PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T138	0-118	Sentence	denotes	Transcription factors RUNX1 and RUNX3 in the induction and suppressive function of Foxp3+ inducible regulatory T cells
T1	0-177	Sentence	denotes	Transcription factors RUNX1 and RUNX3 in the induction and suppressive function of Foxp3+ inducible regulatory T cells RUNX1 and RUNX3 in functional inducible T regulatory cells
T2	180-188	Sentence	denotes	Abstract
T139	189-320	Sentence	denotes	Forkhead box P3 (FOXP3)+CD4+CD25+ inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis.
T3	189-320	Sentence	denotes	Forkhead box P3 (FOXP3)+CD4+CD25+ inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis.
T140	321-487	Sentence	denotes	In this study, we show that the transforming growth factor-β (TGF-β) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4+ T cells.
T4	321-487	Sentence	denotes	In this study, we show that the transforming growth factor-β (TGF-β) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4+ T cells.
T141	488-624	Sentence	denotes	This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter.
T5	488-624	Sentence	denotes	This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter.
T142	625-841	Sentence	denotes	Inactivation of the gene encoding RUNX cofactor core-binding factor-β (CBFβ) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3.
T6	625-841	Sentence	denotes	Inactivation of the gene encoding RUNX cofactor core-binding factor-β (CBFβ) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3.
T143	842-1014	Sentence	denotes	The in vivo conversion of naive CD4+ T cells into Foxp3+ iT reg cells was significantly decreased in adoptively transferred CbfbF/F CD4-cre naive T cells into Rag2−/− mice.
T7	842-1014	Sentence	denotes	The in vivo conversion of naive CD4+ T cells into Foxp3+ iT reg cells was significantly decreased in adoptively transferred CbfbF/F CD4-cre naive T cells into Rag2−/− mice.
T144	1015-1155	Sentence	denotes	Both RUNX1 and RUNX3 siRNA silenced human T reg cells and CbfbF/F CD4-cre mouse T reg cells showed diminished suppressive function in vitro.
T8	1015-1155	Sentence	denotes	Both RUNX1 and RUNX3 siRNA silenced human T reg cells and CbfbF/F CD4-cre mouse T reg cells showed diminished suppressive function in vitro.
T145	1156-1307	Sentence	denotes	Circulating human CD4+ CD25high CD127− T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-β mRNA compared with CD4+CD25− cells.
T9	1156-1307	Sentence	denotes	Circulating human CD4+ CD25high CD127− T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-β mRNA compared with CD4+CD25− cells.
T146	1308-1382	Sentence	denotes	Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells.
T10	1308-1382	Sentence	denotes	Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells.
T147	1383-1531	Sentence	denotes	These data demonstrate Runx transcription factors as a molecular link in TGF-β–induced Foxp3 expression in iT reg cell differentiation and function.
T11	1383-1531	Sentence	denotes	These data demonstrate Runx transcription factors as a molecular link in TGF-β–induced Foxp3 expression in iT reg cell differentiation and function.
T1277	1533-1821	Sentence	denotes	Regulatory T (T reg) cells expressing the transcription factor forkhead box P3 (FOXP3, human; Foxp3, mouse) play an essential role in controlling immune responses to autoantigens, allergens, tumor antigens, transplantation antigens, and infectious agents (Hori et al., 2003; Akdis, 2006).
T12	1533-1821	Sentence	denotes	Regulatory T (T reg) cells expressing the transcription factor forkhead box P3 (FOXP3, human; Foxp3, mouse) play an essential role in controlling immune responses to autoantigens, allergens, tumor antigens, transplantation antigens, and infectious agents (Hori et al., 2003; Akdis, 2006).
T1278	1822-2042	Sentence	denotes	Foxp3 is a member of the forkhead/winged-helix family of transcriptional regulators, and its expression in T reg cells is essential for their development and function (Fontenot et al., 2003; Williams and Rudensky, 2007).
T13	1822-2042	Sentence	denotes	Foxp3 is a member of the forkhead/winged-helix family of transcriptional regulators, and its expression in T reg cells is essential for their development and function (Fontenot et al., 2003; Williams and Rudensky, 2007).
T14	2043-2342	Sentence	denotes	A spontaneous mutation of the X-linked Foxp3 gene in scurfy mice causes an autoimmune-like disease, whereas the mutation in humans leads to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome that is also a severe multiorgan autoimmune disease with hyper-IgE (Ziegler, 2006).
T1279	2043-2625	Sentence	denotes	A spontaneous mutation of the X-linked Foxp3 gene in scurfy mice causes an autoimmune-like disease, whereas the mutation in humans leads to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome that is also a severe multiorgan autoimmune disease with hyper-IgE (Ziegler, 2006). Although the essential role of Foxp3 in central and peripheral tolerance has been extensively studied, its regulation, cooperation with other transcription factors, and how it functions in inducible T reg (iT reg) cells to suppress various target genes is mostly not yet understood.
T15	2343-2625	Sentence	denotes	Although the essential role of Foxp3 in central and peripheral tolerance has been extensively studied, its regulation, cooperation with other transcription factors, and how it functions in inducible T reg (iT reg) cells to suppress various target genes is mostly not yet understood.
T1280	2626-2866	Sentence	denotes	It is known that Foxp3 cooperates with the nuclear factor of activated T cells (NFAT) or nuclear factor-kappa B (NF-κB) to regulate the transcription of different target genes (Schubert et al., 2001; Bettelli et al., 2005; Wu et al., 2006).
T16	2626-2866	Sentence	denotes	It is known that Foxp3 cooperates with the nuclear factor of activated T cells (NFAT) or nuclear factor-kappa B (NF-κB) to regulate the transcription of different target genes (Schubert et al., 2001; Bettelli et al., 2005; Wu et al., 2006).
T1281	2867-2937	Sentence	denotes	The Th2 cytokine IL-4 inhibits FOXP3 expression during T cell priming.
T17	2867-2937	Sentence	denotes	The Th2 cytokine IL-4 inhibits FOXP3 expression during T cell priming.
T1282	2938-3067	Sentence	denotes	GATA3 binds to the FOXP3 promoter and can repress the FOXP3 trans-activation process directly in Th2 cells (Mantel et al., 2007).
T18	2938-3067	Sentence	denotes	GATA3 binds to the FOXP3 promoter and can repress the FOXP3 trans-activation process directly in Th2 cells (Mantel et al., 2007).
T1283	3068-3286	Sentence	denotes	It was further demonstrated that both Th1 and Th2 transcription factors T-bet and GATA3 oppose peripheral induction of Foxp3+ T reg cells in mice through STAT1-, STAT4-, and STAT6-dependent pathways (Wei et al., 2007).
T19	3068-3286	Sentence	denotes	It was further demonstrated that both Th1 and Th2 transcription factors T-bet and GATA3 oppose peripheral induction of Foxp3+ T reg cells in mice through STAT1-, STAT4-, and STAT6-dependent pathways (Wei et al., 2007).
T1284	3287-3610	Sentence	denotes	Although natural T reg (nT reg) cells that differentiate in the thymus are characterized by their stable Foxp3 expression, the generation of iT reg cells specific for allergens, alloantigens, and autoantigens in the periphery has been associated with a transient Foxp3+ phenotype (Fontenot et al., 2003; Hori et al., 2003).
T20	3287-3610	Sentence	denotes	Although natural T reg (nT reg) cells that differentiate in the thymus are characterized by their stable Foxp3 expression, the generation of iT reg cells specific for allergens, alloantigens, and autoantigens in the periphery has been associated with a transient Foxp3+ phenotype (Fontenot et al., 2003; Hori et al., 2003).
T21	3611-3679	Sentence	denotes	The crucial role of TGF-β in their generation has been demonstrated.
T1285	3611-3959	Sentence	denotes	The crucial role of TGF-β in their generation has been demonstrated. The RUNX gene family (Runt-related transcription factor, acute myeloid leukemia [AML], core-binding factor-α [CBFα], and polyoma enhancer-binding protein-2α [PEBP2α]) contains three members, RUNX1 (AML1/CBFA2/PEBP2αB), RUNX2 (AML3/CBFA1/ PEBP2αA), and RUNX3 (AML2/CBFA3/PEBP2αC).
T22	3680-3959	Sentence	denotes	The RUNX gene family (Runt-related transcription factor, acute myeloid leukemia [AML], core-binding factor-α [CBFα], and polyoma enhancer-binding protein-2α [PEBP2α]) contains three members, RUNX1 (AML1/CBFA2/PEBP2αB), RUNX2 (AML3/CBFA1/ PEBP2αA), and RUNX3 (AML2/CBFA3/PEBP2αC).
T1286	3960-4042	Sentence	denotes	They are essential transcriptional regulators of different developmental pathways.
T23	3960-4042	Sentence	denotes	They are essential transcriptional regulators of different developmental pathways.
T1287	4043-4143	Sentence	denotes	RUNX2 is mostly important for bone development and osteoblast differentiation (Komori et al., 1997).
T24	4043-4143	Sentence	denotes	RUNX2 is mostly important for bone development and osteoblast differentiation (Komori et al., 1997).
T1288	4144-4340	Sentence	denotes	RUNX1 plays an important role in hematopoiesis during development, and RUNX3 has important functions in thymogenesis and neurogenesis (Wang et al., 1996; Inoue et al., 2002; Levanon et al., 2002).
T25	4144-4340	Sentence	denotes	RUNX1 plays an important role in hematopoiesis during development, and RUNX3 has important functions in thymogenesis and neurogenesis (Wang et al., 1996; Inoue et al., 2002; Levanon et al., 2002).
T26	4341-4483	Sentence	denotes	RUNX1 and RUNX3 also work together in the establishment of lineage specification of T lymphocytes (Taniuchi et al., 2002; Egawa et al., 2007).
T1289	4341-4780	Sentence	denotes	RUNX1 and RUNX3 also work together in the establishment of lineage specification of T lymphocytes (Taniuchi et al., 2002; Egawa et al., 2007). RUNX1 is a frequent target for chromosomal translocations associated with leukemias (Look, 1997), and RUNX3 methylation and silencing is observed in various human epithelial cancers (Blyth et al., 2005). RUNX family members share the Runt domain, which is responsible for DNA binding (Ito, 1999).
T27	4484-4687	Sentence	denotes	RUNX1 is a frequent target for chromosomal translocations associated with leukemias (Look, 1997), and RUNX3 methylation and silencing is observed in various human epithelial cancers (Blyth et al., 2005).
T28	4688-4780	Sentence	denotes	RUNX family members share the Runt domain, which is responsible for DNA binding (Ito, 1999).
T1290	4781-4881	Sentence	denotes	The Runt domain-containing protein constitutes the α-chain partner of the heterodimeric CBF complex.
T29	4781-4881	Sentence	denotes	The Runt domain-containing protein constitutes the α-chain partner of the heterodimeric CBF complex.
T1291	4882-5089	Sentence	denotes	RUNX proteins heterodimerize with the non–DNA-binding partner, CBFβ, which increases the affinity for DNA binding and stabilizes the complex by preventing ubiquitin-dependent degradation (Wang et al., 1993).
T30	4882-5089	Sentence	denotes	RUNX proteins heterodimerize with the non–DNA-binding partner, CBFβ, which increases the affinity for DNA binding and stabilizes the complex by preventing ubiquitin-dependent degradation (Wang et al., 1993).
T1292	5090-5200	Sentence	denotes	The CBF complexes regulate the expression of cellular genes through binding to promoters or enhancer elements.
T31	5090-5200	Sentence	denotes	The CBF complexes regulate the expression of cellular genes through binding to promoters or enhancer elements.
T1293	5201-5293	Sentence	denotes	The effects of the RUNX–CBFβ complex regulation are clearly cell lineage and stage specific.
T32	5201-5293	Sentence	denotes	The effects of the RUNX–CBFβ complex regulation are clearly cell lineage and stage specific.
T33	5294-5458	Sentence	denotes	They include the crucial choices between cell-cycle exit and continued proliferation, as well as between cell differentiation and self-renewal (Blyth et al., 2005).
T1294	5294-5713	Sentence	denotes	They include the crucial choices between cell-cycle exit and continued proliferation, as well as between cell differentiation and self-renewal (Blyth et al., 2005). Because of the involvement of RUNX mutations in different autoimmune diseases and the known interaction with TGF-β, we investigated the impact of RUNX1 and RUNX3 on the expression of FOXP3 and subsequently on the development and function of iT reg cells.
T34	5459-5713	Sentence	denotes	Because of the involvement of RUNX mutations in different autoimmune diseases and the known interaction with TGF-β, we investigated the impact of RUNX1 and RUNX3 on the expression of FOXP3 and subsequently on the development and function of iT reg cells.
T1295	5714-5856	Sentence	denotes	This study demonstrates that RUNX1 and RUNX3 induced by TGF-β are involved in the development and suppressive function of Foxp3+ iT reg cells.
T35	5714-5856	Sentence	denotes	This study demonstrates that RUNX1 and RUNX3 induced by TGF-β are involved in the development and suppressive function of Foxp3+ iT reg cells.
T36	5858-5865	Sentence	denotes	RESULTS
T3603	5867-5957	Sentence	denotes	The role of RUNX1 and RUNX3 transcription factors in TGF-β–mediated iT reg cell generation
T37	5867-5957	Sentence	denotes	The role of RUNX1 and RUNX3 transcription factors in TGF-β–mediated iT reg cell generation
T3604	5958-6165	Sentence	denotes	To investigate the role of RUNX transcription factors in the development of iT reg cells, we cultured naive CD4+ T cells, isolated from human PBMCs, in conditions that enable the development of iT reg cells.
T38	5958-6165	Sentence	denotes	To investigate the role of RUNX transcription factors in the development of iT reg cells, we cultured naive CD4+ T cells, isolated from human PBMCs, in conditions that enable the development of iT reg cells.
T3605	6166-6288	Sentence	denotes	Stimulation with anti-CD2/3/28 mAbs or TGF-β alone resulted in a minimal up-regulation of RUNX1 and RUNX3 mRNA (Fig. 1 A).
T39	6166-6288	Sentence	denotes	Stimulation with anti-CD2/3/28 mAbs or TGF-β alone resulted in a minimal up-regulation of RUNX1 and RUNX3 mRNA (Fig. 1 A).
T3606	6289-6443	Sentence	denotes	In contrast, the combination of both TGF-β and anti-CD2/3/28 mAbs induced RUNX1 and RUNX3 mRNAs, as well as FOXP3 mRNA, within 48 h in naive CD4+ T cells.
T40	6289-6443	Sentence	denotes	In contrast, the combination of both TGF-β and anti-CD2/3/28 mAbs induced RUNX1 and RUNX3 mRNAs, as well as FOXP3 mRNA, within 48 h in naive CD4+ T cells.
T3607	6444-6649	Sentence	denotes	This result suggested further experiments to investigate whether the up-regulation of RUNX1 and RUNX3 might be a feature of iT reg cells during their development or even a prerequisite for their induction.
T41	6444-6649	Sentence	denotes	This result suggested further experiments to investigate whether the up-regulation of RUNX1 and RUNX3 might be a feature of iT reg cells during their development or even a prerequisite for their induction.
T42	6650-6659	Sentence	denotes	Figure 1.
T28593	6661-6892	Sentence	denotes	RUNX1 and RUNX3 are involved in the induction of Foxp3 in iT reg cells. (A) RUNX1, RUNX3, and FOXP3 mRNA induction in human naive CD4+ T cells after alone or combined anti-CD2/3/28 mAb and TGF-β stimulation in the presence of IL-2.
T43	6661-6892	Sentence	denotes	RUNX1 and RUNX3 are involved in the induction of Foxp3 in iT reg cells. (A) RUNX1, RUNX3, and FOXP3 mRNA induction in human naive CD4+ T cells after alone or combined anti-CD2/3/28 mAb and TGF-β stimulation in the presence of IL-2.
T28594	6893-6957	Sentence	denotes	Real-time PCR of human naive CD4+ T cells after 48 h of culture.
T44	6893-6957	Sentence	denotes	Real-time PCR of human naive CD4+ T cells after 48 h of culture.
T28595	6958-7150	Sentence	denotes	Bars show the mean ± SE of three independent experiments. (B) FOXP3 mRNA induction by anti-CD2/3/28 mAb and TGF-β in human naive CD4+ T cells is reduced after siRNA-mediated RUNX1/3 knockdown.
T45	6958-7150	Sentence	denotes	Bars show the mean ± SE of three independent experiments. (B) FOXP3 mRNA induction by anti-CD2/3/28 mAb and TGF-β in human naive CD4+ T cells is reduced after siRNA-mediated RUNX1/3 knockdown.
T28596	7151-7317	Sentence	denotes	Real-time PCR of RNA from human naive CD4+ T cells, transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and cultured with anti-CD2/3/28, TGF-β and IL-2.
T46	7151-7317	Sentence	denotes	Real-time PCR of RNA from human naive CD4+ T cells, transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and cultured with anti-CD2/3/28, TGF-β and IL-2.
T28597	7318-7475	Sentence	denotes	Bars show the mean ± SD of three independent experiments. (C) FOXP3 mRNA is down-regulated in iT reg cells after siRNA-mediated knockdown of RUNX1 and RUNX3.
T47	7318-7475	Sentence	denotes	Bars show the mean ± SD of three independent experiments. (C) FOXP3 mRNA is down-regulated in iT reg cells after siRNA-mediated knockdown of RUNX1 and RUNX3.
T28598	7476-7702	Sentence	denotes	Real-time PCR for FOXP3, T-bet, GATA3, and RORC2 from human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA or with scrambled siRNA (control) and cultured under iT reg, Th1, Th2, or Th17-driving conditions for 12 d.
T48	7476-7702	Sentence	denotes	Real-time PCR for FOXP3, T-bet, GATA3, and RORC2 from human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA or with scrambled siRNA (control) and cultured under iT reg, Th1, Th2, or Th17-driving conditions for 12 d.
T28599	7703-7855	Sentence	denotes	Bars show the mean ± SD of three independent experiments. (D) FOXP3 protein induction in iT reg cells is reduced after siRNA-mediated RUNX1/3 knockdown.
T49	7703-7855	Sentence	denotes	Bars show the mean ± SD of three independent experiments. (D) FOXP3 protein induction in iT reg cells is reduced after siRNA-mediated RUNX1/3 knockdown.
T28600	7856-8020	Sentence	denotes	Human naive CD4+ T cells were transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and stimulated with anti-CD2/3/28 and TGF-β in the presence of IL-2.
T50	7856-8020	Sentence	denotes	Human naive CD4+ T cells were transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and stimulated with anti-CD2/3/28 and TGF-β in the presence of IL-2.
T28601	8021-8087	Sentence	denotes	CD4 and intracellular FOXP3 analysis by flow cytometry after 72 h.
T51	8021-8087	Sentence	denotes	CD4 and intracellular FOXP3 analysis by flow cytometry after 72 h.
T28602	8088-8134	Sentence	denotes	One of three independent experiments is shown.
T52	8088-8134	Sentence	denotes	One of three independent experiments is shown.
T28603	8135-8231	Sentence	denotes	Statistical differences were verified by the paired Student's t test. , P < 0.05; *, P < 0.01.
T53	8135-8231	Sentence	denotes	Statistical differences were verified by the paired Student's t test. , P < 0.05; *, P < 0.01.
T3608	8234-8392	Sentence	denotes	To test this hypothesis, RUNX1 and RUNX3 expression was knocked down in human naive CD4+ T cells by transfection of small interfering RNAs (siRNAs; Fig. 1 B).
T54	8234-8392	Sentence	denotes	To test this hypothesis, RUNX1 and RUNX3 expression was knocked down in human naive CD4+ T cells by transfection of small interfering RNAs (siRNAs; Fig. 1 B).
T3609	8393-8573	Sentence	denotes	Deficiency of RUNX1 or RUNX3 resulted in markedly reduced TGF-β–mediated induction of FOXP3 mRNA in naive CD4+ T cells compared with control cells transfected with scrambled siRNA.
T55	8393-8573	Sentence	denotes	Deficiency of RUNX1 or RUNX3 resulted in markedly reduced TGF-β–mediated induction of FOXP3 mRNA in naive CD4+ T cells compared with control cells transfected with scrambled siRNA.
T3610	8574-8740	Sentence	denotes	The level of FOXP3 mRNA was further reduced when both RUNX1 and RUNX3 were knocked down in naive CD4+ T cells during their differentiation to iT reg cells (Fig. 1 B).
T56	8574-8740	Sentence	denotes	The level of FOXP3 mRNA was further reduced when both RUNX1 and RUNX3 were knocked down in naive CD4+ T cells during their differentiation to iT reg cells (Fig. 1 B).
T3611	8741-8893	Sentence	denotes	The influence of RUNX1 and RUNX3 on the development of other T cell subsets and their specific transcription factor expression was further investigated.
T57	8741-8893	Sentence	denotes	The influence of RUNX1 and RUNX3 on the development of other T cell subsets and their specific transcription factor expression was further investigated.
T3612	8894-9100	Sentence	denotes	Naive CD4+ T cells were cultured under Th1, Th2, T reg cell, and Th17 differentiation conditions and the mRNA expression of the predominant transcription factor for each cell type was subsequently analyzed.
T58	8894-9100	Sentence	denotes	Naive CD4+ T cells were cultured under Th1, Th2, T reg cell, and Th17 differentiation conditions and the mRNA expression of the predominant transcription factor for each cell type was subsequently analyzed.
T3613	9101-9298	Sentence	denotes	We observed no change in GATA3 expression in Th2 cells, T-bet expression in Th1 cells, or RORC2 mRNA expression in Th17 cells in which RUNX1 and RUNX3 were knocked down compared with control cells.
T59	9101-9298	Sentence	denotes	We observed no change in GATA3 expression in Th2 cells, T-bet expression in Th1 cells, or RORC2 mRNA expression in Th17 cells in which RUNX1 and RUNX3 were knocked down compared with control cells.
T3614	9299-9436	Sentence	denotes	On the contrary, FOXP3 mRNA was significantly decreased in RUNX1- and RUNX3-deficient T reg cells compared with control cells (Fig. 1 C).
T60	9299-9436	Sentence	denotes	On the contrary, FOXP3 mRNA was significantly decreased in RUNX1- and RUNX3-deficient T reg cells compared with control cells (Fig. 1 C).
T3615	9437-9596	Sentence	denotes	The effect of RUNX silencing on the expression level of intracellular FOXP3 during naive CD4+ T cell differentiation to iT reg was evaluated by flow cytometry.
T61	9437-9596	Sentence	denotes	The effect of RUNX silencing on the expression level of intracellular FOXP3 during naive CD4+ T cell differentiation to iT reg was evaluated by flow cytometry.
T3616	9597-9651	Sentence	denotes	FOXP3 was only slightly reduced after RUNX1 silencing.
T62	9597-9651	Sentence	denotes	FOXP3 was only slightly reduced after RUNX1 silencing.
T3617	9652-9706	Sentence	denotes	Transfection of siRNA for RUNX3 had a stronger effect.
T63	9652-9706	Sentence	denotes	Transfection of siRNA for RUNX3 had a stronger effect.
T3618	9707-9809	Sentence	denotes	The most striking FOXP3 reduction was observed when RUNX1 and RUNX3 were silenced together (Fig. 1 D).
T64	9707-9809	Sentence	denotes	The most striking FOXP3 reduction was observed when RUNX1 and RUNX3 were silenced together (Fig. 1 D).
T3619	9810-9884	Sentence	denotes	Similar results were obtained in total CD4+ T cells (Fig. S1 and Fig. S2).
T65	9810-9884	Sentence	denotes	Similar results were obtained in total CD4+ T cells (Fig. S1 and Fig. S2).
T3620	9885-10030	Sentence	denotes	The increased impact of combined RUNX1 and RUNX3 knockdown implies that RUNX1 and RUNX3 might have redundant functions in the induction of FOXP3.
T66	9885-10030	Sentence	denotes	The increased impact of combined RUNX1 and RUNX3 knockdown implies that RUNX1 and RUNX3 might have redundant functions in the induction of FOXP3.
T3621	10031-10286	Sentence	denotes	In addition, the levels of IL-4, IL-5, IL-10, IL-13, and IFN-γ in control siRNA-transfected or RUNX1 and RUNX3 siRNA-transfected CD4+ T cells that were cultured with or without anti-CD2/3/28 mAb and TGF-β did not show any significant difference (Fig. S3).
T67	10031-10286	Sentence	denotes	In addition, the levels of IL-4, IL-5, IL-10, IL-13, and IFN-γ in control siRNA-transfected or RUNX1 and RUNX3 siRNA-transfected CD4+ T cells that were cultured with or without anti-CD2/3/28 mAb and TGF-β did not show any significant difference (Fig. S3).
T3622	10287-10484	Sentence	denotes	To determine whether RUNX1 and RUNX3 are also expressed in human T reg cells in vivo, we isolated peripheral blood CD4+ CD127− CD25high T reg cells and compared them with CD4+ CD127+ CD25− T cells.
T68	10287-10484	Sentence	denotes	To determine whether RUNX1 and RUNX3 are also expressed in human T reg cells in vivo, we isolated peripheral blood CD4+ CD127− CD25high T reg cells and compared them with CD4+ CD127+ CD25− T cells.
T3623	10485-10591	Sentence	denotes	Circulating T reg cells expressed significantly higher levels of RUNX3 mRNA compared with CD4+CD25− cells.
T69	10485-10591	Sentence	denotes	Circulating T reg cells expressed significantly higher levels of RUNX3 mRNA compared with CD4+CD25− cells.
T3624	10592-10692	Sentence	denotes	As expected, IL-10, TGF-β, and FOXP3 mRNAs are also expressed in circulating T reg cells (Fig. 2 A).
T70	10592-10692	Sentence	denotes	As expected, IL-10, TGF-β, and FOXP3 mRNAs are also expressed in circulating T reg cells (Fig. 2 A).
T3625	10693-10773	Sentence	denotes	There was no difference in RUNX1 mRNA expression between these two cell subsets.
T71	10693-10773	Sentence	denotes	There was no difference in RUNX1 mRNA expression between these two cell subsets.
T3626	10774-10895	Sentence	denotes	We also performed an analysis of human tonsils, which contain high numbers of FOXP3+ T reg cells (Verhagen et al., 2006).
T72	10774-10895	Sentence	denotes	We also performed an analysis of human tonsils, which contain high numbers of FOXP3+ T reg cells (Verhagen et al., 2006).
T3627	10896-11099	Sentence	denotes	Staining of tonsil sections for FOXP3 and RUNX3 demonstrated in vivo coexpression of these two molecules in a subset of T reg cells, whereas there was low RUNX1 expression in all tonsil cells (Fig. 2 B).
T73	10896-11099	Sentence	denotes	Staining of tonsil sections for FOXP3 and RUNX3 demonstrated in vivo coexpression of these two molecules in a subset of T reg cells, whereas there was low RUNX1 expression in all tonsil cells (Fig. 2 B).
T74	11100-11109	Sentence	denotes	Figure 2.
T29703	11111-11452	Sentence	denotes	RUNX1 and RUNX3 expression in Foxp3+ T reg cells and in human CD4+, CD127−, CD25high cells. (A) Real-time PCR analysis of CD4+, CD127−, and CD25high T reg cells and CD4+, CD127+, and CD25neg T cells isolated from human peripheral blood showed an increased expression of IL-10, TGF-β, FOXP3, and RUNX3 mRNA in CD25+ compared with CD25− cells.
T75	11111-11452	Sentence	denotes	RUNX1 and RUNX3 expression in Foxp3+ T reg cells and in human CD4+, CD127−, CD25high cells. (A) Real-time PCR analysis of CD4+, CD127−, and CD25high T reg cells and CD4+, CD127+, and CD25neg T cells isolated from human peripheral blood showed an increased expression of IL-10, TGF-β, FOXP3, and RUNX3 mRNA in CD25+ compared with CD25− cells.
T29704	11453-11574	Sentence	denotes	Bars show the mean ± SE of three independent experiments. (B) Human tonsil sections were analyzed by confocal microscopy.
T76	11453-11574	Sentence	denotes	Bars show the mean ± SE of three independent experiments. (B) Human tonsil sections were analyzed by confocal microscopy.
T29705	11575-11658	Sentence	denotes	Tissue sections were stained for FOXP3, RUNX1, RUNX3, and DAPI or isotype controls.
T77	11575-11658	Sentence	denotes	Tissue sections were stained for FOXP3, RUNX1, RUNX3, and DAPI or isotype controls.
T29706	11659-11754	Sentence	denotes	HEK cells RUNX1-transfected or not transfected served as additional control for RUNX1 staining.
T78	11659-11754	Sentence	denotes	HEK cells RUNX1-transfected or not transfected served as additional control for RUNX1 staining.
T29707	11755-11843	Sentence	denotes	Data shown are representative from one of the three tissue samples with similar results.
T79	11755-11843	Sentence	denotes	Data shown are representative from one of the three tissue samples with similar results.
T29708	11844-11855	Sentence	denotes	Bars, 5 µm.
T80	11844-11855	Sentence	denotes	Bars, 5 µm.
T29709	11856-11952	Sentence	denotes	Statistical differences were verified by the paired Student's t test. , P < 0.05; *, P < 0.01.
T81	11856-11952	Sentence	denotes	Statistical differences were verified by the paired Student's t test. , P < 0.05; *, P < 0.01.
T6067	11954-11996	Sentence	denotes	RUNX1 and RUNX3 bind to the FOXP3 promoter
T82	11954-11996	Sentence	denotes	RUNX1 and RUNX3 bind to the FOXP3 promoter
T6068	11997-12180	Sentence	denotes	Transcription element search system analysis of the human FOXP3 promoter predicted 3 putative RUNX binding sites at 333, 287, and 53 bp upstream of the transcription start site (TSS).
T83	11997-12180	Sentence	denotes	Transcription element search system analysis of the human FOXP3 promoter predicted 3 putative RUNX binding sites at 333, 287, and 53 bp upstream of the transcription start site (TSS).
