miR-124 Inhibits STAT3 Signaling to Enhance T Cell–Mediated Immune Clearance of Glioma

Abstract

MicroRNAs (miRs) have been shown to modulate critical gene transcripts involved in tumorigenesis, but their role in tumor-mediated immune suppression is largely unknown. On the basis of miRNA gene expression in gliomas using tissue microarrays, in situ hybridization, and molecular modeling, miR-124 was identified as a lead candidate for modulating signal transducer and activator of transcription 3 (STAT3) signaling, a key pathway mediating immune suppression in the tumor microenvironment. miR-124 is absent in all grades and pathological types of gliomas. Upon up regulating miR-124 in glioma cancer stem cells (gCSCs), the STAT3 pathway was inhibited, and miR-124 reversed gCSC-mediated immune suppression of T-cell proliferation and induction of Foxp3+ regulatory T-cells (Tregs). Treatment of T-cells from immunosuppressed glioblastoma patients with miR-124 induced marked effector response including up regulation of IL-2, IFN-γ, and tumor necrosis factor (TNF)-α. Both systemic administration of miR-124 or adoptive miR-124-transfected T-cell transfers exerted potent anti-glioma therapeutic effects in clonotypic and genetically engineered murine models of glioblastoma and enhanced effector responses in the local tumor microenvironment. These therapeutic effects were ablated in both CD4+ and CD8+ depleted mice and nude mouse systems, indicating that the therapeutic effect of miR-124 depends on the presence of a T-cell-mediated antitumor immune response. Our findings highlight the potential application of miR-124 as a novel immunotherapeutic agent for neoplasms and serve as a model for identifying miRNAs that can be exploited as immune therapeutics.

The tumor microenvironment disarms CD8+ T lymphocyte function via a miR-26a-EZH2 axis

“…6A and B). Consequently, based on in vitro cytotoxicity assays with gp100 [25][26][27][28][29][30][31][32][33] peptidepulsed EL4 lymphoma cells, the tumor-specific killing capacity of miR-26a Decoy pMel-1 cells was significantly improved (Fig. 6C).…”

“…pMel-1 transgenic CD8 C T cells were primed in vitro with their cognate antigen (gp100 [25][26][27][28][29][30][31][32][33] peptide) and transduced with retrovirus expressing mock or miR-26a Decoy . Two days after initial activation, CD8 C T cells were deprived of antigen, or treated with CM (B16 cells derived) for an additional 48 h. As expected, even under optimal priming conditions, miR-26a Decoy was capable of enhancing IFNg and granzyme B protein production by CTLs, based on both population percentage and per-cell production.…”

“…Cells were cultured in complete medium consisting of RPMI-1640 (Hyclone, CA, USA) supplemented with 10% FBS (Hyclone, CA, USA), 2 mM L-glutamine, 1% sodium pyruvate, 1% nonessential amino acids, 0.1% HEPES, 1% penicillin, 1% streptomycin, and 0.1% b-mercaptoethanol (Hyclone, CA, USA). To activate CD8 C T cells, the cells were seeded into tissue culture plates with plate-bound anti-CD3 (5 mg/mL, Biolegend, CA, USA) and anti-CD28 (5 mg/mL, Biolegend, CA, USA) antibodies, or in the presence of 5 mM gp100 [25][26][27][28][29][30][31][32][33] peptide with antigen-presenting cells (for pMel-1 CD8 C T cell activation). In EZH2 inhibition experiments, pMel-1 CTLs were pretreated with 1-10 nM of the EZH2 inhibitor GSK-343 (Selleck Chemicals, TX, USA) or an ethanol vehicle control for 48 h in vitro before flow cytometry analysis or intratumoral injection.…”

“…In vitro cytotoxicity assays pMel-1 CD8 C T cells were stimulated with gp100 [25][26][27][28][29][30][31][32][33] and transduced with mock or miR-26a decoy vector. EL4 cells were loaded with 10 mM of gp100 [25][26][27][28][29][30][31][32][33] overnight, then labeled with Cell Tracker Orange (Invitrogen, Carlsbad, CA) (loaded cells: 5 mM, unloaded cells: 0.5 mM), Peptide-loaded and unloaded cells were used as target or control cells, respectively, by mixing them at a 1:1 ratio before co-culture with CTLs at the indicated ratio in a humidified 37 C incubator.…”

“…Ectopic expression of miR-124 in CTLs enhanced effector function in the TME and exerted potent therapeutic efficacy in a murine model of glioblastoma. 25 Also, in the context of ovarian cancer, deprivation of glucose in memory T cells elevated expression of miR-101 and miR-26a, resulting in diminished functionality and survival capacity of CTLs. 26 These studies indicate that miRNAs are common targets for TME reprogramming of CTL function, and that identifying key miRNAs in CTLs would benefit the design of novel immunotherapy strategies.…”