PubAnnotation

Id	Subject	Object	Predicate	Lexical cue
T58	0-106	Sentence	denotes	GATA3-Driven Th2 Responses Inhibit TGF-β1–Induced FOXP3 Expression and the Formation of Regulatory T Cells
T1	0-147	Sentence	denotes	GATA3-Driven Th2 Responses Inhibit TGF-β1–Induced FOXP3 Expression and the Formation of Regulatory T Cells GATA3 Inhibits FOXP3 and Inducible Tregs
T2	150-158	Sentence	denotes	Abstract
T59	159-387	Sentence	denotes	Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes.
T3	159-387	Sentence	denotes	Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes.
T60	388-575	Sentence	denotes	FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown.
T4	388-575	Sentence	denotes	FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown.
T61	576-686	Sentence	denotes	We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway.
T5	576-686	Sentence	denotes	We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway.
T62	687-782	Sentence	denotes	We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3.
T6	687-782	Sentence	denotes	We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3.
T63	783-1004	Sentence	denotes	This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-β–mediated FOXP3 induction.
T7	783-1004	Sentence	denotes	This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-β–mediated FOXP3 induction.
T64	1005-1127	Sentence	denotes	This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process.
T8	1005-1127	Sentence	denotes	This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process.
T65	1128-1244	Sentence	denotes	Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response.
T9	1128-1244	Sentence	denotes	Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response.
T66	1245-1493	Sentence	denotes	IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.
T10	1245-1493	Sentence	denotes	IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.
T11	1495-1509	Sentence	denotes	Author Summary
T12	1511-1724	Sentence	denotes	Specific immune responses against foreign or autologous antigens are driven by specialized epitope-specific T cells, whose numbers expand upon recognition of antigen found on professional antigen-presenting cells.
T13	1725-1944	Sentence	denotes	The subsequent maturation process involves the differentiation of certain T cell phenotypes such as pro-inflammatory cells (Th1, Th2, Th17) or regulatory T (Treg) cells, which serve to keep the immune response in check.
T14	1945-2077	Sentence	denotes	The current study focuses on the role of two key transcription factors—FOXP3 and GATA3—in controlling the commitment of these cells.
T15	2078-2209	Sentence	denotes	We demonstrate that the Th2 cytokine IL-4 inhibits the induction of FOXP3 and thus inhibits the generation of inducible Treg cells.
T16	2210-2328	Sentence	denotes	We show that IL-4–induced GATA3 mediates FOXP3 inhibition by directly binding to a GATA element in the FOXP3 promoter.
T17	2329-2544	Sentence	denotes	We hypothesize that therapeutic agents aimed at neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and thus promotion of tolerance in allergies and other Th2-dominated diseases.
T18	2546-2619	Sentence	denotes	It is shown that Th2 responses prevent the generation of inducible Tregs.
T19	2620-2722	Sentence	denotes	This is mediated by IL-4 induction of GATA3, which binds directly to and represses the FOXP3 promoter.
T20	2723-2834	Sentence	denotes	This mechanism is likely to be relevant in the induction of immunotolerance, particularly in allergic diseases.
T21	2836-2848	Sentence	denotes	Introduction
T975	2849-2997	Sentence	denotes	Effective immune responses are characterized by T cell activation, which directs adaptive and innate immune responses to kill pathogens efficiently.
T22	2849-2997	Sentence	denotes	Effective immune responses are characterized by T cell activation, which directs adaptive and innate immune responses to kill pathogens efficiently.
T976	2998-3181	Sentence	denotes	Dependent on the pathogen, T cells differentiate into different subtypes, such as Th1 or Th2 cells, which are most efficient in defeating microbial or parasitic invaders respectively.
T23	2998-3181	Sentence	denotes	Dependent on the pathogen, T cells differentiate into different subtypes, such as Th1 or Th2 cells, which are most efficient in defeating microbial or parasitic invaders respectively.
T977	3182-3341	Sentence	denotes	A hallmark of Th1 and Th2 differentiation pathways is the exclusiveness of the individual phenotype leading to either Th1 or Th2, but not to mixed populations.
T24	3182-3341	Sentence	denotes	A hallmark of Th1 and Th2 differentiation pathways is the exclusiveness of the individual phenotype leading to either Th1 or Th2, but not to mixed populations.
T978	3342-3482	Sentence	denotes	The exclusiveness of this mechanism is provided by a polarization process, where Th2 differentiation inhibits Th1 commitment and vice versa.
T25	3342-3482	Sentence	denotes	The exclusiveness of this mechanism is provided by a polarization process, where Th2 differentiation inhibits Th1 commitment and vice versa.
T979	3483-3619	Sentence	denotes	Specifically interleukin 4 (IL-4)–induced STAT6 and GATA3 inhibit differentiation into Th1 cells in the early phase of commitment [1,2].
T26	3483-3619	Sentence	denotes	Specifically interleukin 4 (IL-4)–induced STAT6 and GATA3 inhibit differentiation into Th1 cells in the early phase of commitment [1,2].
T980	3620-3819	Sentence	denotes	GATA3 is sufficient to induce a Th2 phenotype [3] and acts not only through the induction of IL-4, IL-5 and IL-13, the Th2 cytokines, but also through the inhibition of Th1 cell-specific factors [3].
T27	3620-3819	Sentence	denotes	GATA3 is sufficient to induce a Th2 phenotype [3] and acts not only through the induction of IL-4, IL-5 and IL-13, the Th2 cytokines, but also through the inhibition of Th1 cell-specific factors [3].
T981	3820-4074	Sentence	denotes	Recently, it was shown that T-bet directly modulates GATA3 function, suggesting that transcription factors compete in the early differentiation phase of T cells, potentially integrating environmental signals to finally imprint the T cell phenotype [4,5].
T28	3820-4074	Sentence	denotes	Recently, it was shown that T-bet directly modulates GATA3 function, suggesting that transcription factors compete in the early differentiation phase of T cells, potentially integrating environmental signals to finally imprint the T cell phenotype [4,5].
T982	4075-4250	Sentence	denotes	A GATA3-dominated immune response has been shown to be essential in airway hyperresonsiveness [6] and IL-4–dominated responses can break antigen-specific immune tolerance [7].
T29	4075-4250	Sentence	denotes	A GATA3-dominated immune response has been shown to be essential in airway hyperresonsiveness [6] and IL-4–dominated responses can break antigen-specific immune tolerance [7].
T983	4251-4430	Sentence	denotes	Overexpression of a dominant negative form of GATA3 [8] or treatment with antisense-mediated GATA3 blockade [9] decreased the severity of the allergic airway hyper-responsiveness.
T30	4251-4430	Sentence	denotes	Overexpression of a dominant negative form of GATA3 [8] or treatment with antisense-mediated GATA3 blockade [9] decreased the severity of the allergic airway hyper-responsiveness.
T984	4431-4570	Sentence	denotes	The discovery of regulatory T (Treg) cells highlights another phenotype of T cells, which is essential for tolerance against self-antigens.
T31	4431-4570	Sentence	denotes	The discovery of regulatory T (Treg) cells highlights another phenotype of T cells, which is essential for tolerance against self-antigens.
T985	4571-4743	Sentence	denotes	Naturally occurring, thymus-derived Treg (nTreg) cells are generated in the thymus and are assumed to protect against the activity of autoreactive T cells in the periphery.
T32	4571-4743	Sentence	denotes	Naturally occurring, thymus-derived Treg (nTreg) cells are generated in the thymus and are assumed to protect against the activity of autoreactive T cells in the periphery.
T986	4744-4903	Sentence	denotes	These cells express the forkhead transcription factor FOXP3 and constitutively express CD25 on their surface, but they lack expression of Th1 or Th2 cytokines.
T33	4744-4903	Sentence	denotes	These cells express the forkhead transcription factor FOXP3 and constitutively express CD25 on their surface, but they lack expression of Th1 or Th2 cytokines.
T987	4904-5047	Sentence	denotes	Particularly interesting are those Treg cells that are generated in the periphery and thus are potential targets for therapeutic interventions.
T34	4904-5047	Sentence	denotes	Particularly interesting are those Treg cells that are generated in the periphery and thus are potential targets for therapeutic interventions.
T988	5048-5117	Sentence	denotes	These induced Treg (iTreg) cells were reported to express FOXP3 [10].
T35	5048-5117	Sentence	denotes	These induced Treg (iTreg) cells were reported to express FOXP3 [10].
T989	5118-5355	Sentence	denotes	The exact mechanisms of iTreg generation are unclear, but T cell receptor (TCR) triggering has been shown to induce FOXP3 expression and suppressive cells in human [11,12], however the phenotype appears to be of transient nature [13,14].
T36	5118-5355	Sentence	denotes	The exact mechanisms of iTreg generation are unclear, but T cell receptor (TCR) triggering has been shown to induce FOXP3 expression and suppressive cells in human [11,12], however the phenotype appears to be of transient nature [13,14].
T990	5356-5625	Sentence	denotes	TGF-β has been demonstrated to be important for the persistent induction of these cells in vitro and in vivo, since animals lacking the TGF-βRII on T cells have fewer peripherally iTreg cells [15] and suffer from a T cell–dependent multiorgan inflammatory disease [16].
T37	5356-5625	Sentence	denotes	TGF-β has been demonstrated to be important for the persistent induction of these cells in vitro and in vivo, since animals lacking the TGF-βRII on T cells have fewer peripherally iTreg cells [15] and suffer from a T cell–dependent multiorgan inflammatory disease [16].
T991	5626-5791	Sentence	denotes	Although the effect of TGF-β on natural and inducible Treg cell induction has been demonstrated repeatedly [15,17], its molecular mechanisms remain to be identified.
T38	5626-5791	Sentence	denotes	Although the effect of TGF-β on natural and inducible Treg cell induction has been demonstrated repeatedly [15,17], its molecular mechanisms remain to be identified.
T992	5792-5963	Sentence	denotes	The current study provides evidence that GATA3 and FOXP3 play a competitive role in iTreg cell commitment as T-bet and GATA3 for Th1 and Th2 differentiation, respectively.
T39	5792-5963	Sentence	denotes	The current study provides evidence that GATA3 and FOXP3 play a competitive role in iTreg cell commitment as T-bet and GATA3 for Th1 and Th2 differentiation, respectively.
T993	5964-6097	Sentence	denotes	We show that GATA3 inhibits FOXP3 induction and that IL-4 limits FOXP3 expression in a GATA3-mediated way, both in vitro and in vivo.
T40	5964-6097	Sentence	denotes	We show that GATA3 inhibits FOXP3 induction and that IL-4 limits FOXP3 expression in a GATA3-mediated way, both in vitro and in vivo.
T994	6098-6273	Sentence	denotes	We also show that GATA3 directly binds to the FOXP3 promoter and thereby prevents the induction of this gene, demonstrating that Th2 differentiation overrules iTreg induction.
T41	6098-6273	Sentence	denotes	We also show that GATA3 directly binds to the FOXP3 promoter and thereby prevents the induction of this gene, demonstrating that Th2 differentiation overrules iTreg induction.
T42	6275-6282	Sentence	denotes	Results
T2865	6284-6317	Sentence	denotes	FOXP3 Induction in T Cell Subsets
T43	6284-6317	Sentence	denotes	FOXP3 Induction in T Cell Subsets
T2866	6318-6452	Sentence	denotes	It is assumed that FOXP3 expression can be induced in nonregulatory T cells, which is an important step in iTreg cell differentiation.
T44	6318-6452	Sentence	denotes	It is assumed that FOXP3 expression can be induced in nonregulatory T cells, which is an important step in iTreg cell differentiation.
T2867	6453-6538	Sentence	denotes	However, it is not known if all CD4+ T cells have the same capacity to express FOXP3.
T45	6453-6538	Sentence	denotes	However, it is not known if all CD4+ T cells have the same capacity to express FOXP3.
T2868	6539-6918	Sentence	denotes	To investigate whether FOXP3 can be expressed by any T cell subset or if expression is restricted to a distinct lineage, FOXP3 mRNA expression was analyzed in freshly isolated T cells such as CD25-depleted CD4+ cells, CD45RA+ naive or CD45RO+ memory T cells (Figure 1A), as well as T cells driven in vitro toward Th1, Th2, or iTreg phenotypes (Figure 1B; phenotype on Figure S1).
T46	6539-6918	Sentence	denotes	To investigate whether FOXP3 can be expressed by any T cell subset or if expression is restricted to a distinct lineage, FOXP3 mRNA expression was analyzed in freshly isolated T cells such as CD25-depleted CD4+ cells, CD45RA+ naive or CD45RO+ memory T cells (Figure 1A), as well as T cells driven in vitro toward Th1, Th2, or iTreg phenotypes (Figure 1B; phenotype on Figure S1).
T2869	6919-7074	Sentence	denotes	The CD4+CD25–, CD45RA+, CD45RO+, and CD4+CD45RO+CD25– were able to significantly induce FOXP3 mRNA up to 30-fold upon TCR activation and addition of TGF-β.
T47	6919-7074	Sentence	denotes	The CD4+CD25–, CD45RA+, CD45RO+, and CD4+CD45RO+CD25– were able to significantly induce FOXP3 mRNA up to 30-fold upon TCR activation and addition of TGF-β.
T2870	7075-7116	Sentence	denotes	Th1 cells showed only a 10-fold increase.
T48	7075-7116	Sentence	denotes	Th1 cells showed only a 10-fold increase.
T2871	7117-7211	Sentence	denotes	In contrast, Th2 cells stimulated under the same conditions did not increase FOXP3 expression.
T49	7117-7211	Sentence	denotes	In contrast, Th2 cells stimulated under the same conditions did not increase FOXP3 expression.
T2872	7212-7376	Sentence	denotes	The in vitro generated iTreg cells were unable to further up-regulate FOXP3, which was already at high levels under the resting conditions (Figure 1B, right panel).
T50	7212-7376	Sentence	denotes	The in vitro generated iTreg cells were unable to further up-regulate FOXP3, which was already at high levels under the resting conditions (Figure 1B, right panel).
T51	7377-7427	Sentence	denotes	Figure 1 Th2 Cells Cannot Induce FOXP3 Expression
T23457	7387-7427	Sentence	denotes	Th2 Cells Cannot Induce FOXP3 Expression
T2873	7387-8083	Sentence	denotes	Th2 Cells Cannot Induce FOXP3 Expression (A) Human T cells were activated with plate-bound anti-CD3/CD28 with or without TGF-β as indicated on the x-axis of (B). Cells were harvested after 5 days and FOXP3 mRNA was quantified by real-time PCR. Bars show the mean ± SD of 4 independent experiments. (B) In vitro differentiated Th1, Th2, or iTreg cells were activated with anti-CD3/CD28, TGF-β, or anti-IL-4 as indicated. The phenotype of these cells was confirmed by FACS and proliferation analysis (Figure S1). Bars show the mean ± SD of four independent experiments. Th2 cells are known to produce IL-4 upon activation, which may interact with TGF-β signaling and thus prevent FOXP3 induction.
T23458	7428-7548	Sentence	denotes	(A) Human T cells were activated with plate-bound anti-CD3/CD28 with or without TGF-β as indicated on the x-axis of (B).
T52	7428-7548	Sentence	denotes	(A) Human T cells were activated with plate-bound anti-CD3/CD28 with or without TGF-β as indicated on the x-axis of (B).
T23459	7549-7630	Sentence	denotes	Cells were harvested after 5 days and FOXP3 mRNA was quantified by real-time PCR.
T53	7549-7630	Sentence	denotes	Cells were harvested after 5 days and FOXP3 mRNA was quantified by real-time PCR.
T23460	7631-7684	Sentence	denotes	Bars show the mean ± SD of 4 independent experiments.
T54	7631-7684	Sentence	denotes	Bars show the mean ± SD of 4 independent experiments.
T23461	7685-7806	Sentence	denotes	(B) In vitro differentiated Th1, Th2, or iTreg cells were activated with anti-CD3/CD28, TGF-β, or anti-IL-4 as indicated.
T55	7685-7806	Sentence	denotes	(B) In vitro differentiated Th1, Th2, or iTreg cells were activated with anti-CD3/CD28, TGF-β, or anti-IL-4 as indicated.
T23462	7807-7897	Sentence	denotes	The phenotype of these cells was confirmed by FACS and proliferation analysis (Figure S1).
T56	7807-7897	Sentence	denotes	The phenotype of these cells was confirmed by FACS and proliferation analysis (Figure S1).
T23463	7898-7954	Sentence	denotes	Bars show the mean ± SD of four independent experiments.
T57	7898-7954	Sentence	denotes	Bars show the mean ± SD of four independent experiments.
T58	7957-8083	Sentence	denotes	Th2 cells are known to produce IL-4 upon activation, which may interact with TGF-β signaling and thus prevent FOXP3 induction.