T6069	12181-12259	Sentence	denotes	All three binding sites are conserved between human, mouse, and rat (Fig. S4).
T84	12181-12259	Sentence	denotes	All three binding sites are conserved between human, mouse, and rat (Fig. S4).
T6070	12260-12382	Sentence	denotes	To verify the putative binding sites in the FOXP3 promoter, we transiently transfected HEK293T cells with RUNX1 and RUNX3.
T85	12260-12382	Sentence	denotes	To verify the putative binding sites in the FOXP3 promoter, we transiently transfected HEK293T cells with RUNX1 and RUNX3.
T6071	12383-12587	Sentence	denotes	After the pull-down with oligonucleotides containing the wild-type binding sequences, but not mutant sequences, RUNX binding to the FOXP3 promoter oligonucleotides was detected by Western blot (Fig. 3 A).
T86	12383-12587	Sentence	denotes	After the pull-down with oligonucleotides containing the wild-type binding sequences, but not mutant sequences, RUNX binding to the FOXP3 promoter oligonucleotides was detected by Western blot (Fig. 3 A).
T6072	12588-12738	Sentence	denotes	To confirm these results and test the ability of single binding site sequences to bind either RUNX1 or RUNX3, we used the promoter enzyme immunoassay.
T87	12588-12738	Sentence	denotes	To confirm these results and test the ability of single binding site sequences to bind either RUNX1 or RUNX3, we used the promoter enzyme immunoassay.
T6073	12739-12862	Sentence	denotes	Cell lysates were obtained from HEK293T cells that had been transiently transfected with RUNX1 or RUNX3 expression vectors.
T88	12739-12862	Sentence	denotes	Cell lysates were obtained from HEK293T cells that had been transiently transfected with RUNX1 or RUNX3 expression vectors.
T6074	12863-13079	Sentence	denotes	The biotinylated FOXP3 promoter oligonucleotides were linked to a streptavidin-coated microtiter plate, and bound RUNX1 or RUNX3 was detected by using anti-RUNX antibodies and a peroxidase-labeled secondary antibody.
T89	12863-13079	Sentence	denotes	The biotinylated FOXP3 promoter oligonucleotides were linked to a streptavidin-coated microtiter plate, and bound RUNX1 or RUNX3 was detected by using anti-RUNX antibodies and a peroxidase-labeled secondary antibody.
T6075	13080-13316	Sentence	denotes	We showed binding of RUNX1 and RUNX3 to the mixture of all three oligonucleotides containing the binding sites, whereas there was no binding detectable when a combination of the mutated oligonucleotides was used in the assay (Fig. 3 B).
T90	13080-13316	Sentence	denotes	We showed binding of RUNX1 and RUNX3 to the mixture of all three oligonucleotides containing the binding sites, whereas there was no binding detectable when a combination of the mutated oligonucleotides was used in the assay (Fig. 3 B).
T6076	13317-13520	Sentence	denotes	Although there was a similar and high degree of binding to the −333 and −287 sites, a lower degree of binding was detected when the oligonucleotide containing the −53 site in the Foxp3 promoter was used.
T91	13317-13520	Sentence	denotes	Although there was a similar and high degree of binding to the −333 and −287 sites, a lower degree of binding was detected when the oligonucleotide containing the −53 site in the Foxp3 promoter was used.
T6077	13521-13593	Sentence	denotes	This effect was observed both for binding to RUNX1 and RUNX3 (Fig. 3 B).
T92	13521-13593	Sentence	denotes	This effect was observed both for binding to RUNX1 and RUNX3 (Fig. 3 B).
T6078	13594-13715	Sentence	denotes	The binding to the two single binding sites at −333 and −287 was comparable to the mixture of all three oligonucleotides.
T93	13594-13715	Sentence	denotes	The binding to the two single binding sites at −333 and −287 was comparable to the mixture of all three oligonucleotides.
T6079	13716-13915	Sentence	denotes	Chromatin immunoprecipitation (ChIP) assay results confirmed the binding of RUNX1 and RUNX3 complexes containing CBFβ to FOXP3 promoter during the differentiation of naive T cells toward T reg cells.
T94	13716-13915	Sentence	denotes	Chromatin immunoprecipitation (ChIP) assay results confirmed the binding of RUNX1 and RUNX3 complexes containing CBFβ to FOXP3 promoter during the differentiation of naive T cells toward T reg cells.
T6080	13916-14027	Sentence	denotes	Here, naive CD4+ T cells were cultured with IL-2, anti-CD2/3/28 mAb, and TGF-β as a Foxp3-inducing stimulation.
T95	13916-14027	Sentence	denotes	Here, naive CD4+ T cells were cultured with IL-2, anti-CD2/3/28 mAb, and TGF-β as a Foxp3-inducing stimulation.
T6081	14028-14230	Sentence	denotes	Amplification of PCR products from the FOXP3 promoter region with the predicted RUNX binding sites showed that RUNX1, RUNX3, and CBFβ were immunoprecipitated together with the FOXP3 promoter (Fig. 3 C).
T96	14028-14230	Sentence	denotes	Amplification of PCR products from the FOXP3 promoter region with the predicted RUNX binding sites showed that RUNX1, RUNX3, and CBFβ were immunoprecipitated together with the FOXP3 promoter (Fig. 3 C).
T6082	14231-14365	Sentence	denotes	Negative control primer targeting open reading frame-free intergenic DNA, IGX1A did not show any significant change in site occupancy.
T97	14231-14365	Sentence	denotes	Negative control primer targeting open reading frame-free intergenic DNA, IGX1A did not show any significant change in site occupancy.
T98	14366-14375	Sentence	denotes	Figure 3.
T30287	14377-14520	Sentence	denotes	Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown.
T99	14377-14520	Sentence	denotes	Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown.
T30288	14521-14689	Sentence	denotes	The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides.
T100	14521-14689	Sentence	denotes	The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides.
T30289	14690-14776	Sentence	denotes	Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides.
T101	14690-14776	Sentence	denotes	Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides.
T30290	14777-14945	Sentence	denotes	The precipitated oligonucleotide–transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies.
T102	14777-14945	Sentence	denotes	The precipitated oligonucleotide–transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies.
T30291	14946-15083	Sentence	denotes	A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used.
T103	14946-15083	Sentence	denotes	A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used.
T30292	15084-15276	Sentence	denotes	Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter.
T104	15084-15276	Sentence	denotes	Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter.
T30293	15277-15554	Sentence	denotes	Bars show mean ± SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFβ to the human FOXP3 promoter in naive CD4+ T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-β.
T105	15277-15554	Sentence	denotes	Bars show mean ± SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFβ to the human FOXP3 promoter in naive CD4+ T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-β.
T30294	15555-15667	Sentence	denotes	There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used.
T106	15555-15667	Sentence	denotes	There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used.
T30295	15668-15733	Sentence	denotes	The results are normalized to input and isotype control antibody.
T107	15668-15733	Sentence	denotes	The results are normalized to input and isotype control antibody.
T30296	15734-15787	Sentence	denotes	Bars show mean ± SE of three independent experiments.
T108	15734-15787	Sentence	denotes	Bars show mean ± SE of three independent experiments.
T30297	15788-15869	Sentence	denotes	Statistical differences were verified by the paired Student's t test. *, P < 0.05
T109	15788-15869	Sentence	denotes	Statistical differences were verified by the paired Student's t test. *, P < 0.05
T7554	15871-15956	Sentence	denotes	Regulation of FOXP3 promoter activity and FOXP3 protein expression by RUNX1 and RUNX3
T110	15871-15956	Sentence	denotes	Regulation of FOXP3 promoter activity and FOXP3 protein expression by RUNX1 and RUNX3
T7555	15957-16197	Sentence	denotes	To investigate the effect of RUNX1 and RUNX3 binding to the RUNX binding sites in the FOXP3 promoter, we transfected human peripheral blood CD4+ T cells with a FOXP3 promoter luciferase reporter vector and RUNX1 or RUNX3 expression vectors.
T111	15957-16197	Sentence	denotes	To investigate the effect of RUNX1 and RUNX3 binding to the RUNX binding sites in the FOXP3 promoter, we transfected human peripheral blood CD4+ T cells with a FOXP3 promoter luciferase reporter vector and RUNX1 or RUNX3 expression vectors.
T7556	16198-16379	Sentence	denotes	An increase in luciferase activity was observed only when the FOXP3 promoter (−511 to +176) luciferase construct was cotransfected with RUNX1 or RUNX3 expression vectors (Fig. 4 A).
T112	16198-16379	Sentence	denotes	An increase in luciferase activity was observed only when the FOXP3 promoter (−511 to +176) luciferase construct was cotransfected with RUNX1 or RUNX3 expression vectors (Fig. 4 A).
T7557	16380-16475	Sentence	denotes	The increase in promoter activity was greater upon cotransfection of RUNX3 compared with RUNX1.
T113	16380-16475	Sentence	denotes	The increase in promoter activity was greater upon cotransfection of RUNX3 compared with RUNX1.
T7558	16476-16622	Sentence	denotes	Luciferase expression was abrogated when the Runx binding sites in the FOXP3 promoter (−511 to +176) luciferase construct were mutated (Fig. 4 A).
T114	16476-16622	Sentence	denotes	Luciferase expression was abrogated when the Runx binding sites in the FOXP3 promoter (−511 to +176) luciferase construct were mutated (Fig. 4 A).
T7559	16623-16787	Sentence	denotes	In these experiments, the overexpression of RUNX1 and RUNX3 eliminated the need of TGF-β for FOXP3 promoter activation and PMA/ionomycin stimulation was sufficient.
T115	16623-16787	Sentence	denotes	In these experiments, the overexpression of RUNX1 and RUNX3 eliminated the need of TGF-β for FOXP3 promoter activation and PMA/ionomycin stimulation was sufficient.
T116	16788-16797	Sentence	denotes	Figure 4.
T30975	16799-17116	Sentence	denotes	Regulation of FOXP3 promoter activity by RUNX1 and RUNX3. (A) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the wild-type or mutated FOXP3 promoter region (FOXP3 −511/+176) fused to the luciferase reporter gene together with a GFP, RUNX1, or RUNX3 expression vector.
T117	16799-17116	Sentence	denotes	Regulation of FOXP3 promoter activity by RUNX1 and RUNX3. (A) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the wild-type or mutated FOXP3 promoter region (FOXP3 −511/+176) fused to the luciferase reporter gene together with a GFP, RUNX1, or RUNX3 expression vector.
T30976	17117-17683	Sentence	denotes	Bars show the mean luciferase activity ± SE measured as arbitrary light units of three independent experiments. (B) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the putative FOXP3 promoter region (FOXP3 −511/+176) fused to the luciferase reporter gene, or with a vector containing the putative FOXP3 promoter region (FOXP3 −511/+176) with single RUNX binding sites mutated (53, 287, or 333) or with the combination of two or three RUNX binding sites mutated (53, 287, or 333) fused to the luciferase reporter gene.
T118	17117-17683	Sentence	denotes	Bars show the mean luciferase activity ± SE measured as arbitrary light units of three independent experiments. (B) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the putative FOXP3 promoter region (FOXP3 −511/+176) fused to the luciferase reporter gene, or with a vector containing the putative FOXP3 promoter region (FOXP3 −511/+176) with single RUNX binding sites mutated (53, 287, or 333) or with the combination of two or three RUNX binding sites mutated (53, 287, or 333) fused to the luciferase reporter gene.
T30977	17684-17795	Sentence	denotes	Bars show the mean luciferase activity ± SD measured as arbitrary light units of three independent experiments.
T119	17684-17795	Sentence	denotes	Bars show the mean luciferase activity ± SD measured as arbitrary light units of three independent experiments.
T7560	17798-17938	Sentence	denotes	To examine the role of each of the three RUNX binding sites for the FOXP3 promoter activity, we mutated each individually or in combination.
T120	17798-17938	Sentence	denotes	To examine the role of each of the three RUNX binding sites for the FOXP3 promoter activity, we mutated each individually or in combination.
T7561	17939-18103	Sentence	denotes	No reduction in luciferase activity was observed when the −53 site was mutated and only a slight reduction when either the −287 or −333 site was mutated (Fig. 4 B).
T121	17939-18103	Sentence	denotes	No reduction in luciferase activity was observed when the −53 site was mutated and only a slight reduction when either the −287 or −333 site was mutated (Fig. 4 B).
T7562	18104-18495	Sentence	denotes	However, mutating the −53 site in combination with one of the other two sites led to a significant decrease in luciferase activity, with the greatest reduction observed when all three binding sites were mutated (Fig. 4 B), suggesting that the identified binding sites have redundant functions and RUNX binding to more than one site is necessary for the full activation of the FOXP3 promoter.
T122	18104-18495	Sentence	denotes	However, mutating the −53 site in combination with one of the other two sites led to a significant decrease in luciferase activity, with the greatest reduction observed when all three binding sites were mutated (Fig. 4 B), suggesting that the identified binding sites have redundant functions and RUNX binding to more than one site is necessary for the full activation of the FOXP3 promoter.
T7563	18496-18678	Sentence	denotes	Supporting these findings, the overexpression of RUNX1 in human primary CD4+ T cells resulted in significantly elevated levels of FOXP3 protein measured by flow cytometry after 48 h.
T123	18496-18678	Sentence	denotes	Supporting these findings, the overexpression of RUNX1 in human primary CD4+ T cells resulted in significantly elevated levels of FOXP3 protein measured by flow cytometry after 48 h.
T7564	18679-18783	Sentence	denotes	This was achieved without any requirement for anti-CD3, anti-CD28 stimulation, or the presence of TGF-β.
T124	18679-18783	Sentence	denotes	This was achieved without any requirement for anti-CD3, anti-CD28 stimulation, or the presence of TGF-β.
T7565	18784-18926	Sentence	denotes	Although there was a trend, the transfection of CD4+ T cells with RUNX3 did not lead to statistically significant increase in FOXP3 (Fig. S5).
T125	18784-18926	Sentence	denotes	Although there was a trend, the transfection of CD4+ T cells with RUNX3 did not lead to statistically significant increase in FOXP3 (Fig. S5).
T9034	18928-18966	Sentence	denotes	Role of CBFβ in the induction of Foxp3
T126	18928-18966	Sentence	denotes	Role of CBFβ in the induction of Foxp3
T9035	18967-19093	Sentence	denotes	CBFβ, a common cofactor of all RUNX proteins, stabilizes and increases the binding of the runt domain to target DNA sequences.
T127	18967-19093	Sentence	denotes	CBFβ, a common cofactor of all RUNX proteins, stabilizes and increases the binding of the runt domain to target DNA sequences.
T9036	19094-19290	Sentence	denotes	To target all Runx proteins that might be involved in the induction of Foxp3, we used mice in which loxP-flanked Cbfb alleles were inactivated in T cells through expression of a CD4-cre transgene.
T128	19094-19290	Sentence	denotes	To target all Runx proteins that might be involved in the induction of Foxp3, we used mice in which loxP-flanked Cbfb alleles were inactivated in T cells through expression of a CD4-cre transgene.
T9037	19291-19388	Sentence	denotes	Retinoic acid and TGF-β synergize in the induction of Foxp3 in naive T cells (Kang et al., 2007).
T129	19291-19388	Sentence	denotes	Retinoic acid and TGF-β synergize in the induction of Foxp3 in naive T cells (Kang et al., 2007).
T9038	19389-19656	Sentence	denotes	To investigate whether Runx-mediated induction of Foxp3 is dependent on the expression of CBFβ, naive CD4+ CD8− T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were stimulated with anti-CD3/28 mAbs, retinoic acid, and increasing concentrations of TGF-β.
T130	19389-19656	Sentence	denotes	To investigate whether Runx-mediated induction of Foxp3 is dependent on the expression of CBFβ, naive CD4+ CD8− T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were stimulated with anti-CD3/28 mAbs, retinoic acid, and increasing concentrations of TGF-β.
T9039	19657-19788	Sentence	denotes	After 3 d in culture, the cells were restimulated with PMA and ionomycin and analyzed for intracellular Foxp3 and IFN-γ expression.
T131	19657-19788	Sentence	denotes	After 3 d in culture, the cells were restimulated with PMA and ionomycin and analyzed for intracellular Foxp3 and IFN-γ expression.
T9040	19789-19922	Sentence	denotes	TGF-β induced Foxp3 in CbfbF/+ CD4-cre cells in a dose dependent manner, and this was significantly reduced in CbfbF/F CD4-cre cells.
T132	19789-19922	Sentence	denotes	TGF-β induced Foxp3 in CbfbF/+ CD4-cre cells in a dose dependent manner, and this was significantly reduced in CbfbF/F CD4-cre cells.
T9041	19923-20100	Sentence	denotes	Retinoic acid enhanced Foxp3 expression even in 20 pg/ml of TGF-β and more than 95% of the CD4+ T cells from CbfbF/+ CD4-cre mice became Foxp3+ in 100 and 500 pg/ml TGF-β doses.
T133	19923-20100	Sentence	denotes	Retinoic acid enhanced Foxp3 expression even in 20 pg/ml of TGF-β and more than 95% of the CD4+ T cells from CbfbF/+ CD4-cre mice became Foxp3+ in 100 and 500 pg/ml TGF-β doses.
T9042	20101-20341	Sentence	denotes	The induction of Foxp3 was again significantly lower in CbfbF/F CD4-cre CD4+ T cells even in the presence of retinoic acid, demonstrating that deficiency in Runx binding to DNA affects the TGF-β induction of Foxp3 in T reg cells (Fig. 5 A).
T134	20101-20341	Sentence	denotes	The induction of Foxp3 was again significantly lower in CbfbF/F CD4-cre CD4+ T cells even in the presence of retinoic acid, demonstrating that deficiency in Runx binding to DNA affects the TGF-β induction of Foxp3 in T reg cells (Fig. 5 A).
T9043	20342-20450	Sentence	denotes	There was no difference in the induction of Foxp3 when endogenous IL-4 and IFN-γ were neutralized (Fig. S6).
T135	20342-20450	Sentence	denotes	There was no difference in the induction of Foxp3 when endogenous IL-4 and IFN-γ were neutralized (Fig. S6).
T136	20451-20460	Sentence	denotes	Figure 5.
T31435	20462-20786	Sentence	denotes	Diminished capacity of Cbfb-deficient CD4-cre mice T cells in the generation of Foxp3+ CD4+ T cells. (A) FACS-purified naive CD4+ CD8− T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, ± 10 nM retinoic acid (RA), and increasing concentrations of TGF-β.
T137	20462-20786	Sentence	denotes	Diminished capacity of Cbfb-deficient CD4-cre mice T cells in the generation of Foxp3+ CD4+ T cells. (A) FACS-purified naive CD4+ CD8− T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, ± 10 nM retinoic acid (RA), and increasing concentrations of TGF-β.
T31436	20787-20922	Sentence	denotes	After 3 d in culture, the cells were restimulated with PMA + ionomycin, and then analyzed for intracellular Foxp3 and IFN-γ expression.
T138	20787-20922	Sentence	denotes	After 3 d in culture, the cells were restimulated with PMA + ionomycin, and then analyzed for intracellular Foxp3 and IFN-γ expression.
T31437	20923-21131	Sentence	denotes	One of five experiments is shown. (B) Naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice (5 × 106 cells per transfer).
T139	20923-21131	Sentence	denotes	One of five experiments is shown. (B) Naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice (5 × 106 cells per transfer).
T31438	21132-21292	Sentence	denotes	6 wk later, TCRβ+CD4+ cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine (LP) were analyzed for Foxp3-GFP expression.
T140	21132-21292	Sentence	denotes	6 wk later, TCRβ+CD4+ cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine (LP) were analyzed for Foxp3-GFP expression.
T31439	21293-21396	Sentence	denotes	Results from one of four CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice with same findings are shown.
T141	21293-21396	Sentence	denotes	Results from one of four CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice with same findings are shown.
T142	21397-21443	Sentence	denotes	The data from four sets of mice is shown in C.
T31440	21397-21532	Sentence	denotes	The data from four sets of mice is shown in C. Statistical analysis was performed with Mann-Whitney U test. *, P < 0.05 between groups.
T143	21444-21532	Sentence	denotes	Statistical analysis was performed with Mann-Whitney U test. *, P < 0.05 between groups.
T9044	21535-21660	Sentence	denotes	The importance of RUNX transcription factors for the in vivo conversion of naive CD4+ T cells into iT reg cells was examined.
T144	21535-21660	Sentence	denotes	The importance of RUNX transcription factors for the in vivo conversion of naive CD4+ T cells into iT reg cells was examined.
T9045	21661-21791	Sentence	denotes	Control CbfbF/F or CbfbF/F CD4-cre naive T cells, harboring a Foxp3-IRES-GFP allele were adoptively transferred into Rag2−/− mice.
T145	21661-21791	Sentence	denotes	Control CbfbF/F or CbfbF/F CD4-cre naive T cells, harboring a Foxp3-IRES-GFP allele were adoptively transferred into Rag2−/− mice.
T9046	21792-21943	Sentence	denotes	6 wk later, CD4+ T cells in spleen, mesenteric lymph node, and lamina propria of the small intestine were analyzed for Foxp3-GFP expression (Fig. 5 B).
T146	21792-21943	Sentence	denotes	6 wk later, CD4+ T cells in spleen, mesenteric lymph node, and lamina propria of the small intestine were analyzed for Foxp3-GFP expression (Fig. 5 B).
T9047	21944-22179	Sentence	denotes	There was a consistently lower percentage of CD4+ T cells that had developed Foxp3 expression in the mesenteric lymph node and lamina propria of mice transferred with CbfbF/F CD4-cre cells compared with control cells (Fig. 5, B and C).
T147	21944-22179	Sentence	denotes	There was a consistently lower percentage of CD4+ T cells that had developed Foxp3 expression in the mesenteric lymph node and lamina propria of mice transferred with CbfbF/F CD4-cre cells compared with control cells (Fig. 5, B and C).
T9048	22180-22282	Sentence	denotes	These data affirm the significance of RUNX proteins for the in vivo generation of CD4+ Foxp3+ T cells.
T148	22180-22282	Sentence	denotes	These data affirm the significance of RUNX proteins for the in vivo generation of CD4+ Foxp3+ T cells.
T10570	22284-22377	Sentence	denotes	CBFβ, RUNX1, and RUNX3 are important for the suppressive activity of CD25+ Foxp3+ T reg cells
T149	22284-22377	Sentence	denotes	CBFβ, RUNX1, and RUNX3 are important for the suppressive activity of CD25+ Foxp3+ T reg cells
T10571	22378-22604	Sentence	denotes	Even 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.
T150	22378-22604	Sentence	denotes	Even 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.
T10572	22605-22818	Sentence	denotes	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.
T151	22605-22818	Sentence	denotes	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.
T10573	22819-22962	Sentence	denotes	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.
T152	22819-22962	Sentence	denotes	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.
T10574	22963-23074	Sentence	denotes	The cells were then incubated with inactivated splenocytes and stimulated with anti-CD3 mAb for four more days.
T153	22963-23074	Sentence	denotes	The cells were then incubated with inactivated splenocytes and stimulated with anti-CD3 mAb for four more days.
T10575	23075-23132	Sentence	denotes	CD45.1+ cells were analyzed for CFSE dilution (Fig. 6 A).
T154	23075-23132	Sentence	denotes	CD45.1+ cells were analyzed for CFSE dilution (Fig. 6 A).
T10576	23133-23345	Sentence	denotes	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).
T155	23133-23345	Sentence	denotes	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).
T10577	23346-23560	Sentence	denotes	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).
T156	23346-23560	Sentence	denotes	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).
T10578	23561-23688	Sentence	denotes	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.
T157	23561-23688	Sentence	denotes	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.
T10579	23689-23790	Sentence	denotes	No suppression could be observed in all control groups without TGF-β at all tested ratios (Fig. 6 C).
T158	23689-23790	Sentence	denotes	No suppression could be observed in all control groups without TGF-β at all tested ratios (Fig. 6 C).
T10580	23791-23923	Sentence	denotes	The decreased suppression capacity of CbfbF/F CD4-cre iT reg cells was unlikely to be caused by decreased survival or proliferation.
T159	23791-23923	Sentence	denotes	The decreased suppression capacity of CbfbF/F CD4-cre iT reg cells was unlikely to be caused by decreased survival or proliferation.
T10581	23924-24144	Sentence	denotes	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).
T160	23924-24144	Sentence	denotes	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).
T161	24145-24154	Sentence	denotes	Figure 6.
T32169	24156-24333	Sentence	denotes	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.
T162	24156-24333	Sentence	denotes	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.
T32170	24334-24597	Sentence	denotes	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.
T163	24334-24597	Sentence	denotes	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.
T32171	24598-24719	Sentence	denotes	After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios.
T164	24598-24719	Sentence	denotes	After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios.
T32172	24720-24792	Sentence	denotes	These were then incubated with inactivated splenocytes and anti-CD3 mAb.
T165	24720-24792	Sentence	denotes	These were then incubated with inactivated splenocytes and anti-CD3 mAb.
T32173	24793-25015	Sentence	denotes	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.
T166	24793-25015	Sentence	denotes	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.
T32174	25016-25078	Sentence	denotes	Four days later CD45.1+ cells were analyzed for CFSE dilution.
T167	25016-25078	Sentence	denotes	Four days later CD45.1+ cells were analyzed for CFSE dilution.
T32175	25079-25223	Sentence	denotes	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.
T168	25079-25223	Sentence	denotes	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.
T32176	25224-25519	Sentence	denotes	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.
T169	25224-25519	Sentence	denotes	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.
T32177	25520-25614	Sentence	denotes	The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry.
T170	25520-25614	Sentence	denotes	The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry.
T32178	25615-25684	Sentence	denotes	The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5.
T171	25615-25684	Sentence	denotes	The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5.
T32179	25685-25717	Sentence	denotes	One of two experiments is shown.
T172	25685-25717	Sentence	denotes	One of two experiments is shown.
T10582	25720-25848	Sentence	denotes	In addition, we tested the requirement of RUNX1 and RUNX3 for the development of the suppressive capacity in human iT reg cells.
T173	25720-25848	Sentence	denotes	In addition, we tested the requirement of RUNX1 and RUNX3 for the development of the suppressive capacity in human iT reg cells.
T10583	25849-25986	Sentence	denotes	We isolated human naive CD4+ T cells and transfected them with a combination of RUNX1 and RUNX3 siRNA, or with a scrambled control siRNA.
T174	25849-25986	Sentence	denotes	We isolated human naive CD4+ T cells and transfected them with a combination of RUNX1 and RUNX3 siRNA, or with a scrambled control siRNA.
T10584	25987-26121	Sentence	denotes	Cells were cultured under T reg conditions, and then mixed with CFSE-labeled autologous CD4+ T cells and stimulated with anti-CD3 mAb.
T175	25987-26121	Sentence	denotes	Cells were cultured under T reg conditions, and then mixed with CFSE-labeled autologous CD4+ T cells and stimulated with anti-CD3 mAb.
T10585	26122-26380	Sentence	denotes	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).
T176	26122-26380	Sentence	denotes	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).
T10586	26381-26563	Sentence	denotes	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.
T177	26381-26563	Sentence	denotes	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.
