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Suppression of Tregs by anti-glucocorticoid induced TNF receptor antibody enhances the antitumor immunity of interferon-α gene therapy for pancreatic cancer.

Aida K, Miyakawa R, Suzuki K, Narumi K, Udagawa T, Yamamoto Y, Chikaraishi T, Yoshida T, Aoki K - Cancer Sci. (2014)

Bottom Line: First we showed that an intraperitoneal administration of an agonistic anti-glucocorticoid induced TNF receptor (GITR) monoclonal antibody (mAb), which is reported to suppress the function of Tregs, significantly inhibited subcutaneous tumor growth in a murine pancreatic cancer model.The CCR5 expression on Tregs was downregulated in the antibody-treated mice, which may explain the decrease of tumor-infiltrating Tregs.The combination of Treg-suppression by GITR mAb and the tumor immunity induction by IFN-α gene therapy could be a promising therapeutic strategy for pancreatic cancer.

View Article: PubMed Central - PubMed

Affiliation: Division of Gene and Immune Medicine, National Cancer Center Research Institute, Tokyo, Japan; Department of Urology, St. Marianna University, Kanagawa, Japan.

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The combination therapy suppresses the accumulation of Tregs in tumors. Thirteen days after the injection of adenoviruses, fresh frozen sections of subcutaneous tumors were processed for immunohistochemistry. Data are representative of three separate experiments with similar results. (a) Immunostaining of CD4+ T cells. Rt.: right leg, Lt.: left leg. (b) Immunostaining of CD8+ T cells. (c) Immunostaining of Foxp3+ cells. (d) Relative ratio of CD4+ or CD8+ T cells per Foxp3+ cells in tumors. The number of CD4+ (left panel) and CD8+ T cells (right panel) in tumors was compared with that of Foxp3+ cells. (e) Proliferative status of CD4+ and CD8+ T cells infiltrated into Pan02 tumors. Tumors were harvested at 2 weeks after the combination therapy, processed into single-cell suspension, and the frequencies of Ki-67+ cells per CD4+ or CD8+ T cells were analyzed by flow-cytometry with PE-anti mouse Ki-67 antibody (eBioscience, San Diego, CA, USA; n = 3). We gated the lymphocyte regions in fluorescence-activated cell sorting (FACS) plots, and developed spots in two dimensions. A hundred thousand live cells were analyzed.
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fig05: The combination therapy suppresses the accumulation of Tregs in tumors. Thirteen days after the injection of adenoviruses, fresh frozen sections of subcutaneous tumors were processed for immunohistochemistry. Data are representative of three separate experiments with similar results. (a) Immunostaining of CD4+ T cells. Rt.: right leg, Lt.: left leg. (b) Immunostaining of CD8+ T cells. (c) Immunostaining of Foxp3+ cells. (d) Relative ratio of CD4+ or CD8+ T cells per Foxp3+ cells in tumors. The number of CD4+ (left panel) and CD8+ T cells (right panel) in tumors was compared with that of Foxp3+ cells. (e) Proliferative status of CD4+ and CD8+ T cells infiltrated into Pan02 tumors. Tumors were harvested at 2 weeks after the combination therapy, processed into single-cell suspension, and the frequencies of Ki-67+ cells per CD4+ or CD8+ T cells were analyzed by flow-cytometry with PE-anti mouse Ki-67 antibody (eBioscience, San Diego, CA, USA; n = 3). We gated the lymphocyte regions in fluorescence-activated cell sorting (FACS) plots, and developed spots in two dimensions. A hundred thousand live cells were analyzed.

Mentions: It is known that a large number of tumor-infiltrating lymphocytes (TILs) result in a better antitumor effect for cancer patients.(25) To examine whether an increase of TILs in treated tumors is related to tumor growth suppression, immunohistochemical staining of CD4- and CD8-positive cells was performed in the Pan02 tumors 13 days after the injection of Ad-mIFN. The intratumoral IFN-α expression significantly increased the infiltration of CD4+ and CD8+ T cells in both vector-injected and vector-uninjected tumors, and the anti-GITR mAb also increased the number of CD4+ cells in the both tumors (Fig. 5a,b). Furthermore, the combination therapy of anti-GITR mAb and IFN-α gene transfer further increased the infiltration of CD4+ and CD8+ cells compared with the Ad-mIFN or anti-GITR mAb alone groups (Fig. 5a,b). Next, the infiltration of Foxp3+ cells was assessed in the treated Pan02 tumors. Interferon-α gene transfer and anti-GITR mAb administration decreased the number of Foxp3+ cells in the tumors, and the combination therapy markedly decreased Foxp3+ cells (Fig. 5c). Since a high effector T cells/Tregs ratio is associated with a favorable prognosis for cancer patients,(26) we calculated the ratio of CD4+ or CD8+ cells per Foxp3+ cells in the tumors. The ratios were markedly high in the combination therapy-treated tumors (Fig. 5d).