T2874	8084-8233	Sentence	denotes	However, the neutralization of IL-4 with a blocking IL-4 antibody did not rescue FOXP3 expression in the differentiated Th2 cells (unpublished data).
T59	8084-8233	Sentence	denotes	However, the neutralization of IL-4 with a blocking IL-4 antibody did not rescue FOXP3 expression in the differentiated Th2 cells (unpublished data).
T2875	8234-8326	Sentence	denotes	These data demonstrated that Th2 cells have a limited capacity to express FOXP3 (Figure 1B).
T60	8234-8326	Sentence	denotes	These data demonstrated that Th2 cells have a limited capacity to express FOXP3 (Figure 1B).
T2876	8327-8477	Sentence	denotes	The inability of Th2 cells to express FOXP3 was also documented at the single-cell level, confirming that Th2 cells lack FOXP3 expression (Figure 2A).
T61	8327-8477	Sentence	denotes	The inability of Th2 cells to express FOXP3 was also documented at the single-cell level, confirming that Th2 cells lack FOXP3 expression (Figure 2A).
T2877	8478-8533	Sentence	denotes	Only iTreg cells expressed FOXP3 in resting conditions.
T62	8478-8533	Sentence	denotes	Only iTreg cells expressed FOXP3 in resting conditions.
T2878	8534-8637	Sentence	denotes	Interestingly, we observed that resting iTreg cells express FOXP3 but show low CD25 surface expression.
T63	8534-8637	Sentence	denotes	Interestingly, we observed that resting iTreg cells express FOXP3 but show low CD25 surface expression.
T2879	8638-8786	Sentence	denotes	Repeated exposure to TGF-β did not further increase the FOXP3 expression in the iTreg lineage but transiently induced FOXP3 expression in Th1 cells.
T64	8638-8786	Sentence	denotes	Repeated exposure to TGF-β did not further increase the FOXP3 expression in the iTreg lineage but transiently induced FOXP3 expression in Th1 cells.
T2880	8787-8980	Sentence	denotes	Naturally occurring Th2 cells such as CRTH2+ T cells, T cells isolated according to their IL-4 secretion, or an IL-4–producing T cell clone (BR8) were also lacking FOXP3 expression (Figure 2B).
T65	8787-8980	Sentence	denotes	Naturally occurring Th2 cells such as CRTH2+ T cells, T cells isolated according to their IL-4 secretion, or an IL-4–producing T cell clone (BR8) were also lacking FOXP3 expression (Figure 2B).
T2881	8981-9091	Sentence	denotes	Furthermore TGF-β–mediated FOXP3 induction failed in these cells in contrast to the naive T cells (Figure 2B).
T66	8981-9091	Sentence	denotes	Furthermore TGF-β–mediated FOXP3 induction failed in these cells in contrast to the naive T cells (Figure 2B).
T2882	9092-9255	Sentence	denotes	Because IL-4 is the key Th2 cytokine, the expression of IL-4 and FOXP3 in freshly isolated CD4+ T cells was analyzed by fluorescence activated cell sorting (FACS).
T67	9092-9255	Sentence	denotes	Because IL-4 is the key Th2 cytokine, the expression of IL-4 and FOXP3 in freshly isolated CD4+ T cells was analyzed by fluorescence activated cell sorting (FACS).
T2883	9256-9378	Sentence	denotes	IL-4–expressing cells were most abundant among CD45RO+CD25– cells, which did not co-express FOXP3 (Figure 3A, left panel).
T68	9256-9378	Sentence	denotes	IL-4–expressing cells were most abundant among CD45RO+CD25– cells, which did not co-express FOXP3 (Figure 3A, left panel).
T2884	9379-9483	Sentence	denotes	In contrast, CD45RO+CD25+ cells abundantly expressed FOXP3, while lacking IL-4 (Figure 3A, right panel).
T69	9379-9483	Sentence	denotes	In contrast, CD45RO+CD25+ cells abundantly expressed FOXP3, while lacking IL-4 (Figure 3A, right panel).
T2885	9484-9604	Sentence	denotes	As shown in Figure 3B, the frequency of the IL-4+ cells was always below 1% in the FOXP3+ cells close to the background.
T70	9484-9604	Sentence	denotes	As shown in Figure 3B, the frequency of the IL-4+ cells was always below 1% in the FOXP3+ cells close to the background.
T2886	9605-9740	Sentence	denotes	The IL-4+ cells were confined to the FOXP3– cells, as shown for the CD45RO+CD25–, CD45RO+CD25+, and CD45RO+CD25+high cells (Figure 3B).
T71	9605-9740	Sentence	denotes	The IL-4+ cells were confined to the FOXP3– cells, as shown for the CD45RO+CD25–, CD45RO+CD25+, and CD45RO+CD25+high cells (Figure 3B).
T2887	9741-9840	Sentence	denotes	In addition, neither the Th2 clone (BR8) nor CRTH2 cells significantly expressed FOXP3 (Figure 3C).
T72	9741-9840	Sentence	denotes	In addition, neither the Th2 clone (BR8) nor CRTH2 cells significantly expressed FOXP3 (Figure 3C).
T2888	9841-10013	Sentence	denotes	Cells enriched for their IL-4 secretion using the magnetic cell isolation technology contained some FOXP3-expressing cells, but importantly, the expression did not overlap.
T73	9841-10013	Sentence	denotes	Cells enriched for their IL-4 secretion using the magnetic cell isolation technology contained some FOXP3-expressing cells, but importantly, the expression did not overlap.
T2889	10014-10129	Sentence	denotes	Taken together, these data indicate that FOXP3 was not expressed by Th2 cells and was not inducible in those cells.
T74	10014-10129	Sentence	denotes	Taken together, these data indicate that FOXP3 was not expressed by Th2 cells and was not inducible in those cells.
T75	10130-10170	Sentence	denotes	Figure 2 Th2 Cells Do Not Express FOXP3
T23808	10140-10170	Sentence	denotes	Th2 Cells Do Not Express FOXP3
T23809	10171-10348	Sentence	denotes	(A) Intracellular FACS analysis of FOXP3 expression in Th1, Th2, or iTreg-differentiated cells (two rounds, phenotype see Figure S1), rested or activated, with or without TGF-β.
T76	10171-10348	Sentence	denotes	(A) Intracellular FACS analysis of FOXP3 expression in Th1, Th2, or iTreg-differentiated cells (two rounds, phenotype see Figure S1), rested or activated, with or without TGF-β.
T23810	10349-10400	Sentence	denotes	FOXP3 expression was measured after 5 d in culture.
T77	10349-10400	Sentence	denotes	FOXP3 expression was measured after 5 d in culture.
T23811	10401-10467	Sentence	denotes	The dot blots are representative of three independent experiments.
T78	10401-10467	Sentence	denotes	The dot blots are representative of three independent experiments.
T23812	10468-10555	Sentence	denotes	(B) Shows the same experimental setup, but naturally occurring Th2 cells were analyzed.
T79	10468-10555	Sentence	denotes	(B) Shows the same experimental setup, but naturally occurring Th2 cells were analyzed.
T23813	10556-10613	Sentence	denotes	Data are representative of three independent experiments.
T80	10556-10613	Sentence	denotes	Data are representative of three independent experiments.
T81	10614-10663	Sentence	denotes	Figure 3 Th2- or IL-4–Producing Cells Lack FOXP3
T24125	10624-10663	Sentence	denotes	Th2- or IL-4–Producing Cells Lack FOXP3
T24126	10664-10761	Sentence	denotes	(A) FACS analysis of intracellular FOXP3 and IL-4 expression following PMA/Ionomycin stimulation.
T82	10664-10761	Sentence	denotes	(A) FACS analysis of intracellular FOXP3 and IL-4 expression following PMA/Ionomycin stimulation.
T24127	10762-10999	Sentence	denotes	CD4+ T cells were gated on the basis of CD45RO and CD25 surface expression (upper panel), and gated cells are shown below for the CD45RO+CD25− (A, left panel), the CD45RO+CD25+ (right panel), and the CD45RO−CD25− subsets (central panel).
T83	10762-10999	Sentence	denotes	CD4+ T cells were gated on the basis of CD45RO and CD25 surface expression (upper panel), and gated cells are shown below for the CD45RO+CD25− (A, left panel), the CD45RO+CD25+ (right panel), and the CD45RO−CD25− subsets (central panel).
T24128	11000-11109	Sentence	denotes	A statistical analysis of eight independent donors after subtraction of the isotype control are shown in (B).
T84	11000-11109	Sentence	denotes	A statistical analysis of eight independent donors after subtraction of the isotype control are shown in (B).
T24129	11110-11165	Sentence	denotes	The dotted gray line indicates the IC background level.
T85	11110-11165	Sentence	denotes	The dotted gray line indicates the IC background level.
T24130	11166-11343	Sentence	denotes	The error bars show the error of the mean. (C) Similarly, a Th2 clone (BR8), CRTH2+ Th2 cells, IL-4-secreting cells, and memory T cells (CD45RO) were stained for FOXP3 and IL-4.
T86	11166-11343	Sentence	denotes	The error bars show the error of the mean. (C) Similarly, a Th2 clone (BR8), CRTH2+ Th2 cells, IL-4-secreting cells, and memory T cells (CD45RO) were stained for FOXP3 and IL-4.
T24131	11344-11401	Sentence	denotes	Data are representative of three independent experiments.
T87	11344-11401	Sentence	denotes	Data are representative of three independent experiments.
T5207	11403-11452	Sentence	denotes	FOXP3 and GATA3 Kinetics in Differentiating Cells
T88	11403-11452	Sentence	denotes	FOXP3 and GATA3 Kinetics in Differentiating Cells
T5208	11453-11623	Sentence	denotes	The limited capacity of differentiated effector cells to induce FOXP3 expression suggests that iTreg induction has to occur before effector T cell differentiation occurs.
T89	11453-11623	Sentence	denotes	The limited capacity of differentiated effector cells to induce FOXP3 expression suggests that iTreg induction has to occur before effector T cell differentiation occurs.
T5209	11624-11814	Sentence	denotes	Therefore, we analyzed the expression of FOXP3 and GATA3 during the differentiation of naive CD4 T cells into Th0 (neutral, anti-IL-4, anti-IFN-γ, and anti IL-12), Th2, and iTreg phenotypes.
T90	11624-11814	Sentence	denotes	Therefore, we analyzed the expression of FOXP3 and GATA3 during the differentiation of naive CD4 T cells into Th0 (neutral, anti-IL-4, anti-IFN-γ, and anti IL-12), Th2, and iTreg phenotypes.
T5210	11815-12000	Sentence	denotes	After initiation of the differentiation process, FOXP3 and GATA3 showed a similar expression kinetic within the first 3 d, which are considered to be critical in T cell commitment [18].
T91	11815-12000	Sentence	denotes	After initiation of the differentiation process, FOXP3 and GATA3 showed a similar expression kinetic within the first 3 d, which are considered to be critical in T cell commitment [18].
T5211	12001-12111	Sentence	denotes	Under Th2 differentiation conditions, FOXP3 mRNA expression increased only marginally (Figure 4A, left panel).
T92	12001-12111	Sentence	denotes	Under Th2 differentiation conditions, FOXP3 mRNA expression increased only marginally (Figure 4A, left panel).
T5212	12112-12322	Sentence	denotes	Thus, although GATA3 and FOXP3 showed similar kinetics, their expression polarizes at the end of the differentiation process when cells were cultured towards Th2 or iTreg cells, respectively (Figure 4A and 4B).
T93	12112-12322	Sentence	denotes	Thus, although GATA3 and FOXP3 showed similar kinetics, their expression polarizes at the end of the differentiation process when cells were cultured towards Th2 or iTreg cells, respectively (Figure 4A and 4B).
T5213	12323-12633	Sentence	denotes	Interestingly, the Th0 cells were expressing more FOXP3 than the Th2 cells, but expressed low levels of GATA3; however, the protein expression slightly differed from mRNA expression, suggesting also posttranslational regulation and degradation as potential additional mechanisms in the differentiation process.
T94	12323-12633	Sentence	denotes	Interestingly, the Th0 cells were expressing more FOXP3 than the Th2 cells, but expressed low levels of GATA3; however, the protein expression slightly differed from mRNA expression, suggesting also posttranslational regulation and degradation as potential additional mechanisms in the differentiation process.
T5214	12634-12798	Sentence	denotes	The phenotype of iTreg cells included an anergic phenotype upon anti-CD3 re-stimulation (Figure S1A), CD103, CTLA-4, GITR, and PD-1 surface expression (Figure S1B).
T95	12634-12798	Sentence	denotes	The phenotype of iTreg cells included an anergic phenotype upon anti-CD3 re-stimulation (Figure S1A), CD103, CTLA-4, GITR, and PD-1 surface expression (Figure S1B).
T5215	12799-13018	Sentence	denotes	On the single-cell level, it can be seen that cells progress through a transition phase, where GATA3 and FOXP3 expression coexist to some degree in the same cells, which is resolved in iTreg cells after 7 d (Figure 4B).
T96	12799-13018	Sentence	denotes	On the single-cell level, it can be seen that cells progress through a transition phase, where GATA3 and FOXP3 expression coexist to some degree in the same cells, which is resolved in iTreg cells after 7 d (Figure 4B).
T5216	13019-13202	Sentence	denotes	Taken together, these data demonstrated that Th2 cells have lost their capacity to express FOXP3 and showed that Th2 and iTreg cells arise from two different differentiation pathways.
T97	13019-13202	Sentence	denotes	Taken together, these data demonstrated that Th2 cells have lost their capacity to express FOXP3 and showed that Th2 and iTreg cells arise from two different differentiation pathways.
T98	13203-13263	Sentence	denotes	Figure 4 FOXP3 Induction During the Differentiation Process
T24699	13213-13263	Sentence	denotes	FOXP3 Induction During the Differentiation Process
T24700	13264-13394	Sentence	denotes	(A) Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-β (5 ng/ml) or IL-4 (25 ng/ml).
T99	13264-13394	Sentence	denotes	(A) Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-β (5 ng/ml) or IL-4 (25 ng/ml).
T24701	13395-13518	Sentence	denotes	The cells were harvested at different time points, and mRNA was quantified by real-time PCR for FOXP3 and GATA3 expression.
T100	13395-13518	Sentence	denotes	The cells were harvested at different time points, and mRNA was quantified by real-time PCR for FOXP3 and GATA3 expression.
T24702	13519-13576	Sentence	denotes	Bars show the mean ± SD of three independent experiments.
T101	13519-13576	Sentence	denotes	Bars show the mean ± SD of three independent experiments.
T24703	13578-13725	Sentence	denotes	(B) Intracellular GATA3 and FOXP3 staining is shown after exposure of CD4+CD45RA+ T cells to differentiating conditions as in part A of the figure.
T102	13578-13725	Sentence	denotes	(B) Intracellular GATA3 and FOXP3 staining is shown after exposure of CD4+CD45RA+ T cells to differentiating conditions as in part A of the figure.
T24704	13726-13783	Sentence	denotes	Data are representative of three independent experiments.
T103	13726-13783	Sentence	denotes	Data are representative of three independent experiments.
T6377	13785-13830	Sentence	denotes	IL-4 Inhibits TGF-β-Mediated iTreg Commitment
T104	13785-13830	Sentence	denotes	IL-4 Inhibits TGF-β-Mediated iTreg Commitment
T6378	13831-13928	Sentence	denotes	IL-4 induces differentiation of naive T cells, upon antigen encounter, into the Th2 cell lineage.
T105	13831-13928	Sentence	denotes	IL-4 induces differentiation of naive T cells, upon antigen encounter, into the Th2 cell lineage.
T6379	13929-14041	Sentence	denotes	We therefore asked whether IL-4 is able to inhibit TGF-β induction of FOXP3 during the priming of naive T cells.
T106	13929-14041	Sentence	denotes	We therefore asked whether IL-4 is able to inhibit TGF-β induction of FOXP3 during the priming of naive T cells.
T6380	14042-14175	Sentence	denotes	Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-β and/or IL-4 and harvested after 5 d.
T107	14042-14175	Sentence	denotes	Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-β and/or IL-4 and harvested after 5 d.
T6381	14176-14312	Sentence	denotes	IL-4 efficiently repressed the TGF-β–mediated induction of FOXP3 expression (Figure 5A) in a concentration-dependent manner (Figure 5B).
T108	14176-14312	Sentence	denotes	IL-4 efficiently repressed the TGF-β–mediated induction of FOXP3 expression (Figure 5A) in a concentration-dependent manner (Figure 5B).
T6382	14313-14409	Sentence	denotes	Low levels of GATA3 were induced also in the absence of IL-4, as it was previously observed [3].
T109	14313-14409	Sentence	denotes	Low levels of GATA3 were induced also in the absence of IL-4, as it was previously observed [3].
T6383	14410-14493	Sentence	denotes	However, at low concentration, IL-4 was able to marginally induce FOXP3 expression.
T110	14410-14493	Sentence	denotes	However, at low concentration, IL-4 was able to marginally induce FOXP3 expression.
T6384	14494-14597	Sentence	denotes	Of note, GATA3 was also induced in the presence of TGF-β at high IL-4 concentration (Figure 5A and 5B).
T111	14494-14597	Sentence	denotes	Of note, GATA3 was also induced in the presence of TGF-β at high IL-4 concentration (Figure 5A and 5B).
T6385	14598-14914	Sentence	denotes	The IL-4–mediated prevention of FOXP3 expression was not caused by interferences of the receptor signaling, because the phosphorylation of SMAD2 or STAT6 was not affected by the addition of IL-4 and/or TGF-β, which demonstrates that IL-4 as well as TGF-β signaling were functional under these conditions (Figure 5C).
