PMC:2806624 / 22284-26995 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2806624","sourcedb":"PMC","sourceid":"2806624","source_url":"http://www.ncbi.nlm.nih.gov/pmc/2806624","text":"CBFβ, RUNX1, and RUNX3 are important for the suppressive activity of CD25+ Foxp3+ T reg cells\nEven though Cbfb-deficient CD4+ T cells had impaired induction of Foxp3 after stimulation with anti-CD3/28 mAbs and TGF-β, sufficient numbers of Foxp3+ cells could be generated to permit analysis of their suppressive activity. Purified naive CD4+ T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice (Cd45.2) harboring a Foxp3-ires-GFP allele (Bettelli et al., 2006) were stimulated in vitro with anti-CD3/28 mAbs, IL-2, and TGF-β. After 3 d, Foxp3+-GFP+ cells were sorted by flow cytometry and mixed with CFSE-labeled naive CD45.1+ CD4+ cells at ratios of 1:4, 1:2, and 1:1. The cells were then incubated with inactivated splenocytes and stimulated with anti-CD3 mAb for four more days. CD45.1+ cells were analyzed for CFSE dilution (Fig. 6 A). CbfbF/+ CD4-cre CD4+ T cells activated in the presence of TGF-β showed a clear suppression of T cell proliferation that became even more apparent when an increased ratio of FOXP3+/CD25− cells was used (Fig. 6 B). The suppression was significantly reduced when cells from CbfbF/F CD4-cre mice were used, demonstrating that TGF-β–induced Runx complexes are important for the suppressive activity of Foxp3+ T reg cells (Fig. 6 B). As a control, CbfbF/+ CD4-cre CD4+ T cells activated in absence of TGF-β were mixed with CFSE-labeled naive CD45.1+ CD4+ cells. No suppression could be observed in all control groups without TGF-β at all tested ratios (Fig. 6 C). The decreased suppression capacity of CbfbF/F CD4-cre iT reg cells was unlikely to be caused by decreased survival or proliferation. Foxp3+ and Foxp3− cells generated from both CbfbF/F CD4-cre and control cells all displayed similar proliferation rates and cell death as measured by CFSE dilution and annexin V staining, respectively (Fig. S7, A and B).\nFigure 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. In addition, we tested the requirement of RUNX1 and RUNX3 for the development of the suppressive capacity in human iT reg cells. We isolated human naive CD4+ T cells and transfected them with a combination of RUNX1 and RUNX3 siRNA, or with a scrambled control siRNA. Cells were cultured under T reg conditions, and then mixed with CFSE-labeled autologous CD4+ T cells and stimulated with anti-CD3 mAb. Cells in which RUNX1 and RUNX3 were knocked down showed markedly lower suppressive activity compared with control iT reg cells at a T reg/CD4+ T responder cell ratio of 1:20, but not when the T reg/CD4+ T responder cell ratio was increased to 1:5 (Fig. 6 D). These results demonstrate the important role of RUNX1 and RUNX3 not only for the induction of FOXP3, but also for the suppressive capacity of iT reg cells both in humans and in mice. The data suggest both quantity and quality of T reg cells are hampered. Reduced intrinsic suppressive capacity of iT reg cells was demonstrated in mice, because Foxp3-GFP+ cells were FACS sorted and same numbers of iT reg cells are included in control experiments. In the suppression experiment with human cells, reduced suppressive activity was caused by reduced FOXP3 expression in T cells by siRNA inhibition of RUNX1 and RUNX3.","divisions":[{"label":"Title","span":{"begin":0,"end":93}},{"label":"Figure 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