T10587	26564-26635	Sentence	denotes	The data suggest both quantity and quality of T reg cells are hampered.
T178	26564-26635	Sentence	denotes	The data suggest both quantity and quality of T reg cells are hampered.
T10588	26636-26828	Sentence	denotes	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.
T179	26636-26828	Sentence	denotes	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.
T10589	26829-26995	Sentence	denotes	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.
T180	26829-26995	Sentence	denotes	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.
T181	26997-27007	Sentence	denotes	DISCUSSION
T12845	27008-27145	Sentence	denotes	This study demonstrates that RUNX transcription factors 1 and 3 play an important role in the generation of FOXP3+ iT reg cells by TGF-β.
T182	27008-27145	Sentence	denotes	This study demonstrates that RUNX transcription factors 1 and 3 play an important role in the generation of FOXP3+ iT reg cells by TGF-β.
T12846	27146-27255	Sentence	denotes	TGF-β mediates RUNX induction and FOXP3 is efficiently up-regulated by RUNX1 and RUNX3 in human CD4+ T cells.
T183	27146-27255	Sentence	denotes	TGF-β mediates RUNX induction and FOXP3 is efficiently up-regulated by RUNX1 and RUNX3 in human CD4+ T cells.
T12847	27256-27331	Sentence	denotes	There are three putative RUNX binding sites in the proximal FOXP3 promoter.
T184	27256-27331	Sentence	denotes	There are three putative RUNX binding sites in the proximal FOXP3 promoter.
T12848	27332-27391	Sentence	denotes	One binding site was predicted as a binding site for RUNX2.
T185	27332-27391	Sentence	denotes	One binding site was predicted as a binding site for RUNX2.
T12849	27392-27580	Sentence	denotes	Promoter enzyme immunoassay results showed that binding of RUNX1 and RUNX3 also occurred at this site (as well as at the other two), which were initially identified as RUNX1 binding sites.
T186	27392-27580	Sentence	denotes	Promoter enzyme immunoassay results showed that binding of RUNX1 and RUNX3 also occurred at this site (as well as at the other two), which were initially identified as RUNX1 binding sites.
T12850	27581-27769	Sentence	denotes	This finding is not surprising because RUNX proteins bind to promoter or enhancer elements of their target genes via the runt domain, which is conserved between members of the RUNX family.
T187	27581-27769	Sentence	denotes	This finding is not surprising because RUNX proteins bind to promoter or enhancer elements of their target genes via the runt domain, which is conserved between members of the RUNX family.
T12851	27770-27957	Sentence	denotes	The RUNX protein that actually induces the expression of FOXP3 might therefore be dependent on the availability of the specific RUNX family member at certain stages of T cell development.
T188	27770-27957	Sentence	denotes	The RUNX protein that actually induces the expression of FOXP3 might therefore be dependent on the availability of the specific RUNX family member at certain stages of T cell development.
T12852	27958-28118	Sentence	denotes	RUNX proteins are able to increase or inhibit transcriptional activity of their target genes depending on the cell type and the target gene (Otto et al., 2003).
T189	27958-28118	Sentence	denotes	RUNX proteins are able to increase or inhibit transcriptional activity of their target genes depending on the cell type and the target gene (Otto et al., 2003).
T12853	28119-28320	Sentence	denotes	Mutation of only one of the three binding sites had only a little effect on the promoter activity; however, when two binding sites were mutated, the FOXP3 promoter activity dropped to a greater extent.
T190	28119-28320	Sentence	denotes	Mutation of only one of the three binding sites had only a little effect on the promoter activity; however, when two binding sites were mutated, the FOXP3 promoter activity dropped to a greater extent.
T12854	28321-28401	Sentence	denotes	The most striking effect was observed when all three binding sites were mutated.
T191	28321-28401	Sentence	denotes	The most striking effect was observed when all three binding sites were mutated.
T12855	28402-28568	Sentence	denotes	We therefore assume that these binding sites have partially redundant functions, but binding to at least two sites seems to be necessary for full promoter activation.
T192	28402-28568	Sentence	denotes	We therefore assume that these binding sites have partially redundant functions, but binding to at least two sites seems to be necessary for full promoter activation.
T12856	28569-28680	Sentence	denotes	TGF-β promotes or inhibits the proliferation, differentiation, and survival of a wide array of different cells.
T193	28569-28680	Sentence	denotes	TGF-β promotes or inhibits the proliferation, differentiation, and survival of a wide array of different cells.
T12857	28681-28807	Sentence	denotes	It is also produced in activated T cells and it inhibits T cell proliferation (Kehrl et al., 1986; Siegel and Massagué, 2003).
T194	28681-28807	Sentence	denotes	It is also produced in activated T cells and it inhibits T cell proliferation (Kehrl et al., 1986; Siegel and Massagué, 2003).
T12858	28808-28970	Sentence	denotes	It was shown that TGF-β is mandatory for the maintenance of peripheral T reg cells and their expression of Foxp3 (Marie et al., 2005; Rubtsov and Rudensky, 2007).
T195	28808-28970	Sentence	denotes	It was shown that TGF-β is mandatory for the maintenance of peripheral T reg cells and their expression of Foxp3 (Marie et al., 2005; Rubtsov and Rudensky, 2007).
T12859	28971-29078	Sentence	denotes	RUNX transcription factors are targets of the TGF-β superfamily and they are involved in the TGF-β pathway.
T196	28971-29078	Sentence	denotes	RUNX transcription factors are targets of the TGF-β superfamily and they are involved in the TGF-β pathway.
T12860	29079-29172	Sentence	denotes	They interact directly with regulatory SMADs (Miyazawa et al., 2002; Ito and Miyazono, 2003).
T197	29079-29172	Sentence	denotes	They interact directly with regulatory SMADs (Miyazawa et al., 2002; Ito and Miyazono, 2003).
T12861	29173-29342	Sentence	denotes	TGF-β can activate RUNX genes at the transcriptional level, and at the posttranscriptional level through activation or stabilization of RUNX proteins (Jin et al., 2004).
T198	29173-29342	Sentence	denotes	TGF-β can activate RUNX genes at the transcriptional level, and at the posttranscriptional level through activation or stabilization of RUNX proteins (Jin et al., 2004).
T12862	29343-29506	Sentence	denotes	It was shown that RUNX2 regulates the expression of TGF-β type I receptor (Ji et al., 2001), suggesting that other mechanisms for their function could be involved.
T199	29343-29506	Sentence	denotes	It was shown that RUNX2 regulates the expression of TGF-β type I receptor (Ji et al., 2001), suggesting that other mechanisms for their function could be involved.
T12863	29507-29599	Sentence	denotes	The fusion proteins RUNX1-EVI1 and RUNX1-ETO block TGF-β inhibition of leukemic cell growth.
T200	29507-29599	Sentence	denotes	The fusion proteins RUNX1-EVI1 and RUNX1-ETO block TGF-β inhibition of leukemic cell growth.
T12864	29600-29807	Sentence	denotes	RUNX3 plays an important role in TGF-β–mediated growth control in epithelial cells, as loss of RUNX3 leads to decreased sensitivity to TGF-β and hyperproliferation of the gastric mucosa (Blyth et al., 2005).
T201	29600-29807	Sentence	denotes	RUNX3 plays an important role in TGF-β–mediated growth control in epithelial cells, as loss of RUNX3 leads to decreased sensitivity to TGF-β and hyperproliferation of the gastric mucosa (Blyth et al., 2005).
T12865	29808-29954	Sentence	denotes	The present study demonstrates that RUNX3 expression is more dominant in circulating human T reg cells and tonsil T reg cells compared with RUNX1.
T202	29808-29954	Sentence	denotes	The present study demonstrates that RUNX3 expression is more dominant in circulating human T reg cells and tonsil T reg cells compared with RUNX1.
T12866	29955-30045	Sentence	denotes	This could be dependent on the stage of the cells and organ from which they were isolated.
T203	29955-30045	Sentence	denotes	This could be dependent on the stage of the cells and organ from which they were isolated.
T12867	30046-30230	Sentence	denotes	We observed that single siRNA interference of either RUNX1 or RUNX3 alone shows a slight decrease in Foxp3+ T reg cell induction, which could be caused by redundancy of these proteins.
T204	30046-30230	Sentence	denotes	We observed that single siRNA interference of either RUNX1 or RUNX3 alone shows a slight decrease in Foxp3+ T reg cell induction, which could be caused by redundancy of these proteins.
T12868	30231-30287	Sentence	denotes	For this reason, we decided to use CbfβF/F CD4-cre mice.
T205	30231-30287	Sentence	denotes	For this reason, we decided to use CbfβF/F CD4-cre mice.
T12869	30288-30399	Sentence	denotes	Foxp3 induction by TGF-β is reduced in CD4+ T cells of CbfbF/F CD4-cre mice compared with CbfbF/+ CD4-cre mice.
T206	30288-30399	Sentence	denotes	Foxp3 induction by TGF-β is reduced in CD4+ T cells of CbfbF/F CD4-cre mice compared with CbfbF/+ CD4-cre mice.
T12870	30400-30495	Sentence	denotes	Retinoic acid is secreted by a subset of dendritic cells in the gut-associated lymphoid tissue.
T207	30400-30495	Sentence	denotes	Retinoic acid is secreted by a subset of dendritic cells in the gut-associated lymphoid tissue.
T12871	30496-30645	Sentence	denotes	It inhibits the IL-6–driven induction of Th17 cells and facilitates the differentiation of naive T cells to Foxp3+ T reg cells (Mucida et al., 2007).
T208	30496-30645	Sentence	denotes	It inhibits the IL-6–driven induction of Th17 cells and facilitates the differentiation of naive T cells to Foxp3+ T reg cells (Mucida et al., 2007).
T12872	30646-30806	Sentence	denotes	We observed an increased number of Foxp3+ cells by retinoic acid and TGF-β compared with TGF-β treatment alone in CbfbF/+ CD4-cre mice and CbfbF/F CD4-cre mice.
T209	30646-30806	Sentence	denotes	We observed an increased number of Foxp3+ cells by retinoic acid and TGF-β compared with TGF-β treatment alone in CbfbF/+ CD4-cre mice and CbfbF/F CD4-cre mice.
T12873	30807-30925	Sentence	denotes	In addition, we showed a defective in vivo generation of T reg cells from Cbfb-deficient CD4+ T cells in Rag2−/− mice.
T210	30807-30925	Sentence	denotes	In addition, we showed a defective in vivo generation of T reg cells from Cbfb-deficient CD4+ T cells in Rag2−/− mice.
T12874	30926-31102	Sentence	denotes	These data in mice confirm the human data that RUNX proteins play an important role for TGF-β–dependent FOXP3 induction, as well as in the suppressive capacity of iT reg cells.
T211	30926-31102	Sentence	denotes	These data in mice confirm the human data that RUNX proteins play an important role for TGF-β–dependent FOXP3 induction, as well as in the suppressive capacity of iT reg cells.
T12875	31103-31317	Sentence	denotes	As an additional support for this concept, the overexpression of RUNX1 induced increased FOXP3 protein expression without any requirement of TGF-β and anti-CD3 and anti-CD28 stimulation in human primary CD4+ cells.
T212	31103-31317	Sentence	denotes	As an additional support for this concept, the overexpression of RUNX1 induced increased FOXP3 protein expression without any requirement of TGF-β and anti-CD3 and anti-CD28 stimulation in human primary CD4+ cells.
T12876	31318-31436	Sentence	denotes	In both human and mouse systems, reduced Foxp3 expression was associated with reduced T reg cell suppressive activity.
T213	31318-31436	Sentence	denotes	In both human and mouse systems, reduced Foxp3 expression was associated with reduced T reg cell suppressive activity.
T12877	31437-31557	Sentence	denotes	In a recent study, the role of Runx–CBFβ was investigated in nT reg cell development in the thymus (Rudra et al., 2009).
T214	31437-31557	Sentence	denotes	In a recent study, the role of Runx–CBFβ was investigated in nT reg cell development in the thymus (Rudra et al., 2009).
T12878	31558-31662	Sentence	denotes	It was reported that Foxp3 expression in nT reg cells is unstable in the absence of Runx–CBFβ complexes.
T215	31558-31662	Sentence	denotes	It was reported that Foxp3 expression in nT reg cells is unstable in the absence of Runx–CBFβ complexes.
T12879	31663-31820	Sentence	denotes	Cbfb-deficient nT reg cells progressively lose Foxp3 upon division, and there is no evidence of increased death of Cbfb-deficient nT reg cells in that study.
T216	31663-31820	Sentence	denotes	Cbfb-deficient nT reg cells progressively lose Foxp3 upon division, and there is no evidence of increased death of Cbfb-deficient nT reg cells in that study.
T12880	31821-32048	Sentence	denotes	The experiments in Cbfb-deficient CD4-cre T cells in mice and the knockdown experiments in humans in this study suggest that the induction of Foxp3 expression is a major contributing factor in the in vivo conversion experiment.
T217	31821-32048	Sentence	denotes	The experiments in Cbfb-deficient CD4-cre T cells in mice and the knockdown experiments in humans in this study suggest that the induction of Foxp3 expression is a major contributing factor in the in vivo conversion experiment.
T12881	32049-32139	Sentence	denotes	Here, we observed that there is a twofold increased Foxp3+ iT reg cell generation in vivo.
T218	32049-32139	Sentence	denotes	Here, we observed that there is a twofold increased Foxp3+ iT reg cell generation in vivo.
T12882	32140-32291	Sentence	denotes	This is in the same range with previously published studies targeting different mechanisms in Foxp3 induction (Maynard et al., 2007; Sun et al., 2007).
T219	32140-32291	Sentence	denotes	This is in the same range with previously published studies targeting different mechanisms in Foxp3 induction (Maynard et al., 2007; Sun et al., 2007).
T12883	32292-32562	Sentence	denotes	Whether the diminished capacity of Cbfb-deficient CD4-cre T cells in the generation of Foxp3 may be caused by peripheral expansion of Cbfb-deficient non–T reg cells or survival problems faced by Cbfb-deficient iT reg cells after Foxp3 induction remains to be elucidated.
T220	32292-32562	Sentence	denotes	Whether the diminished capacity of Cbfb-deficient CD4-cre T cells in the generation of Foxp3 may be caused by peripheral expansion of Cbfb-deficient non–T reg cells or survival problems faced by Cbfb-deficient iT reg cells after Foxp3 induction remains to be elucidated.
T12884	32563-32674	Sentence	denotes	The involvement of RUNX proteins in autoimmune diseases has been previously suggested (Alarcón-Riquelme, 2003).
T221	32563-32674	Sentence	denotes	The involvement of RUNX proteins in autoimmune diseases has been previously suggested (Alarcón-Riquelme, 2003).
T12885	32675-32859	Sentence	denotes	A mutation in the RUNX1 binding site in the promoter of programmed cell death 1 gene (PDCD-1) has been implicated in systemic lupus erythematosus pathogenesis (Prokunina et al., 2002).
T222	32675-32859	Sentence	denotes	A mutation in the RUNX1 binding site in the promoter of programmed cell death 1 gene (PDCD-1) has been implicated in systemic lupus erythematosus pathogenesis (Prokunina et al., 2002).
T12886	32860-33105	Sentence	denotes	Polymorphisms that alter RUNX1 binding to other genes have also been described in rheumatoid arthritis linkage at 5q31 in Japanese patients (Tokuhiro et al., 2003) and in a psoriasis linkage at 17q25 (Prokunina et al., 2002; Helms et al., 2003).
T223	32860-33105	Sentence	denotes	Polymorphisms that alter RUNX1 binding to other genes have also been described in rheumatoid arthritis linkage at 5q31 in Japanese patients (Tokuhiro et al., 2003) and in a psoriasis linkage at 17q25 (Prokunina et al., 2002; Helms et al., 2003).
T12887	33106-33239	Sentence	denotes	RUNX3-deficient mice spontaneously develop inflammatory bowel disease and hyperplastic gastritis-like lesions (Brenner et al., 2004).
T224	33106-33239	Sentence	denotes	RUNX3-deficient mice spontaneously develop inflammatory bowel disease and hyperplastic gastritis-like lesions (Brenner et al., 2004).
T12888	33240-33354	Sentence	denotes	These disease symptoms resemble those occurring after depletion of Foxp3-expressing T reg cells (Sakaguchi, 2004).
T225	33240-33354	Sentence	denotes	These disease symptoms resemble those occurring after depletion of Foxp3-expressing T reg cells (Sakaguchi, 2004).
T12889	33355-33579	Sentence	denotes	Derepression of Th2 cytokines might also account for some of the observed disease symptoms, as it was shown that T-bet first induces Runx3 in Th1 cells and then partners with Runx3 to direct lineage-specific gene activation.
T226	33355-33579	Sentence	denotes	Derepression of Th2 cytokines might also account for some of the observed disease symptoms, as it was shown that T-bet first induces Runx3 in Th1 cells and then partners with Runx3 to direct lineage-specific gene activation.
T12890	33580-33731	Sentence	denotes	Runx3/Cbfβ are both required for the activation of the Ifng gene and silencing of the Il4 gene in Th1 cells (Djuretic et al., 2007; Naoe et al., 2007).
T227	33580-33731	Sentence	denotes	Runx3/Cbfβ are both required for the activation of the Ifng gene and silencing of the Il4 gene in Th1 cells (Djuretic et al., 2007; Naoe et al., 2007).
T12891	33732-33848	Sentence	denotes	Runx proteins also play an essential role during T lymphocyte differentiation in the thymus (Taniuchi et al., 2002).
T228	33732-33848	Sentence	denotes	Runx proteins also play an essential role during T lymphocyte differentiation in the thymus (Taniuchi et al., 2002).
T12892	33849-34065	Sentence	denotes	Runx1 regulates the transitions of developing thymocytes from the CD4− CD8− double-negative stage to the CD4+ CD8+ double-positive stage and from the DP stage to the mature single-positive stage (Egawa et al., 2007).
T229	33849-34065	Sentence	denotes	Runx1 regulates the transitions of developing thymocytes from the CD4− CD8− double-negative stage to the CD4+ CD8+ double-positive stage and from the DP stage to the mature single-positive stage (Egawa et al., 2007).
T12893	34066-34207	Sentence	denotes	Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4+ helper and CD8+ cytotoxic T cell lineages.
T230	34066-34207	Sentence	denotes	Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4+ helper and CD8+ cytotoxic T cell lineages.
T12894	34208-34363	Sentence	denotes	In addition, inactivation of both Runx1 and Runx3 at the double-positive stages resulted in a severe blockage in the development of CD8+ mature thymocytes.
T231	34208-34363	Sentence	denotes	In addition, inactivation of both Runx1 and Runx3 at the double-positive stages resulted in a severe blockage in the development of CD8+ mature thymocytes.
T12895	34364-34607	Sentence	denotes	These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells, and suggest that they may play a role in nT reg cell development, which remains to be elucidated.
T232	34364-34607	Sentence	denotes	These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells, and suggest that they may play a role in nT reg cell development, which remains to be elucidated.
T12896	34608-34755	Sentence	denotes	Furthermore, it was shown that Runx1 activates IL-2 and IFN-γ gene expression in conventional CD4+ T cells by binding to their respective promoter.
T233	34608-34755	Sentence	denotes	Furthermore, it was shown that Runx1 activates IL-2 and IFN-γ gene expression in conventional CD4+ T cells by binding to their respective promoter.
T12897	34756-34991	Sentence	denotes	RUNX1 interacts physically with Foxp3 protein, and it was demonstrated that this interaction might be responsible for the suppression of IL-2 and IFN-γ production and up-regulation of T reg cell–associated molecules (Ono et al., 2007).
T234	34756-34991	Sentence	denotes	RUNX1 interacts physically with Foxp3 protein, and it was demonstrated that this interaction might be responsible for the suppression of IL-2 and IFN-γ production and up-regulation of T reg cell–associated molecules (Ono et al., 2007).
T12898	34992-35058	Sentence	denotes	It has been shown that Foxp3 also influences Th17 differentiation.
T235	34992-35058	Sentence	denotes	It has been shown that Foxp3 also influences Th17 differentiation.
T12899	35059-35177	Sentence	denotes	Specifically, Foxp3 physically interacts with RORγt, and this interaction inhibits RORγt function (Zhou et al., 2008).
T236	35059-35177	Sentence	denotes	Specifically, Foxp3 physically interacts with RORγt, and this interaction inhibits RORγt function (Zhou et al., 2008).
T12900	35178-35388	Sentence	denotes	This relationship of RORγt and Foxp3 and probably yet unknown mechanisms might be the basis of the observation that the differentiation of Th17 cells and T reg cells is often reciprocal (Bettelli et al., 2006).
T237	35178-35388	Sentence	denotes	This relationship of RORγt and Foxp3 and probably yet unknown mechanisms might be the basis of the observation that the differentiation of Th17 cells and T reg cells is often reciprocal (Bettelli et al., 2006).
T12901	35389-35560	Sentence	denotes	Recently, data suggests that Runx1 may also be involved in regulating Il17 transcription, functioning in complex with RORγt to activate transcription (Zhang et al., 2008).
T238	35389-35560	Sentence	denotes	Recently, data suggests that Runx1 may also be involved in regulating Il17 transcription, functioning in complex with RORγt to activate transcription (Zhang et al., 2008).
T12902	35561-35695	Sentence	denotes	The Runx3-deficient mice develop spontaneous Th2-dominated autoimmune colitis and asthma (Brenner et al., 2004; Fainaru et al., 2005).
T239	35561-35695	Sentence	denotes	The Runx3-deficient mice develop spontaneous Th2-dominated autoimmune colitis and asthma (Brenner et al., 2004; Fainaru et al., 2005).
T12903	35696-35882	Sentence	denotes	Cbfbf/f Cd4 mice also show a spontaneous Th2 dominated disease, with increased serum IgA, IgG1, and IgE titers and lymphocyte and eosinophil infiltration of the lung (Naoe et al., 2007).
T240	35696-35882	Sentence	denotes	Cbfbf/f Cd4 mice also show a spontaneous Th2 dominated disease, with increased serum IgA, IgG1, and IgE titers and lymphocyte and eosinophil infiltration of the lung (Naoe et al., 2007).
T12904	35883-36041	Sentence	denotes	All these phenotypes were previously attributed to a loss of Th2 silencing whereas our findings additionally suggest that loss of T reg function plays a role.
T241	35883-36041	Sentence	denotes	All these phenotypes were previously attributed to a loss of Th2 silencing whereas our findings additionally suggest that loss of T reg function plays a role.
T12905	36042-36123	Sentence	denotes	We have shown a link between Foxp3 induction in iT reg cells and RUNX1 and RUNX3.
T242	36042-36123	Sentence	denotes	We have shown a link between Foxp3 induction in iT reg cells and RUNX1 and RUNX3.
T12906	36124-36285	Sentence	denotes	RUNX proteins play a central role in pathways regulating cell growth and differentiation, and their interaction with the TGF-β pathway is of particular interest.
T243	36124-36285	Sentence	denotes	RUNX proteins play a central role in pathways regulating cell growth and differentiation, and their interaction with the TGF-β pathway is of particular interest.
T12907	36286-36591	Sentence	denotes	Foxp3 protein interacts not only with RUNX proteins but also with several other transcriptional partners, such as NFAT and possibly NF-κB; with histone acetyl transferases, such as TIP60; and histone deacetyl transferase (HDAC) complexes, such as HDAC7 and HDAC9 (Wu et al., 2006; Sakaguchi et al., 2008).
T244	36286-36591	Sentence	denotes	Foxp3 protein interacts not only with RUNX proteins but also with several other transcriptional partners, such as NFAT and possibly NF-κB; with histone acetyl transferases, such as TIP60; and histone deacetyl transferase (HDAC) complexes, such as HDAC7 and HDAC9 (Wu et al., 2006; Sakaguchi et al., 2008).
T12908	36592-36830	Sentence	denotes	NFAT forms a complex with AP-1 and NF-κB and regulates the expression of IL-2, IL-4, IFN-γ, and CTLA4 in conventional T cells, which leads to the activation and differentiation to effector T cells (Dolganov et al., 1996; Hu et al., 2007).
T245	36592-36830	Sentence	denotes	NFAT forms a complex with AP-1 and NF-κB and regulates the expression of IL-2, IL-4, IFN-γ, and CTLA4 in conventional T cells, which leads to the activation and differentiation to effector T cells (Dolganov et al., 1996; Hu et al., 2007).
T12909	36831-36974	Sentence	denotes	The NFAT–AP-1 complex also binds to the Foxp3 promoter after TCR triggering and regulates its gene expression positively (Mantel et al., 2006).
T246	36831-36974	Sentence	denotes	The NFAT–AP-1 complex also binds to the Foxp3 promoter after TCR triggering and regulates its gene expression positively (Mantel et al., 2006).
T12910	36975-37180	Sentence	denotes	It was shown that NFAT and Smad3 cooperate to induce Foxp3 expression through its enhancer (Tone et al., 2008), but no TGF-β response element was identified in the Foxp3 gene or in the surrounding regions.
T247	36975-37180	Sentence	denotes	It was shown that NFAT and Smad3 cooperate to induce Foxp3 expression through its enhancer (Tone et al., 2008), but no TGF-β response element was identified in the Foxp3 gene or in the surrounding regions.
T12911	37181-37417	Sentence	denotes	The initial induction of RUNX1 and RUNX3 and the subsequent binding of these transcription factors to the Foxp3 promoter that we showed here might explain the relatively late induction of Foxp3 mRNA that peaks 24–48 h after stimulation.
T248	37181-37417	Sentence	denotes	The initial induction of RUNX1 and RUNX3 and the subsequent binding of these transcription factors to the Foxp3 promoter that we showed here might explain the relatively late induction of Foxp3 mRNA that peaks 24–48 h after stimulation.
T12912	37418-37519	Sentence	denotes	The interaction of Foxp3 and NFAT is dependent on their cooperative binding to DNA (Wu et al., 2006).
T249	37418-37519	Sentence	denotes	The interaction of Foxp3 and NFAT is dependent on their cooperative binding to DNA (Wu et al., 2006).
T12913	37520-37711	Sentence	denotes	RUNX1 alone, or together with its interacting partners p300 and CREB-binding protein, may cooperate with the NFAT transcription complex to activate the IL-2 promoter (Sakaguchi et al., 2008).
T250	37520-37711	Sentence	denotes	RUNX1 alone, or together with its interacting partners p300 and CREB-binding protein, may cooperate with the NFAT transcription complex to activate the IL-2 promoter (Sakaguchi et al., 2008).
T12914	37712-37867	Sentence	denotes	Similar to this interaction, NFAT may also cooperate with RUNX1 or RUNX3 to activate Foxp3, but further studies are necessary to elaborate on this concept.
T251	37712-37867	Sentence	denotes	Similar to this interaction, NFAT may also cooperate with RUNX1 or RUNX3 to activate Foxp3, but further studies are necessary to elaborate on this concept.
T12915	37868-37972	Sentence	denotes	In conclusion, our findings elucidate the role of RUNX proteins in iT reg cell development and function.
T252	37868-37972	Sentence	denotes	In conclusion, our findings elucidate the role of RUNX proteins in iT reg cell development and function.
T12916	37973-38150	Sentence	denotes	The induction of the transcription factors RUNX1 and RUNX3 by TGF-β and the subsequent up-regulation of Foxp3 play a role in iT reg cell generation and its suppressive capacity.
T253	37973-38150	Sentence	denotes	The induction of the transcription factors RUNX1 and RUNX3 by TGF-β and the subsequent up-regulation of Foxp3 play a role in iT reg cell generation and its suppressive capacity.
T254	38152-38173	Sentence	denotes	MATERIALS AND METHODS
T18671	38175-38180	Sentence	denotes	Mice.
T255	38175-38180	Sentence	denotes	Mice.
T18672	38181-38289	Sentence	denotes	CbfbF/F CD4-cre and Foxp3GFP mice have previously been described (Bettelli et al., 2006; Naoe et al., 2007).
T256	38181-38289	Sentence	denotes	CbfbF/F CD4-cre and Foxp3GFP mice have previously been described (Bettelli et al., 2006; Naoe et al., 2007).
T18673	38290-38396	Sentence	denotes	Cd45.1 and Rag2−/− mice were purchased from Jackson ImmunoResearch Laboratories and Taconic, respectively.
T257	38290-38396	Sentence	denotes	Cd45.1 and Rag2−/− mice were purchased from Jackson ImmunoResearch Laboratories and Taconic, respectively.
T18674	38397-38578	Sentence	denotes	For the in vivo Foxp3 conversion assay, naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice.
T258	38397-38578	Sentence	denotes	For the in vivo Foxp3 conversion assay, naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice.

