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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 anti-GITR mAb was then combined with the intratumoral injection of the IFN-α-adenovirus vector.The CCR5 expression on Tregs was downregulated in the antibody-treated mice, which may explain the decrease of tumor-infiltrating Tregs.

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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Preferential suppression of CCR5 expression on CD4+Foxp3+ cells by anti-glucocorticoid induced TNF receptor monoclonal antibody (anti-GITR mAb). (a) Expression of CCR5 ligands in Pan02 tumors. Total RNA was extracted from Pan02 subcutaneous tumor and subjected to RT-PCR analysis with CCL3, CCL4 and CCL5 specific primers. (b) CCR5+ Tregs in the spleens and tumors. Spleens and tumors were harvested at 3 weeks after tumor inoculation, processed into single-cell suspension, and the percentage of CCR5+CD4+Foxp3+ cells was analyzed by flow-cytometry (n = 4). We gated the CD4+ regions in fluorescence-activated cell sorting (FACS) plots, and developed spots in two dimensions of CCR5 and Foxp3. In the tumors, a hundred thousand live cells were analyzed. (c) Suppression of CCR5 expression on CD4+Foxp3+ cells by anti-GITR mAb. Splenocytes were processed into single-cell suspension, and the cells were analyzed with anti-CD4, anti-Foxp3 and anti-CCR5 antibodies by flow-cytometry (n = 4). (d) Preferential suppression of CCR5 expression on Tregs by anti-GITR mAb. The splenocytes were isolated from the spleens of naïve C57BL/6 mice, and were cultured with anti-GITR mAb at 1 μg/mL for 5 h. The cells were analyzed with anti-CD4, anti-Foxp3 and anti-CCR5 antibodies by flow-cytometry (n = 4).
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fig06: Preferential suppression of CCR5 expression on CD4+Foxp3+ cells by anti-glucocorticoid induced TNF receptor monoclonal antibody (anti-GITR mAb). (a) Expression of CCR5 ligands in Pan02 tumors. Total RNA was extracted from Pan02 subcutaneous tumor and subjected to RT-PCR analysis with CCL3, CCL4 and CCL5 specific primers. (b) CCR5+ Tregs in the spleens and tumors. Spleens and tumors were harvested at 3 weeks after tumor inoculation, processed into single-cell suspension, and the percentage of CCR5+CD4+Foxp3+ cells was analyzed by flow-cytometry (n = 4). We gated the CD4+ regions in fluorescence-activated cell sorting (FACS) plots, and developed spots in two dimensions of CCR5 and Foxp3. In the tumors, a hundred thousand live cells were analyzed. (c) Suppression of CCR5 expression on CD4+Foxp3+ cells by anti-GITR mAb. Splenocytes were processed into single-cell suspension, and the cells were analyzed with anti-CD4, anti-Foxp3 and anti-CCR5 antibodies by flow-cytometry (n = 4). (d) Preferential suppression of CCR5 expression on Tregs by anti-GITR mAb. The splenocytes were isolated from the spleens of naïve C57BL/6 mice, and were cultured with anti-GITR mAb at 1 μg/mL for 5 h. The cells were analyzed with anti-CD4, anti-Foxp3 and anti-CCR5 antibodies by flow-cytometry (n = 4).

Mentions: It was reported that CD4+Foxp3+ Tregs preferentially expressed CCR5 compared with CD4+Foxp3− effector T cells in a murine model of pancreatic cancer.(27) To understand the mechanism of decreased Treg-accumulation in the tumors, the effect of GITR mAb on CCR5 expression of Tregs was analyzed. We first examined the expression of CCR5 ligands such as CCL3, CCL4 and CCL5 in Pan02 subcutaneous tumors. The RT-PCR analysis showed that the Pan02 tumor expressed CCL3, CCL4 and CCL5, whereas the pancreas did not express the ligands except for CCL5 (Fig. 6a). Next, we analyzed the CCR5 expression on Tregs infiltrated into the tumors. Flow-cytometry showed that the percentage of CCR5+ Tregs in the spleens was approximately 7%, whereas that in the tumors was more than 30% (Fig. 6b), suggesting that a particular population of Tregs employed the interaction of CCR5-CCR5 ligands to infiltrate into Pan02 tumors. Then, we examined whether the anti-GITR mAb suppresses the CCR5 expression on Tregs. Flow-cytometry showed that the administration of anti-GITR mAb significantly decreased the CCR5 expression level on Tregs in the spleen, which may explain the reduced number of Tregs infiltrating in the tumors (Fig. 6c). Furthermore, to examine the effect of GITR stimulation on CCR5 expression of Tregs and non-Tregs in vitro, the splenocytes were cultured with DTA-1 antibody at 1 μg/mL for 5 h. Flow-cytometry showed that the frequency of CCR5+ cells per CD4+Foxp3− T cells was decreased approximately 80% of DTA-1-untreated cells, whereas that of CCR5+ cells per Foxp3+ Tregs was decreased <40% (Fig. 6d), suggesting that the GITR mAb suppressed the CCR5 expression on CD4+Foxp3+ Tregs more efficiently than that on CD4+ Foxp3− T cells.