T112	14598-14914	Sentence	denotes	The IL-4–mediated prevention of FOXP3 expression was not caused by interferences of the receptor signaling, because the phosphorylation of SMAD2 or STAT6 was not affected by the addition of IL-4 and/or TGF-β, which demonstrates that IL-4 as well as TGF-β signaling were functional under these conditions (Figure 5C).
T6386	14915-15052	Sentence	denotes	Increasing amounts of IL-4 increase intracellular GATA3, whereas FOXP3 decreased, which is consistent with the mRNA analysis (Figure 5D).
T113	14915-15052	Sentence	denotes	Increasing amounts of IL-4 increase intracellular GATA3, whereas FOXP3 decreased, which is consistent with the mRNA analysis (Figure 5D).
T6387	15053-15162	Sentence	denotes	Furthermore, injection of IL-4 into wild-type B6 mice decreased the inducible or natural Treg number in vivo.
T114	15053-15162	Sentence	denotes	Furthermore, injection of IL-4 into wild-type B6 mice decreased the inducible or natural Treg number in vivo.
T6388	15163-15279	Sentence	denotes	A distinction of the Treg subsets is not possible, because recently activated iTreg cells also express surface CD25.
T115	15163-15279	Sentence	denotes	A distinction of the Treg subsets is not possible, because recently activated iTreg cells also express surface CD25.
T6389	15280-15466	Sentence	denotes	We used complexes of recombinant mouse IL-4 (rmIL-4) plus anti-IL-4 monoclonal antibodies (mAbs), which have been shown to dramatically increase the potency of the cytokine in vivo [19].
T116	15280-15466	Sentence	denotes	We used complexes of recombinant mouse IL-4 (rmIL-4) plus anti-IL-4 monoclonal antibodies (mAbs), which have been shown to dramatically increase the potency of the cytokine in vivo [19].
T6390	15467-15622	Sentence	denotes	In these mice, the percentage of CD4+CD25+ and FOXP3+ T cells dramatically decreased when the antibody-cytokine immune complexes were injected (Figure S2).
T117	15467-15622	Sentence	denotes	In these mice, the percentage of CD4+CD25+ and FOXP3+ T cells dramatically decreased when the antibody-cytokine immune complexes were injected (Figure S2).
T6391	15623-15921	Sentence	denotes	Upon administration of rmIL-4 plus anti-IL-4 mAb complexes, the total number of CD4+CD25+ T cell, as well as the Foxp3+ T cells diminished by half (Figure S2G and S2H), confirming that the lower percentage was not due to an increase in the CD4+CD25– cells, but a real decrease of CD4+CD25+ T cells.
T118	15623-15921	Sentence	denotes	Upon administration of rmIL-4 plus anti-IL-4 mAb complexes, the total number of CD4+CD25+ T cell, as well as the Foxp3+ T cells diminished by half (Figure S2G and S2H), confirming that the lower percentage was not due to an increase in the CD4+CD25– cells, but a real decrease of CD4+CD25+ T cells.
T6392	15922-16042	Sentence	denotes	In conclusion, IL-4 negatively regulates the natural or inducible Treg cell turnover not only in vitro but also in vivo.
T119	15922-16042	Sentence	denotes	In conclusion, IL-4 negatively regulates the natural or inducible Treg cell turnover not only in vitro but also in vivo.
T6393	16043-16240	Sentence	denotes	To study the effects of IL-4 on already-existing human natural or inducible Treg cells, we exposed sorted CD25+ T cells (nTreg cells) to IL-4 and analyzed FOXP3 expression and suppressive capacity.
T120	16043-16240	Sentence	denotes	To study the effects of IL-4 on already-existing human natural or inducible Treg cells, we exposed sorted CD25+ T cells (nTreg cells) to IL-4 and analyzed FOXP3 expression and suppressive capacity.
T6394	16241-16385	Sentence	denotes	In already-existing Treg cells, IL-4 failed to inhibit FOXP3 expression (Figure S3A), and the suppressive capacity was not altered (Figure S3C).
T121	16241-16385	Sentence	denotes	In already-existing Treg cells, IL-4 failed to inhibit FOXP3 expression (Figure S3A), and the suppressive capacity was not altered (Figure S3C).
T6395	16386-16487	Sentence	denotes	Similarly pre-existing iTreg cells did not decrease FOXP3 expression upon IL-4 exposure (Figure S3B).
T122	16386-16487	Sentence	denotes	Similarly pre-existing iTreg cells did not decrease FOXP3 expression upon IL-4 exposure (Figure S3B).
T123	16488-16531	Sentence	denotes	Figure 5 Effect of IL-4 on FOXP3 Induction
T25148	16498-16531	Sentence	denotes	Effect of IL-4 on FOXP3 Induction
T25149	16532-16720	Sentence	denotes	(A) A statistical analysis was performed with six donors on day 5 (TGF-β (10 ng/ml) and with or without IL-4 (100 ng/ml)); Shown is the mean, and error bars indicated the SD of six donors.
T124	16532-16720	Sentence	denotes	(A) A statistical analysis was performed with six donors on day 5 (TGF-β (10 ng/ml) and with or without IL-4 (100 ng/ml)); Shown is the mean, and error bars indicated the SD of six donors.
T25150	16721-16779	Sentence	denotes	Statistical analysis was performed using the Dunnett test.
T125	16721-16779	Sentence	denotes	Statistical analysis was performed using the Dunnett test.
T25151	16780-16864	Sentence	denotes	Statistical significance is indicated by asterisks (p ≤ 0.05, *p ≤ 0.01, Dunnett).
T126	16780-16864	Sentence	denotes	Statistical significance is indicated by asterisks (p ≤ 0.05, *p ≤ 0.01, Dunnett).
T25152	16865-17020	Sentence	denotes	(B) CD4+CD45RA+ cells were activated in the presence of a constant concentration of TGF-β (5 ng/ml) with an increasing concentration of IL-4, as indicated.
T127	16865-17020	Sentence	denotes	(B) CD4+CD45RA+ cells were activated in the presence of a constant concentration of TGF-β (5 ng/ml) with an increasing concentration of IL-4, as indicated.
T25153	17021-17076	Sentence	denotes	Cells were harvested for mRNA quantification after 5 d.
T128	17021-17076	Sentence	denotes	Cells were harvested for mRNA quantification after 5 d.
T25154	17077-17207	Sentence	denotes	(C) CD4+CD45RA+ cells were stimulated in vitro with plate-bound anti-CD3/CD28, TGF-β (10ng/ml), and IL-4 (100 ng/ml) as indicated.
T129	17077-17207	Sentence	denotes	(C) CD4+CD45RA+ cells were stimulated in vitro with plate-bound anti-CD3/CD28, TGF-β (10ng/ml), and IL-4 (100 ng/ml) as indicated.
T25155	17208-17310	Sentence	denotes	After 1 h, cell lysates were prepared and analyzed by Western blot for phosphorylated SMAD2 and STAT6.
T130	17208-17310	Sentence	denotes	After 1 h, cell lysates were prepared and analyzed by Western blot for phosphorylated SMAD2 and STAT6.
T25156	17311-17360	Sentence	denotes	Total STAT6 and GAPDH served as internal control.
T131	17311-17360	Sentence	denotes	Total STAT6 and GAPDH served as internal control.
T132	17361-17485	Sentence	denotes	(D) Intracellular GATA3 and FOXP3 staining are shown after exposure of CD4+CD45RA+ T cells to IL-4 as described for panel B.
T25157	17361-17543	Sentence	denotes	(D) Intracellular GATA3 and FOXP3 staining are shown after exposure of CD4+CD45RA+ T cells to IL-4 as described for panel B. Data are representative of three independent experiments.
T133	17486-17543	Sentence	denotes	Data are representative of three independent experiments.
T8261	17545-17588	Sentence	denotes	The Role of TGF-β in T Cell Differentiation
T134	17545-17588	Sentence	denotes	The Role of TGF-β in T Cell Differentiation
T8262	17589-17816	Sentence	denotes	Although TGF-β–reduced CD25, IL-4 expression, and CD25 expression (Figure 6B), IL-4 significantly inhibited TGF-β–mediated induction of FOXP3 in naive T cells driven toward FOXP3+ T cells, as shown by FACS analysis (Figure 6A).
T135	17589-17816	Sentence	denotes	Although TGF-β–reduced CD25, IL-4 expression, and CD25 expression (Figure 6B), IL-4 significantly inhibited TGF-β–mediated induction of FOXP3 in naive T cells driven toward FOXP3+ T cells, as shown by FACS analysis (Figure 6A).
T8263	17817-17981	Sentence	denotes	It is known that IL-4 is a potent growth factor and may therefore favor the proliferation of FOXP3– cells and thus decrease the relative percentage of FOXP3+ cells.
T136	17817-17981	Sentence	denotes	It is known that IL-4 is a potent growth factor and may therefore favor the proliferation of FOXP3– cells and thus decrease the relative percentage of FOXP3+ cells.
T8264	17982-18139	Sentence	denotes	However, analysis of cell division kinetics by CFSE-labeling demonstrated that IL-4 did not differentially promote cell growth of FOXP3+ over that of FOXP3–.
T137	17982-18139	Sentence	denotes	However, analysis of cell division kinetics by CFSE-labeling demonstrated that IL-4 did not differentially promote cell growth of FOXP3+ over that of FOXP3–.
T8265	18140-18217	Sentence	denotes	In fact both populations showed similarly enhanced proliferation (Figure 6A).
T138	18140-18217	Sentence	denotes	In fact both populations showed similarly enhanced proliferation (Figure 6A).
T8266	18218-18507	Sentence	denotes	Furthermore the TGF-β–mediated induction of FOXP3 expression was not caused by overgrowth of a CD25–FOXP3+ minority, since the number of FOXP3+ cells was low/absent in the purified CD4+CD45RA+ T cells (between 0% and 1%), and the FOXP3+ cells were not confined to the highly divided cells.
T139	18218-18507	Sentence	denotes	Furthermore the TGF-β–mediated induction of FOXP3 expression was not caused by overgrowth of a CD25–FOXP3+ minority, since the number of FOXP3+ cells was low/absent in the purified CD4+CD45RA+ T cells (between 0% and 1%), and the FOXP3+ cells were not confined to the highly divided cells.
T8267	18508-18654	Sentence	denotes	CD25 was down-regulated in TGF-β–treated cells compare to activated T cells, which was even more pronounced in cells cultured with TGF-β and IL-4.
T140	18508-18654	Sentence	denotes	CD25 was down-regulated in TGF-β–treated cells compare to activated T cells, which was even more pronounced in cells cultured with TGF-β and IL-4.
T141	18655-18710	Sentence	denotes	Figure 6 IL-4 Inhibits TGF-β–Mediated iTreg Commitment
T25857	18665-18710	Sentence	denotes	IL-4 Inhibits TGF-β–Mediated iTreg Commitment
T25858	18711-18819	Sentence	denotes	CFSE-labeled CD4+CD45RA+ cells were activated with plate-bound anti-CD3/CD28, TGF-β, and IL-4, as indicated.
T142	18711-18819	Sentence	denotes	CFSE-labeled CD4+CD45RA+ cells were activated with plate-bound anti-CD3/CD28, TGF-β, and IL-4, as indicated.
T25859	18820-18953	Sentence	denotes	After 5 d, cells were analyzed by flow cytometry (A) and results of six independent experiments are shown in the bar graph below (B).
T143	18820-18953	Sentence	denotes	After 5 d, cells were analyzed by flow cytometry (A) and results of six independent experiments are shown in the bar graph below (B).
T25860	18954-19219	Sentence	denotes	Statistical significance (one-way Anova, Newman-Keuls) is indicated by asterisks (p ≤ 0.01, *p ≤ 0.001). (C) Kinetic analysis of intracellular GATA3 and FOXP3 staining is shown in panel C following exposure of CD4+CD45RA+ T cells to anti-CD3/28, IL-4 and TGF-β.
T144	18954-19219	Sentence	denotes	Statistical significance (one-way Anova, Newman-Keuls) is indicated by asterisks (p ≤ 0.01, *p ≤ 0.001). (C) Kinetic analysis of intracellular GATA3 and FOXP3 staining is shown in panel C following exposure of CD4+CD45RA+ T cells to anti-CD3/28, IL-4 and TGF-β.
T25861	19220-19277	Sentence	denotes	Data are representative of three independent experiments.
T145	19220-19277	Sentence	denotes	Data are representative of three independent experiments.
T8268	19280-19378	Sentence	denotes	The addition of IL-4 to iTreg-driving conditions decreased the number of FOXP3+ cells (Figure 6B).
T146	19280-19378	Sentence	denotes	The addition of IL-4 to iTreg-driving conditions decreased the number of FOXP3+ cells (Figure 6B).
T8269	19379-19502	Sentence	denotes	In line with the previous findings, the IL-4–producing cells and the FOXP3 expressing cells are nonoverlapping populations.
T147	19379-19502	Sentence	denotes	In line with the previous findings, the IL-4–producing cells and the FOXP3 expressing cells are nonoverlapping populations.
T8270	19503-19624	Sentence	denotes	Since FOXP3 is known to act as a repressor of cytokine expression [20], we therefore analyzed GATA3 and FOXP3 expression.
T148	19503-19624	Sentence	denotes	Since FOXP3 is known to act as a repressor of cytokine expression [20], we therefore analyzed GATA3 and FOXP3 expression.
T8271	19625-20016	Sentence	denotes	The expression kinetic of naive T cells exposed to IL-4 and TGF-β demonstrated that GATA3 and FOXP3 are initially found in separate populations (day 2), but transiently co-express both factors (days 4–8), before establishing separate populations at the end of the differentiation process (day 10; Figure 6C), suggesting that GATA3 inhibits the development of iTreg cells by repressing FOXP3.
T149	19625-20016	Sentence	denotes	The expression kinetic of naive T cells exposed to IL-4 and TGF-β demonstrated that GATA3 and FOXP3 are initially found in separate populations (day 2), but transiently co-express both factors (days 4–8), before establishing separate populations at the end of the differentiation process (day 10; Figure 6C), suggesting that GATA3 inhibits the development of iTreg cells by repressing FOXP3.
T8272	20017-20240	Sentence	denotes	These results showed that IL-4 acts in vitro as an inhibitor of FOXP3 expression, without interfering with TGF-β signaling, probably acting at the level of transcription factors, and possibly by a GATA3-dependent mechanism.
T150	20017-20240	Sentence	denotes	These results showed that IL-4 acts in vitro as an inhibitor of FOXP3 expression, without interfering with TGF-β signaling, probably acting at the level of transcription factors, and possibly by a GATA3-dependent mechanism.
T9839	20242-20291	Sentence	denotes	GATA3 Is a Negative Regulator of FOXP3 Expression
T151	20242-20291	Sentence	denotes	GATA3 Is a Negative Regulator of FOXP3 Expression
T9840	20292-20424	Sentence	denotes	FOXP3 expression decreased once GATA3 expression is high; therefore, we hypothesized a potential role for GATA3 in repressing FOXP3.
T152	20292-20424	Sentence	denotes	FOXP3 expression decreased once GATA3 expression is high; therefore, we hypothesized a potential role for GATA3 in repressing FOXP3.
T9841	20425-20668	Sentence	denotes	Besides GATA3′s well-known positive effect on gene regulation, GATA3′s repressive capabilities were previously shown to restrict Th1 commitment by inhibiting STAT4 expression [2,21], and therefore GATA3 prevents differentiation into Th1 cells.
T153	20425-20668	Sentence	denotes	Besides GATA3′s well-known positive effect on gene regulation, GATA3′s repressive capabilities were previously shown to restrict Th1 commitment by inhibiting STAT4 expression [2,21], and therefore GATA3 prevents differentiation into Th1 cells.
T9842	20669-20894	Sentence	denotes	To investigate whether GATA3 can directly inhibit FOXP3 induction, we transduced GATA3 or a truncated GATA3 lacking the DNA-binding domain in human primary CD4+CD45RA+ T cells using a TAT-fused, recombinantly expressed GATA3.
T154	20669-20894	Sentence	denotes	To investigate whether GATA3 can directly inhibit FOXP3 induction, we transduced GATA3 or a truncated GATA3 lacking the DNA-binding domain in human primary CD4+CD45RA+ T cells using a TAT-fused, recombinantly expressed GATA3.
T9843	20895-21003	Sentence	denotes	After transduction, the cells were activated with soluble anti-CD3/CD28 in the presence or absence of TGF-β.
T155	20895-21003	Sentence	denotes	After transduction, the cells were activated with soluble anti-CD3/CD28 in the presence or absence of TGF-β.
T9844	21004-21134	Sentence	denotes	TAT-GATA3 was successfully transduced in a homogeneous and dose-dependent manner into human CD4+ T cells (Figure 7A, upper panel).
T156	21004-21134	Sentence	denotes	TAT-GATA3 was successfully transduced in a homogeneous and dose-dependent manner into human CD4+ T cells (Figure 7A, upper panel).
T9845	21135-21356	Sentence	denotes	TAT-GATA3 reduced FOXP3 expression in a dose-dependent manner, whereas a DNA-binding domain truncated version (TAT-ΔDBD-GATA3) did not affect FOXP3 expression as compared with expression in untransduced cells (Figure 7A).
T157	21135-21356	Sentence	denotes	TAT-GATA3 reduced FOXP3 expression in a dose-dependent manner, whereas a DNA-binding domain truncated version (TAT-ΔDBD-GATA3) did not affect FOXP3 expression as compared with expression in untransduced cells (Figure 7A).
T9846	21357-21556	Sentence	denotes	In addition, we analyzed the inhibitory effect of GATA3 on FOXP3 in transgenic DO11.10 mice, constitutively overexpressing GATA3 under the control of the CD2 locus control region (DO11.10xCD2-GATA3).
T158	21357-21556	Sentence	denotes	In addition, we analyzed the inhibitory effect of GATA3 on FOXP3 in transgenic DO11.10 mice, constitutively overexpressing GATA3 under the control of the CD2 locus control region (DO11.10xCD2-GATA3).
T9847	21557-21670	Sentence	denotes	The thymic selection into the CD4 lineage is largely intact in DO11.10xCD2-GATA3 (RW Hendriks, unpublished data).
T159	21557-21670	Sentence	denotes	The thymic selection into the CD4 lineage is largely intact in DO11.10xCD2-GATA3 (RW Hendriks, unpublished data).
