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. 2016 Dec 1;7(12):e2489. doi: 10.1038/cddis.2016.375

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Copyright © 2016 The Author(s)

Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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MRC2 is required for the differentiation and immunosuppressive function of T_reg cells in endometriosis. (a) Complete gating strategy of T_reg cells from the co-culture system. Gate R2 is inclusive of gate R1; cells of gate R2 represent CD4⁺ cells. Flow cytometric analyses CD4⁺Foxp3⁺ T cells in the co-culture system contained MRC2-silenced ESCs or vector-treated ESCs. Numbers in quadrants indicate the percentage of T_reg cells. (b) Quantification of CD4^low and CD4^high T_reg cells in a. Values indicate mean±S.D., n=6, ***P<0.001, ****P<0.0001, two-tailed, paired t-test. (c) Expression of TGF-β₁ (n=5), IL-10 (n=4), CTLA-4 (n=7), and CD39 (n=6) in CD^high T_reg cells shown in (a). Values indicate mean±S.D., *P<0.05, **P<0.01, two-tailed, paired t-test. (d) Flow cytometric analysis was performed to determine the expression of TGF-β₁, IL-10, CTLA-4, and CD39 in CD4^high T_reg cells of si-MRC2 group shown in (a). (e) MFI of TGF-β₁ (n=6), IL-10 (n=8), CTLA-4 (n=7) and CD39 (n=7) in CD4^high T_reg cells of si-MRC2 group shown in a. Values indicate mean±S.D., **P<0.01, ***P<0.001, ****P<0.0001, two-tailed, paired t-test. (f) Flow cytometric analysis was performed to determine the proliferation of T_eff cells (CFSE-labeled) from cultured with vector-pretreated-ESCs-educated-, or MRC2-silenced ESCs-educated T_reg cells. Numbers in quadrants indicate the percentage of divided cells and division index. Vector group contains T_eff cells and vector-pretreated-ESCs-educated T_reg cells; si-MRC2 group contains T_eff cells and MRC2-silenced ESCs-educated T_reg cells. Peaks represent generations of cells. Salmon peak represents parental cells, magenta peaks represent daughter cells of T_eff cells. (g) Quantification of the divided percentage of T_eff cells shown in (f). Values indicate mean±S.D., n=6, *P<0.05, two-tailed, paired t-test. (h) Flow cytometric analyses peritoneal CD4⁺Foxp3⁺ T cells in vivo from vector-administered or MRC2 shRNA-administered group. Numbers in quadrants indicate the percentage of T_reg cells. (i) Flow cytometric analysis was performed to determine the percentage of peritoneal CD4⁺Foxp3⁺ T cells of vector group or si-MRC2 group in vivo. Values indicate mean±S.D., n=13, ****P<0.0001, two-tailed, unpaired t-test. (j) Flow cytometric analyses expression of TGF-β₁ and CTLA-4 of peritoneal CD4⁺Foxp3⁺ T cells in vivo from vector-administered or MRC2 shRNA-administered group. Numbers in quadrants indicate the percentage of cells. (k) Expression of TGF-β₁ and CTLA-4 in peritoneal T_reg cells in vivo shown in (j). Values indicate mean±S.D., n=7, ***P<0.001, two-tailed, unpaired t-test. (l) Flow cytometric analyses the percentage of CD4⁺Foxp3⁺ T cells in ectopic lesions in vivo from vector-administered or MRC2 shRNA-administered group. Numbers in quadrants indicate the percentage of cells. (m) Quantification of T_reg cells shown in (l). Values indicate mean±S.D., n=6, *P<0.05, two-tailed, unpaired t-test. si-MRC2, silenced-MRC2; CD4^hi T_reg, CD4^high T_reg

MRC2 is required for the differentiation and immunosuppressive function of T_reg cells in endometriosis. (a) Complete gating strategy of T_reg cells from the co-culture system. Gate R2 is inclusive of gate R1; cells of gate R2 represent CD4⁺ cells. Flow cytometric analyses CD4⁺Foxp3⁺ T cells in the co-culture system contained MRC2-silenced ESCs or vector-treated ESCs. Numbers in quadrants indicate the percentage of T_reg cells. (b) Quantification of CD4^low and CD4^high T_reg cells in a. Values indicate mean±S.D., n=6, ***P<0.001, ****P<0.0001, two-tailed, paired t-test. (c) Expression of TGF-β₁ (n=5), IL-10 (n=4), CTLA-4 (n=7), and CD39 (n=6) in CD^high T_reg cells shown in (a). Values indicate mean±S.D., *P<0.05, **P<0.01, two-tailed, paired t-test. (d) Flow cytometric analysis was performed to determine the expression of TGF-β₁, IL-10, CTLA-4, and CD39 in CD4^high T_reg cells of si-MRC2 group shown in (a). (e) MFI of TGF-β₁ (n=6), IL-10 (n=8), CTLA-4 (n=7) and CD39 (n=7) in CD4^high T_reg cells of si-MRC2 group shown in a. Values indicate mean±S.D., **P<0.01, ***P<0.001, ****P<0.0001, two-tailed, paired t-test. (f) Flow cytometric analysis was performed to determine the proliferation of T_eff cells (CFSE-labeled) from cultured with vector-pretreated-ESCs-educated-, or MRC2-silenced ESCs-educated T_reg cells. Numbers in quadrants indicate the percentage of divided cells and division index. Vector group contains T_eff cells and vector-pretreated-ESCs-educated T_reg cells; si-MRC2 group contains T_eff cells and MRC2-silenced ESCs-educated T_reg cells. Peaks represent generations of cells. Salmon peak represents parental cells, magenta peaks represent daughter cells of T_eff cells. (g) Quantification of the divided percentage of T_eff cells shown in (f). Values indicate mean±S.D., n=6, *P<0.05, two-tailed, paired t-test. (h) Flow cytometric analyses peritoneal CD4⁺Foxp3⁺ T cells in vivo from vector-administered or MRC2 shRNA-administered group. Numbers in quadrants indicate the percentage of T_reg cells. (i) Flow cytometric analysis was performed to determine the percentage of peritoneal CD4⁺Foxp3⁺ T cells of vector group or si-MRC2 group in vivo. Values indicate mean±S.D., n=13, ****P<0.0001, two-tailed, unpaired t-test. (j) Flow cytometric analyses expression of TGF-β₁ and CTLA-4 of peritoneal CD4⁺Foxp3⁺ T cells in vivo from vector-administered or MRC2 shRNA-administered group. Numbers in quadrants indicate the percentage of cells. (k) Expression of TGF-β₁ and CTLA-4 in peritoneal T_reg cells in vivo shown in (j). Values indicate mean±S.D., n=7, ***P<0.001, two-tailed, unpaired t-test. (l) Flow cytometric analyses the percentage of CD4⁺Foxp3⁺ T cells in ectopic lesions in vivo from vector-administered or MRC2 shRNA-administered group. Numbers in quadrants indicate the percentage of cells. (m) Quantification of T_reg cells shown in (l). Values indicate mean±S.D., n=6, *P<0.05, two-tailed, unpaired t-test. si-MRC2, silenced-MRC2; CD4^hi T_reg, CD4^high T_reg