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6. CbfbF/F CD4-cre mouse cells and iT reg cells generated from human naive CD4+ T cells undergoing siRNA-mediated RUNX1 and RUNX3 knock down show a diminished suppressive activity. Experimental setup (A) and results of the mouse suppression assay (B), FACS-purified naive CD4+8− T cells from CbfbF/F CD4-cre (left) and control CbfbF/+ CD4-cre mice (Cd45.2; right) were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, and 2.5 ng/ml TGF-β. After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. These were then incubated with inactivated splenocytes and anti-CD3 mAb. After a further 4 d, CD45.1+ cells were analyzed for CFSE dilution. (C) As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-β were mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. Four days later CD45.1+ cells were analyzed for CFSE dilution. The median division number (of the naive CD45.1+ CD4+ cells in the cultures containing Foxp3-GFP+ cells) is indicated in each of the histograms. One of three experiments is shown. (D) Human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA and cultured under iT reg differentiating conditions were used in an in vitro suppression assay, cultured together with autologous CFSE-labeled CD4+ T cells, and stimulated with anti-CD3 mAb. The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry. The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5. One of two experiments is shown. "}

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6. CbfbF/F CD4-cre mouse cells and iT reg cells generated from human naive CD4+ T cells undergoing siRNA-mediated RUNX1 and RUNX3 knock down show a diminished suppressive activity. Experimental setup (A) and results of the mouse suppression assay (B), FACS-purified naive CD4+8− T cells from CbfbF/F CD4-cre (left) and control CbfbF/+ CD4-cre mice (Cd45.2; right) were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, and 2.5 ng/ml TGF-β. After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. These were then incubated with inactivated splenocytes and anti-CD3 mAb. After a further 4 d, CD45.1+ cells were analyzed for CFSE dilution. (C) As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-β were mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. Four days later CD45.1+ cells were analyzed for CFSE dilution. The median division number (of the naive CD45.1+ CD4+ cells in the cultures containing Foxp3-GFP+ cells) is indicated in each of the histograms. One of three experiments is shown. (D) Human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA and cultured under iT reg differentiating conditions were used in an in vitro suppression assay, cultured together with autologous CFSE-labeled CD4+ T cells, and stimulated with anti-CD3 mAb. The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry. The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5. One of two experiments is shown. "}

{"project":"GO-BP","denotations":[{"id":"T32885","span":{"begin":757,"end":772},"obj":"http://purl.obolibrary.org/obo/GO_0001775"}],"text":"Figure 6. CbfbF/F CD4-cre mouse cells and iT reg cells generated from human naive CD4+ T cells undergoing siRNA-mediated RUNX1 and RUNX3 knock down show a diminished suppressive activity. Experimental setup (A) and results of the mouse suppression assay (B), FACS-purified naive CD4+8− T cells from CbfbF/F CD4-cre (left) and control CbfbF/+ CD4-cre mice (Cd45.2; right) were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, and 2.5 ng/ml TGF-β. After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. These were then incubated with inactivated splenocytes and anti-CD3 mAb. After a further 4 d, CD45.1+ cells were analyzed for CFSE dilution. (C) As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-β were mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. Four days later CD45.1+ cells were analyzed for CFSE dilution. The median division number (of the naive CD45.1+ CD4+ cells in the cultures containing Foxp3-GFP+ cells) is indicated in each of the histograms. One of three experiments is shown. (D) Human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA and cultured under iT reg differentiating conditions were used in an in vitro suppression assay, cultured together with autologous CFSE-labeled CD4+ T cells, and stimulated with anti-CD3 mAb. The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry. The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5. One of two experiments is shown. "}

{"project":"GO-MF","denotations":[{"id":"T32886","span":{"begin":426,"end":430},"obj":"http://purl.obolibrary.org/obo/GO_0005134"}],"text":"Figure 6. CbfbF/F CD4-cre mouse cells and iT reg cells generated from human naive CD4+ T cells undergoing siRNA-mediated RUNX1 and RUNX3 knock down show a diminished suppressive activity. Experimental setup (A) and results of the mouse suppression assay (B), FACS-purified naive CD4+8− T cells from CbfbF/F CD4-cre (left) and control CbfbF/+ CD4-cre mice (Cd45.2; right) were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, and 2.5 ng/ml TGF-β. After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. These were then incubated with inactivated splenocytes and anti-CD3 mAb. After a further 4 d, CD45.1+ cells were analyzed for CFSE dilution. (C) As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-β were mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. Four days later CD45.1+ cells were analyzed for CFSE dilution. The median division number (of the naive CD45.1+ CD4+ cells in the cultures containing Foxp3-GFP+ cells) is indicated in each of the histograms. One of three experiments is shown. (D) Human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA and cultured under iT reg differentiating conditions were used in an in vitro suppression assay, cultured together with autologous CFSE-labeled CD4+ T cells, and stimulated with anti-CD3 mAb. The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry. The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5. One of two experiments is shown. "}

{"project":"GO-CC","denotations":[{"id":"T32887","span":{"begin":33,"end":38},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32888","span":{"begin":50,"end":55},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32889","span":{"begin":90,"end":95},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32890","span":{"begin":289,"end":294},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32891","span":{"begin":475,"end":480},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32892","span":{"begin":544,"end":549},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32893","span":{"begin":677,"end":682},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32894","span":{"begin":757,"end":762},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32895","span":{"begin":840,"end":845},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32896","span":{"begin":895,"end":900},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32897","span":{"begin":988,"end":993},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32898","span":{"begin":1032,"end":1037},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32899","span":{"begin":1137,"end":1142},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32900","span":{"begin":1334,"end":1339},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32901","span":{"begin":1422,"end":1427},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T32902","span":{"begin":1497,"end":1501},"obj":"http://purl.obolibrary.org/obo/GO_0005623"}],"text":"Figure 6. CbfbF/F CD4-cre mouse cells and iT reg cells generated from human naive CD4+ T cells undergoing siRNA-mediated RUNX1 and RUNX3 knock down show a diminished suppressive activity. Experimental setup (A) and results of the mouse suppression assay (B), FACS-purified naive CD4+8− T cells from CbfbF/F CD4-cre (left) and control CbfbF/+ CD4-cre mice (Cd45.2; right) were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, and 2.5 ng/ml TGF-β. After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. These were then incubated with inactivated splenocytes and anti-CD3 mAb. After a further 4 d, CD45.1+ cells were analyzed for CFSE dilution. (C) As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-β were mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios. Four days later CD45.1+ cells were analyzed for CFSE dilution. The median division number (of the naive CD45.1+ CD4+ cells in the cultures containing Foxp3-GFP+ cells) is indicated in each of the histograms. One of three experiments is shown. (D) Human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA and cultured under iT reg differentiating conditions were used in an in vitro suppression assay, cultured together with autologous CFSE-labeled CD4+ T cells, and stimulated with anti-CD3 mAb. The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry. The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5. One of two experiments is shown. "}

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