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. 2012 Oct;9(4):827-43.
doi: 10.1007/s13311-012-0144-7.

Gene Therapy-Mediated Reprogramming Tumor Infiltrating T Cells Using IL-2 and Inhibiting NF-κB Signaling Improves the Efficacy of Immunotherapy in a Brain Cancer Model

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Free PMC article

Gene Therapy-Mediated Reprogramming Tumor Infiltrating T Cells Using IL-2 and Inhibiting NF-κB Signaling Improves the Efficacy of Immunotherapy in a Brain Cancer Model

Yohei Mineharu et al. Neurotherapeutics. .
Free PMC article

Abstract

Immune-mediated gene therapy using adenovirus expressing Flt3 ligand and thymidine kinase followed by ganciclovir administration (Flt3/TK) effectively elicits tumor regression in preclinical glioma models. Herein, we assessed new strategies to optimize Flt3L/TK therapeutic efficacy in a refractory RG2 orthotopic glioblastoma model. Specifically, we aimed to optimize the therapeutic efficacy of Flt3L/TK treatment in the RG2 model by overexpressing the following genes within the brain tumor microenvironment: 1) a TK mutant with enhanced cytotoxicity (SR39 mutant TK), 2) Flt3L-IgG fusion protein that has a longer half-life, 3) CD40L to stimulate DC maturation, 4) T helper cell type 1 polarizing dendritic cell cytokines interleukin-12 or C-X-C motif ligand 10 chemokine (CXCL)-10, 5) C-C motif ligand 2 chemokine (CCL2) or C-C motif ligand 3 chemokine (CCL3) to enhance dendritic cell recruitment into the tumor microenvironment, 6) T helper cell type 1 cytokines interferon-γ or interleukin-2 to enhance effector T-cell functions, and 7) IκBα or p65RHD (nuclear factor kappa-B [NF-κB] inhibitors) to suppress the function of Foxp3+ Tregs and enhanced effector T-cell functions. Anti-tumor immunity and tumor specific effector T-cell functions were assessed by cytotoxic T lymphocyte assay and intracellular IFN-γ staining. Our data showed that overexpression of interferon-γ or interleukin-2, or inhibition of the nuclear factor kappa-B within the tumor microenvironment, enhanced cytotoxic T lymphocyte-mediated immune responses and successfully extended the median survival of rats bearing intracranial RG2 when combined with Flt3L/TK. These findings indicate that enhancement of T-cell functions constitutes a critical therapeutic target to overcome immune evasion and enhance therapeutic efficacy for brain cancer. In addition, our study provides novel targets to be used in combination with immune-therapeutic strategies for glioblastoma, which are currently being tested in the clinic.