T138

0-118

Sentence

denotes

Transcription factors RUNX1 and RUNX3 in the induction and suppressive function of Foxp3+ inducible regulatory T cells

T1

0-177

Sentence

denotes

Transcription factors RUNX1 and RUNX3 in the induction and suppressive function of Foxp3+ inducible regulatory T cells RUNX1 and RUNX3 in functional inducible T regulatory cells

T2

180-188

Sentence

denotes

Abstract

T139

189-320

Sentence

denotes

Forkhead box P3 (FOXP3)+CD4+CD25+ inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis.

T3

189-320

Sentence

denotes

Forkhead box P3 (FOXP3)+CD4+CD25+ inducible regulatory T (iT reg) cells play an important role in immune tolerance and homeostasis.

T140

321-487

Sentence

denotes

In this study, we show that the transforming growth factor-β (TGF-β) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4+ T cells.

T4

321-487

Sentence

denotes

In this study, we show that the transforming growth factor-β (TGF-β) induces the expression of the Runt-related transcription factors RUNX1 and RUNX3 in CD4+ T cells.

T141

488-624

Sentence

denotes

This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter.

T5

488-624

Sentence

denotes

This induction seems to be a prerequisite for the binding of RUNX1 and RUNX3 to three putative RUNX binding sites in the FOXP3 promoter.

T142

625-841

Sentence

denotes

Inactivation of the gene encoding RUNX cofactor core-binding factor-β (CBFβ) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3.

T6

625-841

Sentence

denotes

Inactivation of the gene encoding RUNX cofactor core-binding factor-β (CBFβ) in mice and small interfering RNA (siRNA)-mediated suppression of RUNX1 and RUNX3 in human T cells resulted in reduced expression of Foxp3.

T143

842-1014

Sentence

denotes

The in vivo conversion of naive CD4+ T cells into Foxp3+ iT reg cells was significantly decreased in adoptively transferred CbfbF/F CD4-cre naive T cells into Rag2−/− mice.

T7

842-1014

Sentence

denotes

The in vivo conversion of naive CD4+ T cells into Foxp3+ iT reg cells was significantly decreased in adoptively transferred CbfbF/F CD4-cre naive T cells into Rag2−/− mice.

T144

1015-1155

Sentence

denotes

Both RUNX1 and RUNX3 siRNA silenced human T reg cells and CbfbF/F CD4-cre mouse T reg cells showed diminished suppressive function in vitro.

T8

1015-1155

Sentence

denotes

Both RUNX1 and RUNX3 siRNA silenced human T reg cells and CbfbF/F CD4-cre mouse T reg cells showed diminished suppressive function in vitro.

T145

1156-1307

Sentence

denotes

Circulating human CD4+ CD25high CD127− T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-β mRNA compared with CD4+CD25− cells.

T9

1156-1307

Sentence

denotes

Circulating human CD4+ CD25high CD127− T reg cells significantly expressed higher levels of RUNX3, FOXP3, and TGF-β mRNA compared with CD4+CD25− cells.

T146

1308-1382

Sentence

denotes

Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells.

T10

1308-1382

Sentence

denotes

Furthermore, FOXP3 and RUNX3 were colocalized in human tonsil T reg cells.

T147

1383-1531

Sentence

denotes

These data demonstrate Runx transcription factors as a molecular link in TGF-β–induced Foxp3 expression in iT reg cell differentiation and function.

T11

1383-1531

Sentence

denotes

These data demonstrate Runx transcription factors as a molecular link in TGF-β–induced Foxp3 expression in iT reg cell differentiation and function.

T1277

1533-1821

Sentence

denotes

Regulatory T (T reg) cells expressing the transcription factor forkhead box P3 (FOXP3, human; Foxp3, mouse) play an essential role in controlling immune responses to autoantigens, allergens, tumor antigens, transplantation antigens, and infectious agents (Hori et al., 2003; Akdis, 2006).

T12

1533-1821

Sentence

denotes

Regulatory T (T reg) cells expressing the transcription factor forkhead box P3 (FOXP3, human; Foxp3, mouse) play an essential role in controlling immune responses to autoantigens, allergens, tumor antigens, transplantation antigens, and infectious agents (Hori et al., 2003; Akdis, 2006).

T1278

1822-2042

Sentence

denotes

Foxp3 is a member of the forkhead/winged-helix family of transcriptional regulators, and its expression in T reg cells is essential for their development and function (Fontenot et al., 2003; Williams and Rudensky, 2007).

T13

1822-2042

Sentence

denotes

Foxp3 is a member of the forkhead/winged-helix family of transcriptional regulators, and its expression in T reg cells is essential for their development and function (Fontenot et al., 2003; Williams and Rudensky, 2007).

T14

2043-2342

Sentence

denotes

A spontaneous mutation of the X-linked Foxp3 gene in scurfy mice causes an autoimmune-like disease, whereas the mutation in humans leads to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome that is also a severe multiorgan autoimmune disease with hyper-IgE (Ziegler, 2006).

T1279

2043-2625

Sentence

denotes

A spontaneous mutation of the X-linked Foxp3 gene in scurfy mice causes an autoimmune-like disease, whereas the mutation in humans leads to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome that is also a severe multiorgan autoimmune disease with hyper-IgE (Ziegler, 2006). Although the essential role of Foxp3 in central and peripheral tolerance has been extensively studied, its regulation, cooperation with other transcription factors, and how it functions in inducible T reg (iT reg) cells to suppress various target genes is mostly not yet understood.

T15

2343-2625

Sentence

denotes

Although the essential role of Foxp3 in central and peripheral tolerance has been extensively studied, its regulation, cooperation with other transcription factors, and how it functions in inducible T reg (iT reg) cells to suppress various target genes is mostly not yet understood.

T1280

2626-2866

Sentence

denotes

It is known that Foxp3 cooperates with the nuclear factor of activated T cells (NFAT) or nuclear factor-kappa B (NF-κB) to regulate the transcription of different target genes (Schubert et al., 2001; Bettelli et al., 2005; Wu et al., 2006).

T16

2626-2866

Sentence

denotes

It is known that Foxp3 cooperates with the nuclear factor of activated T cells (NFAT) or nuclear factor-kappa B (NF-κB) to regulate the transcription of different target genes (Schubert et al., 2001; Bettelli et al., 2005; Wu et al., 2006).

T1281

2867-2937

Sentence

denotes

The Th2 cytokine IL-4 inhibits FOXP3 expression during T cell priming.

T17

2867-2937

Sentence

denotes

The Th2 cytokine IL-4 inhibits FOXP3 expression during T cell priming.

T1282

2938-3067

Sentence

denotes

GATA3 binds to the FOXP3 promoter and can repress the FOXP3 trans-activation process directly in Th2 cells (Mantel et al., 2007).

T18

2938-3067

Sentence

denotes

GATA3 binds to the FOXP3 promoter and can repress the FOXP3 trans-activation process directly in Th2 cells (Mantel et al., 2007).

T1283

3068-3286

Sentence

denotes

It was further demonstrated that both Th1 and Th2 transcription factors T-bet and GATA3 oppose peripheral induction of Foxp3+ T reg cells in mice through STAT1-, STAT4-, and STAT6-dependent pathways (Wei et al., 2007).

T19

3068-3286

Sentence

denotes

It was further demonstrated that both Th1 and Th2 transcription factors T-bet and GATA3 oppose peripheral induction of Foxp3+ T reg cells in mice through STAT1-, STAT4-, and STAT6-dependent pathways (Wei et al., 2007).

T1284

3287-3610

Sentence

denotes

Although natural T reg (nT reg) cells that differentiate in the thymus are characterized by their stable Foxp3 expression, the generation of iT reg cells specific for allergens, alloantigens, and autoantigens in the periphery has been associated with a transient Foxp3+ phenotype (Fontenot et al., 2003; Hori et al., 2003).

T20

3287-3610

Sentence

denotes

Although natural T reg (nT reg) cells that differentiate in the thymus are characterized by their stable Foxp3 expression, the generation of iT reg cells specific for allergens, alloantigens, and autoantigens in the periphery has been associated with a transient Foxp3+ phenotype (Fontenot et al., 2003; Hori et al., 2003).

T21

3611-3679

Sentence

denotes

The crucial role of TGF-β in their generation has been demonstrated.

T1285

3611-3959

Sentence

denotes

The crucial role of TGF-β in their generation has been demonstrated. The RUNX gene family (Runt-related transcription factor, acute myeloid leukemia [AML], core-binding factor-α [CBFα], and polyoma enhancer-binding protein-2α [PEBP2α]) contains three members, RUNX1 (AML1/CBFA2/PEBP2αB), RUNX2 (AML3/CBFA1/ PEBP2αA), and RUNX3 (AML2/CBFA3/PEBP2αC).

T22

3680-3959

Sentence

denotes

The RUNX gene family (Runt-related transcription factor, acute myeloid leukemia [AML], core-binding factor-α [CBFα], and polyoma enhancer-binding protein-2α [PEBP2α]) contains three members, RUNX1 (AML1/CBFA2/PEBP2αB), RUNX2 (AML3/CBFA1/ PEBP2αA), and RUNX3 (AML2/CBFA3/PEBP2αC).

T1286

3960-4042

Sentence

denotes

They are essential transcriptional regulators of different developmental pathways.

T23

3960-4042

Sentence

denotes

They are essential transcriptional regulators of different developmental pathways.

T1287

4043-4143

Sentence

denotes

RUNX2 is mostly important for bone development and osteoblast differentiation (Komori et al., 1997).

T24

4043-4143

Sentence

denotes

RUNX2 is mostly important for bone development and osteoblast differentiation (Komori et al., 1997).

T1288

4144-4340

Sentence

denotes

RUNX1 plays an important role in hematopoiesis during development, and RUNX3 has important functions in thymogenesis and neurogenesis (Wang et al., 1996; Inoue et al., 2002; Levanon et al., 2002).

T25

4144-4340

Sentence

denotes

RUNX1 plays an important role in hematopoiesis during development, and RUNX3 has important functions in thymogenesis and neurogenesis (Wang et al., 1996; Inoue et al., 2002; Levanon et al., 2002).

T26

4341-4483

Sentence

denotes

RUNX1 and RUNX3 also work together in the establishment of lineage specification of T lymphocytes (Taniuchi et al., 2002; Egawa et al., 2007).

T1289

4341-4780

Sentence

denotes

RUNX1 and RUNX3 also work together in the establishment of lineage specification of T lymphocytes (Taniuchi et al., 2002; Egawa et al., 2007). RUNX1 is a frequent target for chromosomal translocations associated with leukemias (Look, 1997), and RUNX3 methylation and silencing is observed in various human epithelial cancers (Blyth et al., 2005). RUNX family members share the Runt domain, which is responsible for DNA binding (Ito, 1999).

T27

4484-4687

Sentence

denotes

RUNX1 is a frequent target for chromosomal translocations associated with leukemias (Look, 1997), and RUNX3 methylation and silencing is observed in various human epithelial cancers (Blyth et al., 2005).

T28

4688-4780

Sentence

denotes

RUNX family members share the Runt domain, which is responsible for DNA binding (Ito, 1999).

T1290

4781-4881

Sentence

denotes

The Runt domain-containing protein constitutes the α-chain partner of the heterodimeric CBF complex.

T29

4781-4881

Sentence

denotes

The Runt domain-containing protein constitutes the α-chain partner of the heterodimeric CBF complex.

T1291

4882-5089

Sentence

denotes

RUNX proteins heterodimerize with the non–DNA-binding partner, CBFβ, which increases the affinity for DNA binding and stabilizes the complex by preventing ubiquitin-dependent degradation (Wang et al., 1993).

T30

4882-5089

Sentence

denotes

RUNX proteins heterodimerize with the non–DNA-binding partner, CBFβ, which increases the affinity for DNA binding and stabilizes the complex by preventing ubiquitin-dependent degradation (Wang et al., 1993).

T1292

5090-5200

Sentence

denotes

The CBF complexes regulate the expression of cellular genes through binding to promoters or enhancer elements.

T31

5090-5200

Sentence

denotes

The CBF complexes regulate the expression of cellular genes through binding to promoters or enhancer elements.

T1293

5201-5293

Sentence

denotes

The effects of the RUNX–CBFβ complex regulation are clearly cell lineage and stage specific.

T32

5201-5293

Sentence

denotes

The effects of the RUNX–CBFβ complex regulation are clearly cell lineage and stage specific.

T33

5294-5458

Sentence

denotes

They include the crucial choices between cell-cycle exit and continued proliferation, as well as between cell differentiation and self-renewal (Blyth et al., 2005).

T1294

5294-5713

Sentence

denotes

They include the crucial choices between cell-cycle exit and continued proliferation, as well as between cell differentiation and self-renewal (Blyth et al., 2005). Because of the involvement of RUNX mutations in different autoimmune diseases and the known interaction with TGF-β, we investigated the impact of RUNX1 and RUNX3 on the expression of FOXP3 and subsequently on the development and function of iT reg cells.

T34

5459-5713

Sentence

denotes

Because of the involvement of RUNX mutations in different autoimmune diseases and the known interaction with TGF-β, we investigated the impact of RUNX1 and RUNX3 on the expression of FOXP3 and subsequently on the development and function of iT reg cells.

T1295

5714-5856

Sentence

denotes

This study demonstrates that RUNX1 and RUNX3 induced by TGF-β are involved in the development and suppressive function of Foxp3+ iT reg cells.

T35

5714-5856

Sentence

denotes

This study demonstrates that RUNX1 and RUNX3 induced by TGF-β are involved in the development and suppressive function of Foxp3+ iT reg cells.

T36

5858-5865

Sentence

denotes

RESULTS

T3603

5867-5957

Sentence

denotes

The role of RUNX1 and RUNX3 transcription factors in TGF-β–mediated iT reg cell generation

T37

5867-5957

Sentence

denotes

The role of RUNX1 and RUNX3 transcription factors in TGF-β–mediated iT reg cell generation

T3604

5958-6165

Sentence

denotes

To investigate the role of RUNX transcription factors in the development of iT reg cells, we cultured naive CD4+ T cells, isolated from human PBMCs, in conditions that enable the development of iT reg cells.

T38

5958-6165

Sentence

denotes

To investigate the role of RUNX transcription factors in the development of iT reg cells, we cultured naive CD4+ T cells, isolated from human PBMCs, in conditions that enable the development of iT reg cells.

T3605

6166-6288

Sentence

denotes

Stimulation with anti-CD2/3/28 mAbs or TGF-β alone resulted in a minimal up-regulation of RUNX1 and RUNX3 mRNA (Fig. 1 A).

T39

6166-6288

Sentence

denotes

Stimulation with anti-CD2/3/28 mAbs or TGF-β alone resulted in a minimal up-regulation of RUNX1 and RUNX3 mRNA (Fig. 1 A).

T3606

6289-6443

Sentence

denotes

In contrast, the combination of both TGF-β and anti-CD2/3/28 mAbs induced RUNX1 and RUNX3 mRNAs, as well as FOXP3 mRNA, within 48 h in naive CD4+ T cells.

T40

6289-6443

Sentence

denotes

In contrast, the combination of both TGF-β and anti-CD2/3/28 mAbs induced RUNX1 and RUNX3 mRNAs, as well as FOXP3 mRNA, within 48 h in naive CD4+ T cells.

T3607

6444-6649

Sentence

denotes

This result suggested further experiments to investigate whether the up-regulation of RUNX1 and RUNX3 might be a feature of iT reg cells during their development or even a prerequisite for their induction.

T41

6444-6649

Sentence

denotes

This result suggested further experiments to investigate whether the up-regulation of RUNX1 and RUNX3 might be a feature of iT reg cells during their development or even a prerequisite for their induction.

T42

6650-6659

Sentence

denotes

Figure 1.

T28593

6661-6892

Sentence

denotes

RUNX1 and RUNX3 are involved in the induction of Foxp3 in iT reg cells. (A) RUNX1, RUNX3, and FOXP3 mRNA induction in human naive CD4+ T cells after alone or combined anti-CD2/3/28 mAb and TGF-β stimulation in the presence of IL-2.

T43

6661-6892

Sentence

denotes

RUNX1 and RUNX3 are involved in the induction of Foxp3 in iT reg cells. (A) RUNX1, RUNX3, and FOXP3 mRNA induction in human naive CD4+ T cells after alone or combined anti-CD2/3/28 mAb and TGF-β stimulation in the presence of IL-2.

T28594

6893-6957

Sentence

denotes

Real-time PCR of human naive CD4+ T cells after 48 h of culture.

T44

6893-6957

Sentence

denotes

Real-time PCR of human naive CD4+ T cells after 48 h of culture.

T28595

6958-7150

Sentence

denotes

Bars show the mean ± SE of three independent experiments. (B) FOXP3 mRNA induction by anti-CD2/3/28 mAb and TGF-β in human naive CD4+ T cells is reduced after siRNA-mediated RUNX1/3 knockdown.

T45

6958-7150

Sentence

denotes

Bars show the mean ± SE of three independent experiments. (B) FOXP3 mRNA induction by anti-CD2/3/28 mAb and TGF-β in human naive CD4+ T cells is reduced after siRNA-mediated RUNX1/3 knockdown.

T28596

7151-7317

Sentence

denotes

Real-time PCR of RNA from human naive CD4+ T cells, transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and cultured with anti-CD2/3/28, TGF-β and IL-2.

T46

7151-7317

Sentence

denotes

Real-time PCR of RNA from human naive CD4+ T cells, transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and cultured with anti-CD2/3/28, TGF-β and IL-2.

T28597

7318-7475

Sentence

denotes

Bars show the mean ± SD of three independent experiments. (C) FOXP3 mRNA is down-regulated in iT reg cells after siRNA-mediated knockdown of RUNX1 and RUNX3.

T47

7318-7475

Sentence

denotes

Bars show the mean ± SD of three independent experiments. (C) FOXP3 mRNA is down-regulated in iT reg cells after siRNA-mediated knockdown of RUNX1 and RUNX3.

T28598

7476-7702

Sentence

denotes

Real-time PCR for FOXP3, T-bet, GATA3, and RORC2 from human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA or with scrambled siRNA (control) and cultured under iT reg, Th1, Th2, or Th17-driving conditions for 12 d.

T48

7476-7702

Sentence

denotes

Real-time PCR for FOXP3, T-bet, GATA3, and RORC2 from human naive CD4+ T cells, transfected with RUNX1 and RUNX3 siRNA or with scrambled siRNA (control) and cultured under iT reg, Th1, Th2, or Th17-driving conditions for 12 d.

T28599

7703-7855

Sentence

denotes

Bars show the mean ± SD of three independent experiments. (D) FOXP3 protein induction in iT reg cells is reduced after siRNA-mediated RUNX1/3 knockdown.

T49

7703-7855

Sentence

denotes

Bars show the mean ± SD of three independent experiments. (D) FOXP3 protein induction in iT reg cells is reduced after siRNA-mediated RUNX1/3 knockdown.

T28600

7856-8020

Sentence

denotes

Human naive CD4+ T cells were transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and stimulated with anti-CD2/3/28 and TGF-β in the presence of IL-2.

T50

7856-8020

Sentence

denotes

Human naive CD4+ T cells were transfected with RUNX1 and/or RUNX3 siRNA or with a control siRNA and stimulated with anti-CD2/3/28 and TGF-β in the presence of IL-2.

T28601

8021-8087

Sentence

denotes

CD4 and intracellular FOXP3 analysis by flow cytometry after 72 h.

T51

8021-8087

Sentence

denotes

CD4 and intracellular FOXP3 analysis by flow cytometry after 72 h.

T28602

8088-8134

Sentence

denotes

One of three independent experiments is shown.

T52

8088-8134

Sentence

denotes

One of three independent experiments is shown.

T28603

8135-8231

Sentence

denotes

Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01.

T53

8135-8231

Sentence

denotes

Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01.

T3608

8234-8392

Sentence

denotes

To test this hypothesis, RUNX1 and RUNX3 expression was knocked down in human naive CD4+ T cells by transfection of small interfering RNAs (siRNAs; Fig. 1 B).

T54

8234-8392

Sentence

denotes

To test this hypothesis, RUNX1 and RUNX3 expression was knocked down in human naive CD4+ T cells by transfection of small interfering RNAs (siRNAs; Fig. 1 B).

T3609

8393-8573

Sentence

denotes

Deficiency of RUNX1 or RUNX3 resulted in markedly reduced TGF-β–mediated induction of FOXP3 mRNA in naive CD4+ T cells compared with control cells transfected with scrambled siRNA.

T55

8393-8573

Sentence

denotes

Deficiency of RUNX1 or RUNX3 resulted in markedly reduced TGF-β–mediated induction of FOXP3 mRNA in naive CD4+ T cells compared with control cells transfected with scrambled siRNA.

T3610

8574-8740

Sentence

denotes

The level of FOXP3 mRNA was further reduced when both RUNX1 and RUNX3 were knocked down in naive CD4+ T cells during their differentiation to iT reg cells (Fig. 1 B).

T56

8574-8740

Sentence

denotes

The level of FOXP3 mRNA was further reduced when both RUNX1 and RUNX3 were knocked down in naive CD4+ T cells during their differentiation to iT reg cells (Fig. 1 B).

T3611

8741-8893

Sentence

denotes

The influence of RUNX1 and RUNX3 on the development of other T cell subsets and their specific transcription factor expression was further investigated.

T57

8741-8893

Sentence

denotes

The influence of RUNX1 and RUNX3 on the development of other T cell subsets and their specific transcription factor expression was further investigated.

T3612

8894-9100

Sentence

denotes

Naive CD4+ T cells were cultured under Th1, Th2, T reg cell, and Th17 differentiation conditions and the mRNA expression of the predominant transcription factor for each cell type was subsequently analyzed.

T58

8894-9100

Sentence

denotes

Naive CD4+ T cells were cultured under Th1, Th2, T reg cell, and Th17 differentiation conditions and the mRNA expression of the predominant transcription factor for each cell type was subsequently analyzed.

T3613

9101-9298

Sentence

denotes

We observed no change in GATA3 expression in Th2 cells, T-bet expression in Th1 cells, or RORC2 mRNA expression in Th17 cells in which RUNX1 and RUNX3 were knocked down compared with control cells.

T59

9101-9298

Sentence

denotes

We observed no change in GATA3 expression in Th2 cells, T-bet expression in Th1 cells, or RORC2 mRNA expression in Th17 cells in which RUNX1 and RUNX3 were knocked down compared with control cells.

T3614

9299-9436

Sentence

denotes

On the contrary, FOXP3 mRNA was significantly decreased in RUNX1- and RUNX3-deficient T reg cells compared with control cells (Fig. 1 C).

T60

9299-9436

Sentence

denotes

On the contrary, FOXP3 mRNA was significantly decreased in RUNX1- and RUNX3-deficient T reg cells compared with control cells (Fig. 1 C).

T3615

9437-9596

Sentence

denotes

The effect of RUNX silencing on the expression level of intracellular FOXP3 during naive CD4+ T cell differentiation to iT reg was evaluated by flow cytometry.

T61

9437-9596

Sentence

denotes

The effect of RUNX silencing on the expression level of intracellular FOXP3 during naive CD4+ T cell differentiation to iT reg was evaluated by flow cytometry.

T3616

9597-9651

Sentence

denotes

FOXP3 was only slightly reduced after RUNX1 silencing.

T62

9597-9651

Sentence

denotes

FOXP3 was only slightly reduced after RUNX1 silencing.

T3617

9652-9706

Sentence

denotes

Transfection of siRNA for RUNX3 had a stronger effect.

T63

9652-9706

Sentence

denotes

Transfection of siRNA for RUNX3 had a stronger effect.

T3618

9707-9809

Sentence

denotes

The most striking FOXP3 reduction was observed when RUNX1 and RUNX3 were silenced together (Fig. 1 D).

T64

9707-9809

Sentence

denotes

The most striking FOXP3 reduction was observed when RUNX1 and RUNX3 were silenced together (Fig. 1 D).

T3619

9810-9884

Sentence

denotes

Similar results were obtained in total CD4+ T cells (Fig. S1 and Fig. S2).

T65

9810-9884

Sentence

denotes

Similar results were obtained in total CD4+ T cells (Fig. S1 and Fig. S2).

T3620

9885-10030

Sentence

denotes

The increased impact of combined RUNX1 and RUNX3 knockdown implies that RUNX1 and RUNX3 might have redundant functions in the induction of FOXP3.

T66

9885-10030

Sentence

denotes

The increased impact of combined RUNX1 and RUNX3 knockdown implies that RUNX1 and RUNX3 might have redundant functions in the induction of FOXP3.

T3621

10031-10286

Sentence

denotes

In addition, the levels of IL-4, IL-5, IL-10, IL-13, and IFN-γ in control siRNA-transfected or RUNX1 and RUNX3 siRNA-transfected CD4+ T cells that were cultured with or without anti-CD2/3/28 mAb and TGF-β did not show any significant difference (Fig. S3).

T67

10031-10286

Sentence

denotes

In addition, the levels of IL-4, IL-5, IL-10, IL-13, and IFN-γ in control siRNA-transfected or RUNX1 and RUNX3 siRNA-transfected CD4+ T cells that were cultured with or without anti-CD2/3/28 mAb and TGF-β did not show any significant difference (Fig. S3).

T3622

10287-10484

Sentence

denotes

To determine whether RUNX1 and RUNX3 are also expressed in human T reg cells in vivo, we isolated peripheral blood CD4+ CD127− CD25high T reg cells and compared them with CD4+ CD127+ CD25− T cells.

T68

10287-10484

Sentence

denotes

To determine whether RUNX1 and RUNX3 are also expressed in human T reg cells in vivo, we isolated peripheral blood CD4+ CD127− CD25high T reg cells and compared them with CD4+ CD127+ CD25− T cells.

T3623

10485-10591

Sentence

denotes

Circulating T reg cells expressed significantly higher levels of RUNX3 mRNA compared with CD4+CD25− cells.

T69

10485-10591

Sentence

denotes

Circulating T reg cells expressed significantly higher levels of RUNX3 mRNA compared with CD4+CD25− cells.

T3624

10592-10692

Sentence

denotes

As expected, IL-10, TGF-β, and FOXP3 mRNAs are also expressed in circulating T reg cells (Fig. 2 A).

T70

10592-10692

Sentence

denotes

As expected, IL-10, TGF-β, and FOXP3 mRNAs are also expressed in circulating T reg cells (Fig. 2 A).

T3625

10693-10773

Sentence

denotes

There was no difference in RUNX1 mRNA expression between these two cell subsets.

T71

10693-10773

Sentence

denotes

There was no difference in RUNX1 mRNA expression between these two cell subsets.

T3626

10774-10895

Sentence

denotes

We also performed an analysis of human tonsils, which contain high numbers of FOXP3+ T reg cells (Verhagen et al., 2006).

T72

10774-10895

Sentence

denotes

We also performed an analysis of human tonsils, which contain high numbers of FOXP3+ T reg cells (Verhagen et al., 2006).

T3627

10896-11099

Sentence

denotes

Staining of tonsil sections for FOXP3 and RUNX3 demonstrated in vivo coexpression of these two molecules in a subset of T reg cells, whereas there was low RUNX1 expression in all tonsil cells (Fig. 2 B).

T73

10896-11099

Sentence

denotes

Staining of tonsil sections for FOXP3 and RUNX3 demonstrated in vivo coexpression of these two molecules in a subset of T reg cells, whereas there was low RUNX1 expression in all tonsil cells (Fig. 2 B).

T74

11100-11109

Sentence

denotes

Figure 2.

T29703

11111-11452

Sentence

denotes

RUNX1 and RUNX3 expression in Foxp3+ T reg cells and in human CD4+, CD127−, CD25high cells. (A) Real-time PCR analysis of CD4+, CD127−, and CD25high T reg cells and CD4+, CD127+, and CD25neg T cells isolated from human peripheral blood showed an increased expression of IL-10, TGF-β, FOXP3, and RUNX3 mRNA in CD25+ compared with CD25− cells.

T75

11111-11452

Sentence

denotes

RUNX1 and RUNX3 expression in Foxp3+ T reg cells and in human CD4+, CD127−, CD25high cells. (A) Real-time PCR analysis of CD4+, CD127−, and CD25high T reg cells and CD4+, CD127+, and CD25neg T cells isolated from human peripheral blood showed an increased expression of IL-10, TGF-β, FOXP3, and RUNX3 mRNA in CD25+ compared with CD25− cells.

T29704

11453-11574

Sentence

denotes

Bars show the mean ± SE of three independent experiments. (B) Human tonsil sections were analyzed by confocal microscopy.

