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Systemic CD8+ T cell-mediated tumoricidal effects by intratumoral treatment of oncolytic herpes simplex virus with the agonistic monoclonal antibody for murine glucocorticoid-induced tumor necrosis factor receptor.

Ishihara M, Seo N, Mitsui J, Muraoka D, Tanaka M, Mineno J, Ikeda H, Shiku H - PLoS ONE (2014)

Bottom Line: In this study, we examined the tumoricidal effects of oncolytic HF10, a naturally occurring mutant of herpes simplex virus type-1, combined with an agonistic DTA-1 monoclonal antibody specific for the glucocorticoid-induced tumor necrosis factor receptor.The kinetics and immunological mechanisms of DTA-1 in HF10 infection were examined using flow cytometry and immunohistochemistry.Studies using Fc-digested DTA-1 and Fcγ receptor knockout mice demonstrated the direct participation of DTA-1 in regulatory T cell depletion by antibody-dependent cellular cytotoxicity primarily via macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan.

ABSTRACT
Oncolytic virotherapy combined with immunomodulators is a novel noninvasive strategy for cancer treatment. In this study, we examined the tumoricidal effects of oncolytic HF10, a naturally occurring mutant of herpes simplex virus type-1, combined with an agonistic DTA-1 monoclonal antibody specific for the glucocorticoid-induced tumor necrosis factor receptor. Two murine tumor models were used to evaluate the therapeutic efficacies of HF10 virotherapy combined with DTA-1. The kinetics and immunological mechanisms of DTA-1 in HF10 infection were examined using flow cytometry and immunohistochemistry. Intratumoral administration of HF10 in combination with DTA-1 at a low dose resulted in a more vigorous attenuation of growth of the untreated contralateral as well as the treated tumors than treatment with either HF10 or DTA-1 alone. An accumulation of CD8(+) T cells, including tumor- and herpes simplex virus type-1-specific populations, and a decrease in the number of CD4(+) Foxp3(+) T regulatory cells were seen in both HF10- and DTA-1-treated tumors. Studies using Fc-digested DTA-1 and Fcγ receptor knockout mice demonstrated the direct participation of DTA-1 in regulatory T cell depletion by antibody-dependent cellular cytotoxicity primarily via macrophages. These results indicated the potential therapeutic efficacy of a glucocorticoid-induced tumor necrosis factor receptor-specific monoclonal antibody in oncolytic virotherapy at local tumor sites.

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DTA-1-mediated depletion of tumor-infiltrating CD4+ Foxp3+ Treg cells by ADCC.(A) DTA-1- (dotted line) or isotype control (solid line)-treated CMS5a and murine GITR gene-transfected CMS5a (CMS5a/GITR) cells were stained with a FITC-conjugated anti-rat IgG (H+L) antibody and analyzed by flow cytometry. (B) CFSE-labeled CMS5a and CMS5a/GITR cells were used as targets. The mixture of IFN-γ-activated RAW264.7 cells (effector cells) and target cells were incubated for 12 hrs with control IgG or DTA hrs with control IgG or DTA-1 at effector/target ratios of 5 and 15. (C) Frequency of Foxp3+ cells in tumor-infiltrating CD4+ cell population at 3 days after i.t. DTA-1 or Fc-digested DTA-1 (DTA-1 Fab) treatment was measured by flow cytometric analysis. (D) Frequency of Foxp3+ cells in tumor-infiltrating CD4+ cell population at 3 days after DTA-1 i.t. treatment in wild-type or FcRγ KO mice was measured by flow cytometric analysis. By Student's t-test, the decrease in the frequency of Foxp3+ cells in DTA-1-treated CT26/NY-ESO-1 tumors of wild type mice, but not FcRγ KO mice (N.S.: Not significant), was significantly different from untreated control group. (E) Frozen sections of CT26/NY-ESO-1 tumors obtained at 3 days after DTA-1 i.t. treatment in wild type and FcRγ KO mice were stained with FITC-anti-Foxp3 and PE-anti-CD8α mAbs, and DAPI.
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pone-0104669-g006: DTA-1-mediated depletion of tumor-infiltrating CD4+ Foxp3+ Treg cells by ADCC.(A) DTA-1- (dotted line) or isotype control (solid line)-treated CMS5a and murine GITR gene-transfected CMS5a (CMS5a/GITR) cells were stained with a FITC-conjugated anti-rat IgG (H+L) antibody and analyzed by flow cytometry. (B) CFSE-labeled CMS5a and CMS5a/GITR cells were used as targets. The mixture of IFN-γ-activated RAW264.7 cells (effector cells) and target cells were incubated for 12 hrs with control IgG or DTA hrs with control IgG or DTA-1 at effector/target ratios of 5 and 15. (C) Frequency of Foxp3+ cells in tumor-infiltrating CD4+ cell population at 3 days after i.t. DTA-1 or Fc-digested DTA-1 (DTA-1 Fab) treatment was measured by flow cytometric analysis. (D) Frequency of Foxp3+ cells in tumor-infiltrating CD4+ cell population at 3 days after DTA-1 i.t. treatment in wild-type or FcRγ KO mice was measured by flow cytometric analysis. By Student's t-test, the decrease in the frequency of Foxp3+ cells in DTA-1-treated CT26/NY-ESO-1 tumors of wild type mice, but not FcRγ KO mice (N.S.: Not significant), was significantly different from untreated control group. (E) Frozen sections of CT26/NY-ESO-1 tumors obtained at 3 days after DTA-1 i.t. treatment in wild type and FcRγ KO mice were stained with FITC-anti-Foxp3 and PE-anti-CD8α mAbs, and DAPI.