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)

Preferential suppression of CCR5 expression on CD4+Foxp3+ cells by anti-glucocorticoid induced TNF receptor monoclonal antibody (anti-GITR mAb). (a) Expression of CCR5 ligands in Pan02 tumors. Total RNA was extracted from Pan02 subcutaneous tumor and subjected to RT-PCR analysis with CCL3, CCL4 and CCL5 specific primers. (b) CCR5+ Tregs in the spleens and tumors. Spleens and tumors were harvested at 3 weeks after tumor inoculation, processed into single-cell suspension, and the percentage of CCR5+CD4+Foxp3+ cells was analyzed by flow-cytometry (n = 4). We gated the CD4+ regions in fluorescence-activated cell sorting (FACS) plots, and developed spots in two dimensions of CCR5 and Foxp3. In the tumors, a hundred thousand live cells were analyzed. (c) Suppression of CCR5 expression on CD4+Foxp3+ cells by anti-GITR mAb. Splenocytes were processed into single-cell suspension, and the cells were analyzed with anti-CD4, anti-Foxp3 and anti-CCR5 antibodies by flow-cytometry (n = 4). (d) Preferential suppression of CCR5 expression on Tregs by anti-GITR mAb. The splenocytes were isolated from the spleens of naïve C57BL/6 mice, and were cultured with anti-GITR mAb at 1 μg/mL for 5 h. The cells were analyzed with anti-CD4, anti-Foxp3 and anti-CCR5 antibodies by flow-cytometry (n = 4).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig06: Preferential suppression of CCR5 expression on CD4+Foxp3+ cells by anti-glucocorticoid induced TNF receptor monoclonal antibody (anti-GITR mAb). (a) Expression of CCR5 ligands in Pan02 tumors. Total RNA was extracted from Pan02 subcutaneous tumor and subjected to RT-PCR analysis with CCL3, CCL4 and CCL5 specific primers. (b) CCR5+ Tregs in the spleens and tumors. Spleens and tumors were harvested at 3 weeks after tumor inoculation, processed into single-cell suspension, and the percentage of CCR5+CD4+Foxp3+ cells was analyzed by flow-cytometry (n = 4). We gated the CD4+ regions in fluorescence-activated cell sorting (FACS) plots, and developed spots in two dimensions of CCR5 and Foxp3. In the tumors, a hundred thousand live cells were analyzed. (c) Suppression of CCR5 expression on CD4+Foxp3+ cells by anti-GITR mAb. Splenocytes were processed into single-cell suspension, and the cells were analyzed with anti-CD4, anti-Foxp3 and anti-CCR5 antibodies by flow-cytometry (n = 4). (d) Preferential suppression of CCR5 expression on Tregs by anti-GITR mAb. The splenocytes were isolated from the spleens of naïve C57BL/6 mice, and were cultured with anti-GITR mAb at 1 μg/mL for 5 h. The cells were analyzed with anti-CD4, anti-Foxp3 and anti-CCR5 antibodies by flow-cytometry (n = 4).
Mentions: It was reported that CD4+Foxp3+ Tregs preferentially expressed CCR5 compared with CD4+Foxp3− effector T cells in a murine model of pancreatic cancer.(27) To understand the mechanism of decreased Treg-accumulation in the tumors, the effect of GITR mAb on CCR5 expression of Tregs was analyzed. We first examined the expression of CCR5 ligands such as CCL3, CCL4 and CCL5 in Pan02 subcutaneous tumors. The RT-PCR analysis showed that the Pan02 tumor expressed CCL3, CCL4 and CCL5, whereas the pancreas did not express the ligands except for CCL5 (Fig. 6a). Next, we analyzed the CCR5 expression on Tregs infiltrated into the tumors. Flow-cytometry showed that the percentage of CCR5+ Tregs in the spleens was approximately 7%, whereas that in the tumors was more than 30% (Fig. 6b), suggesting that a particular population of Tregs employed the interaction of CCR5-CCR5 ligands to infiltrate into Pan02 tumors. Then, we examined whether the anti-GITR mAb suppresses the CCR5 expression on Tregs. Flow-cytometry showed that the administration of anti-GITR mAb significantly decreased the CCR5 expression level on Tregs in the spleen, which may explain the reduced number of Tregs infiltrating in the tumors (Fig. 6c). Furthermore, to examine the effect of GITR stimulation on CCR5 expression of Tregs and non-Tregs in vitro, the splenocytes were cultured with DTA-1 antibody at 1 μg/mL for 5 h. Flow-cytometry showed that the frequency of CCR5+ cells per CD4+Foxp3− T cells was decreased approximately 80% of DTA-1-untreated cells, whereas that of CCR5+ cells per Foxp3+ Tregs was decreased <40% (Fig. 6d), suggesting that the GITR mAb suppressed the CCR5 expression on CD4+Foxp3+ Tregs more efficiently than that on CD4+ Foxp3− T cells.

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 anti-GITR mAb was then combined with the intratumoral injection of the IFN-α-adenovirus vector.The CCR5 expression on Tregs was downregulated in the antibody-treated mice, which may explain the decrease of tumor-infiltrating Tregs.

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