T76

11453-11574

Sentence

denotes

Bars show the mean ± SE of three independent experiments. (B) Human tonsil sections were analyzed by confocal microscopy.

T29705

11575-11658

Sentence

denotes

Tissue sections were stained for FOXP3, RUNX1, RUNX3, and DAPI or isotype controls.

T77

11575-11658

Sentence

denotes

Tissue sections were stained for FOXP3, RUNX1, RUNX3, and DAPI or isotype controls.

T29706

11659-11754

Sentence

denotes

HEK cells RUNX1-transfected or not transfected served as additional control for RUNX1 staining.

T78

11659-11754

Sentence

denotes

HEK cells RUNX1-transfected or not transfected served as additional control for RUNX1 staining.

T29707

11755-11843

Sentence

denotes

Data shown are representative from one of the three tissue samples with similar results.

T79

11755-11843

Sentence

denotes

Data shown are representative from one of the three tissue samples with similar results.

T29708

11844-11855

Sentence

denotes

Bars, 5 µm.

T80

11844-11855

Sentence

denotes

Bars, 5 µm.

T29709

11856-11952

Sentence

denotes

Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01.

T81

11856-11952

Sentence

denotes

Statistical differences were verified by the paired Student's t test. *, P < 0.05; **, P < 0.01.

T6067

11954-11996

Sentence

denotes

RUNX1 and RUNX3 bind to the FOXP3 promoter

T82

11954-11996

Sentence

denotes

RUNX1 and RUNX3 bind to the FOXP3 promoter

T6068

11997-12180

Sentence

denotes

Transcription element search system analysis of the human FOXP3 promoter predicted 3 putative RUNX binding sites at 333, 287, and 53 bp upstream of the transcription start site (TSS).

T83

11997-12180

Sentence

denotes

Transcription element search system analysis of the human FOXP3 promoter predicted 3 putative RUNX binding sites at 333, 287, and 53 bp upstream of the transcription start site (TSS).

T6069

12181-12259

Sentence

denotes

All three binding sites are conserved between human, mouse, and rat (Fig. S4).

T84

12181-12259

Sentence

denotes

All three binding sites are conserved between human, mouse, and rat (Fig. S4).

T6070

12260-12382

Sentence

denotes

To verify the putative binding sites in the FOXP3 promoter, we transiently transfected HEK293T cells with RUNX1 and RUNX3.

T85

12260-12382

Sentence

denotes

To verify the putative binding sites in the FOXP3 promoter, we transiently transfected HEK293T cells with RUNX1 and RUNX3.

T6071

12383-12587

Sentence

denotes

After the pull-down with oligonucleotides containing the wild-type binding sequences, but not mutant sequences, RUNX binding to the FOXP3 promoter oligonucleotides was detected by Western blot (Fig. 3 A).

T86

12383-12587

Sentence

denotes

After the pull-down with oligonucleotides containing the wild-type binding sequences, but not mutant sequences, RUNX binding to the FOXP3 promoter oligonucleotides was detected by Western blot (Fig. 3 A).

T6072

12588-12738

Sentence

denotes

To confirm these results and test the ability of single binding site sequences to bind either RUNX1 or RUNX3, we used the promoter enzyme immunoassay.

T87

12588-12738

Sentence

denotes

To confirm these results and test the ability of single binding site sequences to bind either RUNX1 or RUNX3, we used the promoter enzyme immunoassay.

T6073

12739-12862

Sentence

denotes

Cell lysates were obtained from HEK293T cells that had been transiently transfected with RUNX1 or RUNX3 expression vectors.

T88

12739-12862

Sentence

denotes

Cell lysates were obtained from HEK293T cells that had been transiently transfected with RUNX1 or RUNX3 expression vectors.

T6074

12863-13079

Sentence

denotes

The biotinylated FOXP3 promoter oligonucleotides were linked to a streptavidin-coated microtiter plate, and bound RUNX1 or RUNX3 was detected by using anti-RUNX antibodies and a peroxidase-labeled secondary antibody.

T89

12863-13079

Sentence

denotes

The biotinylated FOXP3 promoter oligonucleotides were linked to a streptavidin-coated microtiter plate, and bound RUNX1 or RUNX3 was detected by using anti-RUNX antibodies and a peroxidase-labeled secondary antibody.

T6075

13080-13316

Sentence

denotes

We showed binding of RUNX1 and RUNX3 to the mixture of all three oligonucleotides containing the binding sites, whereas there was no binding detectable when a combination of the mutated oligonucleotides was used in the assay (Fig. 3 B).

T90

13080-13316

Sentence

denotes

We showed binding of RUNX1 and RUNX3 to the mixture of all three oligonucleotides containing the binding sites, whereas there was no binding detectable when a combination of the mutated oligonucleotides was used in the assay (Fig. 3 B).

T6076

13317-13520

Sentence

denotes

Although there was a similar and high degree of binding to the −333 and −287 sites, a lower degree of binding was detected when the oligonucleotide containing the −53 site in the Foxp3 promoter was used.

T91

13317-13520

Sentence

denotes

Although there was a similar and high degree of binding to the −333 and −287 sites, a lower degree of binding was detected when the oligonucleotide containing the −53 site in the Foxp3 promoter was used.

T6077

13521-13593

Sentence

denotes

This effect was observed both for binding to RUNX1 and RUNX3 (Fig. 3 B).

T92

13521-13593

Sentence

denotes

This effect was observed both for binding to RUNX1 and RUNX3 (Fig. 3 B).

T6078

13594-13715

Sentence

denotes

The binding to the two single binding sites at −333 and −287 was comparable to the mixture of all three oligonucleotides.

T93

13594-13715

Sentence

denotes

The binding to the two single binding sites at −333 and −287 was comparable to the mixture of all three oligonucleotides.

T6079

13716-13915

Sentence

denotes

Chromatin immunoprecipitation (ChIP) assay results confirmed the binding of RUNX1 and RUNX3 complexes containing CBFβ to FOXP3 promoter during the differentiation of naive T cells toward T reg cells.

T94

13716-13915

Sentence

denotes

Chromatin immunoprecipitation (ChIP) assay results confirmed the binding of RUNX1 and RUNX3 complexes containing CBFβ to FOXP3 promoter during the differentiation of naive T cells toward T reg cells.

T6080

13916-14027

Sentence

denotes

Here, naive CD4+ T cells were cultured with IL-2, anti-CD2/3/28 mAb, and TGF-β as a Foxp3-inducing stimulation.

T95

13916-14027

Sentence

denotes

Here, naive CD4+ T cells were cultured with IL-2, anti-CD2/3/28 mAb, and TGF-β as a Foxp3-inducing stimulation.

T6081

14028-14230

Sentence

denotes

Amplification of PCR products from the FOXP3 promoter region with the predicted RUNX binding sites showed that RUNX1, RUNX3, and CBFβ were immunoprecipitated together with the FOXP3 promoter (Fig. 3 C).

T96

14028-14230

Sentence

denotes

Amplification of PCR products from the FOXP3 promoter region with the predicted RUNX binding sites showed that RUNX1, RUNX3, and CBFβ were immunoprecipitated together with the FOXP3 promoter (Fig. 3 C).

T6082

14231-14365

Sentence

denotes

Negative control primer targeting open reading frame-free intergenic DNA, IGX1A did not show any significant change in site occupancy.

T97

14231-14365

Sentence

denotes

Negative control primer targeting open reading frame-free intergenic DNA, IGX1A did not show any significant change in site occupancy.

T98

14366-14375

Sentence

denotes

Figure 3.

T30287

14377-14520

Sentence

denotes

Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown.

T99

14377-14520

Sentence

denotes

Binding of RUNX1 and RUNX3 proteins to the predicted binding sites in the FOXP3 promoter. (A) Mutated and wild-type oligonucleotides are shown.

T30288

14521-14689

Sentence

denotes

The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides.

T100

14521-14689

Sentence

denotes

The predicted RUNX binding sites are accentuated (boxed and in green letters) and stars mark mutations introduced into the binding site of the control oligonucleotides.

T30289

14690-14776

Sentence

denotes

Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides.

T101

14690-14776

Sentence

denotes

Nuclear extracts from HEK293T cells were incubated with biotinylated oligonucleotides.

T30290

14777-14945

Sentence

denotes

The precipitated oligonucleotide–transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies.

T102

14777-14945

Sentence

denotes

The precipitated oligonucleotide–transcription factor complexes were separated by SDS-PAGE and identified by Western blotting with anti-RUNX1 and anti-RUNX3 antibodies.

T30291

14946-15083

Sentence

denotes

A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used.

T103

14946-15083

Sentence

denotes

A mixture of all three oligonucleotides with the predicted binding sites or with the inserted mutation into the predicted sites was used.

T30292

15084-15276

Sentence

denotes

Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter.

T104

15084-15276

Sentence

denotes

Data shown are one representative of three independent experiments with similar results. (B) Promoter enzyme immunoassay using wild-type and mutated oligonucleotides within the FOXP3 promoter.

T30293

15277-15554

Sentence

denotes

Bars show mean ± SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFβ to the human FOXP3 promoter in naive CD4+ T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-β.

T105

15277-15554

Sentence

denotes

Bars show mean ± SE of three independent experiments. (C) Chromatin immunoprecipitation assay results show binding of RUNX1 and RUNX3 complexes containing CBFβ to the human FOXP3 promoter in naive CD4+ T cells that were cultured with IL-2 together with anti-CD2/3/28 and TGF-β.

T30294

15555-15667

Sentence

denotes

There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used.

T106

15555-15667

Sentence

denotes

There was no change in site occupancy in all immunoprecipitations when IGX1A negative control primers were used.

T30295

15668-15733

Sentence

denotes

The results are normalized to input and isotype control antibody.

T107

15668-15733

Sentence

denotes

The results are normalized to input and isotype control antibody.

T30296

15734-15787

Sentence

denotes

Bars show mean ± SE of three independent experiments.

T108

15734-15787

Sentence

denotes

Bars show mean ± SE of three independent experiments.

T30297

15788-15869

Sentence

denotes

Statistical differences were verified by the paired Student's t test. *, P < 0.05

T109

15788-15869

Sentence

denotes

Statistical differences were verified by the paired Student's t test. *, P < 0.05

T7554

15871-15956

Sentence

denotes

Regulation of FOXP3 promoter activity and FOXP3 protein expression by RUNX1 and RUNX3

T110

15871-15956

Sentence

denotes

Regulation of FOXP3 promoter activity and FOXP3 protein expression by RUNX1 and RUNX3

T7555

15957-16197

Sentence

denotes

To investigate the effect of RUNX1 and RUNX3 binding to the RUNX binding sites in the FOXP3 promoter, we transfected human peripheral blood CD4+ T cells with a FOXP3 promoter luciferase reporter vector and RUNX1 or RUNX3 expression vectors.

T111

15957-16197

Sentence

denotes

To investigate the effect of RUNX1 and RUNX3 binding to the RUNX binding sites in the FOXP3 promoter, we transfected human peripheral blood CD4+ T cells with a FOXP3 promoter luciferase reporter vector and RUNX1 or RUNX3 expression vectors.

T7556

16198-16379

Sentence

denotes

An increase in luciferase activity was observed only when the FOXP3 promoter (−511 to +176) luciferase construct was cotransfected with RUNX1 or RUNX3 expression vectors (Fig. 4 A).

T112

16198-16379

Sentence

denotes

An increase in luciferase activity was observed only when the FOXP3 promoter (−511 to +176) luciferase construct was cotransfected with RUNX1 or RUNX3 expression vectors (Fig. 4 A).

T7557

16380-16475

Sentence

denotes

The increase in promoter activity was greater upon cotransfection of RUNX3 compared with RUNX1.

T113

16380-16475

Sentence

denotes

The increase in promoter activity was greater upon cotransfection of RUNX3 compared with RUNX1.

T7558

16476-16622

Sentence

denotes

Luciferase expression was abrogated when the Runx binding sites in the FOXP3 promoter (−511 to +176) luciferase construct were mutated (Fig. 4 A).

T114

16476-16622

Sentence

denotes

Luciferase expression was abrogated when the Runx binding sites in the FOXP3 promoter (−511 to +176) luciferase construct were mutated (Fig. 4 A).

T7559

16623-16787

Sentence

denotes

In these experiments, the overexpression of RUNX1 and RUNX3 eliminated the need of TGF-β for FOXP3 promoter activation and PMA/ionomycin stimulation was sufficient.

T115

16623-16787

Sentence

denotes

In these experiments, the overexpression of RUNX1 and RUNX3 eliminated the need of TGF-β for FOXP3 promoter activation and PMA/ionomycin stimulation was sufficient.

T116

16788-16797

Sentence

denotes

Figure 4.

T30975

16799-17116

Sentence

denotes

Regulation of FOXP3 promoter activity by RUNX1 and RUNX3. (A) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the wild-type or mutated FOXP3 promoter region (FOXP3 −511/+176) fused to the luciferase reporter gene together with a GFP, RUNX1, or RUNX3 expression vector.

T117

16799-17116

Sentence

denotes

Regulation of FOXP3 promoter activity by RUNX1 and RUNX3. (A) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the wild-type or mutated FOXP3 promoter region (FOXP3 −511/+176) fused to the luciferase reporter gene together with a GFP, RUNX1, or RUNX3 expression vector.

T30976

17117-17683

Sentence

denotes

Bars show the mean luciferase activity ± SE measured as arbitrary light units of three independent experiments. (B) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the putative FOXP3 promoter region (FOXP3 −511/+176) fused to the luciferase reporter gene, or with a vector containing the putative FOXP3 promoter region (FOXP3 −511/+176) with single RUNX binding sites mutated (53, 287, or 333) or with the combination of two or three RUNX binding sites mutated (53, 287, or 333) fused to the luciferase reporter gene.

T118

17117-17683

Sentence

denotes

Bars show the mean luciferase activity ± SE measured as arbitrary light units of three independent experiments. (B) Human primary CD4+ cells were transfected with an empty vector (pGL3 Basic), a vector containing the putative FOXP3 promoter region (FOXP3 −511/+176) fused to the luciferase reporter gene, or with a vector containing the putative FOXP3 promoter region (FOXP3 −511/+176) with single RUNX binding sites mutated (53, 287, or 333) or with the combination of two or three RUNX binding sites mutated (53, 287, or 333) fused to the luciferase reporter gene.

T30977

17684-17795

Sentence

denotes

Bars show the mean luciferase activity ± SD measured as arbitrary light units of three independent experiments.

T119

17684-17795

Sentence

denotes

Bars show the mean luciferase activity ± SD measured as arbitrary light units of three independent experiments.

T7560

17798-17938

Sentence

denotes

To examine the role of each of the three RUNX binding sites for the FOXP3 promoter activity, we mutated each individually or in combination.

T120

17798-17938

Sentence

denotes

To examine the role of each of the three RUNX binding sites for the FOXP3 promoter activity, we mutated each individually or in combination.

T7561

17939-18103

Sentence

denotes

No reduction in luciferase activity was observed when the −53 site was mutated and only a slight reduction when either the −287 or −333 site was mutated (Fig. 4 B).

T121

17939-18103

Sentence

denotes

No reduction in luciferase activity was observed when the −53 site was mutated and only a slight reduction when either the −287 or −333 site was mutated (Fig. 4 B).

T7562

18104-18495

Sentence

denotes

However, mutating the −53 site in combination with one of the other two sites led to a significant decrease in luciferase activity, with the greatest reduction observed when all three binding sites were mutated (Fig. 4 B), suggesting that the identified binding sites have redundant functions and RUNX binding to more than one site is necessary for the full activation of the FOXP3 promoter.

T122

18104-18495

Sentence

denotes

However, mutating the −53 site in combination with one of the other two sites led to a significant decrease in luciferase activity, with the greatest reduction observed when all three binding sites were mutated (Fig. 4 B), suggesting that the identified binding sites have redundant functions and RUNX binding to more than one site is necessary for the full activation of the FOXP3 promoter.

T7563

18496-18678

Sentence

denotes

Supporting these findings, the overexpression of RUNX1 in human primary CD4+ T cells resulted in significantly elevated levels of FOXP3 protein measured by flow cytometry after 48 h.

T123

18496-18678

Sentence

denotes

Supporting these findings, the overexpression of RUNX1 in human primary CD4+ T cells resulted in significantly elevated levels of FOXP3 protein measured by flow cytometry after 48 h.

T7564

18679-18783

Sentence

denotes

This was achieved without any requirement for anti-CD3, anti-CD28 stimulation, or the presence of TGF-β.

T124

18679-18783

Sentence

denotes

This was achieved without any requirement for anti-CD3, anti-CD28 stimulation, or the presence of TGF-β.

T7565

18784-18926

Sentence

denotes

Although there was a trend, the transfection of CD4+ T cells with RUNX3 did not lead to statistically significant increase in FOXP3 (Fig. S5).

T125

18784-18926

Sentence

denotes

Although there was a trend, the transfection of CD4+ T cells with RUNX3 did not lead to statistically significant increase in FOXP3 (Fig. S5).

T9034

18928-18966

Sentence

denotes

Role of CBFβ in the induction of Foxp3

T126

18928-18966

Sentence

denotes

Role of CBFβ in the induction of Foxp3

T9035

18967-19093

Sentence

denotes

CBFβ, a common cofactor of all RUNX proteins, stabilizes and increases the binding of the runt domain to target DNA sequences.

T127

18967-19093

Sentence

denotes

CBFβ, a common cofactor of all RUNX proteins, stabilizes and increases the binding of the runt domain to target DNA sequences.

T9036

19094-19290

Sentence

denotes

To target all Runx proteins that might be involved in the induction of Foxp3, we used mice in which loxP-flanked Cbfb alleles were inactivated in T cells through expression of a CD4-cre transgene.

T128

19094-19290

Sentence

denotes

To target all Runx proteins that might be involved in the induction of Foxp3, we used mice in which loxP-flanked Cbfb alleles were inactivated in T cells through expression of a CD4-cre transgene.

T9037

19291-19388

Sentence

denotes

Retinoic acid and TGF-β synergize in the induction of Foxp3 in naive T cells (Kang et al., 2007).

T129

19291-19388

Sentence

denotes

Retinoic acid and TGF-β synergize in the induction of Foxp3 in naive T cells (Kang et al., 2007).

T9038

19389-19656

Sentence

denotes

To investigate whether Runx-mediated induction of Foxp3 is dependent on the expression of CBFβ, naive CD4+ CD8− T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were stimulated with anti-CD3/28 mAbs, retinoic acid, and increasing concentrations of TGF-β.

T130

19389-19656

Sentence

denotes

To investigate whether Runx-mediated induction of Foxp3 is dependent on the expression of CBFβ, naive CD4+ CD8− T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were stimulated with anti-CD3/28 mAbs, retinoic acid, and increasing concentrations of TGF-β.

T9039

19657-19788

Sentence

denotes

After 3 d in culture, the cells were restimulated with PMA and ionomycin and analyzed for intracellular Foxp3 and IFN-γ expression.

T131

19657-19788

Sentence

denotes

After 3 d in culture, the cells were restimulated with PMA and ionomycin and analyzed for intracellular Foxp3 and IFN-γ expression.

T9040

19789-19922

Sentence

denotes

TGF-β induced Foxp3 in CbfbF/+ CD4-cre cells in a dose dependent manner, and this was significantly reduced in CbfbF/F CD4-cre cells.

T132

19789-19922

Sentence

denotes

TGF-β induced Foxp3 in CbfbF/+ CD4-cre cells in a dose dependent manner, and this was significantly reduced in CbfbF/F CD4-cre cells.

T9041

19923-20100

Sentence

denotes

Retinoic acid enhanced Foxp3 expression even in 20 pg/ml of TGF-β and more than 95% of the CD4+ T cells from CbfbF/+ CD4-cre mice became Foxp3+ in 100 and 500 pg/ml TGF-β doses.

T133

19923-20100

Sentence

denotes

Retinoic acid enhanced Foxp3 expression even in 20 pg/ml of TGF-β and more than 95% of the CD4+ T cells from CbfbF/+ CD4-cre mice became Foxp3+ in 100 and 500 pg/ml TGF-β doses.

T9042

20101-20341

Sentence

denotes

The induction of Foxp3 was again significantly lower in CbfbF/F CD4-cre CD4+ T cells even in the presence of retinoic acid, demonstrating that deficiency in Runx binding to DNA affects the TGF-β induction of Foxp3 in T reg cells (Fig. 5 A).

T134

20101-20341

Sentence

denotes

The induction of Foxp3 was again significantly lower in CbfbF/F CD4-cre CD4+ T cells even in the presence of retinoic acid, demonstrating that deficiency in Runx binding to DNA affects the TGF-β induction of Foxp3 in T reg cells (Fig. 5 A).

T9043

20342-20450

Sentence

denotes

There was no difference in the induction of Foxp3 when endogenous IL-4 and IFN-γ were neutralized (Fig. S6).

T135

20342-20450

Sentence

denotes

There was no difference in the induction of Foxp3 when endogenous IL-4 and IFN-γ were neutralized (Fig. S6).

T136

20451-20460

Sentence

denotes

Figure 5.

T31435

20462-20786

Sentence

denotes

Diminished capacity of Cbfb-deficient CD4-cre mice T cells in the generation of Foxp3+ CD4+ T cells. (A) FACS-purified naive CD4+ CD8− T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, ± 10 nM retinoic acid (RA), and increasing concentrations of TGF-β.

T137

20462-20786

Sentence

denotes

Diminished capacity of Cbfb-deficient CD4-cre mice T cells in the generation of Foxp3+ CD4+ T cells. (A) FACS-purified naive CD4+ CD8− T cells from CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice were activated in vitro with anti-CD3/28 mAb, 50 U/ml IL-2, ± 10 nM retinoic acid (RA), and increasing concentrations of TGF-β.

T31436

20787-20922

Sentence

denotes

After 3 d in culture, the cells were restimulated with PMA + ionomycin, and then analyzed for intracellular Foxp3 and IFN-γ expression.

T138

20787-20922

Sentence

denotes

After 3 d in culture, the cells were restimulated with PMA + ionomycin, and then analyzed for intracellular Foxp3 and IFN-γ expression.

T31437

20923-21131

Sentence

denotes

One of five experiments is shown. (B) Naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice (5 × 106 cells per transfer).

T139

20923-21131

Sentence

denotes

One of five experiments is shown. (B) Naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice (5 × 106 cells per transfer).

T31438

21132-21292

Sentence

denotes

6 wk later, TCRβ+CD4+ cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine (LP) were analyzed for Foxp3-GFP expression.

T140

21132-21292

Sentence

denotes

6 wk later, TCRβ+CD4+ cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine (LP) were analyzed for Foxp3-GFP expression.

T31439

21293-21396

Sentence

denotes

Results from one of four CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice with same findings are shown.

T141

21293-21396

Sentence

denotes

Results from one of four CbfbF/F CD4-cre and control CbfbF/+ CD4-cre mice with same findings are shown.

T142

21397-21443

Sentence

denotes

The data from four sets of mice is shown in C.

T31440

21397-21532

Sentence

denotes

The data from four sets of mice is shown in C. Statistical analysis was performed with Mann-Whitney U test. *, P < 0.05 between groups.

T143

21444-21532

Sentence

denotes

Statistical analysis was performed with Mann-Whitney U test. *, P < 0.05 between groups.

T9044

21535-21660

Sentence

denotes

The importance of RUNX transcription factors for the in vivo conversion of naive CD4+ T cells into iT reg cells was examined.

T144

21535-21660

Sentence

denotes

The importance of RUNX transcription factors for the in vivo conversion of naive CD4+ T cells into iT reg cells was examined.

T9045

21661-21791

Sentence

denotes

Control CbfbF/F or CbfbF/F CD4-cre naive T cells, harboring a Foxp3-IRES-GFP allele were adoptively transferred into Rag2−/− mice.

T145

21661-21791

Sentence

denotes

Control CbfbF/F or CbfbF/F CD4-cre naive T cells, harboring a Foxp3-IRES-GFP allele were adoptively transferred into Rag2−/− mice.

T9046

21792-21943

Sentence

denotes

6 wk later, CD4+ T cells in spleen, mesenteric lymph node, and lamina propria of the small intestine were analyzed for Foxp3-GFP expression (Fig. 5 B).

T146

21792-21943

Sentence

denotes

6 wk later, CD4+ T cells in spleen, mesenteric lymph node, and lamina propria of the small intestine were analyzed for Foxp3-GFP expression (Fig. 5 B).

T9047

21944-22179

Sentence

denotes

There was a consistently lower percentage of CD4+ T cells that had developed Foxp3 expression in the mesenteric lymph node and lamina propria of mice transferred with CbfbF/F CD4-cre cells compared with control cells (Fig. 5, B and C).

T147

21944-22179

Sentence

denotes

There was a consistently lower percentage of CD4+ T cells that had developed Foxp3 expression in the mesenteric lymph node and lamina propria of mice transferred with CbfbF/F CD4-cre cells compared with control cells (Fig. 5, B and C).

T9048

22180-22282

Sentence

denotes

These data affirm the significance of RUNX proteins for the in vivo generation of CD4+ Foxp3+ T cells.

T148

22180-22282

Sentence

denotes

These data affirm the significance of RUNX proteins for the in vivo generation of CD4+ Foxp3+ T cells.

T10570

22284-22377

Sentence

denotes

CBFβ, RUNX1, and RUNX3 are important for the suppressive activity of CD25+ Foxp3+ T reg cells

T149

22284-22377

Sentence

denotes

CBFβ, RUNX1, and RUNX3 are important for the suppressive activity of CD25+ Foxp3+ T reg cells

T10571

22378-22604

Sentence

denotes

Even 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.

T150

22378-22604

Sentence

denotes

Even 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.

T10572

22605-22818

Sentence

denotes

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.

T151

22605-22818

Sentence

denotes

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.

T10573

22819-22962

Sentence

denotes

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.

T152

22819-22962

Sentence

denotes

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.

T10574

22963-23074

Sentence

denotes

The cells were then incubated with inactivated splenocytes and stimulated with anti-CD3 mAb for four more days.

T153

22963-23074

Sentence

denotes

The cells were then incubated with inactivated splenocytes and stimulated with anti-CD3 mAb for four more days.

T10575

23075-23132

Sentence

denotes

CD45.1+ cells were analyzed for CFSE dilution (Fig. 6 A).

T154

23075-23132

Sentence

denotes

CD45.1+ cells were analyzed for CFSE dilution (Fig. 6 A).

T10576

23133-23345

Sentence

denotes

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).

T155

23133-23345

Sentence

denotes

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).

T10577

23346-23560

Sentence

denotes

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).

T156

23346-23560

Sentence

denotes

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).

T10578

23561-23688

Sentence

denotes

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.

T157

23561-23688

Sentence

denotes

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.

T10579

23689-23790

Sentence

denotes

No suppression could be observed in all control groups without TGF-β at all tested ratios (Fig. 6 C).

T158

23689-23790

Sentence

denotes

No suppression could be observed in all control groups without TGF-β at all tested ratios (Fig. 6 C).

T10580

23791-23923

Sentence

denotes

The decreased suppression capacity of CbfbF/F CD4-cre iT reg cells was unlikely to be caused by decreased survival or proliferation.

T159

23791-23923

Sentence

denotes

The decreased suppression capacity of CbfbF/F CD4-cre iT reg cells was unlikely to be caused by decreased survival or proliferation.

T10581

23924-24144

Sentence

denotes

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).

T160

23924-24144

Sentence

denotes

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).

T161

24145-24154

Sentence

denotes

Figure 6.

T32169

24156-24333

Sentence

denotes

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.

T162

24156-24333

Sentence

denotes

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.

T32170

24334-24597

Sentence

denotes

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.

T163

24334-24597

Sentence

denotes

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.

T32171

24598-24719

Sentence

denotes

After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios.

T164

24598-24719

Sentence

denotes

After 3 d, Foxp3-GFP+ cells were FACS-sorted and mixed with CFSE-loaded naive CD45.1+ CD4+ cells at the indicated ratios.

T32172

24720-24792

Sentence

denotes

These were then incubated with inactivated splenocytes and anti-CD3 mAb.

T165

24720-24792

Sentence

denotes

These were then incubated with inactivated splenocytes and anti-CD3 mAb.

T32173

24793-25015

Sentence

denotes

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.

T166

24793-25015

Sentence

denotes

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.

T32174

25016-25078

Sentence

denotes

Four days later CD45.1+ cells were analyzed for CFSE dilution.

T167

25016-25078

Sentence

denotes

Four days later CD45.1+ cells were analyzed for CFSE dilution.

T32175

25079-25223

Sentence

denotes

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.