T9848	21671-21773	Sentence	denotes	These mice develop lymphomas at an older age, but signs of autoimmune disease were not described [22].
T160	21671-21773	Sentence	denotes	These mice develop lymphomas at an older age, but signs of autoimmune disease were not described [22].
T9849	21774-21956	Sentence	denotes	To investigate the effect of GATA3 on iTreg, CD4+CD62L+CD25– cells were isolated, activated with OVA in the presence or absence of TGF-β, and Foxp3 expression was analyzed after 4 d.
T161	21774-21956	Sentence	denotes	To investigate the effect of GATA3 on iTreg, CD4+CD62L+CD25– cells were isolated, activated with OVA in the presence or absence of TGF-β, and Foxp3 expression was analyzed after 4 d.
T9850	21957-22014	Sentence	denotes	The naive CD4+CD25– cells were Foxp3– (unpublished data).
T162	21957-22014	Sentence	denotes	The naive CD4+CD25– cells were Foxp3– (unpublished data).
T9851	22015-22126	Sentence	denotes	As described for the human cells, TGF-β dramatically up-regulated Foxp3 in the DO11.10 littermate control mice.
T163	22015-22126	Sentence	denotes	As described for the human cells, TGF-β dramatically up-regulated Foxp3 in the DO11.10 littermate control mice.
T9852	22127-22269	Sentence	denotes	In contrast, cells from the CD2-GATA3xDO11.10 mice showed dramatically reduced Foxp3 expression when activated with TGF-β and OVA (Figure 7B).
T164	22127-22269	Sentence	denotes	In contrast, cells from the CD2-GATA3xDO11.10 mice showed dramatically reduced Foxp3 expression when activated with TGF-β and OVA (Figure 7B).
T9853	22270-22439	Sentence	denotes	All mice produced similar amounts of TGF-β; in addition, Smad7 was equally expressed [23] in T cells of both mice strains (Figure 7C), indicating intact TGF-β signaling.
T165	22270-22439	Sentence	denotes	All mice produced similar amounts of TGF-β; in addition, Smad7 was equally expressed [23] in T cells of both mice strains (Figure 7C), indicating intact TGF-β signaling.
T166	22440-22504	Sentence	denotes	Figure 7 GATA3 Acts as a Negative Regulator of FOXP3 Expression
T26337	22450-22504	Sentence	denotes	GATA3 Acts as a Negative Regulator of FOXP3 Expression
T26338	22505-22713	Sentence	denotes	(A) Human naive CD4+CDRA+ T cells were transduced with 0, 20, 100, and 500 nM of TAT-GATA3 protein, and intracellular presence of GATA3 was analyzed using FACS following anti-CD3/CD28 activation of the cells.
T167	22505-22713	Sentence	denotes	(A) Human naive CD4+CDRA+ T cells were transduced with 0, 20, 100, and 500 nM of TAT-GATA3 protein, and intracellular presence of GATA3 was analyzed using FACS following anti-CD3/CD28 activation of the cells.
T26339	22714-22840	Sentence	denotes	GFP-positive cells were gated and analyzed for intracellular FOXP3 expression following a 2-d incubation period (lower panel).
T168	22714-22840	Sentence	denotes	GFP-positive cells were gated and analyzed for intracellular FOXP3 expression following a 2-d incubation period (lower panel).
T26340	22841-22897	Sentence	denotes	Data are representative of four independent experiments.
T169	22841-22897	Sentence	denotes	Data are representative of four independent experiments.
T26341	22898-23014	Sentence	denotes	(B) CD4+CD25− T cells were isolated from D011.10 and D011.10xCD2-GATA3 mice and treated with OVA and TGF-β for 96 h.
T170	22898-23014	Sentence	denotes	(B) CD4+CD25− T cells were isolated from D011.10 and D011.10xCD2-GATA3 mice and treated with OVA and TGF-β for 96 h.
T26342	23015-23073	Sentence	denotes	Surface CD4 and intracellular FOXP3 were measured by FACS.
T171	23015-23073	Sentence	denotes	Surface CD4 and intracellular FOXP3 were measured by FACS.
T26343	23074-23137	Sentence	denotes	These data are representative of three independent experiments.
T172	23074-23137	Sentence	denotes	These data are representative of three independent experiments.
T26344	23138-23257	Sentence	denotes	(C) The cells treated as in (B) were harvested and mRNA was quantified by real-time PCR for SMAD7 and TGF-β expression.
T173	23138-23257	Sentence	denotes	(C) The cells treated as in (B) were harvested and mRNA was quantified by real-time PCR for SMAD7 and TGF-β expression.
T26345	23258-23315	Sentence	denotes	Bars show the mean ± SD of three independent experiments.
T174	23258-23315	Sentence	denotes	Bars show the mean ± SD of three independent experiments.
T9854	23318-23455	Sentence	denotes	Taken together, these results demonstrated a repressive role of IL-4-induced GATA3 transcription factor in the generation of iTreg cells.
T175	23318-23455	Sentence	denotes	Taken together, these results demonstrated a repressive role of IL-4-induced GATA3 transcription factor in the generation of iTreg cells.
T11568	23457-23491	Sentence	denotes	GATA3 Represses the FOXP3 Promoter
T176	23457-23491	Sentence	denotes	GATA3 Represses the FOXP3 Promoter
T11569	23492-23669	Sentence	denotes	To investigate the molecular mechanism of GATA3-mediated repression of human FOXP3, the human FOXP3 promoter was studied and a palindromic binding site for GATA3 was discovered.
T177	23492-23669	Sentence	denotes	To investigate the molecular mechanism of GATA3-mediated repression of human FOXP3, the human FOXP3 promoter was studied and a palindromic binding site for GATA3 was discovered.
T11570	23670-23764	Sentence	denotes	The GATA-binding site is located 303 bp upstream from the transcription start site (TSS) [24].
T178	23670-23764	Sentence	denotes	The GATA-binding site is located 303 bp upstream from the transcription start site (TSS) [24].
T11571	23765-23899	Sentence	denotes	This site is highly conserved between humans, mice, and rats (Figure S4) and may therefore play an important role in FOXP3 regulation.
T179	23765-23899	Sentence	denotes	This site is highly conserved between humans, mice, and rats (Figure S4) and may therefore play an important role in FOXP3 regulation.
T11572	23900-23988	Sentence	denotes	The functional relevance of this site was studied using a FOXP3-promoter construct [24].
T180	23900-23988	Sentence	denotes	The functional relevance of this site was studied using a FOXP3-promoter construct [24].
T11573	23989-24190	Sentence	denotes	We transfected human primary CD4+ T cells, in vitro differentiated Th2 cells, and Jurkat cells (Jurkat cells are known to constitutively express GATA3 [25,26]), and we measured FOXP3 promoter activity.
T181	23989-24190	Sentence	denotes	We transfected human primary CD4+ T cells, in vitro differentiated Th2 cells, and Jurkat cells (Jurkat cells are known to constitutively express GATA3 [25,26]), and we measured FOXP3 promoter activity.
T11574	24191-24406	Sentence	denotes	The promoter was not active in the GATA3-expressing cell line Jurkat or in the in vitro-differentiated Th2 cells, whereas the construct was active in the CD4 cells, which express a lower amount of GATA3 (Figure 8A).
T182	24191-24406	Sentence	denotes	The promoter was not active in the GATA3-expressing cell line Jurkat or in the in vitro-differentiated Th2 cells, whereas the construct was active in the CD4 cells, which express a lower amount of GATA3 (Figure 8A).
T11575	24407-24546	Sentence	denotes	Overexpression of GATA3 in naive T cells diminished luciferase activity of the FOXP3 promoter compared with the control vector (Figure 8B).
T183	24407-24546	Sentence	denotes	Overexpression of GATA3 in naive T cells diminished luciferase activity of the FOXP3 promoter compared with the control vector (Figure 8B).
T11576	24547-24667	Sentence	denotes	To further address the function of the GATA3 site, we inserted a site-specific mutation deleting the GATA3-binding site.
T184	24547-24667	Sentence	denotes	To further address the function of the GATA3 site, we inserted a site-specific mutation deleting the GATA3-binding site.
T11577	24668-24901	Sentence	denotes	This mutation increased luciferase activity by 3-fold in memory CD4+CD45RO+ T cells, whereas no difference was observed in naive (GATA3–) CD4+CD45RA+ T cells, revealing a repressor activity of GATA3 on the FOXP3 promoter (Figure 8C).
T185	24668-24901	Sentence	denotes	This mutation increased luciferase activity by 3-fold in memory CD4+CD45RO+ T cells, whereas no difference was observed in naive (GATA3–) CD4+CD45RA+ T cells, revealing a repressor activity of GATA3 on the FOXP3 promoter (Figure 8C).
T11578	24902-24992	Sentence	denotes	Furthermore GATA3 binds directly to the FOXP3 promoter as investigated by pull-down assay.
T186	24902-24992	Sentence	denotes	Furthermore GATA3 binds directly to the FOXP3 promoter as investigated by pull-down assay.
T11579	24993-25246	Sentence	denotes	HEK cells were transiently transfected with GATA3 or a control vector, and increasing amounts of lysates were incubated with oligonucleotides containing the GATA3 site of the FOXP3 promoter or a control oligonucleotide with a mutated GATA3-binding site.
T187	24993-25246	Sentence	denotes	HEK cells were transiently transfected with GATA3 or a control vector, and increasing amounts of lysates were incubated with oligonucleotides containing the GATA3 site of the FOXP3 promoter or a control oligonucleotide with a mutated GATA3-binding site.
T11580	25247-25311	Sentence	denotes	After the pull-down, GATA3 binding was detected by Western blot.
T188	25247-25311	Sentence	denotes	After the pull-down, GATA3 binding was detected by Western blot.
T11581	25312-25398	Sentence	denotes	Similarly, GATA3-expressing Th2 cells and iTreg cells were subjected to this approach.
T189	25312-25398	Sentence	denotes	Similarly, GATA3-expressing Th2 cells and iTreg cells were subjected to this approach.
T11582	25399-25498	Sentence	denotes	Only HEK cells overexpressing GATA3 and Th2 cells showed GATA3-binding activity (Figure 8D and 8E).
T190	25399-25498	Sentence	denotes	Only HEK cells overexpressing GATA3 and Th2 cells showed GATA3-binding activity (Figure 8D and 8E).
T11583	25499-25578	Sentence	denotes	These experiments demonstrated that GATA3 binds the palindromic FOXP3 promoter.
T191	25499-25578	Sentence	denotes	These experiments demonstrated that GATA3 binds the palindromic FOXP3 promoter.
T11584	25579-25811	Sentence	denotes	To gain insights into the in vivo situation, we performed a chromatin immunoprecipitation (ChIP) using an anti-GATA3 antibody and showed that GATA3 binds to the FOXP3 promoter region in Th2 cells, but not in iTreg cells (Figure 7F).
T192	25579-25811	Sentence	denotes	To gain insights into the in vivo situation, we performed a chromatin immunoprecipitation (ChIP) using an anti-GATA3 antibody and showed that GATA3 binds to the FOXP3 promoter region in Th2 cells, but not in iTreg cells (Figure 7F).
T11585	25812-25926	Sentence	denotes	Taken together these data demonstrate that the GATA3-binding to the FOXP3 promoter is repressing FOXP3 expression.
T193	25812-25926	Sentence	denotes	Taken together these data demonstrate that the GATA3-binding to the FOXP3 promoter is repressing FOXP3 expression.
T194	25927-25977	Sentence	denotes	Figure 8 GATA3 Represses the Human FOXP3 Promoter
T26927	25937-25977	Sentence	denotes	GATA3 Represses the Human FOXP3 Promoter
T26928	25978-26176	Sentence	denotes	(A) Jurkat, Th2 cells, and human primary CD4 cells were transfected with an empty vector (pGL3 basic) or a vector containing the putative FOXP3 promoter region fused to the luciferase reporter gene.
T195	25978-26176	Sentence	denotes	(A) Jurkat, Th2 cells, and human primary CD4 cells were transfected with an empty vector (pGL3 basic) or a vector containing the putative FOXP3 promoter region fused to the luciferase reporter gene.
T26929	26177-26330	Sentence	denotes	Bars show the mean ± SD of arbitrary light units normalized for renilla luciferase of four independent experiments; samples were measured in triplicates.
T196	26177-26330	Sentence	denotes	Bars show the mean ± SD of arbitrary light units normalized for renilla luciferase of four independent experiments; samples were measured in triplicates.
T26930	26331-26472	Sentence	denotes	(B) Naive CD4 T cells were transfected with the FOXP3 promoter reporter construct together with a GATA3 expression vector or an empty vector.
T197	26331-26472	Sentence	denotes	(B) Naive CD4 T cells were transfected with the FOXP3 promoter reporter construct together with a GATA3 expression vector or an empty vector.
T26931	26473-26530	Sentence	denotes	Bars show the mean ± SD of three independent experiments.
T198	26473-26530	Sentence	denotes	Bars show the mean ± SD of three independent experiments.
T26932	26531-26716	Sentence	denotes	(C) Naive (left panel) or memory (right panel) CD4 T cells were transfected with wild-type or a GATA3 mutated 511-FOXP3 promoter reporter construct and activated with PMA and ionomycin.
T199	26531-26716	Sentence	denotes	(C) Naive (left panel) or memory (right panel) CD4 T cells were transfected with wild-type or a GATA3 mutated 511-FOXP3 promoter reporter construct and activated with PMA and ionomycin.
T26933	26717-26871	Sentence	denotes	Bars show the mean ± SD of arbitrary light units normalized for renilla luciferase of eight independent experiments; samples were measured in triplicates.
T200	26717-26871	Sentence	denotes	Bars show the mean ± SD of arbitrary light units normalized for renilla luciferase of eight independent experiments; samples were measured in triplicates.
T26934	26872-27063	Sentence	denotes	(D) Nuclear extracts were prepared from HEK cells transfected with GATA3 or an empty vector, (E) Th1, Th2, or iTreg cells and binding factors precipitated using biotinylated oligonucleotides.
T201	26872-27063	Sentence	denotes	(D) Nuclear extracts were prepared from HEK cells transfected with GATA3 or an empty vector, (E) Th1, Th2, or iTreg cells and binding factors precipitated using biotinylated oligonucleotides.
T26935	27064-27149	Sentence	denotes	The oligonucleotides–transcription factor complexes were separated on a SDS-PAGE gel.
T202	27064-27149	Sentence	denotes	The oligonucleotides–transcription factor complexes were separated on a SDS-PAGE gel.
T26936	27150-27251	Sentence	denotes	The amounts of GATA3 protein in the precipitates were assessed by immunoblotting with anti-GATA3 mAb.
T203	27150-27251	Sentence	denotes	The amounts of GATA3 protein in the precipitates were assessed by immunoblotting with anti-GATA3 mAb.
T26937	27252-27301	Sentence	denotes	Total nuclear extracts were also run as controls.
T204	27252-27301	Sentence	denotes	Total nuclear extracts were also run as controls.
T26938	27302-27357	Sentence	denotes	Data are representative of three different experiments.
T205	27302-27357	Sentence	denotes	Data are representative of three different experiments.
T26939	27358-27443	Sentence	denotes	(F) iTreg or Th2 cells were analyzed by ChIP for GATA3 binding to the FOXP3 promoter.
T206	27358-27443	Sentence	denotes	(F) iTreg or Th2 cells were analyzed by ChIP for GATA3 binding to the FOXP3 promoter.
T26940	27444-27551	Sentence	denotes	The “input” represents PCR amplification of the total sample, which was not subjected to any precipitation.
T207	27444-27551	Sentence	denotes	The “input” represents PCR amplification of the total sample, which was not subjected to any precipitation.
T26941	27552-27612	Sentence	denotes	Results are representative of three independent experiments.
T208	27552-27612	Sentence	denotes	Results are representative of three independent experiments.
T209	27614-27624	Sentence	denotes	Discussion
T13433	27625-27796	Sentence	denotes	The current study reveals that FOXP3 induction, an important step in iTreg commitment, is inhibited by GATA3, which is the key regulator for polarization toward Th2 cells.
T210	27625-27796	Sentence	denotes	The current study reveals that FOXP3 induction, an important step in iTreg commitment, is inhibited by GATA3, which is the key regulator for polarization toward Th2 cells.
T13434	27797-27899	Sentence	denotes	After differentiation, the effector Th2 cells become refractory to conversion into a FOXP3+ phenotype.
T211	27797-27899	Sentence	denotes	After differentiation, the effector Th2 cells become refractory to conversion into a FOXP3+ phenotype.
T13435	27900-28003	Sentence	denotes	In accordance with other studies, we found that CD4+CD25– cells were able to up-regulate FOXP3 [12,27].
T212	27900-28003	Sentence	denotes	In accordance with other studies, we found that CD4+CD25– cells were able to up-regulate FOXP3 [12,27].
T13436	28004-28200	Sentence	denotes	Already-committed cells such as memory T cells and Th1 cells showed only moderate and transient FOXP3 induction, which is not sufficient to change the phenotype toward a regulatory T cell profile.
T213	28004-28200	Sentence	denotes	Already-committed cells such as memory T cells and Th1 cells showed only moderate and transient FOXP3 induction, which is not sufficient to change the phenotype toward a regulatory T cell profile.