Figures

Fig. 1
Fig. 1
In vivo tumor growth of RG2 glioma and therapeutic efficacy of Ad-Flt3L + Ad-TK for the tumor. (a-b), In vivo tumor growth rates of intracranial RG2 glioma in rats. Fisher rats were implanted in the striatum with 20,000 syngeneic RG2 cells. Brains were processed for stereology to determine tumor volume 3, 6, and 9 days after tumor implantation. Microphotographs show the appearance of representative brain sections stained with Nissl. (a) Scale bar, 2 mm. Graph shows tumor growth rate. Doubling time and regression coefficient (R2) are indicated. (b) Scale bar, 100 μm. (c) Experimental design to assess therapeutic efficacy of immunogene therapy using adenoviral vectors expressing Flt3 ligand (Ad-Flt3L) and thymidine kinase (Ad-TK) in an orthotopic RG2 glioma model. Fisher rats were implanted with 20,000 RG2 glioma cells in the striatum. After 5 days, rats were treated with Ad vectors followed by ganciclovir (GCV) administration for 7 days. (d) Kaplan-Meier survival curve of rats treated with Ad-TK + Ad-Flt3L (n = 5), Ad-TK (n = 4), Ad-Flt3L (n = 5), or saline (n = 6). *p < 0.05 versus saline; ^p < 0.05 versus Ad-TK; Mantel log-rank test. (e) Micrographs show the neuropathology of rats bearing RG2 tumors treated with Ad-Flt3L + Ad-TK or saline. Rats were perfused 19 days after tumor implantation and brain sections were stained using Nissl, and immunocytochemistry was done using antibodies specific for myelin basic protein (MBP) and tyrosine hydroxlylase (TH). Scale bar, 2 mm
Fig. 2
Fig. 2
SR39 is more cytotoxic than TK and Flt3L-IgG fusion protein has longer serum half-life than Flt3L, but they do not improve the therapeutic efficacy of Ad-Flt3L + Ad-TK. (a) Cytotoxicity of SR39, a mutant form of TK, is higher than TK in vitro. RG2 cells were treated with Ad-0 (adenoviral vector without transgene), Ad-TK, or Ad-SR39 in the presence or absence of gancyclovir (GCV). Cell death in RG2 cells was assessed by Fluorescein Isothiocyanate (FITC)-annexin V and propidium iodide (PI). Cells positive for Annexin-V and/or PI were considered to be dead. The proportion of dead cells is shown. *p < 0.05 versus mock; ^p < 0.05 versus Ad-TK; 2-way analysis of variance (ANOVA) followed by Turkey’s test. (b) Kaplan-Meier survival curve of rats treated with Ad-SR39 + Ad-Flt3L (n = 5), Ad-SR39 (n = 5), Ad-Flt3L (n = 5) or saline (n = 5). *p < 0.05 versus saline; ^p < 0.05 versus Ad-SR39; Mantel log-rank test. (c) Transgene expression of Ad-Flt3L and Ad-Flt3L-IgG2a in vivo. Fisher rats were implanted with 20,000 RG2 cells, and 5 days later they were treated with Ad-Flt3L or Ad-Flt3L-IgG2a. Four days after vector injection, the serum was collected and Flt3L levels were measured by enzyme-linked immunosorbent assay. *p < 0.05 versus saline, ^p < 0.05 versus Ad-Flt3L; 1-way analysis of variance followed by Tukey’s test. (d) Kaplan-Meier survival curve of rats treated with Ad-TK + Ad-Flt3L-IgG2a (n = 5), Ad-TK (n = 5), Ad-Flt3L-IgG2a (n = 5), or saline (n = 5). *p < 0.05 versus saline; ^p < 0.05 versus Ad-TK; Mantel log-rank test
Fig. 3
Fig. 3
Manipulation of Dendritic Cell (DC) functions in combination with Ad-Flt3L + Ad-TK. (a) Fisher rats were implanted with 20,000 RG2 glioma cells in the striatum. After 5 days, rats were treated with Ad vectors followed by gancyclovir (GCV) administration for 7 days. (b-f) Kaplan-Meier survival curve of rats treated with Ad-Flt3L/TK + Ad-CD40L. (b) Ad-Flt3/TK + Ad-IL-12. (c) Ad-Flt3/TK + Ad-CXCL-10. (d) Ad-Flt3L/TK + Ad-CCL3. (e) and Ad-Flt3/TK + Ad-CCL2. (f) Compared with Ad-Flt3L/TK or saline (n = 4-5 for each group) are shown
Fig. 4
Fig. 4
Interferon-gamma (IFN-γ) increases the expression of Major histocompatibility complex (MHC) class I on RG2 glioma cells and enhances the therapeutic efficacy of Ad-Flt3L +Ad-TK. (a) Effect of IFN-γ on the expression of MHC class I and class II complexes on RG2 cells in vitro. RG2 cells were incubated with 10 ng/mL recombinant rat IFN-γ or mock for 48 h. Overlays show the fluorescence intensity of RG2 cells labeled with FITC-MHC I (left panel) or PerCP-MHC II (right panel). Green area represents isotype control. (b) Effect of IFN-γ on the expression of MHC I and MHC II on RG2 cells in vivo. Fisher rats were implanted with 200,000 RG2-citrine cells in the brain, and 5 days later they were treated with Ad-IFN-γ or saline. Four days after the treatment, brains were processed for IHC. Confocal micrographs show the expression of MHC I (red) and MHC II (red) on RG2-citrine cells (green). Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI) (blue); scale bar, 40 μm. (c) Fisher rats were implanted with 20,000 RG2 cells in the striatum, and 5 days later they were treated with Ad vectors. Kaplan-Meier survival curve of rats treated with Ad-Flt3L/TK + Ad-IFN-γ (n = 5), Ad-Flt3L/TK (n = 5), Ad-IFN-γ (n = 5), or saline (n = 5) are shown. *p < 0.05 versus saline, ^p < 0.05 versus Ad-Flt3L/TK; Mantel log rank test
Fig. 5
Fig. 5