T168

25079-25223

Sentence

denotes

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.

T32176

25224-25519

Sentence

denotes

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.

T169

25224-25519

Sentence

denotes

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.

T32177

25520-25614

Sentence

denotes

The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry.

T170

25520-25614

Sentence

denotes

The CFSE dilution of the CD4+ T cell responder cells was analyzed after 5 d by flow cytometry.

T32178

25615-25684

Sentence

denotes

The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5.

T171

25615-25684

Sentence

denotes

The T reg/responder CD4+ T cell ratios used were 1:20, 1:10, and 1:5.

T32179

25685-25717

Sentence

denotes

One of two experiments is shown.

T172

25685-25717

Sentence

denotes

One of two experiments is shown.

T10582

25720-25848

Sentence

denotes

In addition, we tested the requirement of RUNX1 and RUNX3 for the development of the suppressive capacity in human iT reg cells.

T173

25720-25848

Sentence

denotes

In addition, we tested the requirement of RUNX1 and RUNX3 for the development of the suppressive capacity in human iT reg cells.

T10583

25849-25986

Sentence

denotes

We isolated human naive CD4+ T cells and transfected them with a combination of RUNX1 and RUNX3 siRNA, or with a scrambled control siRNA.

T174

25849-25986

Sentence

denotes

We isolated human naive CD4+ T cells and transfected them with a combination of RUNX1 and RUNX3 siRNA, or with a scrambled control siRNA.

T10584

25987-26121

Sentence

denotes

Cells were cultured under T reg conditions, and then mixed with CFSE-labeled autologous CD4+ T cells and stimulated with anti-CD3 mAb.

T175

25987-26121

Sentence

denotes

Cells were cultured under T reg conditions, and then mixed with CFSE-labeled autologous CD4+ T cells and stimulated with anti-CD3 mAb.

T10585

26122-26380

Sentence

denotes

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).

T176

26122-26380

Sentence

denotes

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).

T10586

26381-26563

Sentence

denotes

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.

T177

26381-26563

Sentence

denotes

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.

T10587

26564-26635

Sentence

denotes

The data suggest both quantity and quality of T reg cells are hampered.

T178

26564-26635

Sentence

denotes

The data suggest both quantity and quality of T reg cells are hampered.

T10588

26636-26828

Sentence

denotes

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.

T179

26636-26828

Sentence

denotes

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.

T10589

26829-26995

Sentence

denotes

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.

T180

26829-26995

Sentence

denotes

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.

T181

26997-27007

Sentence

denotes

DISCUSSION

T12845

27008-27145

Sentence

denotes

This study demonstrates that RUNX transcription factors 1 and 3 play an important role in the generation of FOXP3+ iT reg cells by TGF-β.

T182

27008-27145

Sentence

denotes

This study demonstrates that RUNX transcription factors 1 and 3 play an important role in the generation of FOXP3+ iT reg cells by TGF-β.

T12846

27146-27255

Sentence

denotes

TGF-β mediates RUNX induction and FOXP3 is efficiently up-regulated by RUNX1 and RUNX3 in human CD4+ T cells.

T183

27146-27255

Sentence

denotes

TGF-β mediates RUNX induction and FOXP3 is efficiently up-regulated by RUNX1 and RUNX3 in human CD4+ T cells.

T12847

27256-27331

Sentence

denotes

There are three putative RUNX binding sites in the proximal FOXP3 promoter.

T184

27256-27331

Sentence

denotes

There are three putative RUNX binding sites in the proximal FOXP3 promoter.

T12848

27332-27391

Sentence

denotes

One binding site was predicted as a binding site for RUNX2.

T185

27332-27391

Sentence

denotes

One binding site was predicted as a binding site for RUNX2.

T12849

27392-27580

Sentence

denotes

Promoter enzyme immunoassay results showed that binding of RUNX1 and RUNX3 also occurred at this site (as well as at the other two), which were initially identified as RUNX1 binding sites.

T186

27392-27580

Sentence

denotes

Promoter enzyme immunoassay results showed that binding of RUNX1 and RUNX3 also occurred at this site (as well as at the other two), which were initially identified as RUNX1 binding sites.

T12850

27581-27769

Sentence

denotes

This finding is not surprising because RUNX proteins bind to promoter or enhancer elements of their target genes via the runt domain, which is conserved between members of the RUNX family.

T187

27581-27769

Sentence

denotes

This finding is not surprising because RUNX proteins bind to promoter or enhancer elements of their target genes via the runt domain, which is conserved between members of the RUNX family.

T12851

27770-27957

Sentence

denotes

The RUNX protein that actually induces the expression of FOXP3 might therefore be dependent on the availability of the specific RUNX family member at certain stages of T cell development.

T188

27770-27957

Sentence

denotes

The RUNX protein that actually induces the expression of FOXP3 might therefore be dependent on the availability of the specific RUNX family member at certain stages of T cell development.

T12852

27958-28118

Sentence

denotes

RUNX proteins are able to increase or inhibit transcriptional activity of their target genes depending on the cell type and the target gene (Otto et al., 2003).

T189

27958-28118

Sentence

denotes

RUNX proteins are able to increase or inhibit transcriptional activity of their target genes depending on the cell type and the target gene (Otto et al., 2003).

T12853

28119-28320

Sentence

denotes

Mutation of only one of the three binding sites had only a little effect on the promoter activity; however, when two binding sites were mutated, the FOXP3 promoter activity dropped to a greater extent.

T190

28119-28320

Sentence

denotes

Mutation of only one of the three binding sites had only a little effect on the promoter activity; however, when two binding sites were mutated, the FOXP3 promoter activity dropped to a greater extent.

T12854

28321-28401

Sentence

denotes

The most striking effect was observed when all three binding sites were mutated.

T191

28321-28401

Sentence

denotes

The most striking effect was observed when all three binding sites were mutated.

T12855

28402-28568

Sentence

denotes

We therefore assume that these binding sites have partially redundant functions, but binding to at least two sites seems to be necessary for full promoter activation.

T192

28402-28568

Sentence

denotes

We therefore assume that these binding sites have partially redundant functions, but binding to at least two sites seems to be necessary for full promoter activation.

T12856

28569-28680

Sentence

denotes

TGF-β promotes or inhibits the proliferation, differentiation, and survival of a wide array of different cells.

T193

28569-28680

Sentence

denotes

TGF-β promotes or inhibits the proliferation, differentiation, and survival of a wide array of different cells.

T12857

28681-28807

Sentence

denotes

It is also produced in activated T cells and it inhibits T cell proliferation (Kehrl et al., 1986; Siegel and Massagué, 2003).

T194

28681-28807

Sentence

denotes

It is also produced in activated T cells and it inhibits T cell proliferation (Kehrl et al., 1986; Siegel and Massagué, 2003).

T12858

28808-28970

Sentence

denotes

It was shown that TGF-β is mandatory for the maintenance of peripheral T reg cells and their expression of Foxp3 (Marie et al., 2005; Rubtsov and Rudensky, 2007).

T195

28808-28970

Sentence

denotes

It was shown that TGF-β is mandatory for the maintenance of peripheral T reg cells and their expression of Foxp3 (Marie et al., 2005; Rubtsov and Rudensky, 2007).

T12859

28971-29078

Sentence

denotes

RUNX transcription factors are targets of the TGF-β superfamily and they are involved in the TGF-β pathway.

T196

28971-29078

Sentence

denotes

RUNX transcription factors are targets of the TGF-β superfamily and they are involved in the TGF-β pathway.

T12860

29079-29172

Sentence

denotes

They interact directly with regulatory SMADs (Miyazawa et al., 2002; Ito and Miyazono, 2003).

T197

29079-29172

Sentence

denotes

They interact directly with regulatory SMADs (Miyazawa et al., 2002; Ito and Miyazono, 2003).

T12861

29173-29342

Sentence

denotes

TGF-β can activate RUNX genes at the transcriptional level, and at the posttranscriptional level through activation or stabilization of RUNX proteins (Jin et al., 2004).

T198

29173-29342

Sentence

denotes

TGF-β can activate RUNX genes at the transcriptional level, and at the posttranscriptional level through activation or stabilization of RUNX proteins (Jin et al., 2004).

T12862

29343-29506

Sentence

denotes

It was shown that RUNX2 regulates the expression of TGF-β type I receptor (Ji et al., 2001), suggesting that other mechanisms for their function could be involved.

T199

29343-29506

Sentence

denotes

It was shown that RUNX2 regulates the expression of TGF-β type I receptor (Ji et al., 2001), suggesting that other mechanisms for their function could be involved.

T12863

29507-29599

Sentence

denotes

The fusion proteins RUNX1-EVI1 and RUNX1-ETO block TGF-β inhibition of leukemic cell growth.

T200

29507-29599

Sentence

denotes

The fusion proteins RUNX1-EVI1 and RUNX1-ETO block TGF-β inhibition of leukemic cell growth.

T12864

29600-29807

Sentence

denotes

RUNX3 plays an important role in TGF-β–mediated growth control in epithelial cells, as loss of RUNX3 leads to decreased sensitivity to TGF-β and hyperproliferation of the gastric mucosa (Blyth et al., 2005).

T201

29600-29807

Sentence

denotes

RUNX3 plays an important role in TGF-β–mediated growth control in epithelial cells, as loss of RUNX3 leads to decreased sensitivity to TGF-β and hyperproliferation of the gastric mucosa (Blyth et al., 2005).

T12865

29808-29954

Sentence

denotes

The present study demonstrates that RUNX3 expression is more dominant in circulating human T reg cells and tonsil T reg cells compared with RUNX1.

T202

29808-29954

Sentence

denotes

The present study demonstrates that RUNX3 expression is more dominant in circulating human T reg cells and tonsil T reg cells compared with RUNX1.

T12866

29955-30045

Sentence

denotes

This could be dependent on the stage of the cells and organ from which they were isolated.

T203

29955-30045

Sentence

denotes

This could be dependent on the stage of the cells and organ from which they were isolated.

T12867

30046-30230

Sentence

denotes

We observed that single siRNA interference of either RUNX1 or RUNX3 alone shows a slight decrease in Foxp3+ T reg cell induction, which could be caused by redundancy of these proteins.

T204

30046-30230

Sentence

denotes

We observed that single siRNA interference of either RUNX1 or RUNX3 alone shows a slight decrease in Foxp3+ T reg cell induction, which could be caused by redundancy of these proteins.

T12868

30231-30287

Sentence

denotes

For this reason, we decided to use CbfβF/F CD4-cre mice.

T205

30231-30287

Sentence

denotes

For this reason, we decided to use CbfβF/F CD4-cre mice.

T12869

30288-30399

Sentence

denotes

Foxp3 induction by TGF-β is reduced in CD4+ T cells of CbfbF/F CD4-cre mice compared with CbfbF/+ CD4-cre mice.

T206

30288-30399

Sentence

denotes

Foxp3 induction by TGF-β is reduced in CD4+ T cells of CbfbF/F CD4-cre mice compared with CbfbF/+ CD4-cre mice.

T12870

30400-30495

Sentence

denotes

Retinoic acid is secreted by a subset of dendritic cells in the gut-associated lymphoid tissue.

T207

30400-30495

Sentence

denotes

Retinoic acid is secreted by a subset of dendritic cells in the gut-associated lymphoid tissue.

T12871

30496-30645

Sentence

denotes

It inhibits the IL-6–driven induction of Th17 cells and facilitates the differentiation of naive T cells to Foxp3+ T reg cells (Mucida et al., 2007).

T208

30496-30645

Sentence

denotes

It inhibits the IL-6–driven induction of Th17 cells and facilitates the differentiation of naive T cells to Foxp3+ T reg cells (Mucida et al., 2007).

T12872

30646-30806

Sentence

denotes

We observed an increased number of Foxp3+ cells by retinoic acid and TGF-β compared with TGF-β treatment alone in CbfbF/+ CD4-cre mice and CbfbF/F CD4-cre mice.

T209

30646-30806

Sentence

denotes

We observed an increased number of Foxp3+ cells by retinoic acid and TGF-β compared with TGF-β treatment alone in CbfbF/+ CD4-cre mice and CbfbF/F CD4-cre mice.

T12873

30807-30925

Sentence

denotes

In addition, we showed a defective in vivo generation of T reg cells from Cbfb-deficient CD4+ T cells in Rag2−/− mice.

T210

30807-30925

Sentence

denotes

In addition, we showed a defective in vivo generation of T reg cells from Cbfb-deficient CD4+ T cells in Rag2−/− mice.

T12874

30926-31102

Sentence

denotes

These data in mice confirm the human data that RUNX proteins play an important role for TGF-β–dependent FOXP3 induction, as well as in the suppressive capacity of iT reg cells.

T211

30926-31102

Sentence

denotes

These data in mice confirm the human data that RUNX proteins play an important role for TGF-β–dependent FOXP3 induction, as well as in the suppressive capacity of iT reg cells.

T12875

31103-31317

Sentence

denotes

As an additional support for this concept, the overexpression of RUNX1 induced increased FOXP3 protein expression without any requirement of TGF-β and anti-CD3 and anti-CD28 stimulation in human primary CD4+ cells.

T212

31103-31317

Sentence

denotes

As an additional support for this concept, the overexpression of RUNX1 induced increased FOXP3 protein expression without any requirement of TGF-β and anti-CD3 and anti-CD28 stimulation in human primary CD4+ cells.

T12876

31318-31436

Sentence

denotes

In both human and mouse systems, reduced Foxp3 expression was associated with reduced T reg cell suppressive activity.

T213

31318-31436

Sentence

denotes

In both human and mouse systems, reduced Foxp3 expression was associated with reduced T reg cell suppressive activity.

T12877

31437-31557

Sentence

denotes

In a recent study, the role of Runx–CBFβ was investigated in nT reg cell development in the thymus (Rudra et al., 2009).

T214

31437-31557

Sentence

denotes

In a recent study, the role of Runx–CBFβ was investigated in nT reg cell development in the thymus (Rudra et al., 2009).

T12878

31558-31662

Sentence

denotes

It was reported that Foxp3 expression in nT reg cells is unstable in the absence of Runx–CBFβ complexes.

T215

31558-31662

Sentence

denotes

It was reported that Foxp3 expression in nT reg cells is unstable in the absence of Runx–CBFβ complexes.

T12879

31663-31820

Sentence

denotes

Cbfb-deficient nT reg cells progressively lose Foxp3 upon division, and there is no evidence of increased death of Cbfb-deficient nT reg cells in that study.

T216

31663-31820

Sentence

denotes

Cbfb-deficient nT reg cells progressively lose Foxp3 upon division, and there is no evidence of increased death of Cbfb-deficient nT reg cells in that study.

T12880

31821-32048

Sentence

denotes

The experiments in Cbfb-deficient CD4-cre T cells in mice and the knockdown experiments in humans in this study suggest that the induction of Foxp3 expression is a major contributing factor in the in vivo conversion experiment.

T217

31821-32048

Sentence

denotes

The experiments in Cbfb-deficient CD4-cre T cells in mice and the knockdown experiments in humans in this study suggest that the induction of Foxp3 expression is a major contributing factor in the in vivo conversion experiment.

T12881

32049-32139

Sentence

denotes

Here, we observed that there is a twofold increased Foxp3+ iT reg cell generation in vivo.

T218

32049-32139

Sentence

denotes

Here, we observed that there is a twofold increased Foxp3+ iT reg cell generation in vivo.

T12882

32140-32291

Sentence

denotes

This is in the same range with previously published studies targeting different mechanisms in Foxp3 induction (Maynard et al., 2007; Sun et al., 2007).

T219

32140-32291

Sentence

denotes

This is in the same range with previously published studies targeting different mechanisms in Foxp3 induction (Maynard et al., 2007; Sun et al., 2007).

T12883

32292-32562

Sentence

denotes

Whether the diminished capacity of Cbfb-deficient CD4-cre T cells in the generation of Foxp3 may be caused by peripheral expansion of Cbfb-deficient non–T reg cells or survival problems faced by Cbfb-deficient iT reg cells after Foxp3 induction remains to be elucidated.

T220

32292-32562

Sentence

denotes

Whether the diminished capacity of Cbfb-deficient CD4-cre T cells in the generation of Foxp3 may be caused by peripheral expansion of Cbfb-deficient non–T reg cells or survival problems faced by Cbfb-deficient iT reg cells after Foxp3 induction remains to be elucidated.

T12884

32563-32674

Sentence

denotes

The involvement of RUNX proteins in autoimmune diseases has been previously suggested (Alarcón-Riquelme, 2003).

T221

32563-32674

Sentence

denotes

The involvement of RUNX proteins in autoimmune diseases has been previously suggested (Alarcón-Riquelme, 2003).

T12885

32675-32859

Sentence

denotes

A mutation in the RUNX1 binding site in the promoter of programmed cell death 1 gene (PDCD-1) has been implicated in systemic lupus erythematosus pathogenesis (Prokunina et al., 2002).

T222

32675-32859

Sentence

denotes

A mutation in the RUNX1 binding site in the promoter of programmed cell death 1 gene (PDCD-1) has been implicated in systemic lupus erythematosus pathogenesis (Prokunina et al., 2002).

T12886

32860-33105

Sentence

denotes

Polymorphisms that alter RUNX1 binding to other genes have also been described in rheumatoid arthritis linkage at 5q31 in Japanese patients (Tokuhiro et al., 2003) and in a psoriasis linkage at 17q25 (Prokunina et al., 2002; Helms et al., 2003).

T223

32860-33105

Sentence

denotes

Polymorphisms that alter RUNX1 binding to other genes have also been described in rheumatoid arthritis linkage at 5q31 in Japanese patients (Tokuhiro et al., 2003) and in a psoriasis linkage at 17q25 (Prokunina et al., 2002; Helms et al., 2003).

T12887

33106-33239

Sentence

denotes

RUNX3-deficient mice spontaneously develop inflammatory bowel disease and hyperplastic gastritis-like lesions (Brenner et al., 2004).

T224

33106-33239

Sentence

denotes

RUNX3-deficient mice spontaneously develop inflammatory bowel disease and hyperplastic gastritis-like lesions (Brenner et al., 2004).

T12888

33240-33354

Sentence

denotes

These disease symptoms resemble those occurring after depletion of Foxp3-expressing T reg cells (Sakaguchi, 2004).

T225

33240-33354

Sentence

denotes

These disease symptoms resemble those occurring after depletion of Foxp3-expressing T reg cells (Sakaguchi, 2004).

T12889

33355-33579

Sentence

denotes

Derepression of Th2 cytokines might also account for some of the observed disease symptoms, as it was shown that T-bet first induces Runx3 in Th1 cells and then partners with Runx3 to direct lineage-specific gene activation.

T226

33355-33579

Sentence

denotes

Derepression of Th2 cytokines might also account for some of the observed disease symptoms, as it was shown that T-bet first induces Runx3 in Th1 cells and then partners with Runx3 to direct lineage-specific gene activation.

T12890

33580-33731

Sentence

denotes

Runx3/Cbfβ are both required for the activation of the Ifng gene and silencing of the Il4 gene in Th1 cells (Djuretic et al., 2007; Naoe et al., 2007).

T227

33580-33731

Sentence

denotes

Runx3/Cbfβ are both required for the activation of the Ifng gene and silencing of the Il4 gene in Th1 cells (Djuretic et al., 2007; Naoe et al., 2007).

T12891

33732-33848

Sentence

denotes

Runx proteins also play an essential role during T lymphocyte differentiation in the thymus (Taniuchi et al., 2002).

T228

33732-33848

Sentence

denotes

Runx proteins also play an essential role during T lymphocyte differentiation in the thymus (Taniuchi et al., 2002).

T12892

33849-34065

Sentence

denotes

Runx1 regulates the transitions of developing thymocytes from the CD4− CD8− double-negative stage to the CD4+ CD8+ double-positive stage and from the DP stage to the mature single-positive stage (Egawa et al., 2007).

T229

33849-34065

Sentence

denotes

Runx1 regulates the transitions of developing thymocytes from the CD4− CD8− double-negative stage to the CD4+ CD8+ double-positive stage and from the DP stage to the mature single-positive stage (Egawa et al., 2007).

T12893

34066-34207

Sentence

denotes

Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4+ helper and CD8+ cytotoxic T cell lineages.

T230

34066-34207

Sentence

denotes

Runx1 and Runx3 deficiencies caused marked reductions in mature thymocytes and T cells of the CD4+ helper and CD8+ cytotoxic T cell lineages.

T12894

34208-34363

Sentence

denotes

In addition, inactivation of both Runx1 and Runx3 at the double-positive stages resulted in a severe blockage in the development of CD8+ mature thymocytes.

T231

34208-34363

Sentence

denotes

In addition, inactivation of both Runx1 and Runx3 at the double-positive stages resulted in a severe blockage in the development of CD8+ mature thymocytes.

T12895

34364-34607

Sentence

denotes

These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells, and suggest that they may play a role in nT reg cell development, which remains to be elucidated.

T232

34364-34607

Sentence

denotes

These results indicate that Runx proteins have important roles at multiple stages of T cell development and in the homeostasis of mature T cells, and suggest that they may play a role in nT reg cell development, which remains to be elucidated.

T12896

34608-34755

Sentence

denotes

Furthermore, it was shown that Runx1 activates IL-2 and IFN-γ gene expression in conventional CD4+ T cells by binding to their respective promoter.

T233

34608-34755

Sentence

denotes

Furthermore, it was shown that Runx1 activates IL-2 and IFN-γ gene expression in conventional CD4+ T cells by binding to their respective promoter.

T12897

34756-34991

Sentence

denotes

RUNX1 interacts physically with Foxp3 protein, and it was demonstrated that this interaction might be responsible for the suppression of IL-2 and IFN-γ production and up-regulation of T reg cell–associated molecules (Ono et al., 2007).

T234

34756-34991

Sentence

denotes

RUNX1 interacts physically with Foxp3 protein, and it was demonstrated that this interaction might be responsible for the suppression of IL-2 and IFN-γ production and up-regulation of T reg cell–associated molecules (Ono et al., 2007).

T12898

34992-35058

Sentence

denotes

It has been shown that Foxp3 also influences Th17 differentiation.

T235

34992-35058

Sentence

denotes

It has been shown that Foxp3 also influences Th17 differentiation.

T12899

35059-35177

Sentence

denotes

Specifically, Foxp3 physically interacts with RORγt, and this interaction inhibits RORγt function (Zhou et al., 2008).

T236

35059-35177

Sentence

denotes

Specifically, Foxp3 physically interacts with RORγt, and this interaction inhibits RORγt function (Zhou et al., 2008).

T12900

35178-35388

Sentence

denotes

This relationship of RORγt and Foxp3 and probably yet unknown mechanisms might be the basis of the observation that the differentiation of Th17 cells and T reg cells is often reciprocal (Bettelli et al., 2006).

T237

35178-35388

Sentence

denotes

This relationship of RORγt and Foxp3 and probably yet unknown mechanisms might be the basis of the observation that the differentiation of Th17 cells and T reg cells is often reciprocal (Bettelli et al., 2006).

T12901

35389-35560

Sentence

denotes

Recently, data suggests that Runx1 may also be involved in regulating Il17 transcription, functioning in complex with RORγt to activate transcription (Zhang et al., 2008).

T238

35389-35560

Sentence

denotes

Recently, data suggests that Runx1 may also be involved in regulating Il17 transcription, functioning in complex with RORγt to activate transcription (Zhang et al., 2008).

T12902

35561-35695

Sentence

denotes

The Runx3-deficient mice develop spontaneous Th2-dominated autoimmune colitis and asthma (Brenner et al., 2004; Fainaru et al., 2005).

T239

35561-35695

Sentence

denotes

The Runx3-deficient mice develop spontaneous Th2-dominated autoimmune colitis and asthma (Brenner et al., 2004; Fainaru et al., 2005).

T12903

35696-35882

Sentence

denotes

Cbfbf/f Cd4 mice also show a spontaneous Th2 dominated disease, with increased serum IgA, IgG1, and IgE titers and lymphocyte and eosinophil infiltration of the lung (Naoe et al., 2007).

T240

35696-35882

Sentence

denotes

Cbfbf/f Cd4 mice also show a spontaneous Th2 dominated disease, with increased serum IgA, IgG1, and IgE titers and lymphocyte and eosinophil infiltration of the lung (Naoe et al., 2007).

T12904

35883-36041

Sentence

denotes

All these phenotypes were previously attributed to a loss of Th2 silencing whereas our findings additionally suggest that loss of T reg function plays a role.

T241

35883-36041

Sentence

denotes

All these phenotypes were previously attributed to a loss of Th2 silencing whereas our findings additionally suggest that loss of T reg function plays a role.

T12905

36042-36123

Sentence

denotes

We have shown a link between Foxp3 induction in iT reg cells and RUNX1 and RUNX3.

T242

36042-36123

Sentence

denotes

We have shown a link between Foxp3 induction in iT reg cells and RUNX1 and RUNX3.

T12906

36124-36285

Sentence

denotes

RUNX proteins play a central role in pathways regulating cell growth and differentiation, and their interaction with the TGF-β pathway is of particular interest.

T243

36124-36285

Sentence

denotes

RUNX proteins play a central role in pathways regulating cell growth and differentiation, and their interaction with the TGF-β pathway is of particular interest.

T12907

36286-36591

Sentence

denotes

Foxp3 protein interacts not only with RUNX proteins but also with several other transcriptional partners, such as NFAT and possibly NF-κB; with histone acetyl transferases, such as TIP60; and histone deacetyl transferase (HDAC) complexes, such as HDAC7 and HDAC9 (Wu et al., 2006; Sakaguchi et al., 2008).

T244

36286-36591

Sentence

denotes

Foxp3 protein interacts not only with RUNX proteins but also with several other transcriptional partners, such as NFAT and possibly NF-κB; with histone acetyl transferases, such as TIP60; and histone deacetyl transferase (HDAC) complexes, such as HDAC7 and HDAC9 (Wu et al., 2006; Sakaguchi et al., 2008).

T12908

36592-36830

Sentence

denotes

NFAT forms a complex with AP-1 and NF-κB and regulates the expression of IL-2, IL-4, IFN-γ, and CTLA4 in conventional T cells, which leads to the activation and differentiation to effector T cells (Dolganov et al., 1996; Hu et al., 2007).

T245

36592-36830

Sentence

denotes

NFAT forms a complex with AP-1 and NF-κB and regulates the expression of IL-2, IL-4, IFN-γ, and CTLA4 in conventional T cells, which leads to the activation and differentiation to effector T cells (Dolganov et al., 1996; Hu et al., 2007).

T12909

36831-36974

Sentence

denotes

The NFAT–AP-1 complex also binds to the Foxp3 promoter after TCR triggering and regulates its gene expression positively (Mantel et al., 2006).

T246

36831-36974

Sentence

denotes

The NFAT–AP-1 complex also binds to the Foxp3 promoter after TCR triggering and regulates its gene expression positively (Mantel et al., 2006).

T12910

36975-37180

Sentence

denotes

It was shown that NFAT and Smad3 cooperate to induce Foxp3 expression through its enhancer (Tone et al., 2008), but no TGF-β response element was identified in the Foxp3 gene or in the surrounding regions.

T247

36975-37180

Sentence

denotes

It was shown that NFAT and Smad3 cooperate to induce Foxp3 expression through its enhancer (Tone et al., 2008), but no TGF-β response element was identified in the Foxp3 gene or in the surrounding regions.

T12911

37181-37417

Sentence

denotes

The initial induction of RUNX1 and RUNX3 and the subsequent binding of these transcription factors to the Foxp3 promoter that we showed here might explain the relatively late induction of Foxp3 mRNA that peaks 24–48 h after stimulation.

T248

37181-37417

Sentence

denotes

The initial induction of RUNX1 and RUNX3 and the subsequent binding of these transcription factors to the Foxp3 promoter that we showed here might explain the relatively late induction of Foxp3 mRNA that peaks 24–48 h after stimulation.

T12912

37418-37519

Sentence

denotes

The interaction of Foxp3 and NFAT is dependent on their cooperative binding to DNA (Wu et al., 2006).

T249

37418-37519

Sentence

denotes

The interaction of Foxp3 and NFAT is dependent on their cooperative binding to DNA (Wu et al., 2006).

T12913

37520-37711

Sentence

denotes

RUNX1 alone, or together with its interacting partners p300 and CREB-binding protein, may cooperate with the NFAT transcription complex to activate the IL-2 promoter (Sakaguchi et al., 2008).

T250

37520-37711

Sentence

denotes

RUNX1 alone, or together with its interacting partners p300 and CREB-binding protein, may cooperate with the NFAT transcription complex to activate the IL-2 promoter (Sakaguchi et al., 2008).