T13437	28201-28497	Sentence	denotes	In contrast, naive T cells could efficiently up-regulate FOXP3 when treated with TGF-β to induce iTreg cells [10,28–34], suggesting that FOXP3 plays an important role in the early differentiation process and may act in a way similar to that known for the Th1/Th2 decision factors T-bet and GATA3.
T214	28201-28497	Sentence	denotes	In contrast, naive T cells could efficiently up-regulate FOXP3 when treated with TGF-β to induce iTreg cells [10,28–34], suggesting that FOXP3 plays an important role in the early differentiation process and may act in a way similar to that known for the Th1/Th2 decision factors T-bet and GATA3.
T13438	28498-28754	Sentence	denotes	This commitment is characterized by competitive expression of these factors [35,36], which we also observed in differentiating FOXP3+ iTreg cells including a phase of co-expression, which turns into nonoverlapping expression upon completed differentiation.
T215	28498-28754	Sentence	denotes	This commitment is characterized by competitive expression of these factors [35,36], which we also observed in differentiating FOXP3+ iTreg cells including a phase of co-expression, which turns into nonoverlapping expression upon completed differentiation.
T13439	28755-28908	Sentence	denotes	In this competitive process TGF-β appeared to be mandatory for the induction of FOXP3, possibly by keeping the expression of GATA3 and T-bet low [37,38].
T216	28755-28908	Sentence	denotes	In this competitive process TGF-β appeared to be mandatory for the induction of FOXP3, possibly by keeping the expression of GATA3 and T-bet low [37,38].
T13440	28909-29188	Sentence	denotes	In contrast, differentiating naive T cells in the absence of polarization factors (Th0) such as IL-4, IL-12, or TGF-β showed only a transient FOXP3 expression and failed to generate a population of FOXP3-expressing cells, but GATA3 and T-bet were up-regulated (unpublished data).
T217	28909-29188	Sentence	denotes	In contrast, differentiating naive T cells in the absence of polarization factors (Th0) such as IL-4, IL-12, or TGF-β showed only a transient FOXP3 expression and failed to generate a population of FOXP3-expressing cells, but GATA3 and T-bet were up-regulated (unpublished data).
T13441	29189-29386	Sentence	denotes	Interestingly, as we and others previously described, FOXP3-promoting factors, such as dexamethasone [39], CTLA-4 [40], and estrogens [41], are also known as inhibitors of GATA3 expression [42–45].
T218	29189-29386	Sentence	denotes	Interestingly, as we and others previously described, FOXP3-promoting factors, such as dexamethasone [39], CTLA-4 [40], and estrogens [41], are also known as inhibitors of GATA3 expression [42–45].
T13442	29387-29518	Sentence	denotes	Therefore GATA3 not only induces differentiation into Th2 cells but also inhibits FOXP3 expression and commitment into iTreg cells.
T219	29387-29518	Sentence	denotes	Therefore GATA3 not only induces differentiation into Th2 cells but also inhibits FOXP3 expression and commitment into iTreg cells.
T13443	29519-29690	Sentence	denotes	The Th2 cytokine IL-4 but not IL-13 (unpublished data) was able to inhibit TGF-β–mediated FOXP3 induction and therefore prevented conversion into the regulatory phenotype.
T220	29519-29690	Sentence	denotes	The Th2 cytokine IL-4 but not IL-13 (unpublished data) was able to inhibit TGF-β–mediated FOXP3 induction and therefore prevented conversion into the regulatory phenotype.
T13444	29691-29819	Sentence	denotes	To prove the inhibitory effect of IL-4 on inducible or natural Treg commitment in vivo, we treated mice with IL-4 and anti-IL-4.
T221	29691-29819	Sentence	denotes	To prove the inhibitory effect of IL-4 on inducible or natural Treg commitment in vivo, we treated mice with IL-4 and anti-IL-4.
T13445	29820-29892	Sentence	denotes	This has been shown to increase the effect of the cytokine in vivo [19].
T222	29820-29892	Sentence	denotes	This has been shown to increase the effect of the cytokine in vivo [19].
T13446	29893-30035	Sentence	denotes	Only the IL-4/IL-4 mAb complex resulted in a decrease of the amount of natural or inducible Treg (CD25+ and Foxp3+) cells 7 d after treatment.
T223	29893-30035	Sentence	denotes	Only the IL-4/IL-4 mAb complex resulted in a decrease of the amount of natural or inducible Treg (CD25+ and Foxp3+) cells 7 d after treatment.
T13447	30036-30145	Sentence	denotes	Our results suggest that IL-4 is only interfering with the differentiation of naive T cells into iTreg cells.
T224	30036-30145	Sentence	denotes	Our results suggest that IL-4 is only interfering with the differentiation of naive T cells into iTreg cells.
T13448	30146-30458	Sentence	denotes	But since a distinction of nTreg and iTreg cells is currently not possible, because iTreg cells also transiently express CD25 after activation, we cannot exclude that IL-4 may also inhibit Foxp3 expression in nTreg cells in vivo or that additional effects may contribute to the observed drop in Foxp3 expression.
T225	30146-30458	Sentence	denotes	But since a distinction of nTreg and iTreg cells is currently not possible, because iTreg cells also transiently express CD25 after activation, we cannot exclude that IL-4 may also inhibit Foxp3 expression in nTreg cells in vivo or that additional effects may contribute to the observed drop in Foxp3 expression.
T13449	30459-30598	Sentence	denotes	IL-4 has already been shown to negatively regulate the development of naive T cells into Th1 or the IL-17–producing T cells (Th17) [46,47].
T226	30459-30598	Sentence	denotes	IL-4 has already been shown to negatively regulate the development of naive T cells into Th1 or the IL-17–producing T cells (Th17) [46,47].
T13450	30599-30801	Sentence	denotes	Similar effects have been recently described for IL-6, which, combined with TGF-β, inhibits the generation of iTreg cells and induces differentiation into the Th17 cells by an unknown mechanism [48,49].
T227	30599-30801	Sentence	denotes	Similar effects have been recently described for IL-6, which, combined with TGF-β, inhibits the generation of iTreg cells and induces differentiation into the Th17 cells by an unknown mechanism [48,49].
T13451	30802-30905	Sentence	denotes	Thus the polarization into iTreg cells is negatively regulated by the effector cytokines IL-4 and IL-6.
T228	30802-30905	Sentence	denotes	Thus the polarization into iTreg cells is negatively regulated by the effector cytokines IL-4 and IL-6.
T13452	30906-31005	Sentence	denotes	IL-4 has been previously shown to induce the generation of FOXP3+ Treg cells out of CD4+CD25– [50].
T229	30906-31005	Sentence	denotes	IL-4 has been previously shown to induce the generation of FOXP3+ Treg cells out of CD4+CD25– [50].
T13453	31006-31156	Sentence	denotes	In those experiments, the concentrations of IL-4 used were low, and as we also observed, IL-4 at low concentration slightly enhanced FOXP3 expression.
T230	31006-31156	Sentence	denotes	In those experiments, the concentrations of IL-4 used were low, and as we also observed, IL-4 at low concentration slightly enhanced FOXP3 expression.
T13454	31157-31238	Sentence	denotes	Importantly, these concentrations were not sufficient to induce GATA3 expression.
T231	31157-31238	Sentence	denotes	Importantly, these concentrations were not sufficient to induce GATA3 expression.
T13455	31239-31359	Sentence	denotes	IL-4 may favor proliferation of nTreg cells [47] or directly regulate FOXP3 expression in a STAT-dependent fashion [51].
T232	31239-31359	Sentence	denotes	IL-4 may favor proliferation of nTreg cells [47] or directly regulate FOXP3 expression in a STAT-dependent fashion [51].
T13456	31360-31520	Sentence	denotes	Since IL-13 does not effectively reduce FOXP3 and fails to induce GATA3, we hypothesized that the IL-4–dependent inhibition of FOXP3 could be mediated by GATA3.
T233	31360-31520	Sentence	denotes	Since IL-13 does not effectively reduce FOXP3 and fails to induce GATA3, we hypothesized that the IL-4–dependent inhibition of FOXP3 could be mediated by GATA3.
T13457	31521-31660	Sentence	denotes	In fact, GATA3-inducing IL-4 concentrations repressed TGF-β–mediated FOXP3 expression, whereas IL-4 as well as TGF-β signaling were intact.
T234	31521-31660	Sentence	denotes	In fact, GATA3-inducing IL-4 concentrations repressed TGF-β–mediated FOXP3 expression, whereas IL-4 as well as TGF-β signaling were intact.
T13458	31661-31829	Sentence	denotes	This result suggested a competitive mechanism between GATA3 and FOXP3 transcription factors in determining lineage commitment during the early phase of differentiation.
T235	31661-31829	Sentence	denotes	This result suggested a competitive mechanism between GATA3 and FOXP3 transcription factors in determining lineage commitment during the early phase of differentiation.
T13459	31830-31932	Sentence	denotes	Accordingly, we investigated naturally high GATA3-expressing cells and confirmed the absence of FOXP3.
T236	31830-31932	Sentence	denotes	Accordingly, we investigated naturally high GATA3-expressing cells and confirmed the absence of FOXP3.
T13460	31933-32049	Sentence	denotes	Protein transduction of GATA3 into naive T cells inhibited FOXP3 induction in human, differentiating, naive T cells.
T237	31933-32049	Sentence	denotes	Protein transduction of GATA3 into naive T cells inhibited FOXP3 induction in human, differentiating, naive T cells.
T13461	32050-32195	Sentence	denotes	This inhibitory effect of GATA3 was further confirmed in BALB/c transgenic mice, expressing GATA3 in T cells (DO11.10:CD2-GATA3 transgenic mice).
T238	32050-32195	Sentence	denotes	This inhibitory effect of GATA3 was further confirmed in BALB/c transgenic mice, expressing GATA3 in T cells (DO11.10:CD2-GATA3 transgenic mice).
T13462	32196-32371	Sentence	denotes	In line with the transient overexpression of GATA3 in human T cells, cells of these mice failed to induce FOXP3 expression upon exposure with antigen in the presence of TGF-β.
T239	32196-32371	Sentence	denotes	In line with the transient overexpression of GATA3 in human T cells, cells of these mice failed to induce FOXP3 expression upon exposure with antigen in the presence of TGF-β.
T13463	32372-32529	Sentence	denotes	Strikingly, the DO11.10:CD2-GATA3 mice do have peripheral FOXP3+ cells, which however displayed a 10%–25% lower frequency compared to wild-type DO11.10 mice.
T240	32372-32529	Sentence	denotes	Strikingly, the DO11.10:CD2-GATA3 mice do have peripheral FOXP3+ cells, which however displayed a 10%–25% lower frequency compared to wild-type DO11.10 mice.
T13464	32530-32671	Sentence	denotes	Thus GATA3 restrains the development of certain Treg subsets, presumably the inducible, peripheral population and not those of thymic origin.
T241	32530-32671	Sentence	denotes	Thus GATA3 restrains the development of certain Treg subsets, presumably the inducible, peripheral population and not those of thymic origin.
T13465	32672-32801	Sentence	denotes	Thymic T cells undergo a different maturation process, which may explain the insensitivity of nTreg to GATA3 overexpression [52].
T242	32672-32801	Sentence	denotes	Thymic T cells undergo a different maturation process, which may explain the insensitivity of nTreg to GATA3 overexpression [52].
T13466	32802-33172	Sentence	denotes	In contrast to the Th2-differentiating and iTreg-inhibiting function of GATA3 in peripheral T cells, GATA3 acts in the thymus together with other transcription factors such as the Repressor of GATA3 (ROG) in the differentiation process toward CD8 cells [53,54] or participates in complex transcriptional feedback network to regulate sympathoadrenal differentiation [55].
T243	32802-33172	Sentence	denotes	In contrast to the Th2-differentiating and iTreg-inhibiting function of GATA3 in peripheral T cells, GATA3 acts in the thymus together with other transcription factors such as the Repressor of GATA3 (ROG) in the differentiation process toward CD8 cells [53,54] or participates in complex transcriptional feedback network to regulate sympathoadrenal differentiation [55].
T13467	33173-33258	Sentence	denotes	Therefore, the role of GATA3 appears to be tissue specific and cannot be generalized.
T244	33173-33258	Sentence	denotes	Therefore, the role of GATA3 appears to be tissue specific and cannot be generalized.
T13468	33259-33369	Sentence	denotes	Our study demonstrates that GATA3 repressed FOXP3 expression directly by binding to the FOXP3 promoter region.
T245	33259-33369	Sentence	denotes	Our study demonstrates that GATA3 repressed FOXP3 expression directly by binding to the FOXP3 promoter region.
T13469	33370-33525	Sentence	denotes	A palindromic GATA-site is located 303 bp upstream of the TSS in a highly conserved region, which we have previously identified as the FOXP3 promoter [24].
T246	33370-33525	Sentence	denotes	A palindromic GATA-site is located 303 bp upstream of the TSS in a highly conserved region, which we have previously identified as the FOXP3 promoter [24].
T13470	33526-33797	Sentence	denotes	Site-specific mutation of this site increased the activity of the promoter constructs, thus revealing the repressive nature of this GATA element in memory T cells, which naturally express GATA3, whereas no difference was seen in naive T cells, which do not express GATA3.
T247	33526-33797	Sentence	denotes	Site-specific mutation of this site increased the activity of the promoter constructs, thus revealing the repressive nature of this GATA element in memory T cells, which naturally express GATA3, whereas no difference was seen in naive T cells, which do not express GATA3.
T13471	33798-33891	Sentence	denotes	This palindromic GATA element is bound by GATA3 protein as proven with pull-down experiments.
T248	33798-33891	Sentence	denotes	This palindromic GATA element is bound by GATA3 protein as proven with pull-down experiments.
T13472	33892-33976	Sentence	denotes	Furthermore, it is shown by ChIP that GATA3 binds this element also in intact cells.
T249	33892-33976	Sentence	denotes	Furthermore, it is shown by ChIP that GATA3 binds this element also in intact cells.
T13473	33977-34184	Sentence	denotes	It is known that GATA3 can induce transcription by chromatin remodeling [56], by directly transactivating promoters [36], or, as shown in the current study, acts as a repressor of gene expression [21,57–59].
T250	33977-34184	Sentence	denotes	It is known that GATA3 can induce transcription by chromatin remodeling [56], by directly transactivating promoters [36], or, as shown in the current study, acts as a repressor of gene expression [21,57–59].
T13474	34185-34291	Sentence	denotes	Therefore keeping GATA3 expression low might be required to induce efficient FOXP3+ iTreg cell generation.
T251	34185-34291	Sentence	denotes	Therefore keeping GATA3 expression low might be required to induce efficient FOXP3+ iTreg cell generation.
T13475	34292-34581	Sentence	denotes	The molecular interactions enabling GATA3 to inhibit FOXP3 are not identified yet, but the GATA-binding site is located adjacent to positive, inducing sites, composed of AP-1-NFATc2 sites [24], and GATA3 may compete with the binding of AP-1/NFAT to the promoter (unpublished observations).
T252	34292-34581	Sentence	denotes	The molecular interactions enabling GATA3 to inhibit FOXP3 are not identified yet, but the GATA-binding site is located adjacent to positive, inducing sites, composed of AP-1-NFATc2 sites [24], and GATA3 may compete with the binding of AP-1/NFAT to the promoter (unpublished observations).
T13476	34582-34671	Sentence	denotes	In summary, we demonstrated that FOXP3 is negatively regulated by cytokines such as IL-4.
T253	34582-34671	Sentence	denotes	In summary, we demonstrated that FOXP3 is negatively regulated by cytokines such as IL-4.
T13477	34672-34845	Sentence	denotes	GATA3 acts as an inhibitor of FOXP3 expression in early T cell differentiation, as well as in differentiated Th2 cells by directly binding and repressing the FOXP3 promoter.
T254	34672-34845	Sentence	denotes	GATA3 acts as an inhibitor of FOXP3 expression in early T cell differentiation, as well as in differentiated Th2 cells by directly binding and repressing the FOXP3 promoter.
T13478	34846-34954	Sentence	denotes	We therefore describe a new mechanism of how Il-4 avoids tolerance induction by repressing FOXP3 expression.
T255	34846-34954	Sentence	denotes	We therefore describe a new mechanism of how Il-4 avoids tolerance induction by repressing FOXP3 expression.
T13479	34955-35146	Sentence	denotes	These findings will give new perspectives toward understanding molecular mechanisms of iTreg induction and thus pathways of peripheral tolerance induction, particularly in allergy and asthma.
T256	34955-35146	Sentence	denotes	These findings will give new perspectives toward understanding molecular mechanisms of iTreg induction and thus pathways of peripheral tolerance induction, particularly in allergy and asthma.
T257	35148-35169	Sentence	denotes	Materials and Methods
T17772	35171-35176	Sentence	denotes	Mice.
T258	35171-35176	Sentence	denotes	Mice.

