine pathways, CXCL10-CXCR3 and CCL20-CCR6. Mouse Tc17, Tc17-1 and Tc1 cells generated from Pmel-1 mouse-derived naive CD8+ T cells expressed high CCR6 and low CXCR3, high CCR6 and high CXCR3, and little CCR6 and high CXCR3, respectively. Although Tc17 displayed lower levels of antigen-specific cytotoxic activity compared with Tc17-1 and Tc1 in a 4 hr cytotoxic assay, Tc17 killed tumor cells more effectively than the other populations in long-term cultures (4 to 6 days). While Tc1 cells underwent apoptosis due to their exhausted state, Tc17-1 maintained strong cytotoxicity and proliferative activities even after 4 day co-culture with tumor cells. Moreover, the supernatant derived from activated Tc17 and Tc17-1 induced CCL20 and CXCL10 in the GL261 glioma cells while Tc1-derived supernatant induced CXCL10 but not CCL20. These results suggest a possibility that Tc17-1 may elicit persistent anti-CNS tumor responses and induce both CCL20 and CXCL10 in the CNS tumor microenvironment, thereby attracting additional effector cells to the site. In vivo studies in mice bearing CNS gliomas are warranted to determine whether adoptively transferred Tc17-1 infiltrate to the CNS tumor site and mediate superior anti-tumor response compared with Tc1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3507. doi:1538-7445.AM2012-3507