Mentions: To examine whether DTA-1 can mediate ADCC in a murine system, we performed an in vitro ADCC assay using IFN-γ-activated RAW264.7 macrophage cells as an effector and murine GITR gene-transfected CMS5a (CMS5a/GITR) cells (Fig. 6A) as a target. CMS5a/GITR cells were lysed in the presence of DTA-1 in a GITR-specific manner (Fig. 6B). We further investigated in vivo the ADCC effects of DTA-1 using Fc portion-digested DTA-1 (DTA-1 Fab) and FcRγ KO mice. Depletion of CD4+ Foxp3+ Treg cells in CT26/NY-ESO-1 tumors was not observed following i.t. DTA-1 Fab treatment (Fig. 6C). In addition, no significant decreases in the number of CD4+ Foxp3+ Treg cells and accumulation of CD8+ T cells were detected by DTA-1 treatment in FcRγ KO mice, unlike the results from wild-type mice (Fig. 6D, 6E, and S4). These results clearly indicated the direct participation of DTA-1 in Treg cell depletion by ADCC.


Systemic CD8+ T cell-mediated tumoricidal effects by intratumoral treatment of oncolytic herpes simplex virus with the agonistic monoclonal antibody for murine glucocorticoid-induced tumor necrosis factor receptor.

Ishihara M, Seo N, Mitsui J, Muraoka D, Tanaka M, Mineno J, Ikeda H, Shiku H - PLoS ONE (2014)

DTA-1-mediated depletion of tumor-infiltrating CD4+ Foxp3+ Treg cells by ADCC.(A) DTA-1- (dotted line) or isotype control (solid line)-treated CMS5a and murine GITR gene-transfected CMS5a (CMS5a/GITR) cells were stained with a FITC-conjugated anti-rat IgG (H+L) antibody and analyzed by flow cytometry. (B) CFSE-labeled CMS5a and CMS5a/GITR cells were used as targets. The mixture of IFN-γ-activated RAW264.7 cells (effector cells) and target cells were incubated for 12 hrs with control IgG or DTA hrs with control IgG or DTA-1 at effector/target ratios of 5 and 15. (C) Frequency of Foxp3+ cells in tumor-infiltrating CD4+ cell population at 3 days after i.t. DTA-1 or Fc-digested DTA-1 (DTA-1 Fab) treatment was measured by flow cytometric analysis. (D) Frequency of Foxp3+ cells in tumor-infiltrating CD4+ cell population at 3 days after DTA-1 i.t. treatment in wild-type or FcRγ KO mice was measured by flow cytometric analysis. By Student's t-test, the decrease in the frequency of Foxp3+ cells in DTA-1-treated CT26/NY-ESO-1 tumors of wild type mice, but not FcRγ KO mice (N.S.: Not significant), was significantly different from untreated control group. (E) Frozen sections of CT26/NY-ESO-1 tumors obtained at 3 days after DTA-1 i.t. treatment in wild type and FcRγ KO mice were stained with FITC-anti-Foxp3 and PE-anti-CD8α mAbs, and DAPI.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4126744&req=5