T58

0-106

Sentence

denotes

GATA3-Driven Th2 Responses Inhibit TGF-β1–Induced FOXP3 Expression and the Formation of Regulatory T Cells

T1

0-147

Sentence

denotes

GATA3-Driven Th2 Responses Inhibit TGF-β1–Induced FOXP3 Expression and the Formation of Regulatory T Cells GATA3 Inhibits FOXP3 and Inducible Tregs

T2

150-158

Sentence

denotes

Abstract

T59

159-387

Sentence

denotes

Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes.

T3

159-387

Sentence

denotes

Transcription factors act in concert to induce lineage commitment towards Th1, Th2, or T regulatory (Treg) cells, and their counter-regulatory mechanisms were shown to be critical for polarization between Th1 and Th2 phenotypes.

T60

388-575

Sentence

denotes

FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown.

T4

388-575

Sentence

denotes

FOXP3 is an essential transcription factor for natural, thymus-derived (nTreg) and inducible Treg (iTreg) commitment; however, the mechanisms regulating its expression are as yet unknown.

T61

576-686

Sentence

denotes

We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway.

T5

576-686

Sentence

denotes

We describe a mechanism controlling iTreg polarization, which is overruled by the Th2 differentiation pathway.

T62

687-782

Sentence

denotes

We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3.

T6

687-782

Sentence

denotes

We demonstrated that interleukin 4 (IL-4) present at the time of T cell priming inhibits FOXP3.

T63

783-1004

Sentence

denotes

This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-β–mediated FOXP3 induction.

T7

783-1004

Sentence

denotes

This inhibitory mechanism was also confirmed in Th2 cells and in T cells of transgenic mice overexpressing GATA-3 in T cells, which are shown to be deficient in transforming growth factor (TGF)-β–mediated FOXP3 induction.

T64

1005-1127

Sentence

denotes

This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process.

T8

1005-1127

Sentence

denotes

This inhibition is mediated by direct binding of GATA3 to the FOXP3 promoter, which represses its transactivation process.

T65

1128-1244

Sentence

denotes

Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response.

T9

1128-1244

Sentence

denotes

Therefore, this study provides a new understanding of tolerance development, controlled by a type 2 immune response.

T66

1245-1493

Sentence

denotes

IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.

T10

1245-1493

Sentence

denotes

IL-4 treatment in mice reduces iTreg cell frequency, highlighting that therapeutic approaches that target IL-4 or GATA3 might provide new preventive strategies facilitating tolerance induction particularly in Th2-mediated diseases, such as allergy.

T11

1495-1509

Sentence

denotes

Author Summary

T12

1511-1724

Sentence

denotes

Specific immune responses against foreign or autologous antigens are driven by specialized epitope-specific T cells, whose numbers expand upon recognition of antigen found on professional antigen-presenting cells.

T13

1725-1944

Sentence

denotes

The subsequent maturation process involves the differentiation of certain T cell phenotypes such as pro-inflammatory cells (Th1, Th2, Th17) or regulatory T (Treg) cells, which serve to keep the immune response in check.

T14

1945-2077

Sentence

denotes

The current study focuses on the role of two key transcription factors—FOXP3 and GATA3—in controlling the commitment of these cells.

T15

2078-2209

Sentence

denotes

We demonstrate that the Th2 cytokine IL-4 inhibits the induction of FOXP3 and thus inhibits the generation of inducible Treg cells.

T16

2210-2328

Sentence

denotes

We show that IL-4–induced GATA3 mediates FOXP3 inhibition by directly binding to a GATA element in the FOXP3 promoter.

T17

2329-2544

Sentence

denotes

We hypothesize that therapeutic agents aimed at neutralizing IL-4 could be a novel strategy to facilitate inducible Treg cell generation and thus promotion of tolerance in allergies and other Th2-dominated diseases.

T18

2546-2619

Sentence

denotes

It is shown that Th2 responses prevent the generation of inducible Tregs.

T19

2620-2722

Sentence

denotes

This is mediated by IL-4 induction of GATA3, which binds directly to and represses the FOXP3 promoter.

T20

2723-2834

Sentence

denotes

This mechanism is likely to be relevant in the induction of immunotolerance, particularly in allergic diseases.

T21

2836-2848

Sentence

denotes

Introduction

T975

2849-2997

Sentence

denotes

Effective immune responses are characterized by T cell activation, which directs adaptive and innate immune responses to kill pathogens efficiently.

T22

2849-2997

Sentence

denotes

Effective immune responses are characterized by T cell activation, which directs adaptive and innate immune responses to kill pathogens efficiently.

T976

2998-3181

Sentence

denotes

Dependent on the pathogen, T cells differentiate into different subtypes, such as Th1 or Th2 cells, which are most efficient in defeating microbial or parasitic invaders respectively.

T23

2998-3181

Sentence

denotes

Dependent on the pathogen, T cells differentiate into different subtypes, such as Th1 or Th2 cells, which are most efficient in defeating microbial or parasitic invaders respectively.

T977

3182-3341

Sentence

denotes

A hallmark of Th1 and Th2 differentiation pathways is the exclusiveness of the individual phenotype leading to either Th1 or Th2, but not to mixed populations.

T24

3182-3341

Sentence

denotes

A hallmark of Th1 and Th2 differentiation pathways is the exclusiveness of the individual phenotype leading to either Th1 or Th2, but not to mixed populations.

T978

3342-3482

Sentence

denotes

The exclusiveness of this mechanism is provided by a polarization process, where Th2 differentiation inhibits Th1 commitment and vice versa.

T25

3342-3482

Sentence

denotes

The exclusiveness of this mechanism is provided by a polarization process, where Th2 differentiation inhibits Th1 commitment and vice versa.

T979

3483-3619

Sentence

denotes

Specifically interleukin 4 (IL-4)–induced STAT6 and GATA3 inhibit differentiation into Th1 cells in the early phase of commitment [1,2].

T26

3483-3619

Sentence

denotes

Specifically interleukin 4 (IL-4)–induced STAT6 and GATA3 inhibit differentiation into Th1 cells in the early phase of commitment [1,2].

T980

3620-3819

Sentence

denotes

GATA3 is sufficient to induce a Th2 phenotype [3] and acts not only through the induction of IL-4, IL-5 and IL-13, the Th2 cytokines, but also through the inhibition of Th1 cell-specific factors [3].

T27

3620-3819

Sentence

denotes

GATA3 is sufficient to induce a Th2 phenotype [3] and acts not only through the induction of IL-4, IL-5 and IL-13, the Th2 cytokines, but also through the inhibition of Th1 cell-specific factors [3].

T981

3820-4074

Sentence

denotes

Recently, it was shown that T-bet directly modulates GATA3 function, suggesting that transcription factors compete in the early differentiation phase of T cells, potentially integrating environmental signals to finally imprint the T cell phenotype [4,5].

T28

3820-4074

Sentence

denotes

Recently, it was shown that T-bet directly modulates GATA3 function, suggesting that transcription factors compete in the early differentiation phase of T cells, potentially integrating environmental signals to finally imprint the T cell phenotype [4,5].

T982

4075-4250

Sentence

denotes

A GATA3-dominated immune response has been shown to be essential in airway hyperresonsiveness [6] and IL-4–dominated responses can break antigen-specific immune tolerance [7].

T29

4075-4250

Sentence

denotes

A GATA3-dominated immune response has been shown to be essential in airway hyperresonsiveness [6] and IL-4–dominated responses can break antigen-specific immune tolerance [7].

T983

4251-4430

Sentence

denotes

Overexpression of a dominant negative form of GATA3 [8] or treatment with antisense-mediated GATA3 blockade [9] decreased the severity of the allergic airway hyper-responsiveness.

T30

4251-4430

Sentence

denotes

Overexpression of a dominant negative form of GATA3 [8] or treatment with antisense-mediated GATA3 blockade [9] decreased the severity of the allergic airway hyper-responsiveness.

T984

4431-4570

Sentence

denotes

The discovery of regulatory T (Treg) cells highlights another phenotype of T cells, which is essential for tolerance against self-antigens.

T31

4431-4570

Sentence

denotes

The discovery of regulatory T (Treg) cells highlights another phenotype of T cells, which is essential for tolerance against self-antigens.

T985

4571-4743

Sentence

denotes

Naturally occurring, thymus-derived Treg (nTreg) cells are generated in the thymus and are assumed to protect against the activity of autoreactive T cells in the periphery.

T32

4571-4743

Sentence

denotes

Naturally occurring, thymus-derived Treg (nTreg) cells are generated in the thymus and are assumed to protect against the activity of autoreactive T cells in the periphery.

T986

4744-4903

Sentence

denotes

These cells express the forkhead transcription factor FOXP3 and constitutively express CD25 on their surface, but they lack expression of Th1 or Th2 cytokines.

T33

4744-4903

Sentence

denotes

These cells express the forkhead transcription factor FOXP3 and constitutively express CD25 on their surface, but they lack expression of Th1 or Th2 cytokines.

T987

4904-5047

Sentence

denotes

Particularly interesting are those Treg cells that are generated in the periphery and thus are potential targets for therapeutic interventions.

T34

4904-5047

Sentence

denotes

Particularly interesting are those Treg cells that are generated in the periphery and thus are potential targets for therapeutic interventions.

T988

5048-5117

Sentence

denotes

These induced Treg (iTreg) cells were reported to express FOXP3 [10].

T35

5048-5117

Sentence

denotes

These induced Treg (iTreg) cells were reported to express FOXP3 [10].

T989

5118-5355

Sentence

denotes

The exact mechanisms of iTreg generation are unclear, but T cell receptor (TCR) triggering has been shown to induce FOXP3 expression and suppressive cells in human [11,12], however the phenotype appears to be of transient nature [13,14].

T36

5118-5355

Sentence

denotes

The exact mechanisms of iTreg generation are unclear, but T cell receptor (TCR) triggering has been shown to induce FOXP3 expression and suppressive cells in human [11,12], however the phenotype appears to be of transient nature [13,14].

T990

5356-5625

Sentence

denotes

TGF-β has been demonstrated to be important for the persistent induction of these cells in vitro and in vivo, since animals lacking the TGF-βRII on T cells have fewer peripherally iTreg cells [15] and suffer from a T cell–dependent multiorgan inflammatory disease [16].

T37

5356-5625

Sentence

denotes

TGF-β has been demonstrated to be important for the persistent induction of these cells in vitro and in vivo, since animals lacking the TGF-βRII on T cells have fewer peripherally iTreg cells [15] and suffer from a T cell–dependent multiorgan inflammatory disease [16].

T991

5626-5791

Sentence

denotes

Although the effect of TGF-β on natural and inducible Treg cell induction has been demonstrated repeatedly [15,17], its molecular mechanisms remain to be identified.

T38

5626-5791

Sentence

denotes

Although the effect of TGF-β on natural and inducible Treg cell induction has been demonstrated repeatedly [15,17], its molecular mechanisms remain to be identified.

T992

5792-5963

Sentence

denotes

The current study provides evidence that GATA3 and FOXP3 play a competitive role in iTreg cell commitment as T-bet and GATA3 for Th1 and Th2 differentiation, respectively.

T39

5792-5963

Sentence

denotes

The current study provides evidence that GATA3 and FOXP3 play a competitive role in iTreg cell commitment as T-bet and GATA3 for Th1 and Th2 differentiation, respectively.

T993

5964-6097

Sentence

denotes

We show that GATA3 inhibits FOXP3 induction and that IL-4 limits FOXP3 expression in a GATA3-mediated way, both in vitro and in vivo.

T40

5964-6097

Sentence

denotes

We show that GATA3 inhibits FOXP3 induction and that IL-4 limits FOXP3 expression in a GATA3-mediated way, both in vitro and in vivo.

T994

6098-6273

Sentence

denotes

We also show that GATA3 directly binds to the FOXP3 promoter and thereby prevents the induction of this gene, demonstrating that Th2 differentiation overrules iTreg induction.

T41

6098-6273

Sentence

denotes

We also show that GATA3 directly binds to the FOXP3 promoter and thereby prevents the induction of this gene, demonstrating that Th2 differentiation overrules iTreg induction.

T42

6275-6282

Sentence

denotes

Results

T2865

6284-6317

Sentence

denotes

FOXP3 Induction in T Cell Subsets

T43

6284-6317

Sentence

denotes

FOXP3 Induction in T Cell Subsets

T2866

6318-6452

Sentence

denotes

It is assumed that FOXP3 expression can be induced in nonregulatory T cells, which is an important step in iTreg cell differentiation.

T44

6318-6452

Sentence

denotes

It is assumed that FOXP3 expression can be induced in nonregulatory T cells, which is an important step in iTreg cell differentiation.

T2867

6453-6538

Sentence

denotes

However, it is not known if all CD4+ T cells have the same capacity to express FOXP3.

T45

6453-6538

Sentence

denotes

However, it is not known if all CD4+ T cells have the same capacity to express FOXP3.

T2868

6539-6918

Sentence

denotes

To investigate whether FOXP3 can be expressed by any T cell subset or if expression is restricted to a distinct lineage, FOXP3 mRNA expression was analyzed in freshly isolated T cells such as CD25-depleted CD4+ cells, CD45RA+ naive or CD45RO+ memory T cells (Figure 1A), as well as T cells driven in vitro toward Th1, Th2, or iTreg phenotypes (Figure 1B; phenotype on Figure S1).

T46

6539-6918

Sentence

denotes

To investigate whether FOXP3 can be expressed by any T cell subset or if expression is restricted to a distinct lineage, FOXP3 mRNA expression was analyzed in freshly isolated T cells such as CD25-depleted CD4+ cells, CD45RA+ naive or CD45RO+ memory T cells (Figure 1A), as well as T cells driven in vitro toward Th1, Th2, or iTreg phenotypes (Figure 1B; phenotype on Figure S1).

T2869

6919-7074

Sentence

denotes

The CD4+CD25–, CD45RA+, CD45RO+, and CD4+CD45RO+CD25– were able to significantly induce FOXP3 mRNA up to 30-fold upon TCR activation and addition of TGF-β.

T47

6919-7074

Sentence

denotes

The CD4+CD25–, CD45RA+, CD45RO+, and CD4+CD45RO+CD25– were able to significantly induce FOXP3 mRNA up to 30-fold upon TCR activation and addition of TGF-β.

T2870

7075-7116

Sentence

denotes

Th1 cells showed only a 10-fold increase.

T48

7075-7116

Sentence

denotes

Th1 cells showed only a 10-fold increase.

T2871

7117-7211

Sentence

denotes

In contrast, Th2 cells stimulated under the same conditions did not increase FOXP3 expression.

T49

7117-7211

Sentence

denotes

In contrast, Th2 cells stimulated under the same conditions did not increase FOXP3 expression.

T2872

7212-7376

Sentence

denotes

The in vitro generated iTreg cells were unable to further up-regulate FOXP3, which was already at high levels under the resting conditions (Figure 1B, right panel).

T50

7212-7376

Sentence

denotes

The in vitro generated iTreg cells were unable to further up-regulate FOXP3, which was already at high levels under the resting conditions (Figure 1B, right panel).

T51

7377-7427

Sentence

denotes

Figure 1 Th2 Cells Cannot Induce FOXP3 Expression

T23457

7387-7427

Sentence

denotes

Th2 Cells Cannot Induce FOXP3 Expression

T2873

7387-8083

Sentence

denotes

Th2 Cells Cannot Induce FOXP3 Expression (A) Human T cells were activated with plate-bound anti-CD3/CD28 with or without TGF-β as indicated on the x-axis of (B). Cells were harvested after 5 days and FOXP3 mRNA was quantified by real-time PCR. Bars show the mean ± SD of 4 independent experiments. (B) In vitro differentiated Th1, Th2, or iTreg cells were activated with anti-CD3/CD28, TGF-β, or anti-IL-4 as indicated. The phenotype of these cells was confirmed by FACS and proliferation analysis (Figure S1). Bars show the mean ± SD of four independent experiments. Th2 cells are known to produce IL-4 upon activation, which may interact with TGF-β signaling and thus prevent FOXP3 induction.

T23458

7428-7548

Sentence

denotes

(A) Human T cells were activated with plate-bound anti-CD3/CD28 with or without TGF-β as indicated on the x-axis of (B).

T52

7428-7548

Sentence

denotes

(A) Human T cells were activated with plate-bound anti-CD3/CD28 with or without TGF-β as indicated on the x-axis of (B).

T23459

7549-7630

Sentence

denotes

Cells were harvested after 5 days and FOXP3 mRNA was quantified by real-time PCR.

T53

7549-7630

Sentence

denotes

Cells were harvested after 5 days and FOXP3 mRNA was quantified by real-time PCR.

T23460

7631-7684

Sentence

denotes

Bars show the mean ± SD of 4 independent experiments.

T54

7631-7684

Sentence

denotes

Bars show the mean ± SD of 4 independent experiments.

T23461

7685-7806

Sentence

denotes

(B) In vitro differentiated Th1, Th2, or iTreg cells were activated with anti-CD3/CD28, TGF-β, or anti-IL-4 as indicated.