T12914

37712-37867

Sentence

denotes

Similar to this interaction, NFAT may also cooperate with RUNX1 or RUNX3 to activate Foxp3, but further studies are necessary to elaborate on this concept.

T251

37712-37867

Sentence

denotes

Similar to this interaction, NFAT may also cooperate with RUNX1 or RUNX3 to activate Foxp3, but further studies are necessary to elaborate on this concept.

T12915

37868-37972

Sentence

denotes

In conclusion, our findings elucidate the role of RUNX proteins in iT reg cell development and function.

T252

37868-37972

Sentence

denotes

In conclusion, our findings elucidate the role of RUNX proteins in iT reg cell development and function.

T12916

37973-38150

Sentence

denotes

The induction of the transcription factors RUNX1 and RUNX3 by TGF-β and the subsequent up-regulation of Foxp3 play a role in iT reg cell generation and its suppressive capacity.

T253

37973-38150

Sentence

denotes

The induction of the transcription factors RUNX1 and RUNX3 by TGF-β and the subsequent up-regulation of Foxp3 play a role in iT reg cell generation and its suppressive capacity.

T254

38152-38173

Sentence

denotes

MATERIALS AND METHODS

T18671

38175-38180

Sentence

denotes

Mice.

T255

38175-38180

Sentence

denotes

Mice.

T18672

38181-38289

Sentence

denotes

CbfbF/F CD4-cre and Foxp3GFP mice have previously been described (Bettelli et al., 2006; Naoe et al., 2007).

T256

38181-38289

Sentence

denotes

CbfbF/F CD4-cre and Foxp3GFP mice have previously been described (Bettelli et al., 2006; Naoe et al., 2007).

T18673

38290-38396

Sentence

denotes

Cd45.1 and Rag2−/− mice were purchased from Jackson ImmunoResearch Laboratories and Taconic, respectively.

T257

38290-38396

Sentence

denotes

Cd45.1 and Rag2−/− mice were purchased from Jackson ImmunoResearch Laboratories and Taconic, respectively.

T18674

38397-38578

Sentence

denotes

For the in vivo Foxp3 conversion assay, naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice.

T258

38397-38578

Sentence

denotes

For the in vivo Foxp3 conversion assay, naive CD4+ T cells from Cbfb CD4-cre or control mice (harboring a Foxp3-IRES-GFP allele) were adoptively transferred into Rag-deficient mice.

T18675

38579-38616

Sentence

denotes

5 × 106 cells were used per transfer.

T259

38579-38616

Sentence

denotes

5 × 106 cells were used per transfer.

T18676

38617-38778

Sentence

denotes

6 wk later, TCRβ+CD4+ gated cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine were analyzed for Foxp3-GFP expression.

T260

38617-38778

Sentence

denotes

6 wk later, TCRβ+CD4+ gated cells from the spleen, mesenteric lymph node (MLN), and lamina propria of the small intestine were analyzed for Foxp3-GFP expression.

T18677

38779-38851

Sentence

denotes

All analyses and experiments were performed on animals at 6–8 wk of age.

T261

38779-38851

Sentence

denotes

All analyses and experiments were performed on animals at 6–8 wk of age.

T18678

38852-39105

Sentence

denotes

Animals were housed under specific pathogen–free conditions at the animal facility of the Skirball Institute, and experiments were performed in accordance with approved protocols for the New York University Institutional Animal Care and Usage Committee.

T262

38852-39105

Sentence

denotes

Animals were housed under specific pathogen–free conditions at the animal facility of the Skirball Institute, and experiments were performed in accordance with approved protocols for the New York University Institutional Animal Care and Usage Committee.

T19191

39107-39164

Sentence

denotes

Isolation of PBMCs, CD4+ T cells, and culture conditions.

T263

39107-39164

Sentence

denotes

Isolation of PBMCs, CD4+ T cells, and culture conditions.

T19192

39165-39373

Sentence

denotes

Human PBMCs were isolated by Ficoll (Biochrom) density gradient centrifugation and CD4+ T cells were then isolated using the Dynal CD4+ Isolation kit (Invitrogen) according to the manufacturer's instructions.

T264

39165-39373

Sentence

denotes

Human PBMCs were isolated by Ficoll (Biochrom) density gradient centrifugation and CD4+ T cells were then isolated using the Dynal CD4+ Isolation kit (Invitrogen) according to the manufacturer's instructions.

T19193

39374-39453

Sentence

denotes

The purity of CD4+ T cells was initially tested by flow cytometry and was ≥95%.

T265

39374-39453

Sentence

denotes

The purity of CD4+ T cells was initially tested by flow cytometry and was ≥95%.

T19194

39454-39808

Sentence

denotes

Cells were stimulated with the following combination of mAbs to T cell surface molecules (Meiler et al., 2008): anti-CD2 (clone 4B2 and 6G4; 0.5 µg/ml), anti-CD3 (clone OKT3; 0.5 µg/ml), and anti-CD28 mAb (clone B7G5; 0.5 µg/ml; all from Sanquin) and cultured in serum-free AIM-V medium (Life Technologies) with the addition of 1 nmol/liter IL-2 (Roche).

T266

39454-39808

Sentence

denotes

Cells were stimulated with the following combination of mAbs to T cell surface molecules (Meiler et al., 2008): anti-CD2 (clone 4B2 and 6G4; 0.5 µg/ml), anti-CD3 (clone OKT3; 0.5 µg/ml), and anti-CD28 mAb (clone B7G5; 0.5 µg/ml; all from Sanquin) and cultured in serum-free AIM-V medium (Life Technologies) with the addition of 1 nmol/liter IL-2 (Roche).

T19195

39809-39874

Sentence

denotes

TGF-β (R&D Systems) was used at 5 ng/ml, if not stated otherwise.

T267

39809-39874

Sentence

denotes

TGF-β (R&D Systems) was used at 5 ng/ml, if not stated otherwise.

T19196

39875-39955

Sentence

denotes

A combination of PMA (25 ng/ml) and ionomycin (1 mg/ml; Sigma-Aldrich) was used.

T268

39875-39955

Sentence

denotes

A combination of PMA (25 ng/ml) and ionomycin (1 mg/ml; Sigma-Aldrich) was used.

T19197

39956-40132

Sentence

denotes

Human CD4+ CD127− CD25high and CD4+ CD127+ CD25neg cells were purified by flow cytometry using anti-CD127, anti–CD25-PC5 (Beckman Coulter), and anti–CD4-FITC antibodies (Dako).

T269

39956-40132

Sentence

denotes

Human CD4+ CD127− CD25high and CD4+ CD127+ CD25neg cells were purified by flow cytometry using anti-CD127, anti–CD25-PC5 (Beckman Coulter), and anti–CD4-FITC antibodies (Dako).

T19198

40133-40394

Sentence

denotes

Mouse naive (CD62Lhi44lo25−) CD4+ T cells were purified by flow cytometry and activated in vitro with 5 µg/ml plate-bound anti-CD3 and 1 µg/ml soluble anti-CD28 antibodies (eBioscience) in RPMI supplemented with 10% FCS, 5 mM β-mercaptoethanol, and antibiotics.

T270

40133-40394

Sentence

denotes

Mouse naive (CD62Lhi44lo25−) CD4+ T cells were purified by flow cytometry and activated in vitro with 5 µg/ml plate-bound anti-CD3 and 1 µg/ml soluble anti-CD28 antibodies (eBioscience) in RPMI supplemented with 10% FCS, 5 mM β-mercaptoethanol, and antibiotics.

T19199

40395-40494

Sentence

denotes

Neutralizing anti–IFN-γ and anti–IL-4 mAbs (BD) were used at 1 µg/ml concentrations when indicated.

T271

40395-40494

Sentence

denotes

Neutralizing anti–IFN-γ and anti–IL-4 mAbs (BD) were used at 1 µg/ml concentrations when indicated.

T19990

40496-40528

Sentence

denotes

In vitro T cell differentiation.

T272

40496-40528

Sentence

denotes

In vitro T cell differentiation.

T19991

40529-40718

Sentence

denotes

CD4+ CD45RA+ magnetically sorted (CD45RO depletion with AutoMACS; Miltenyi Biotec) cells were stimulated with immobilized plate-bound anti-CD3 (1 µg/ml; OKT3; IgG1) and anti-CD28 (2 µg/ml).

T273

40529-40718

Sentence

denotes

CD4+ CD45RA+ magnetically sorted (CD45RO depletion with AutoMACS; Miltenyi Biotec) cells were stimulated with immobilized plate-bound anti-CD3 (1 µg/ml; OKT3; IgG1) and anti-CD28 (2 µg/ml).

T19992

40719-40796

Sentence

denotes

For Th1 differentiation conditions, cells were stimulated with the following:

T274

40719-40796

Sentence

denotes

For Th1 differentiation conditions, cells were stimulated with the following:

T19993

40797-40864

Sentence

denotes

40 ng/ml IL-2, 5 µg/ml anti–IL-4, and 25 ng/ml IL-12 (R&D Systems).

T275

40797-40864

Sentence

denotes

40 ng/ml IL-2, 5 µg/ml anti–IL-4, and 25 ng/ml IL-12 (R&D Systems).

T19994

40865-40926

Sentence

denotes

For Th2 conditions, cells were stimulated with the following:

T276

40865-40926

Sentence

denotes

For Th2 conditions, cells were stimulated with the following:

T19995

40927-40994

Sentence

denotes

40 ng/ml IL-2, 25 ng/ml IL-4, and 5 µg/ml anti–IL-12 (R&D Systems).

T277

40927-40994

Sentence

denotes

40 ng/ml IL-2, 25 ng/ml IL-4, and 5 µg/ml anti–IL-12 (R&D Systems).

T19996

40995-41063

Sentence

denotes

For T reg cell conditions, cells were stimulated with the following:

T278

40995-41063

Sentence

denotes

For T reg cell conditions, cells were stimulated with the following:

T19997

41064-41132

Sentence

denotes

40 ng/ml IL-2, 5 ng/ml TGF-β, 5 µg/ml anti–IL-12, 5 µg/ml anti–IL-4.

T279

41064-41132

Sentence

denotes

40 ng/ml IL-2, 5 ng/ml TGF-β, 5 µg/ml anti–IL-12, 5 µg/ml anti–IL-4.

T19998

41133-41195

Sentence

denotes

For Th17 conditions, cells were stimulated with the following:

T280

41133-41195

Sentence

denotes

For Th17 conditions, cells were stimulated with the following:

T19999

41196-41353

Sentence

denotes

40 ng/ml IL-2, 20 ng/ml IL-6, 5 ng/ml TGF-β, 20 ng/ml IL-23 (Alexis Biochemicals Corp.), 10 ng/ml IL-1β, 5 µg/ml anti-IL-4, and 5 µg/ml anti–IL-12 were used.

T281

41196-41353

Sentence

denotes

40 ng/ml IL-2, 20 ng/ml IL-6, 5 ng/ml TGF-β, 20 ng/ml IL-23 (Alexis Biochemicals Corp.), 10 ng/ml IL-1β, 5 µg/ml anti-IL-4, and 5 µg/ml anti–IL-12 were used.

T20000

41354-41414

Sentence

denotes

Proliferating cells were expanded in medium containing IL-2.

T282

41354-41414

Sentence

denotes

Proliferating cells were expanded in medium containing IL-2.

T20001

41415-41613

Sentence

denotes

The cytokine profile of these cells demonstrated that IFN-γ is the predominant cytokine in Th1 cells, IL-4 and IL-13 in Th2 cells, and IL-17 in Th17 cells (Akdis et al., 2000; Burgler et al., 2009).

T283

41415-41613

Sentence

denotes

The cytokine profile of these cells demonstrated that IFN-γ is the predominant cytokine in Th1 cells, IL-4 and IL-13 in Th2 cells, and IL-17 in Th17 cells (Akdis et al., 2000; Burgler et al., 2009).

T20669

41615-41636

Sentence

denotes

Immunohistochemistry.

T284

41615-41636

Sentence

denotes

Immunohistochemistry.

T20670

41637-41760

Sentence

denotes

Human tonsils were obtained from tonsillectomy samples of hypertrophic and obstructive tonsils without a current infection.

T285

41637-41760

Sentence

denotes

Human tonsils were obtained from tonsillectomy samples of hypertrophic and obstructive tonsils without a current infection.

T20671

41761-41874

Sentence

denotes

Ethical permission was obtained from Cantonal Ethics Commission, and informed consent was obtained from patients.

T286

41761-41874

Sentence

denotes

Ethical permission was obtained from Cantonal Ethics Commission, and informed consent was obtained from patients.

T20672

41875-42082

Sentence

denotes

Paraformaldehyde-fixed tonsil cryosections were stained with unconjugated rabbit IgG polyclonal antibody to human RUNX1 (Santa Cruz Biotechnology, Inc.) or unconjugated mouse IgG1 mAb to human RUNX3 (Abcam).

T287

41875-42082

Sentence

denotes

Paraformaldehyde-fixed tonsil cryosections were stained with unconjugated rabbit IgG polyclonal antibody to human RUNX1 (Santa Cruz Biotechnology, Inc.) or unconjugated mouse IgG1 mAb to human RUNX3 (Abcam).

T20673

42083-42175

Sentence

denotes

After a washing step, the sections were stained with the corresponding secondary antibodies.

T288

42083-42175

Sentence

denotes

After a washing step, the sections were stained with the corresponding secondary antibodies.

T20674

42176-42372

Sentence

denotes

RUNX1-binding antibodies were detected by using Alexa Fluor 633–conjugated goat anti–rabbit IgG and RUNX3-binding antibodies were detected by using Alexa Fluor 532–conjugated goat anti–mouse IgG1.

T289

42176-42372

Sentence

denotes

RUNX1-binding antibodies were detected by using Alexa Fluor 633–conjugated goat anti–rabbit IgG and RUNX3-binding antibodies were detected by using Alexa Fluor 532–conjugated goat anti–mouse IgG1.

T20675

42373-42504

Sentence

denotes

Afterward, the sections were washed and in the case of RUNX3 staining a blocking step with an unconjugated mouse IgG1 mAb was used.

T290

42373-42504

Sentence

denotes

Afterward, the sections were washed and in the case of RUNX3 staining a blocking step with an unconjugated mouse IgG1 mAb was used.

T20676

42505-42653

Sentence

denotes

Finally, the sections were stained with Alexa Fluor 488–conjugated mouse IgG1 mAb to human FOXP3 (eBioscience) or the corresponding isotype control.

T291

42505-42653

Sentence

denotes

Finally, the sections were stained with Alexa Fluor 488–conjugated mouse IgG1 mAb to human FOXP3 (eBioscience) or the corresponding isotype control.

T20677

42654-42772

Sentence

denotes

Tissue sections were stained with DAPI for the demonstration of nuclei and mounted with Prolong antifade (Invitrogen).

T292

42654-42772

Sentence

denotes

Tissue sections were stained with DAPI for the demonstration of nuclei and mounted with Prolong antifade (Invitrogen).

T20678

42773-42888

Sentence

denotes

Images were acquired and analyzed using the confocal microscope DMI 4000B and the TCS SPE system (both from Leica).

T293

42773-42888

Sentence

denotes

Images were acquired and analyzed using the confocal microscope DMI 4000B and the TCS SPE system (both from Leica).

T21238

42890-42918

Sentence

denotes

In vitro suppression assays.

T294

42890-42918

Sentence

denotes

In vitro suppression assays.

T21239

42919-43041

Sentence

denotes

Mouse Foxp3+ CD4+ CD8− T cells were FACS purified based on GFP expressed from a Foxp3-IRES-GFP knock-in allele (Foxp3GFP).

T295

42919-43041

Sentence

denotes

Mouse Foxp3+ CD4+ CD8− T cells were FACS purified based on GFP expressed from a Foxp3-IRES-GFP knock-in allele (Foxp3GFP).

T21240

43042-43167

Sentence

denotes

Naive (CD62Lhi44lo25−) CD4+ T cells (effectors) were FACS-purified from Cd45.1 mice, then loaded with 5 µM CFSE (Invitrogen).

T296

43042-43167

Sentence

denotes

Naive (CD62Lhi44lo25−) CD4+ T cells (effectors) were FACS-purified from Cd45.1 mice, then loaded with 5 µM CFSE (Invitrogen).

T21241

43168-43287

Sentence

denotes

Total splenocytes from C57BL/6 mice inactivated with 50 µg/ml mitomycin C (Sigma-Aldrich) for 45 min were used as APCs.

T297

43168-43287

Sentence

denotes

Total splenocytes from C57BL/6 mice inactivated with 50 µg/ml mitomycin C (Sigma-Aldrich) for 45 min were used as APCs.

T21242

43288-43442

Sentence

denotes

A total of 4 × 105 CD4+ cells (CFSE-loaded CD25− plus Foxp3-GFP+) was mixed with 105 APCs + 1 µg/ml anti-CD3 mAb per well of a 96 well round bottom plate.

T298

43288-43442

Sentence

denotes

A total of 4 × 105 CD4+ cells (CFSE-loaded CD25− plus Foxp3-GFP+) was mixed with 105 APCs + 1 µg/ml anti-CD3 mAb per well of a 96 well round bottom plate.

T21243

43443-43509

Sentence

denotes

Proliferation of the effector cells was analyzed by CFSE dilution.

T299

43443-43509

Sentence

denotes

Proliferation of the effector cells was analyzed by CFSE dilution.

T21244

43510-43591

Sentence

denotes

Apoptosis of the cells was investigated by annexin V staining and flow cytometry.

T300

43510-43591

Sentence

denotes

Apoptosis of the cells was investigated by annexin V staining and flow cytometry.

T21245

43592-43729

Sentence

denotes

Positive and negative control gates were made according to T cells cultured only in the presence of IL-2 without anti-CD3/28 stimulation.

T301

43592-43729

Sentence

denotes

Positive and negative control gates were made according to T cells cultured only in the presence of IL-2 without anti-CD3/28 stimulation.

T21246

43730-43903

Sentence

denotes

Human naive CD4+ T cells were isolated by negative selection by MACS from PBMCs and either transfected with a scrambled siRNA or with a combination of RUNX1 and RUNX3 siRNA.

T302

43730-43903

Sentence

denotes

Human naive CD4+ T cells were isolated by negative selection by MACS from PBMCs and either transfected with a scrambled siRNA or with a combination of RUNX1 and RUNX3 siRNA.

T21247

43904-44089

Sentence

denotes

Cells were then cultured under iT reg cell differentiating conditions and mixed with 2 × 105 autologous irradiated PBMCs that were used as APCs and autologous CFSE-labeled CD4+ T cells.

T303

43904-44089

Sentence

denotes

Cells were then cultured under iT reg cell differentiating conditions and mixed with 2 × 105 autologous irradiated PBMCs that were used as APCs and autologous CFSE-labeled CD4+ T cells.

T21248

44090-44149

Sentence

denotes

T reg cell to responder cell ratio was 1:20, 1:10, and 1:5.

T304

44090-44149

Sentence

denotes

T reg cell to responder cell ratio was 1:20, 1:10, and 1:5.

T21249

44150-44263

Sentence

denotes

To check the proliferation of the CD4+ T cells without suppression, no T reg cells were added in a control group.

T305

44150-44263

Sentence

denotes

To check the proliferation of the CD4+ T cells without suppression, no T reg cells were added in a control group.

T21250

44264-44472

Sentence

denotes

Cells were stimulated with 2.5 µg/ml anti-CD3 mAb, cultured in a 96-well plate and the proliferation of the effector cells was determined by analyzing the CFSE dilution by flow cytometry after 5 d of culture.

T306

44264-44472

Sentence

denotes

Cells were stimulated with 2.5 µg/ml anti-CD3 mAb, cultured in a 96-well plate and the proliferation of the effector cells was determined by analyzing the CFSE dilution by flow cytometry after 5 d of culture.

T21251

44473-44551

Sentence

denotes

Gating on the CD4+CFSE+ T cells enabled the exclusion of APCs and T reg cells.

T307

44473-44551

Sentence

denotes

Gating on the CD4+CFSE+ T cells enabled the exclusion of APCs and T reg cells.

T22103

44553-44652

Sentence

denotes

Cloning of the FOXP3 promoter, construction of mutant FOXP3 promoter, and RUNX expression plasmids.

T308

44553-44652

Sentence

denotes

Cloning of the FOXP3 promoter, construction of mutant FOXP3 promoter, and RUNX expression plasmids.

T22104

44653-44783

Sentence

denotes

The FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate pGL3 FOXP3 -511/+176 (Mantel et al., 2006).

T309

44653-44783

Sentence

denotes

The FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate pGL3 FOXP3 -511/+176 (Mantel et al., 2006).

T22105

44784-45017

Sentence

denotes

Site-directed mutagenesis for the three putative RUNX binding sites in the FOXP3 promoter region was introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions and confirmed by sequencing the DNA.

T310

44784-45017

Sentence

denotes

Site-directed mutagenesis for the three putative RUNX binding sites in the FOXP3 promoter region was introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions and confirmed by sequencing the DNA.

T22106

45018-45497

Sentence

denotes

The following primers and their complementary strands were used: foxp runx-333, forward 5′-CACTTTTGTTTTAAAAACTGTCCTTTCTCATGAGCCCTATTATC-3′; foxp runx-333 reverse 5′-GATAATAGGGCTCATGAGAAAGGACAGTTTTTAAAACAAAAGTG-3′; foxp runx-287 forward 5′-CCTCTCACCTCTGTCCTGAGGGGAAGAAATC-3′; foxp runx-287 reverse 5′-GATTTCTTCCCCTCAGGACAGAGGTGAGAGG-3′; foxp runx-53 forward 5′-GCTTCCACACCGTACAGCGTCCTTTTTCTTCTCGGTATAAAAG-3′; foxp runx-53 reverse 5′-CTTTTATACCGAGAAGAAAAAGGACGCTGTACGGTGTGGAAGC-3′.

T311

45018-45497

Sentence

denotes

The following primers and their complementary strands were used: foxp runx-333, forward 5′-CACTTTTGTTTTAAAAACTGTCCTTTCTCATGAGCCCTATTATC-3′; foxp runx-333 reverse 5′-GATAATAGGGCTCATGAGAAAGGACAGTTTTTAAAACAAAAGTG-3′; foxp runx-287 forward 5′-CCTCTCACCTCTGTCCTGAGGGGAAGAAATC-3′; foxp runx-287 reverse 5′-GATTTCTTCCCCTCAGGACAGAGGTGAGAGG-3′; foxp runx-53 forward 5′-GCTTCCACACCGTACAGCGTCCTTTTTCTTCTCGGTATAAAAG-3′; foxp runx-53 reverse 5′-CTTTTATACCGAGAAGAAAAAGGACGCTGTACGGTGTGGAAGC-3′.

T22107

45498-45652

Sentence

denotes

The human RUNX1 fragment from the Addgene plasmid 12504 (Biggs et al., 2006) pFlagCMV2-AML1B was sub-cloned in the pEGFPN1 vector (Clontech Laboratories).

T312

45498-45652

Sentence

denotes

The human RUNX1 fragment from the Addgene plasmid 12504 (Biggs et al., 2006) pFlagCMV2-AML1B was sub-cloned in the pEGFPN1 vector (Clontech Laboratories).

T313

45653-45712

Sentence

denotes

The RUNX3 vector pCMV human RUNX3, which was a gift from K.

T22108

45653-45860

Sentence

denotes

The RUNX3 vector pCMV human RUNX3, which was a gift from K. Ito (Institute of Molecular and Cell Biology, Proteos, Singapore) was subcloned into pEGFPN1 vector (Clontech Laboratories; Yamamura et al., 2006).

T314

45713-45860

Sentence

denotes

Ito (Institute of Molecular and Cell Biology, Proteos, Singapore) was subcloned into pEGFPN1 vector (Clontech Laboratories; Yamamura et al., 2006).

T22669

45862-45901

Sentence

denotes

Transfections and reporter gene assays.

T315

45862-45901

Sentence

denotes

Transfections and reporter gene assays.

T22670

45902-45959

Sentence

denotes

T cells were rested in serum-free AIM-V medium overnight.

T316

45902-45959

Sentence

denotes

T cells were rested in serum-free AIM-V medium overnight.

T22671

45960-46231

Sentence

denotes

3.5 µg of the FOXP3 promoter luciferase reporter vector or a combination together with the RUNX1, RUNX3 pEGFPN1 vector, and 0.5 µg phRL-TK were added to 3 × 106 CD4+ T cells resuspended in 100 µl of Nucleofector solution (Lonza) and electroporated using the program U-15.

T317

45960-46231

Sentence

denotes

3.5 µg of the FOXP3 promoter luciferase reporter vector or a combination together with the RUNX1, RUNX3 pEGFPN1 vector, and 0.5 µg phRL-TK were added to 3 × 106 CD4+ T cells resuspended in 100 µl of Nucleofector solution (Lonza) and electroporated using the program U-15.

T22672

46232-46443

Sentence

denotes

After a 24-h culture in serum-free conditions and stimuli as indicated in the figures, luciferase activity was measured by the dual luciferase assay system (Promega) according to the manufacturer's instructions.

T318

46232-46443

Sentence

denotes

After a 24-h culture in serum-free conditions and stimuli as indicated in the figures, luciferase activity was measured by the dual luciferase assay system (Promega) according to the manufacturer's instructions.

T22673

46444-46612

Sentence

denotes

PMA/ionomycin was used to stimulate the cells, because the transfection was only transient and the luciferase assay required a strong and fast stimulation of the cells.

T319

46444-46612

Sentence

denotes

PMA/ionomycin was used to stimulate the cells, because the transfection was only transient and the luciferase assay required a strong and fast stimulation of the cells.

T22674

46613-47012

Sentence

denotes

To evaluate the effect of overexpression of RUNX1 or RUNX3 on FOXP3 protein levels, CD4+ T cells were preactivated with 2 µg/ml phytohemagglutinin (Sigma-Aldrich) in serum-free AIM-V medium in the presence of 1 nmol/liter IL-2 (Roche) for 12 h, and then transfected with the vector pEGFPN1 containing the RUNX1 or RUNX3 fragment using the Nucleofector system (Amaxa Biosystems) and the program T-23.

T320

46613-47012

Sentence

denotes

To evaluate the effect of overexpression of RUNX1 or RUNX3 on FOXP3 protein levels, CD4+ T cells were preactivated with 2 µg/ml phytohemagglutinin (Sigma-Aldrich) in serum-free AIM-V medium in the presence of 1 nmol/liter IL-2 (Roche) for 12 h, and then transfected with the vector pEGFPN1 containing the RUNX1 or RUNX3 fragment using the Nucleofector system (Amaxa Biosystems) and the program T-23.

T22675

47013-47129

Sentence

denotes

FOXP3 expression was evaluated by flow cytometry after 48 h of culture in AIM-V medium containing 1 nmol/liter IL-2.

T321

47013-47129

Sentence

denotes

FOXP3 expression was evaluated by flow cytometry after 48 h of culture in AIM-V medium containing 1 nmol/liter IL-2.

T23292

47131-47148

Sentence

denotes

RNA interference.

T322

47131-47148

Sentence

denotes

RNA interference.

T23293

47149-47326

Sentence

denotes

CD4+ or naive CD4+ T cells were resuspended in 100 µl of Nucleofector solution (Lonza) and electroporated with 2 µM siRNA using the Nucleofector technology program U-14 (Lonza).

T323

47149-47326

Sentence

denotes

CD4+ or naive CD4+ T cells were resuspended in 100 µl of Nucleofector solution (Lonza) and electroporated with 2 µM siRNA using the Nucleofector technology program U-14 (Lonza).

T23294

47327-47558

Sentence

denotes

Five different Silencer or Silencer Select Pre-designed siRNAs for RUNX1 (Applied Biosystems) and three Silencer Pre-designed siRNAs for RUNX3 (Applied Biosystems) were tested, and the best was selected for all further experiments.

T324

47327-47558

Sentence

denotes

Five different Silencer or Silencer Select Pre-designed siRNAs for RUNX1 (Applied Biosystems) and three Silencer Pre-designed siRNAs for RUNX3 (Applied Biosystems) were tested, and the best was selected for all further experiments.

T23295

47559-47646

Sentence

denotes

The Silencer Negative Control #1 siRNA (Applied Biosystems) was used for normalization.

T325

47559-47646

Sentence

denotes

The Silencer Negative Control #1 siRNA (Applied Biosystems) was used for normalization.

T23296

47647-47750

Sentence

denotes

Cells were then left unstimulated or were stimulated after 12 h with anti-CD2, anti-CD3, and anti-CD28.