pone-0104669-g006: DTA-1-mediated depletion of tumor-infiltrating CD4+ Foxp3+ Treg cells by ADCC.(A) DTA-1- (dotted line) or isotype control (solid line)-treated CMS5a and murine GITR gene-transfected CMS5a (CMS5a/GITR) cells were stained with a FITC-conjugated anti-rat IgG (H+L) antibody and analyzed by flow cytometry. (B) CFSE-labeled CMS5a and CMS5a/GITR cells were used as targets. The mixture of IFN-γ-activated RAW264.7 cells (effector cells) and target cells were incubated for 12 hrs with control IgG or DTA hrs with control IgG or DTA-1 at effector/target ratios of 5 and 15. (C) Frequency of Foxp3+ cells in tumor-infiltrating CD4+ cell population at 3 days after i.t. DTA-1 or Fc-digested DTA-1 (DTA-1 Fab) treatment was measured by flow cytometric analysis. (D) Frequency of Foxp3+ cells in tumor-infiltrating CD4+ cell population at 3 days after DTA-1 i.t. treatment in wild-type or FcRγ KO mice was measured by flow cytometric analysis. By Student's t-test, the decrease in the frequency of Foxp3+ cells in DTA-1-treated CT26/NY-ESO-1 tumors of wild type mice, but not FcRγ KO mice (N.S.: Not significant), was significantly different from untreated control group. (E) Frozen sections of CT26/NY-ESO-1 tumors obtained at 3 days after DTA-1 i.t. treatment in wild type and FcRγ KO mice were stained with FITC-anti-Foxp3 and PE-anti-CD8α mAbs, and DAPI.
Mentions: To examine whether DTA-1 can mediate ADCC in a murine system, we performed an in vitro ADCC assay using IFN-γ-activated RAW264.7 macrophage cells as an effector and murine GITR gene-transfected CMS5a (CMS5a/GITR) cells (Fig. 6A) as a target. CMS5a/GITR cells were lysed in the presence of DTA-1 in a GITR-specific manner (Fig. 6B). We further investigated in vivo the ADCC effects of DTA-1 using Fc portion-digested DTA-1 (DTA-1 Fab) and FcRγ KO mice. Depletion of CD4+ Foxp3+ Treg cells in CT26/NY-ESO-1 tumors was not observed following i.t. DTA-1 Fab treatment (Fig. 6C). In addition, no significant decreases in the number of CD4+ Foxp3+ Treg cells and accumulation of CD8+ T cells were detected by DTA-1 treatment in FcRγ KO mice, unlike the results from wild-type mice (Fig. 6D, 6E, and S4). These results clearly indicated the direct participation of DTA-1 in Treg cell depletion by ADCC.

Bottom Line: In this study, we examined the tumoricidal effects of oncolytic HF10, a naturally occurring mutant of herpes simplex virus type-1, combined with an agonistic DTA-1 monoclonal antibody specific for the glucocorticoid-induced tumor necrosis factor receptor.The kinetics and immunological mechanisms of DTA-1 in HF10 infection were examined using flow cytometry and immunohistochemistry.Studies using Fc-digested DTA-1 and Fcγ receptor knockout mice demonstrated the direct participation of DTA-1 in regulatory T cell depletion by antibody-dependent cellular cytotoxicity primarily via macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Mie, Japan.

ABSTRACT
Oncolytic virotherapy combined with immunomodulators is a novel noninvasive strategy for cancer treatment. In this study, we examined the tumoricidal effects of oncolytic HF10, a naturally occurring mutant of herpes simplex virus type-1, combined with an agonistic DTA-1 monoclonal antibody specific for the glucocorticoid-induced tumor necrosis factor receptor. Two murine tumor models were used to evaluate the therapeutic efficacies of HF10 virotherapy combined with DTA-1. The kinetics and immunological mechanisms of DTA-1 in HF10 infection were examined using flow cytometry and immunohistochemistry. Intratumoral administration of HF10 in combination with DTA-1 at a low dose resulted in a more vigorous attenuation of growth of the untreated contralateral as well as the treated tumors than treatment with either HF10 or DTA-1 alone. An accumulation of CD8(+) T cells, including tumor- and herpes simplex virus type-1-specific populations, and a decrease in the number of CD4(+) Foxp3(+) T regulatory cells were seen in both HF10- and DTA-1-treated tumors. Studies using Fc-digested DTA-1 and Fcγ receptor knockout mice demonstrated the direct participation of DTA-1 in regulatory T cell depletion by antibody-dependent cellular cytotoxicity primarily via macrophages. These results indicated the potential therapeutic efficacy of a glucocorticoid-induced tumor necrosis factor receptor-specific monoclonal antibody in oncolytic virotherapy at local tumor sites.

Show MeSH
Related in: MedlinePlus