T55

7685-7806

Sentence

denotes

(B) In vitro differentiated Th1, Th2, or iTreg cells were activated with anti-CD3/CD28, TGF-β, or anti-IL-4 as indicated.

T23462

7807-7897

Sentence

denotes

The phenotype of these cells was confirmed by FACS and proliferation analysis (Figure S1).

T56

7807-7897

Sentence

denotes

The phenotype of these cells was confirmed by FACS and proliferation analysis (Figure S1).

T23463

7898-7954

Sentence

denotes

Bars show the mean ± SD of four independent experiments.

T57

7898-7954

Sentence

denotes

Bars show the mean ± SD of four independent experiments.

T58

7957-8083

Sentence

denotes

Th2 cells are known to produce IL-4 upon activation, which may interact with TGF-β signaling and thus prevent FOXP3 induction.

T2874

8084-8233

Sentence

denotes

However, the neutralization of IL-4 with a blocking IL-4 antibody did not rescue FOXP3 expression in the differentiated Th2 cells (unpublished data).

T59

8084-8233

Sentence

denotes

However, the neutralization of IL-4 with a blocking IL-4 antibody did not rescue FOXP3 expression in the differentiated Th2 cells (unpublished data).

T2875

8234-8326

Sentence

denotes

These data demonstrated that Th2 cells have a limited capacity to express FOXP3 (Figure 1B).

T60

8234-8326

Sentence

denotes

These data demonstrated that Th2 cells have a limited capacity to express FOXP3 (Figure 1B).

T2876

8327-8477

Sentence

denotes

The inability of Th2 cells to express FOXP3 was also documented at the single-cell level, confirming that Th2 cells lack FOXP3 expression (Figure 2A).

T61

8327-8477

Sentence

denotes

The inability of Th2 cells to express FOXP3 was also documented at the single-cell level, confirming that Th2 cells lack FOXP3 expression (Figure 2A).

T2877

8478-8533

Sentence

denotes

Only iTreg cells expressed FOXP3 in resting conditions.

T62

8478-8533

Sentence

denotes

Only iTreg cells expressed FOXP3 in resting conditions.

T2878

8534-8637

Sentence

denotes

Interestingly, we observed that resting iTreg cells express FOXP3 but show low CD25 surface expression.

T63

8534-8637

Sentence

denotes

Interestingly, we observed that resting iTreg cells express FOXP3 but show low CD25 surface expression.

T2879

8638-8786

Sentence

denotes

Repeated exposure to TGF-β did not further increase the FOXP3 expression in the iTreg lineage but transiently induced FOXP3 expression in Th1 cells.

T64

8638-8786

Sentence

denotes

Repeated exposure to TGF-β did not further increase the FOXP3 expression in the iTreg lineage but transiently induced FOXP3 expression in Th1 cells.

T2880

8787-8980

Sentence

denotes

Naturally occurring Th2 cells such as CRTH2+ T cells, T cells isolated according to their IL-4 secretion, or an IL-4–producing T cell clone (BR8) were also lacking FOXP3 expression (Figure 2B).

T65

8787-8980

Sentence

denotes

Naturally occurring Th2 cells such as CRTH2+ T cells, T cells isolated according to their IL-4 secretion, or an IL-4–producing T cell clone (BR8) were also lacking FOXP3 expression (Figure 2B).

T2881

8981-9091

Sentence

denotes

Furthermore TGF-β–mediated FOXP3 induction failed in these cells in contrast to the naive T cells (Figure 2B).

T66

8981-9091

Sentence

denotes

Furthermore TGF-β–mediated FOXP3 induction failed in these cells in contrast to the naive T cells (Figure 2B).

T2882

9092-9255

Sentence

denotes

Because IL-4 is the key Th2 cytokine, the expression of IL-4 and FOXP3 in freshly isolated CD4+ T cells was analyzed by fluorescence activated cell sorting (FACS).

T67

9092-9255

Sentence

denotes

Because IL-4 is the key Th2 cytokine, the expression of IL-4 and FOXP3 in freshly isolated CD4+ T cells was analyzed by fluorescence activated cell sorting (FACS).

T2883

9256-9378

Sentence

denotes

IL-4–expressing cells were most abundant among CD45RO+CD25– cells, which did not co-express FOXP3 (Figure 3A, left panel).

T68

9256-9378

Sentence

denotes

IL-4–expressing cells were most abundant among CD45RO+CD25– cells, which did not co-express FOXP3 (Figure 3A, left panel).

T2884

9379-9483

Sentence

denotes

In contrast, CD45RO+CD25+ cells abundantly expressed FOXP3, while lacking IL-4 (Figure 3A, right panel).

T69

9379-9483

Sentence

denotes

In contrast, CD45RO+CD25+ cells abundantly expressed FOXP3, while lacking IL-4 (Figure 3A, right panel).

T2885

9484-9604

Sentence

denotes

As shown in Figure 3B, the frequency of the IL-4+ cells was always below 1% in the FOXP3+ cells close to the background.

T70

9484-9604

Sentence

denotes

As shown in Figure 3B, the frequency of the IL-4+ cells was always below 1% in the FOXP3+ cells close to the background.

T2886

9605-9740

Sentence

denotes

The IL-4+ cells were confined to the FOXP3– cells, as shown for the CD45RO+CD25–, CD45RO+CD25+, and CD45RO+CD25+high cells (Figure 3B).

T71

9605-9740

Sentence

denotes

The IL-4+ cells were confined to the FOXP3– cells, as shown for the CD45RO+CD25–, CD45RO+CD25+, and CD45RO+CD25+high cells (Figure 3B).

T2887

9741-9840

Sentence

denotes

In addition, neither the Th2 clone (BR8) nor CRTH2 cells significantly expressed FOXP3 (Figure 3C).

T72

9741-9840

Sentence

denotes

In addition, neither the Th2 clone (BR8) nor CRTH2 cells significantly expressed FOXP3 (Figure 3C).

T2888

9841-10013

Sentence

denotes

Cells enriched for their IL-4 secretion using the magnetic cell isolation technology contained some FOXP3-expressing cells, but importantly, the expression did not overlap.

T73

9841-10013

Sentence

denotes

Cells enriched for their IL-4 secretion using the magnetic cell isolation technology contained some FOXP3-expressing cells, but importantly, the expression did not overlap.

T2889

10014-10129

Sentence

denotes

Taken together, these data indicate that FOXP3 was not expressed by Th2 cells and was not inducible in those cells.

T74

10014-10129

Sentence

denotes

Taken together, these data indicate that FOXP3 was not expressed by Th2 cells and was not inducible in those cells.

T75

10130-10170

Sentence

denotes

Figure 2 Th2 Cells Do Not Express FOXP3

T23808

10140-10170

Sentence

denotes

Th2 Cells Do Not Express FOXP3

T23809

10171-10348

Sentence

denotes

(A) Intracellular FACS analysis of FOXP3 expression in Th1, Th2, or iTreg-differentiated cells (two rounds, phenotype see Figure S1), rested or activated, with or without TGF-β.

T76

10171-10348

Sentence

denotes

(A) Intracellular FACS analysis of FOXP3 expression in Th1, Th2, or iTreg-differentiated cells (two rounds, phenotype see Figure S1), rested or activated, with or without TGF-β.

T23810

10349-10400

Sentence

denotes

FOXP3 expression was measured after 5 d in culture.

T77

10349-10400

Sentence

denotes

FOXP3 expression was measured after 5 d in culture.

T23811

10401-10467

Sentence

denotes

The dot blots are representative of three independent experiments.

T78

10401-10467

Sentence

denotes

The dot blots are representative of three independent experiments.

T23812

10468-10555

Sentence

denotes

(B) Shows the same experimental setup, but naturally occurring Th2 cells were analyzed.

T79

10468-10555

Sentence

denotes

(B) Shows the same experimental setup, but naturally occurring Th2 cells were analyzed.

T23813

10556-10613

Sentence

denotes

Data are representative of three independent experiments.

T80

10556-10613

Sentence

denotes

Data are representative of three independent experiments.

T81

10614-10663

Sentence

denotes

Figure 3 Th2- or IL-4–Producing Cells Lack FOXP3

T24125

10624-10663

Sentence

denotes

Th2- or IL-4–Producing Cells Lack FOXP3

T24126

10664-10761

Sentence

denotes

(A) FACS analysis of intracellular FOXP3 and IL-4 expression following PMA/Ionomycin stimulation.

T82

10664-10761

Sentence

denotes

(A) FACS analysis of intracellular FOXP3 and IL-4 expression following PMA/Ionomycin stimulation.

T24127

10762-10999

Sentence

denotes

CD4+ T cells were gated on the basis of CD45RO and CD25 surface expression (upper panel), and gated cells are shown below for the CD45RO+CD25− (A, left panel), the CD45RO+CD25+ (right panel), and the CD45RO−CD25− subsets (central panel).

T83

10762-10999

Sentence

denotes

CD4+ T cells were gated on the basis of CD45RO and CD25 surface expression (upper panel), and gated cells are shown below for the CD45RO+CD25− (A, left panel), the CD45RO+CD25+ (right panel), and the CD45RO−CD25− subsets (central panel).

T24128

11000-11109

Sentence

denotes

A statistical analysis of eight independent donors after subtraction of the isotype control are shown in (B).

T84

11000-11109

Sentence

denotes

A statistical analysis of eight independent donors after subtraction of the isotype control are shown in (B).

T24129

11110-11165

Sentence

denotes

The dotted gray line indicates the IC background level.

T85

11110-11165

Sentence

denotes

The dotted gray line indicates the IC background level.

T24130

11166-11343

Sentence

denotes

The error bars show the error of the mean. (C) Similarly, a Th2 clone (BR8), CRTH2+ Th2 cells, IL-4-secreting cells, and memory T cells (CD45RO) were stained for FOXP3 and IL-4.

T86

11166-11343

Sentence

denotes

The error bars show the error of the mean. (C) Similarly, a Th2 clone (BR8), CRTH2+ Th2 cells, IL-4-secreting cells, and memory T cells (CD45RO) were stained for FOXP3 and IL-4.

T24131

11344-11401

Sentence

denotes

Data are representative of three independent experiments.

T87

11344-11401

Sentence

denotes

Data are representative of three independent experiments.

T5207

11403-11452

Sentence

denotes

FOXP3 and GATA3 Kinetics in Differentiating Cells

T88

11403-11452

Sentence

denotes

FOXP3 and GATA3 Kinetics in Differentiating Cells

T5208

11453-11623

Sentence

denotes

The limited capacity of differentiated effector cells to induce FOXP3 expression suggests that iTreg induction has to occur before effector T cell differentiation occurs.

T89

11453-11623

Sentence

denotes

The limited capacity of differentiated effector cells to induce FOXP3 expression suggests that iTreg induction has to occur before effector T cell differentiation occurs.

T5209

11624-11814

Sentence

denotes

Therefore, we analyzed the expression of FOXP3 and GATA3 during the differentiation of naive CD4 T cells into Th0 (neutral, anti-IL-4, anti-IFN-γ, and anti IL-12), Th2, and iTreg phenotypes.

T90

11624-11814

Sentence

denotes

Therefore, we analyzed the expression of FOXP3 and GATA3 during the differentiation of naive CD4 T cells into Th0 (neutral, anti-IL-4, anti-IFN-γ, and anti IL-12), Th2, and iTreg phenotypes.

T5210

11815-12000

Sentence

denotes

After initiation of the differentiation process, FOXP3 and GATA3 showed a similar expression kinetic within the first 3 d, which are considered to be critical in T cell commitment [18].

T91

11815-12000

Sentence

denotes

After initiation of the differentiation process, FOXP3 and GATA3 showed a similar expression kinetic within the first 3 d, which are considered to be critical in T cell commitment [18].

T5211

12001-12111

Sentence

denotes

Under Th2 differentiation conditions, FOXP3 mRNA expression increased only marginally (Figure 4A, left panel).

T92

12001-12111

Sentence

denotes

Under Th2 differentiation conditions, FOXP3 mRNA expression increased only marginally (Figure 4A, left panel).

T5212

12112-12322

Sentence

denotes

Thus, although GATA3 and FOXP3 showed similar kinetics, their expression polarizes at the end of the differentiation process when cells were cultured towards Th2 or iTreg cells, respectively (Figure 4A and 4B).

T93

12112-12322

Sentence

denotes

Thus, although GATA3 and FOXP3 showed similar kinetics, their expression polarizes at the end of the differentiation process when cells were cultured towards Th2 or iTreg cells, respectively (Figure 4A and 4B).

T5213

12323-12633

Sentence

denotes

Interestingly, the Th0 cells were expressing more FOXP3 than the Th2 cells, but expressed low levels of GATA3; however, the protein expression slightly differed from mRNA expression, suggesting also posttranslational regulation and degradation as potential additional mechanisms in the differentiation process.

T94

12323-12633

Sentence

denotes

Interestingly, the Th0 cells were expressing more FOXP3 than the Th2 cells, but expressed low levels of GATA3; however, the protein expression slightly differed from mRNA expression, suggesting also posttranslational regulation and degradation as potential additional mechanisms in the differentiation process.

T5214

12634-12798

Sentence

denotes

The phenotype of iTreg cells included an anergic phenotype upon anti-CD3 re-stimulation (Figure S1A), CD103, CTLA-4, GITR, and PD-1 surface expression (Figure S1B).

T95

12634-12798

Sentence

denotes

The phenotype of iTreg cells included an anergic phenotype upon anti-CD3 re-stimulation (Figure S1A), CD103, CTLA-4, GITR, and PD-1 surface expression (Figure S1B).

T5215

12799-13018

Sentence

denotes

On the single-cell level, it can be seen that cells progress through a transition phase, where GATA3 and FOXP3 expression coexist to some degree in the same cells, which is resolved in iTreg cells after 7 d (Figure 4B).

T96

12799-13018

Sentence

denotes

On the single-cell level, it can be seen that cells progress through a transition phase, where GATA3 and FOXP3 expression coexist to some degree in the same cells, which is resolved in iTreg cells after 7 d (Figure 4B).

T5216

13019-13202

Sentence

denotes

Taken together, these data demonstrated that Th2 cells have lost their capacity to express FOXP3 and showed that Th2 and iTreg cells arise from two different differentiation pathways.

T97

13019-13202

Sentence

denotes

Taken together, these data demonstrated that Th2 cells have lost their capacity to express FOXP3 and showed that Th2 and iTreg cells arise from two different differentiation pathways.

T98

13203-13263

Sentence

denotes

Figure 4 FOXP3 Induction During the Differentiation Process

T24699

13213-13263

Sentence

denotes

FOXP3 Induction During the Differentiation Process

T24700

13264-13394

Sentence

denotes

(A) Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-β (5 ng/ml) or IL-4 (25 ng/ml).

T99

13264-13394

Sentence

denotes

(A) Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-β (5 ng/ml) or IL-4 (25 ng/ml).

T24701

13395-13518

Sentence

denotes

The cells were harvested at different time points, and mRNA was quantified by real-time PCR for FOXP3 and GATA3 expression.

T100

13395-13518

Sentence

denotes

The cells were harvested at different time points, and mRNA was quantified by real-time PCR for FOXP3 and GATA3 expression.

T24702

13519-13576

Sentence

denotes

Bars show the mean ± SD of three independent experiments.

T101

13519-13576

Sentence

denotes

Bars show the mean ± SD of three independent experiments.

T24703

13578-13725

Sentence

denotes

(B) Intracellular GATA3 and FOXP3 staining is shown after exposure of CD4+CD45RA+ T cells to differentiating conditions as in part A of the figure.

T102

13578-13725

Sentence

denotes

(B) Intracellular GATA3 and FOXP3 staining is shown after exposure of CD4+CD45RA+ T cells to differentiating conditions as in part A of the figure.

T24704

13726-13783

Sentence

denotes

Data are representative of three independent experiments.

T103

13726-13783

Sentence

denotes

Data are representative of three independent experiments.

T6377

13785-13830

Sentence

denotes

IL-4 Inhibits TGF-β-Mediated iTreg Commitment

T104

13785-13830

Sentence

denotes

IL-4 Inhibits TGF-β-Mediated iTreg Commitment

T6378

13831-13928

Sentence

denotes

IL-4 induces differentiation of naive T cells, upon antigen encounter, into the Th2 cell lineage.

T105

13831-13928

Sentence

denotes

IL-4 induces differentiation of naive T cells, upon antigen encounter, into the Th2 cell lineage.

T6379

13929-14041

Sentence

denotes

We therefore asked whether IL-4 is able to inhibit TGF-β induction of FOXP3 during the priming of naive T cells.

T106

13929-14041

Sentence

denotes

We therefore asked whether IL-4 is able to inhibit TGF-β induction of FOXP3 during the priming of naive T cells.

T6380

14042-14175

Sentence

denotes

Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-β and/or IL-4 and harvested after 5 d.

T107

14042-14175

Sentence

denotes

Human CD4+CD45RA+ T cells were activated with plate-bound anti-CD3/CD28 in the presence of TGF-β and/or IL-4 and harvested after 5 d.

T6381

14176-14312

Sentence

denotes

IL-4 efficiently repressed the TGF-β–mediated induction of FOXP3 expression (Figure 5A) in a concentration-dependent manner (Figure 5B).

T108

14176-14312

Sentence

denotes

IL-4 efficiently repressed the TGF-β–mediated induction of FOXP3 expression (Figure 5A) in a concentration-dependent manner (Figure 5B).