T326

47647-47750

Sentence

denotes

Cells were then left unstimulated or were stimulated after 12 h with anti-CD2, anti-CD3, and anti-CD28.

T23297

47751-47841

Sentence

denotes

Cells were cultured in serum-free AIM-V medium with the addition of 1 nmol/l IL-2 (Roche).

T327

47751-47841

Sentence

denotes

Cells were cultured in serum-free AIM-V medium with the addition of 1 nmol/l IL-2 (Roche).

T23298

47842-47950

Sentence

denotes

Cells were harvested for mRNA detection of the target genes after 24 h and for protein detection after 48 h.

T328

47842-47950

Sentence

denotes

Cells were harvested for mRNA detection of the target genes after 24 h and for protein detection after 48 h.

T23655

47952-47985

Sentence

denotes

RNA isolation and cDNA synthesis.

T329

47952-47985

Sentence

denotes

RNA isolation and cDNA synthesis.

T23656

47986-48079

Sentence

denotes

RNA was isolated using the RNeasy Mini kit (QIAGEN) according to the manufacturer's protocol.

T330

47986-48079

Sentence

denotes

RNA was isolated using the RNeasy Mini kit (QIAGEN) according to the manufacturer's protocol.

T23657

48080-48243

Sentence

denotes

Reverse transcription of human samples was performed with reverse-transcription reagents (Fermentas) with random hexamers according to the manufacturer's protocol.

T331

48080-48243

Sentence

denotes

Reverse transcription of human samples was performed with reverse-transcription reagents (Fermentas) with random hexamers according to the manufacturer's protocol.

T23818

48245-48259

Sentence

denotes

Real-time PCR.

T332

48245-48259

Sentence

denotes

Real-time PCR.

T23819

48260-48666

Sentence

denotes

PCR primers and probes were designed based on the sequences reported in GenBank with the Primer Express software version 1.2 (Applied Biosystems) as follows: FOXP3 forward primer, 5′-GAAACAGCACATTCCCAGAGTTC-3′; FOXP3 reverse primer, 5′-ATGGCCCAGCGGATGAG-3′; EF-1a forward primer, 5′-CTGAACCATCCAGGCCAAAT-3′; and EF-1a reverse primer, 5′-GCCGTGTGGCAATCCAAT-3′, as previously described (Mantel et al., 2007).

T333

48260-48666

Sentence

denotes

PCR primers and probes were designed based on the sequences reported in GenBank with the Primer Express software version 1.2 (Applied Biosystems) as follows: FOXP3 forward primer, 5′-GAAACAGCACATTCCCAGAGTTC-3′; FOXP3 reverse primer, 5′-ATGGCCCAGCGGATGAG-3′; EF-1a forward primer, 5′-CTGAACCATCCAGGCCAAAT-3′; and EF-1a reverse primer, 5′-GCCGTGTGGCAATCCAAT-3′, as previously described (Mantel et al., 2007).

T23820

48667-48793

Sentence

denotes

GATA3 forward primer, 5′-GCGGGCTCTATCACAAAATGA-3′; and GATA3 reverse primer 5′-GCTCTCCTGGCTGCAGACAGC-3′ (Mantel et al., 2007).

T334

48667-48793

Sentence

denotes

GATA3 forward primer, 5′-GCGGGCTCTATCACAAAATGA-3′; and GATA3 reverse primer 5′-GCTCTCCTGGCTGCAGACAGC-3′ (Mantel et al., 2007).

T23821

48794-49007

Sentence

denotes

T-bet forward primer, 5′-GATGCGCCAGGAAGTTTCAT-3′; T-bet reverse primer, 5′-GCACAATCATCTGGGTCACATT-3′; RORC2 forward primer, 5′-CAGTCATGAGAACACAAATTGAAGTG-3′; and RORC2 reverse primer 5′-CAGGTGATAACCCCGTAGTGGAT-3′.

T335

48794-49007

Sentence

denotes

T-bet forward primer, 5′-GATGCGCCAGGAAGTTTCAT-3′; T-bet reverse primer, 5′-GCACAATCATCTGGGTCACATT-3′; RORC2 forward primer, 5′-CAGTCATGAGAACACAAATTGAAGTG-3′; and RORC2 reverse primer 5′-CAGGTGATAACCCCGTAGTGGAT-3′.

T23822

49008-49207

Sentence

denotes

The prepared cDNAs were amplified using SYBR green PCR master mix (Fermentas) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

T336

49008-49207

Sentence

denotes

The prepared cDNAs were amplified using SYBR green PCR master mix (Fermentas) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

T23823

49208-49437

Sentence

denotes

RUNX1 and RUNX3 mRNA was detected by using TaqMan Gene Expression Assays from Applied Biosystems and used according to the manufacturer's instruction using TaqMan master mix using a 7000 real-time PCR system (Applied Biosystems).

T337

49208-49437

Sentence

denotes

RUNX1 and RUNX3 mRNA was detected by using TaqMan Gene Expression Assays from Applied Biosystems and used according to the manufacturer's instruction using TaqMan master mix using a 7000 real-time PCR system (Applied Biosystems).

T23824

49438-49637

Sentence

denotes

PCR amplification of the housekeeping gene encoding elongation factor (EF)-1α or by using the 18S rRNA Gene Expression Assay (Applied Biosystems) was performed to allow normalization between samples.

T338

49438-49637

Sentence

denotes

PCR amplification of the housekeeping gene encoding elongation factor (EF)-1α or by using the 18S rRNA Gene Expression Assay (Applied Biosystems) was performed to allow normalization between samples.

T23825

49638-49774

Sentence

denotes

Relative quantification and calculation of the range of confidence was performed using the comparative ΔΔCT method (Applied Biosystems).

T339

49638-49774

Sentence

denotes

Relative quantification and calculation of the range of confidence was performed using the comparative ΔΔCT method (Applied Biosystems).

T23826

49775-49932

Sentence

denotes

The percentage of FOXP3 mRNA in siRNA-mediated RUNX knockdown cells was calculated in relation to cells, which were transfected with scrambled control siRNA.

T340

49775-49932

Sentence

denotes

The percentage of FOXP3 mRNA in siRNA-mediated RUNX knockdown cells was calculated in relation to cells, which were transfected with scrambled control siRNA.

T23827

49933-50072

Sentence

denotes

Arbitrary units show the 2−(Δct) values multiplied by 10,000 incorporating the ct values of the gene of interest and the housekeeping gene.

T341

49933-50072

Sentence

denotes

Arbitrary units show the 2−(Δct) values multiplied by 10,000 incorporating the ct values of the gene of interest and the housekeeping gene.

T24562

50074-50089

Sentence

denotes

Flow cytometry.

T342

50074-50089

Sentence

denotes

Flow cytometry.

T24563

50090-50445

Sentence

denotes

For analysis of human FOXP3 expression on the single-cell level, cells were first stained with the monoclonal CD4 mAb (Beckman Coulter), and after fixation and permeabilization, they were incubated with anti–human Foxp3-Alexa Fluor 488 antibody (BioLegend) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL; Beckman Coulter).

T343

50090-50445

Sentence

denotes

For analysis of human FOXP3 expression on the single-cell level, cells were first stained with the monoclonal CD4 mAb (Beckman Coulter), and after fixation and permeabilization, they were incubated with anti–human Foxp3-Alexa Fluor 488 antibody (BioLegend) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL; Beckman Coulter).

T24564

50446-50511

Sentence

denotes

A mouse IgG1 antibody (BioLegend) was used as an isotype control.

T344

50446-50511

Sentence

denotes

A mouse IgG1 antibody (BioLegend) was used as an isotype control.

T24565

50512-50571

Sentence

denotes

Data were analyzed with the CXP software (Beckman Coulter).

T345

50512-50571

Sentence

denotes

Data were analyzed with the CXP software (Beckman Coulter).

T24566

50572-50673

Sentence

denotes

Cells were cultured with IL-2, and then left unstimulated or stimulated with anti-CD2/-CD3/-CD28 mAb.

T346

50572-50673

Sentence

denotes

Cells were cultured with IL-2, and then left unstimulated or stimulated with anti-CD2/-CD3/-CD28 mAb.

T24567

50674-50799

Sentence

denotes

Flow cytometry analyses of the mouse cells were performed on an LSRII (BD) and cell sorting was performed on a FACSAria (BD).

T347

50674-50799

Sentence

denotes

Flow cytometry analyses of the mouse cells were performed on an LSRII (BD) and cell sorting was performed on a FACSAria (BD).

T24568

50800-50875

Sentence

denotes

All antibodies for these experiments were purchased from eBioscience or BD.

T348

50800-50875

Sentence

denotes

All antibodies for these experiments were purchased from eBioscience or BD.

T24942

50877-50894

Sentence

denotes

Western blotting.

T349

50877-50894

Sentence

denotes

Western blotting.

T24943

50895-51071

Sentence

denotes

For human RUNX1 and RUNX3 analysis on the protein level, 106 cells were lysed and loaded next to a protein-mass ladder (Invitrogen) on a NuPAGE 4–12% Bis-Tris gel (Invitrogen).

T350

50895-51071

Sentence

denotes

For human RUNX1 and RUNX3 analysis on the protein level, 106 cells were lysed and loaded next to a protein-mass ladder (Invitrogen) on a NuPAGE 4–12% Bis-Tris gel (Invitrogen).

T24944

51072-51142

Sentence

denotes

The proteins were electroblotted onto a PVDF membrane (GE Healthcare).

T351

51072-51142

Sentence

denotes

The proteins were electroblotted onto a PVDF membrane (GE Healthcare).

T352

51143-51455

Sentence

denotes

Unspecific binding was blocked with 3% milk in TBS Tween, and the membranes were subsequently incubated with a 1:1,000 dilution of rabbit anti-RUNX1 (ab11903; Abcam) or 1:200 dilution of rabbit anti-RUNX3 (H-50; Santa Cruz Biotechnology, Inc.) in blocking buffer containing 3% milk in TBS Tween overnight at 4°C.

T24945

51143-51617

Sentence

denotes

Unspecific binding was blocked with 3% milk in TBS Tween, and the membranes were subsequently incubated with a 1:1,000 dilution of rabbit anti-RUNX1 (ab11903; Abcam) or 1:200 dilution of rabbit anti-RUNX3 (H-50; Santa Cruz Biotechnology, Inc.) in blocking buffer containing 3% milk in TBS Tween overnight at 4°C. The blots were developed using an anti-rabbit IgG HRP-labeled mAb (Cell Signaling Technology) and visualized with a LAS-1000 gel documentation system (Fujifilm).

T353

51456-51617

Sentence

denotes

The blots were developed using an anti-rabbit IgG HRP-labeled mAb (Cell Signaling Technology) and visualized with a LAS-1000 gel documentation system (Fujifilm).

T24946

51618-51850

Sentence

denotes

To confirm sample loading and transfer membranes were incubated in stripping buffer and reblocked for 1 h and reprobed using anti-GAPDH (6C5; Ambion) and developed using an anti–mouse IgG HRP-labeled mAb (Cell Signaling Technology).

T354

51618-51850

Sentence

denotes

To confirm sample loading and transfer membranes were incubated in stripping buffer and reblocked for 1 h and reprobed using anti-GAPDH (6C5; Ambion) and developed using an anti–mouse IgG HRP-labeled mAb (Cell Signaling Technology).

T25364

51852-51868

Sentence

denotes

Pull-down assay.

T355

51852-51868

Sentence

denotes

Pull-down assay.

T25365

51869-52014

Sentence

denotes

HEK293T cells were transfected with RUNX1 or RUNX3 using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction.

T356

51869-52014

Sentence

denotes

HEK293T cells were transfected with RUNX1 or RUNX3 using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instruction.

T25366

52015-52211

Sentence

denotes

Cells were lysed by sonication in HKMG buffer (10 mM Hepes, pH 7.9, 100 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM DTT, 0.5% Nonidet P-40) containing a protease inhibitor cocktail (Roche Diagnostics).

T357

52015-52211

Sentence

denotes

Cells were lysed by sonication in HKMG buffer (10 mM Hepes, pH 7.9, 100 mM KCl, 5 mM MgCl2, 10% glycerol, 1 mM DTT, 0.5% Nonidet P-40) containing a protease inhibitor cocktail (Roche Diagnostics).

T25367

52212-52467

Sentence

denotes

The cell lysate was precleared using streptavidin-agarose beads (GE Healthcare), incubated with biotinylated double-stranded oligonucleotides containing the wild-type or mutated RUNX binding sites, and polydeoxyinosinicdeoxycytidylic acid (Sigma-Aldrich).

T358

52212-52467

Sentence

denotes

The cell lysate was precleared using streptavidin-agarose beads (GE Healthcare), incubated with biotinylated double-stranded oligonucleotides containing the wild-type or mutated RUNX binding sites, and polydeoxyinosinicdeoxycytidylic acid (Sigma-Aldrich).

T25368

52468-52588

Sentence

denotes

A combination of all three oligonucleotides containing the mutated or the wild-type binding sites was used in the assay.

T359

52468-52588

Sentence

denotes

A combination of all three oligonucleotides containing the mutated or the wild-type binding sites was used in the assay.

T25369

52589-52865

Sentence

denotes

DNA-bound proteins were collected with streptavidin-agarose beads, washed with HKMG buffer, and finally resuspended in NuPAGE loading buffer (Invitrogen Life Technologies), heated to 70°C for 10 min, and separated on a NuPAGE 4–12% Bis-Tris gel (Invitrogen Life Technologies).

T360

52589-52865

Sentence

denotes

DNA-bound proteins were collected with streptavidin-agarose beads, washed with HKMG buffer, and finally resuspended in NuPAGE loading buffer (Invitrogen Life Technologies), heated to 70°C for 10 min, and separated on a NuPAGE 4–12% Bis-Tris gel (Invitrogen Life Technologies).

T25370

52866-53015

Sentence

denotes

The proteins were electroblotted onto a PVDF membrane (GE Healthcare) and detected using RUNX1 or RUNX3 antibodies described in the previous section.

T361

52866-53015

Sentence

denotes

The proteins were electroblotted onto a PVDF membrane (GE Healthcare) and detected using RUNX1 or RUNX3 antibodies described in the previous section.

T25839

53017-53046

Sentence

denotes

Promoter enzyme immuno assay.

T362

53017-53046

Sentence

denotes

Promoter enzyme immuno assay.

T25840

53047-53158

Sentence

denotes

As performed in the pull-down assay, HEK293T cells were transfected with RUNX1 or RUNX3 and subsequently lysed.

T363

53047-53158

Sentence

denotes

As performed in the pull-down assay, HEK293T cells were transfected with RUNX1 or RUNX3 and subsequently lysed.

T25841

53159-53208

Sentence

denotes

Insoluble material was removed by centrifugation.

T364

53159-53208

Sentence

denotes

Insoluble material was removed by centrifugation.

T25842

53209-53346

Sentence

denotes

384-well plates, precoated with streptavidin (Thermo Fisher Scientific) were washed 3 times with washing buffer (PBS and 0.05% Tween 20).

T365

53209-53346

Sentence

denotes

384-well plates, precoated with streptavidin (Thermo Fisher Scientific) were washed 3 times with washing buffer (PBS and 0.05% Tween 20).

T25843

53347-53516

Sentence

denotes

Biotinylated FOXP3 promoter/oligonucleotides probes containing the RUNX binding sites were added (1 pmol per well; 50 fmol/µl) and incubated for 1 h at room temperature.

T366

53347-53516

Sentence

denotes

Biotinylated FOXP3 promoter/oligonucleotides probes containing the RUNX binding sites were added (1 pmol per well; 50 fmol/µl) and incubated for 1 h at room temperature.

T25844

53517-53681

Sentence

denotes

Either a combination of all three oligonucleotides containing the mutated or the wild-type binding sites was used or single oligonucleotides were used in the assay.

T367

53517-53681

Sentence

denotes

Either a combination of all three oligonucleotides containing the mutated or the wild-type binding sites was used or single oligonucleotides were used in the assay.

T368

53682-53814

Sentence

denotes

After 3 washing steps with washing buffer, the nuclear extract was added (concentration > 0.2 µg/µl) and incubated overnight at 4°C.

T25845

53682-53911

Sentence

denotes

After 3 washing steps with washing buffer, the nuclear extract was added (concentration > 0.2 µg/µl) and incubated overnight at 4°C. The lysates were incubated with 10 µg of poly-deoxyinosinic-deoxycytidylic acid (Sigma-Aldrich).

T369

53815-53911

Sentence

denotes

The lysates were incubated with 10 µg of poly-deoxyinosinic-deoxycytidylic acid (Sigma-Aldrich).

T25846

53912-54118

Sentence

denotes

The plate was washed with HKMG buffer and incubated with a 1:1000 dilution of rabbit anti-RUNX1 (ab11903, Abcam) or 1:200 dilution of rabbit anti-RUNX3 (H-50, Santa Cruz Biotechnology, Inc.) at 4°C for 2 h.

T370

53912-54118

Sentence

denotes

The plate was washed with HKMG buffer and incubated with a 1:1000 dilution of rabbit anti-RUNX1 (ab11903, Abcam) or 1:200 dilution of rabbit anti-RUNX3 (H-50, Santa Cruz Biotechnology, Inc.) at 4°C for 2 h.

T371

54119-54311

Sentence

denotes

After three washing steps with HKMG buffer, a secondary antibody (anti–rabbit IgG-HRP, 1:3,000 in HKMG buffer, Cell Signaling Technology) was added, and the plate was incubated for 1 h at 4°C.

T25847

54119-54409

Sentence

denotes

After three washing steps with HKMG buffer, a secondary antibody (anti–rabbit IgG-HRP, 1:3,000 in HKMG buffer, Cell Signaling Technology) was added, and the plate was incubated for 1 h at 4°C. The wells were washed 4 times with HKMG buffer before adding the substrate reagent (R&D Systems).

T372

54312-54409

Sentence

denotes

The wells were washed 4 times with HKMG buffer before adding the substrate reagent (R&D Systems).

T25848

54410-54468

Sentence

denotes

The colorimetric reaction was stopped by adding 2 M H2SO4.

T373

54410-54468

Sentence

denotes

The colorimetric reaction was stopped by adding 2 M H2SO4.

T25849

54469-54553

Sentence

denotes

Absorbance at 450 nm was measured using a microplate reader (Berthold Technologies).

T374

54469-54553

Sentence

denotes

Absorbance at 450 nm was measured using a microplate reader (Berthold Technologies).

T26525

54555-54560

Sentence

denotes

ChIP.

T375

54555-54560

Sentence

denotes

ChIP.

T26526

54561-54788

Sentence

denotes

Human naive CD4+ T cells were cultured either with IL-2 only or with IL-2, anti-CD2/3/28, and TGF-β for 72 h, and protein–DNA complexes were fixed by cross-linking with formaldehyde in a final concentration of 1.42% for 15 min.

T376

54561-54788

Sentence

denotes

Human naive CD4+ T cells were cultured either with IL-2 only or with IL-2, anti-CD2/3/28, and TGF-β for 72 h, and protein–DNA complexes were fixed by cross-linking with formaldehyde in a final concentration of 1.42% for 15 min.

T26527

54789-54884

Sentence

denotes

Formaldehyde was quenched with 125 mM glycine for 5 min, and cells were subsequently harvested.

T377

54789-54884

Sentence

denotes

Formaldehyde was quenched with 125 mM glycine for 5 min, and cells were subsequently harvested.

T26528

54885-54998

Sentence

denotes

The ChIP assay was performed as described in the fast chromatin immunoprecipitation method (Nelson et al., 2006).

T378

54885-54998

Sentence

denotes

The ChIP assay was performed as described in the fast chromatin immunoprecipitation method (Nelson et al., 2006).

T26529

54999-55607

Sentence

denotes

Cells were lysed with immunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, NP-40 [0.5% vol/vol]) containing phosphatase (Roche) and protease inhibitors cocktails (Roche), the nuclear pellet was washed, the chromatin was sheared by sonication and incubated with antibodies for RUNX1 (H-65 X; Santa Cruz Biotechnology, Inc.), RUNX3 (H-50 X; Santa Cruz Biotechnology, Inc.), CBFβ (PEBP2β; FL-182 X; Santa Cruz Biotechnology, Inc.), and as controls normal rabbit IgG (Santa Cruz Biotechnology, Inc.), anti-human RNA polymerase II antibody, and mouse control IgG (both from SA Biosciences).

T379

54999-55607

Sentence

denotes

Cells were lysed with immunoprecipitation buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, NP-40 [0.5% vol/vol]) containing phosphatase (Roche) and protease inhibitors cocktails (Roche), the nuclear pellet was washed, the chromatin was sheared by sonication and incubated with antibodies for RUNX1 (H-65 X; Santa Cruz Biotechnology, Inc.), RUNX3 (H-50 X; Santa Cruz Biotechnology, Inc.), CBFβ (PEBP2β; FL-182 X; Santa Cruz Biotechnology, Inc.), and as controls normal rabbit IgG (Santa Cruz Biotechnology, Inc.), anti-human RNA polymerase II antibody, and mouse control IgG (both from SA Biosciences).

T26530

55608-55759

Sentence

denotes

The cleared chromatin was incubated with protein A agarose beads and, after several washing steps, DNA was isolated with 10% (wt/vol) Chelex 100 resin.

T380

55608-55759

Sentence

denotes

The cleared chromatin was incubated with protein A agarose beads and, after several washing steps, DNA was isolated with 10% (wt/vol) Chelex 100 resin.

T26531

55760-55818

Sentence

denotes

Samples were treated with proteinase K at 55°C for 30 min.

T381

55760-55818

Sentence

denotes

Samples were treated with proteinase K at 55°C for 30 min.

T26532

55819-55891

Sentence

denotes

The proteinase K was then inactivated by boiling the samples for 10 min.

T382

55819-55891

Sentence

denotes

The proteinase K was then inactivated by boiling the samples for 10 min.

T26533

55892-55946

Sentence

denotes

The purified DNA was used in a real-time PCR reaction.

T383

55892-55946

Sentence

denotes

The purified DNA was used in a real-time PCR reaction.

T26534

55947-56220

Sentence

denotes

Specific primers for the FOXP3 promoter, spanning the region from −87 to −3, FOXP3 promoter forward primer 5′-AGAGGTCTGCGGCTTCCA-3′, FOXP3 promoter reverse primer 5′-GGAAACTGTCACGTATCAAAAACAA-3′, or control GAPDH primer (SA Biosciences) for the RNA polymerase II were used.

T384

55947-56220

Sentence

denotes

Specific primers for the FOXP3 promoter, spanning the region from −87 to −3, FOXP3 promoter forward primer 5′-AGAGGTCTGCGGCTTCCA-3′, FOXP3 promoter reverse primer 5′-GGAAACTGTCACGTATCAAAAACAA-3′, or control GAPDH primer (SA Biosciences) for the RNA polymerase II were used.

T26535

56221-56369

Sentence

denotes

A negative control PCR for each immunoprecipitation using IGX1A negative control primer targeting ORF-free intergenic DNA (SA Biosciences) was used.

T385

56221-56369

Sentence

denotes

A negative control PCR for each immunoprecipitation using IGX1A negative control primer targeting ORF-free intergenic DNA (SA Biosciences) was used.

T26536

56370-56541

Sentence

denotes

The fold enrichment in site occupancy was calculated incorporating IgG control values and input DNA values using the ChampionChIP qPCR data analysis file (SA Biosciences).

T386

56370-56541

Sentence

denotes

The fold enrichment in site occupancy was calculated incorporating IgG control values and input DNA values using the ChampionChIP qPCR data analysis file (SA Biosciences).

T27094

56543-56577

Sentence

denotes

Quantification of cytokine levels.

T387

56543-56577

Sentence

denotes

Quantification of cytokine levels.

T27095

56578-56690

Sentence

denotes

IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, and IFN-γ secretion was assessed using fluorescent bead-based technology.

T388

56578-56690

Sentence

denotes

IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, and IFN-γ secretion was assessed using fluorescent bead-based technology.

T27096

56691-56816

Sentence

denotes

The Bio-Plex-hu Cytokine Panel, 17-Plex Group 1 was used according to the manufacturer's instructions (Bio-Rad Laboratories).

T389

56691-56816

Sentence

denotes

The Bio-Plex-hu Cytokine Panel, 17-Plex Group 1 was used according to the manufacturer's instructions (Bio-Rad Laboratories).

T27097

56817-56913

Sentence

denotes

Fluorescent signals were read and analyzed using the Bio-Plex 200 System (Bio-Rad Laboratories).

T390

56817-56913

Sentence

denotes

Fluorescent signals were read and analyzed using the Bio-Plex 200 System (Bio-Rad Laboratories).

T27510

56915-56944

Sentence

denotes

Online supplemental material.

T391

56915-56944

Sentence

denotes

Online supplemental material.

T27511

56945-57073

Sentence

denotes

Fig. S1 shows the induction of RUNX1, RUNX3, and FOXP3 mRNA in human CD4+ T cells after anti-CD2/3/28 mAb and TGF-β stimulation.

T392

56945-57073

Sentence

denotes

Fig. S1 shows the induction of RUNX1, RUNX3, and FOXP3 mRNA in human CD4+ T cells after anti-CD2/3/28 mAb and TGF-β stimulation.

T27512

57074-57258

Sentence

denotes

Fig. S2 shows decreased RUNX1 and RUNX3 mRNA and protein expression after siRNA-mediated knockdown and decreased FOXP3 expression in human CD4+ T cells after RUNX1 and RUNX3 knockdown.

T393

57074-57258

Sentence

denotes

Fig. S2 shows decreased RUNX1 and RUNX3 mRNA and protein expression after siRNA-mediated knockdown and decreased FOXP3 expression in human CD4+ T cells after RUNX1 and RUNX3 knockdown.

T27513

57259-57423

Sentence

denotes

Fig. S3 shows the quantification of IL-4, IL-5, IL-10, IL-13, and IFN-γ levels in control siRNA transfected or RUNX1 and RUNX3 siRNA transfected human CD4+ T cells.

T394

57259-57423

Sentence

denotes

Fig. S3 shows the quantification of IL-4, IL-5, IL-10, IL-13, and IFN-γ levels in control siRNA transfected or RUNX1 and RUNX3 siRNA transfected human CD4+ T cells.

T27514

57424-57531

Sentence

denotes

Fig. S4 shows the putative RUNX binding sites in the FOXP3 core promoter sequence of human, mouse, and rat.

T395

57424-57531

Sentence

denotes

Fig. S4 shows the putative RUNX binding sites in the FOXP3 core promoter sequence of human, mouse, and rat.

T27515

57532-57639

Sentence

denotes

Fig. S5 shows the induction of FOXP3 protein after overexpression of RUNX1 and RUNX3 in human CD4+ T cells.

T396

57532-57639

Sentence

denotes

Fig. S5 shows the induction of FOXP3 protein after overexpression of RUNX1 and RUNX3 in human CD4+ T cells.

T27516

57640-57927

Sentence

denotes

Fig. S6 shows that endogenous IL-4 and IFN-γ do not effect Foxp3 expression in naive CD4+ T cells of CbfbF/F CD4-cre and CbfbF/F control mice, which were stimulated with anti-CD3 and anti-CD28 mAbs, IL-2 and TGF-β in the absence or presence of anti-IL-4 and anti-IFN-γ neutralizing mAbs.

T397

57640-57927

Sentence

denotes

Fig. S6 shows that endogenous IL-4 and IFN-γ do not effect Foxp3 expression in naive CD4+ T cells of CbfbF/F CD4-cre and CbfbF/F control mice, which were stimulated with anti-CD3 and anti-CD28 mAbs, IL-2 and TGF-β in the absence or presence of anti-IL-4 and anti-IFN-γ neutralizing mAbs.

T27517

57928-58067

Sentence

denotes

Fig. S7 shows similar cell death (A) and proliferation (B) of Foxp3+ and Foxp3− cells in CbfbF/F CD4-cre and CbfbF/F control mice cultures.

T398

57928-58067

Sentence

denotes

Fig. S7 shows similar cell death (A) and proliferation (B) of Foxp3+ and Foxp3− cells in CbfbF/F CD4-cre and CbfbF/F control mice cultures.

T27518

58068-58166

Sentence

denotes

Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20090596/DC1.

T399

58068-58166

Sentence

denotes

Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20090596/DC1.

PMC:2806624 JSON TXT 21 Projects

Annnotations TAB TSV DIC JSON TextAE Lectin_function IAV-Glycan

PMC:2806624 JSONTXT 21 Projects

Annnotations TAB TSV DIC JSON TextAE Lectin_function IAV-Glycan

PMC:2806624 JSON TXT 21 Projects