Depletion of myeloid-derived suppressor cells during interleukin-12 immunogene therapy does not confer a survival advantage in experimental malignant glioma

Abstract

Myeloid-derived suppressor cells (MDSCs) accumulate in the glioma microenvironment during tumor progression and promote immunosuppression. Interleukin-12 (IL-12) immunogene therapy can alter MDSCs toward an antigen-presenting cell phenotype and these mature cells can have a central role in antigen presentation. It remains unclear, however, how MDSC depletion can affect glioma immunotherapy. In this study, we generated a replication-deficient adenoviral vector, Ad.5/3.cRGD-mIL12p70, that transduces the GL261-based murine glioma cell line, resulting in the induction of biologically active, murine IL12p70 expression. Ex vivo, IL-12 expressed by GL261 cells induced interferon-γ synthesis in CD8(+) T cells (P<0.001), CD4(+) T cells (P=0.009) and natural killer cells (P=0.036). When injected 1 week after tumor implantation, Ad.5/3.cRGD-mIL12p70 successfully prolonged the survival of glioma-bearing mice. Sixty percent of animals treated with IL-12 immunotherapy were long-term survivors over 175 days, whereas all the control group animals expired by 40 days after tumor implantation (P=0.026). Mice receiving Ad.5/3.cRGD-mIL12p70 also accumulated 50% less MDSCs in the brain than the control group (P=0.007). Moreover, in the IL-12 group, MDSCs significantly overexpressed CD80 and major histocompatibility complex class II molecules (P=0.041). Depletion of MDSCs with Gr1(+) antibody had no survival benefit induced by IL-12-mediated immunotherapy. Of note, IL-12 therapy increased the presence of myeloid dendritic cells (mDCs) in the glioma microenvironment (P=0.0069). Ultimately, the data show that in the context of IL-12 immunogene therapy, MDSCs are dispensable and mDCs may provide the majority of antigen presentation in the brain.

Figure 1

In vitro characterization of the gene therapy vector Ad5/3.cRGD.mIL-12 to secrete functional interleukin-12 (IL-12). (a) Enzyme-linked immunosorbent assay (ELISA) was performed to detect secreted interleukin levels from mouse glioma cells. mIL12p70 levels were dependent on the initial quantity of the viral infectious units present in the environment/media. (b) Assessment of the time interval that murine glioma cells are capable of secreting mIL12p70 was performed via an ELISA. (c–e) Functionality assessment of the secreted IL-12 from glioma cells. Supernatant from adenovirus-infected glioma cells was used to incubate splenocytes for 72 h. Induction of interferon-γ (IFN-γ) in CD8 (c), CD4 (d) and natural killer (NK) (e) cells was assessed by flow cytometry after blocking cytokine secretion with Golgi-Plug for 4 h. As a positive control, we used recombinant mouse IL12p70 (rmIL12p70). ND, none detected; *P<0.05.

Figure 2

The survival benefit of Ad.mIL12 gene therapy is associated with alterations in the myeloid-derived suppressor cell (MDSC) population. (a) Intratumoral delivery of Ad.mIL12 1 week after GL261 mouse glioma establishment results in tumor clearance and long-term survivors. (b) Flow cytometry shows that interleukin-12 (IL-12) immunotherapy reduces MDSCs’ presence in the brains of glioma-bearing mice (representative flow plot depicted below the graph). (c–e) Phenotypic changes of MDSCs after IL-12 immunotherapy. (c) Flow cytometry detects increased expression of antigen-presenting and costimulatory molecules such as major histocompatibiltiy complex (MHC) class II and CD80 (representative flow plot depicted below the graph). Quantitative reverse transcription-polymerase chain reaction (RT-PCR) shows reduction of mRNA transcripts for immunosuppressive factors, such as arginase-1 (d) and inducible nitric oxide synthase (iNOS) (e). mRNA transcription was expressed as the percent of control-treated group. *P<0.05.

Figure 3

Depletion of myeloid-derived suppressor cells (MDSCs) by systemic delivery of Gr1 antibody does not alter the effects of intracranial interleukin-12 (IL-12) immunotherapy. (a) MDSC depletion does not affect the survival of animals treated with IL-12 gene therapy. One week after establishing GL261-OVA tumors, mice were injected intracranially with phosphate-buffered saline (PBS), Ad.GFP or Ad.mIL12 with or without MDSC depletion. (b) The effect of Gr1-Ab depletion on induction of CD8 T-cell recruitment in the brain (i) and quantification of immune memory response to OVA in intracranial CD8 T cells (ii). Immune cells from animals bearing orthotopic GL261-OVA glioma and treated with IL-12 in the presence or absence of MDSC depletion were segregated from whole brain homogenate via a Percoll gradient and stimulated as shown in the figure with either control (Sc peptide) or OVA peptide in the presence of costimulatory signals from CD28/CD49b antibodies. Interferon-γ (IFN-γ) induction was quantified by flow cytometry. ND, none detected; NS, nonsignificant difference; *P<0.05.

Figure 4

Myeloid dendritic cells (mDCs) infiltrate glioma at greater numbers after interleukin-12 (IL-12) immunotherapy. (a) Quantification of DC numbers in comparison with myeloid-derived suppressor cells (MDSCs) was carried out by flow cytometry and cell counting. (b) Ad.mIL12 increases the intracranial presence of mDCs (i) without altering their major histocompatibility complex (MHC) class II expression (ii); representative flow plot depicted below the graph). (c) Ad.mIL12 decreases the presence of pDCs (i) while increasing their MHC class II expression (ii; representative flow plot depicted below the graph). NS, nonsignificant difference; *P<0.05.