Suppression of Tregs by anti-glucocorticoid induced TNF receptor antibody enhances the antitumor immunity of interferon-α gene therapy for pancreatic cancer.

Aida K, Miyakawa R, Suzuki K, Narumi K, Udagawa T, Yamamoto Y, Chikaraishi T, Yoshida T, Aoki K - Cancer Sci. (2014)

The combination therapy suppresses the accumulation of Tregs in tumors. Thirteen days after the injection of adenoviruses, fresh frozen sections of subcutaneous tumors were processed for immunohistochemistry. Data are representative of three separate experiments with similar results. (a) Immunostaining of CD4+ T cells. Rt.: right leg, Lt.: left leg. (b) Immunostaining of CD8+ T cells. (c) Immunostaining of Foxp3+ cells. (d) Relative ratio of CD4+ or CD8+ T cells per Foxp3+ cells in tumors. The number of CD4+ (left panel) and CD8+ T cells (right panel) in tumors was compared with that of Foxp3+ cells. (e) Proliferative status of CD4+ and CD8+ T cells infiltrated into Pan02 tumors. Tumors were harvested at 2 weeks after the combination therapy, processed into single-cell suspension, and the frequencies of Ki-67+ cells per CD4+ or CD8+ T cells were analyzed by flow-cytometry with PE-anti mouse Ki-67 antibody (eBioscience, San Diego, CA, USA; n = 3). We gated the lymphocyte regions in fluorescence-activated cell sorting (FACS) plots, and developed spots in two dimensions. A hundred thousand live cells were analyzed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4317823&req=5

fig05: The combination therapy suppresses the accumulation of Tregs in tumors. Thirteen days after the injection of adenoviruses, fresh frozen sections of subcutaneous tumors were processed for immunohistochemistry. Data are representative of three separate experiments with similar results. (a) Immunostaining of CD4+ T cells. Rt.: right leg, Lt.: left leg. (b) Immunostaining of CD8+ T cells. (c) Immunostaining of Foxp3+ cells. (d) Relative ratio of CD4+ or CD8+ T cells per Foxp3+ cells in tumors. The number of CD4+ (left panel) and CD8+ T cells (right panel) in tumors was compared with that of Foxp3+ cells. (e) Proliferative status of CD4+ and CD8+ T cells infiltrated into Pan02 tumors. Tumors were harvested at 2 weeks after the combination therapy, processed into single-cell suspension, and the frequencies of Ki-67+ cells per CD4+ or CD8+ T cells were analyzed by flow-cytometry with PE-anti mouse Ki-67 antibody (eBioscience, San Diego, CA, USA; n = 3). We gated the lymphocyte regions in fluorescence-activated cell sorting (FACS) plots, and developed spots in two dimensions. A hundred thousand live cells were analyzed.
Mentions: It is known that a large number of tumor-infiltrating lymphocytes (TILs) result in a better antitumor effect for cancer patients.(25) To examine whether an increase of TILs in treated tumors is related to tumor growth suppression, immunohistochemical staining of CD4- and CD8-positive cells was performed in the Pan02 tumors 13 days after the injection of Ad-mIFN. The intratumoral IFN-α expression significantly increased the infiltration of CD4+ and CD8+ T cells in both vector-injected and vector-uninjected tumors, and the anti-GITR mAb also increased the number of CD4+ cells in the both tumors (Fig. 5a,b). Furthermore, the combination therapy of anti-GITR mAb and IFN-α gene transfer further increased the infiltration of CD4+ and CD8+ cells compared with the Ad-mIFN or anti-GITR mAb alone groups (Fig. 5a,b). Next, the infiltration of Foxp3+ cells was assessed in the treated Pan02 tumors. Interferon-α gene transfer and anti-GITR mAb administration decreased the number of Foxp3+ cells in the tumors, and the combination therapy markedly decreased Foxp3+ cells (Fig. 5c). Since a high effector T cells/Tregs ratio is associated with a favorable prognosis for cancer patients,(26) we calculated the ratio of CD4+ or CD8+ cells per Foxp3+ cells in the tumors. The ratios were markedly high in the combination therapy-treated tumors (Fig. 5d).

Bottom Line: First we showed that an intraperitoneal administration of an agonistic anti-glucocorticoid induced TNF receptor (GITR) monoclonal antibody (mAb), which is reported to suppress the function of Tregs, significantly inhibited subcutaneous tumor growth in a murine pancreatic cancer model.The CCR5 expression on Tregs was downregulated in the antibody-treated mice, which may explain the decrease of tumor-infiltrating Tregs.The combination of Treg-suppression by GITR mAb and the tumor immunity induction by IFN-α gene therapy could be a promising therapeutic strategy for pancreatic cancer.

View Article: PubMed Central - PubMed

Affiliation: Division of Gene and Immune Medicine, National Cancer Center Research Institute, Tokyo, Japan; Department of Urology, St. Marianna University, Kanagawa, Japan.

Show MeSH
Related in: MedlinePlus