Ad-interleukin (IL)-2 enhances anti-tumor T-cell responses induced by Ad-Flt3L/TK and prolongs the survival of rats bearing RG2 glioma. (a) Experimental design to assess the therapeutic efficacy and anti-tumor immune responses of Ad-IL-2 in combination with Ad-Flt3L/TK. (b) CD8+-immune cell infiltration into the tumor. Seven days after the treatment (12 days after tumor implantation), brains were processed and stained with CD8α. Lower panels are high magnification images of area defined in the red box in the images above. The tumor mass is indicated with a (T). Scale bar, 2 mm (upper panels) and 0.2 mm (lower panels). (c) Flow cytometry analysis of effector and regulatory T cells. Seven days after the treatment with Ad-Flt3L/TK + Ad-IL-2 (F/T+IL-2), Ad-Flt3L/TK (F/T) or saline, immune cells were isolated from brain tumor and incubated for 18 h with PMA, ionomycin, blefedrin A, and monensin. Immune cells were then stained with APC-CD3 and PE-CD8α followed by fixation, permeabilization, and intracellular staining with FITC-IFN-γ. Labeled cells were analyzed by flow cytometry; representative dot plots, and corresponding percentage of CD8+ CD3+ cells producing IFN-γ are shown. For the detection of Tregs, immune cells were isolated from brain tumor and stained with eFluor450-Foxp3, APC-CD4, and PE-CD25. Representative dot plots and corresponding percentage of each quadrant are shown. The column scatter graph shows a percentage of CD4+ CD25+ cells that express Foxp3 for Ad-Flt3L/TK treated rats (F/T) and Ad-Flt3L/TK + Ad-IL-2 (F/T+IL-2). *p < 0.5 versus saline, ^p < 0.05 versus F/T; one-way analysis of variance (ANOVA) followed by Tukey’s test. (d) Cytotoxic T-lymphocyte (CTL) assay. RG2 cells stained with VivoTag-680 were incubated for 5 h at 37 °C with T cells isolated from tumor bearing rats 7 days after the treatment with Ad-Flt3L/TK + Ad-IL-2 or Ad-Flt3L/TK at the indicated ratios. Tumor cell death was assessed by Annexin-V and propidium iodide (PI) followed by flow cytometry. Differences between treatment groups were evaluated by 2-way ANOVA followed by Tukey’s post test. (e) Effect of Ad-IL-2 on the levels of central memory CD8+ T cell. Upper panels show representative dot plots and corresponding percentage of memory T cells (CD45RC- CD8+ CD3+) in the spleen at 28 days after immunogene therapy by F/T or F/T+IL-2. Memory T cells were then gated for the expression of CD62 (lower histograms). The number is the percentage of CD62Lhigh population. The column scatter graph shows the percentage of central memory CD8+ T-cell population (CD62Lhigh CD45RC+) among CD8+ T cells (CD8+ CD3+) for each group. *p < 0.05 versus F/T; Student t test. (f) Kaplan-Meier survival curve of rats treated with Ad-Flt3L/TK + Ad-IL-2 (n = 5), Ad-Flt3L/TK (n = 5), Ad-IL-2 (n = 5), or saline (n = 5) are shown. *p < 0.05 versus saline, ^p < 0.05 versus Ad-Flt3L/TK; Mantel log rank test
Fig. 6
Fig. 6
Nuclear factor kappa-B (NF-κB) inhibition by Ad-IκBα reduces tumor infiltrating Foxp3+ regulatory T cells and enhances anti-tumor T-cell responses. (a) Effect of Ad-IκBα on Foxp3 expression of CD4+ T cells. Immune cells were isolated from brain tumor and stained with eFluor450-Foxp3, APC-CD4, and PE-CD25. Representative dot plots and corresponding percentage of each quadrant are shown. The column scatter graph shows percentage of CD4+ CD25+ cells that express Foxp3 for Ad-Flt3L/TK-treated rats (F/T) and Ad-Flt3L/TK + Ad-IκBα treated rats (F/T+IκB). ^p < 0.05 versus F/T; Student’s t test. (b, c) Flow cytometry analysis measuring interferon (IFN)-γ production by CD4+ T cells (b) and CD8+ T cells (c). Fisher rats were implanted with RG2 tumor cells, and 5 days later they were treated with Ad-Flt3L/TK (F/T) or Ad-FLt3L/TK + Ad- IκBα (F/T+ IκB). Seven days after the treatment, immune cells were isolated from the brain tumor and incubated for 18 h with Phorbol 12-myristate 13-acetate (PMA), ionomycin, blefedrin A, and monensin. Those immune cells were stained with APC-CD3 and PE-CD4 or PE-CD8α followed by fixation, permeabilization, and intracellular staining with FITC-IFN-γ. Representative dot plots and corresponding percentage of IFN-γ+ cells among CD4+ CD3+ cells (b) or CD8+ CD3+ cells (c) are shown. *p < 0.05 versus saline; ^p < 0.05 versus Ad-Flt3L/TK; Student’s t test. (d) Cytolytic activity of T-lymphocytes from cervical draining lymph nodes. RG2 cells stained with VivoTag-680 were incubated with lymphocyte isolated from rats 7 days after the treatment with Ad-Flt3L/TK+Ad-IL-2 or Ad-Flt3L/TK for 5 h at the indicated ratios. Tumor cell death was assessed by flow cytometry with FITC-Annexin-V and Propidium Iodide (PI). The difference between the treatment groups was evaluated by 2-way analysis of variance followed by Turkey’s post-hoc test. (e, f) Kaplan-Meier survival curve of rats treated with Ad-Flt3L/TK + Ad-IκBα (n = 5), Ad-Flt3L/TK + Ad-p65RHD (n = 5), Ad-Flt3L/TK (n = 5), or saline (n = 5) are shown. *p < 0.05 versus saline; ^p < 0.05 versus Ad-Flt3L/TK; Mantel log rank test

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