T6382

14313-14409

Sentence

denotes

Low levels of GATA3 were induced also in the absence of IL-4, as it was previously observed [3].

T109

14313-14409

Sentence

denotes

Low levels of GATA3 were induced also in the absence of IL-4, as it was previously observed [3].

T6383

14410-14493

Sentence

denotes

However, at low concentration, IL-4 was able to marginally induce FOXP3 expression.

T110

14410-14493

Sentence

denotes

However, at low concentration, IL-4 was able to marginally induce FOXP3 expression.

T6384

14494-14597

Sentence

denotes

Of note, GATA3 was also induced in the presence of TGF-β at high IL-4 concentration (Figure 5A and 5B).

T111

14494-14597

Sentence

denotes

Of note, GATA3 was also induced in the presence of TGF-β at high IL-4 concentration (Figure 5A and 5B).

T6385

14598-14914

Sentence

denotes

The IL-4–mediated prevention of FOXP3 expression was not caused by interferences of the receptor signaling, because the phosphorylation of SMAD2 or STAT6 was not affected by the addition of IL-4 and/or TGF-β, which demonstrates that IL-4 as well as TGF-β signaling were functional under these conditions (Figure 5C).

T112

14598-14914

Sentence

denotes

The IL-4–mediated prevention of FOXP3 expression was not caused by interferences of the receptor signaling, because the phosphorylation of SMAD2 or STAT6 was not affected by the addition of IL-4 and/or TGF-β, which demonstrates that IL-4 as well as TGF-β signaling were functional under these conditions (Figure 5C).

T6386

14915-15052

Sentence

denotes

Increasing amounts of IL-4 increase intracellular GATA3, whereas FOXP3 decreased, which is consistent with the mRNA analysis (Figure 5D).

T113

14915-15052

Sentence

denotes

Increasing amounts of IL-4 increase intracellular GATA3, whereas FOXP3 decreased, which is consistent with the mRNA analysis (Figure 5D).

T6387

15053-15162

Sentence

denotes

Furthermore, injection of IL-4 into wild-type B6 mice decreased the inducible or natural Treg number in vivo.

T114

15053-15162

Sentence

denotes

Furthermore, injection of IL-4 into wild-type B6 mice decreased the inducible or natural Treg number in vivo.

T6388

15163-15279

Sentence

denotes

A distinction of the Treg subsets is not possible, because recently activated iTreg cells also express surface CD25.

T115

15163-15279

Sentence

denotes

A distinction of the Treg subsets is not possible, because recently activated iTreg cells also express surface CD25.

T6389

15280-15466

Sentence

denotes

We used complexes of recombinant mouse IL-4 (rmIL-4) plus anti-IL-4 monoclonal antibodies (mAbs), which have been shown to dramatically increase the potency of the cytokine in vivo [19].

T116

15280-15466

Sentence

denotes

We used complexes of recombinant mouse IL-4 (rmIL-4) plus anti-IL-4 monoclonal antibodies (mAbs), which have been shown to dramatically increase the potency of the cytokine in vivo [19].

T6390

15467-15622

Sentence

denotes

In these mice, the percentage of CD4+CD25+ and FOXP3+ T cells dramatically decreased when the antibody-cytokine immune complexes were injected (Figure S2).

T117

15467-15622

Sentence

denotes

In these mice, the percentage of CD4+CD25+ and FOXP3+ T cells dramatically decreased when the antibody-cytokine immune complexes were injected (Figure S2).

T6391

15623-15921

Sentence

denotes

Upon administration of rmIL-4 plus anti-IL-4 mAb complexes, the total number of CD4+CD25+ T cell, as well as the Foxp3+ T cells diminished by half (Figure S2G and S2H), confirming that the lower percentage was not due to an increase in the CD4+CD25– cells, but a real decrease of CD4+CD25+ T cells.

T118

15623-15921

Sentence

denotes

Upon administration of rmIL-4 plus anti-IL-4 mAb complexes, the total number of CD4+CD25+ T cell, as well as the Foxp3+ T cells diminished by half (Figure S2G and S2H), confirming that the lower percentage was not due to an increase in the CD4+CD25– cells, but a real decrease of CD4+CD25+ T cells.

T6392

15922-16042

Sentence

denotes

In conclusion, IL-4 negatively regulates the natural or inducible Treg cell turnover not only in vitro but also in vivo.

T119

15922-16042

Sentence

denotes

In conclusion, IL-4 negatively regulates the natural or inducible Treg cell turnover not only in vitro but also in vivo.

T6393

16043-16240

Sentence

denotes

To study the effects of IL-4 on already-existing human natural or inducible Treg cells, we exposed sorted CD25+ T cells (nTreg cells) to IL-4 and analyzed FOXP3 expression and suppressive capacity.

T120

16043-16240

Sentence

denotes

To study the effects of IL-4 on already-existing human natural or inducible Treg cells, we exposed sorted CD25+ T cells (nTreg cells) to IL-4 and analyzed FOXP3 expression and suppressive capacity.

T6394

16241-16385

Sentence

denotes

In already-existing Treg cells, IL-4 failed to inhibit FOXP3 expression (Figure S3A), and the suppressive capacity was not altered (Figure S3C).

T121

16241-16385

Sentence

denotes

In already-existing Treg cells, IL-4 failed to inhibit FOXP3 expression (Figure S3A), and the suppressive capacity was not altered (Figure S3C).

T6395

16386-16487

Sentence

denotes

Similarly pre-existing iTreg cells did not decrease FOXP3 expression upon IL-4 exposure (Figure S3B).

T122

16386-16487

Sentence

denotes

Similarly pre-existing iTreg cells did not decrease FOXP3 expression upon IL-4 exposure (Figure S3B).

T123

16488-16531

Sentence

denotes

Figure 5 Effect of IL-4 on FOXP3 Induction

T25148

16498-16531

Sentence

denotes

Effect of IL-4 on FOXP3 Induction

T25149

16532-16720

Sentence

denotes

(A) A statistical analysis was performed with six donors on day 5 (TGF-β (10 ng/ml) and with or without IL-4 (100 ng/ml)); Shown is the mean, and error bars indicated the SD of six donors.

T124

16532-16720

Sentence

denotes

(A) A statistical analysis was performed with six donors on day 5 (TGF-β (10 ng/ml) and with or without IL-4 (100 ng/ml)); Shown is the mean, and error bars indicated the SD of six donors.

T25150

16721-16779

Sentence

denotes

Statistical analysis was performed using the Dunnett test.

T125

16721-16779

Sentence

denotes

Statistical analysis was performed using the Dunnett test.

T25151

16780-16864

Sentence

denotes

Statistical significance is indicated by asterisks (*p ≤ 0.05, **p ≤ 0.01, Dunnett).

T126

16780-16864

Sentence

denotes

Statistical significance is indicated by asterisks (*p ≤ 0.05, **p ≤ 0.01, Dunnett).

T25152

16865-17020

Sentence

denotes

(B) CD4+CD45RA+ cells were activated in the presence of a constant concentration of TGF-β (5 ng/ml) with an increasing concentration of IL-4, as indicated.

T127

16865-17020

Sentence

denotes

(B) CD4+CD45RA+ cells were activated in the presence of a constant concentration of TGF-β (5 ng/ml) with an increasing concentration of IL-4, as indicated.

T25153

17021-17076

Sentence

denotes

Cells were harvested for mRNA quantification after 5 d.

T128

17021-17076

Sentence

denotes

Cells were harvested for mRNA quantification after 5 d.

T25154

17077-17207

Sentence

denotes

(C) CD4+CD45RA+ cells were stimulated in vitro with plate-bound anti-CD3/CD28, TGF-β (10ng/ml), and IL-4 (100 ng/ml) as indicated.

T129

17077-17207

Sentence

denotes

(C) CD4+CD45RA+ cells were stimulated in vitro with plate-bound anti-CD3/CD28, TGF-β (10ng/ml), and IL-4 (100 ng/ml) as indicated.

T25155

17208-17310

Sentence

denotes

After 1 h, cell lysates were prepared and analyzed by Western blot for phosphorylated SMAD2 and STAT6.

T130

17208-17310

Sentence

denotes

After 1 h, cell lysates were prepared and analyzed by Western blot for phosphorylated SMAD2 and STAT6.

T25156

17311-17360

Sentence

denotes

Total STAT6 and GAPDH served as internal control.

T131

17311-17360

Sentence

denotes

Total STAT6 and GAPDH served as internal control.

T132

17361-17485

Sentence

denotes

(D) Intracellular GATA3 and FOXP3 staining are shown after exposure of CD4+CD45RA+ T cells to IL-4 as described for panel B.

T25157

17361-17543

Sentence

denotes

(D) Intracellular GATA3 and FOXP3 staining are shown after exposure of CD4+CD45RA+ T cells to IL-4 as described for panel B. Data are representative of three independent experiments.

T133

17486-17543

Sentence

denotes

Data are representative of three independent experiments.

T8261

17545-17588

Sentence

denotes

The Role of TGF-β in T Cell Differentiation

T134

17545-17588

Sentence

denotes

The Role of TGF-β in T Cell Differentiation

T8262

17589-17816

Sentence

denotes

Although TGF-β–reduced CD25, IL-4 expression, and CD25 expression (Figure 6B), IL-4 significantly inhibited TGF-β–mediated induction of FOXP3 in naive T cells driven toward FOXP3+ T cells, as shown by FACS analysis (Figure 6A).

T135

17589-17816

Sentence

denotes

Although TGF-β–reduced CD25, IL-4 expression, and CD25 expression (Figure 6B), IL-4 significantly inhibited TGF-β–mediated induction of FOXP3 in naive T cells driven toward FOXP3+ T cells, as shown by FACS analysis (Figure 6A).

T8263

17817-17981

Sentence

denotes

It is known that IL-4 is a potent growth factor and may therefore favor the proliferation of FOXP3– cells and thus decrease the relative percentage of FOXP3+ cells.

T136

17817-17981

Sentence

denotes

It is known that IL-4 is a potent growth factor and may therefore favor the proliferation of FOXP3– cells and thus decrease the relative percentage of FOXP3+ cells.

T8264

17982-18139

Sentence

denotes

However, analysis of cell division kinetics by CFSE-labeling demonstrated that IL-4 did not differentially promote cell growth of FOXP3+ over that of FOXP3–.

T137

17982-18139

Sentence

denotes

However, analysis of cell division kinetics by CFSE-labeling demonstrated that IL-4 did not differentially promote cell growth of FOXP3+ over that of FOXP3–.

T8265

18140-18217

Sentence

denotes

In fact both populations showed similarly enhanced proliferation (Figure 6A).

T138

18140-18217

Sentence

denotes

In fact both populations showed similarly enhanced proliferation (Figure 6A).

T8266

18218-18507

Sentence

denotes

Furthermore the TGF-β–mediated induction of FOXP3 expression was not caused by overgrowth of a CD25–FOXP3+ minority, since the number of FOXP3+ cells was low/absent in the purified CD4+CD45RA+ T cells (between 0% and 1%), and the FOXP3+ cells were not confined to the highly divided cells.

T139

18218-18507

Sentence

denotes

Furthermore the TGF-β–mediated induction of FOXP3 expression was not caused by overgrowth of a CD25–FOXP3+ minority, since the number of FOXP3+ cells was low/absent in the purified CD4+CD45RA+ T cells (between 0% and 1%), and the FOXP3+ cells were not confined to the highly divided cells.

T8267

18508-18654

Sentence

denotes

CD25 was down-regulated in TGF-β–treated cells compare to activated T cells, which was even more pronounced in cells cultured with TGF-β and IL-4.

T140

18508-18654

Sentence

denotes

CD25 was down-regulated in TGF-β–treated cells compare to activated T cells, which was even more pronounced in cells cultured with TGF-β and IL-4.

T141

18655-18710

Sentence

denotes

Figure 6 IL-4 Inhibits TGF-β–Mediated iTreg Commitment

T25857

18665-18710

Sentence

denotes

IL-4 Inhibits TGF-β–Mediated iTreg Commitment

T25858

18711-18819

Sentence

denotes

CFSE-labeled CD4+CD45RA+ cells were activated with plate-bound anti-CD3/CD28, TGF-β, and IL-4, as indicated.

T142

18711-18819

Sentence

denotes

CFSE-labeled CD4+CD45RA+ cells were activated with plate-bound anti-CD3/CD28, TGF-β, and IL-4, as indicated.

T25859

18820-18953

Sentence

denotes

After 5 d, cells were analyzed by flow cytometry (A) and results of six independent experiments are shown in the bar graph below (B).

T143

18820-18953

Sentence

denotes

After 5 d, cells were analyzed by flow cytometry (A) and results of six independent experiments are shown in the bar graph below (B).

T25860

18954-19219

Sentence

denotes

Statistical significance (one-way Anova, Newman-Keuls) is indicated by asterisks (**p ≤ 0.01, ***p ≤ 0.001). (C) Kinetic analysis of intracellular GATA3 and FOXP3 staining is shown in panel C following exposure of CD4+CD45RA+ T cells to anti-CD3/28, IL-4 and TGF-β.

T144

18954-19219

Sentence

denotes

Statistical significance (one-way Anova, Newman-Keuls) is indicated by asterisks (**p ≤ 0.01, ***p ≤ 0.001). (C) Kinetic analysis of intracellular GATA3 and FOXP3 staining is shown in panel C following exposure of CD4+CD45RA+ T cells to anti-CD3/28, IL-4 and TGF-β.

T25861

19220-19277

Sentence

denotes

Data are representative of three independent experiments.

T145

19220-19277

Sentence

denotes

Data are representative of three independent experiments.

T8268

19280-19378

Sentence

denotes

The addition of IL-4 to iTreg-driving conditions decreased the number of FOXP3+ cells (Figure 6B).

T146

19280-19378

Sentence

denotes

The addition of IL-4 to iTreg-driving conditions decreased the number of FOXP3+ cells (Figure 6B).

T8269

19379-19502

Sentence

denotes

In line with the previous findings, the IL-4–producing cells and the FOXP3 expressing cells are nonoverlapping populations.

T147

19379-19502

Sentence

denotes

In line with the previous findings, the IL-4–producing cells and the FOXP3 expressing cells are nonoverlapping populations.

T8270

19503-19624

Sentence

denotes

Since FOXP3 is known to act as a repressor of cytokine expression [20], we therefore analyzed GATA3 and FOXP3 expression.

T148

19503-19624

Sentence

denotes

Since FOXP3 is known to act as a repressor of cytokine expression [20], we therefore analyzed GATA3 and FOXP3 expression.

T8271

19625-20016

Sentence

denotes

The expression kinetic of naive T cells exposed to IL-4 and TGF-β demonstrated that GATA3 and FOXP3 are initially found in separate populations (day 2), but transiently co-express both factors (days 4–8), before establishing separate populations at the end of the differentiation process (day 10; Figure 6C), suggesting that GATA3 inhibits the development of iTreg cells by repressing FOXP3.

T149

19625-20016

Sentence

denotes

The expression kinetic of naive T cells exposed to IL-4 and TGF-β demonstrated that GATA3 and FOXP3 are initially found in separate populations (day 2), but transiently co-express both factors (days 4–8), before establishing separate populations at the end of the differentiation process (day 10; Figure 6C), suggesting that GATA3 inhibits the development of iTreg cells by repressing FOXP3.

T8272

20017-20240

Sentence

denotes

These results showed that IL-4 acts in vitro as an inhibitor of FOXP3 expression, without interfering with TGF-β signaling, probably acting at the level of transcription factors, and possibly by a GATA3-dependent mechanism.

T150

20017-20240

Sentence

denotes

These results showed that IL-4 acts in vitro as an inhibitor of FOXP3 expression, without interfering with TGF-β signaling, probably acting at the level of transcription factors, and possibly by a GATA3-dependent mechanism.

T9839

20242-20291

Sentence

denotes

GATA3 Is a Negative Regulator of FOXP3 Expression

T151

20242-20291

Sentence

denotes

GATA3 Is a Negative Regulator of FOXP3 Expression

T9840

20292-20424

Sentence

denotes

FOXP3 expression decreased once GATA3 expression is high; therefore, we hypothesized a potential role for GATA3 in repressing FOXP3.

T152

20292-20424

Sentence

denotes

FOXP3 expression decreased once GATA3 expression is high; therefore, we hypothesized a potential role for GATA3 in repressing FOXP3.

T9841

20425-20668

Sentence

denotes

Besides GATA3′s well-known positive effect on gene regulation, GATA3′s repressive capabilities were previously shown to restrict Th1 commitment by inhibiting STAT4 expression [2,21], and therefore GATA3 prevents differentiation into Th1 cells.

T153

20425-20668

Sentence

denotes

Besides GATA3′s well-known positive effect on gene regulation, GATA3′s repressive capabilities were previously shown to restrict Th1 commitment by inhibiting STAT4 expression [2,21], and therefore GATA3 prevents differentiation into Th1 cells.

T9842

20669-20894

Sentence

denotes

To investigate whether GATA3 can directly inhibit FOXP3 induction, we transduced GATA3 or a truncated GATA3 lacking the DNA-binding domain in human primary CD4+CD45RA+ T cells using a TAT-fused, recombinantly expressed GATA3.

T154

20669-20894

Sentence

denotes

To investigate whether GATA3 can directly inhibit FOXP3 induction, we transduced GATA3 or a truncated GATA3 lacking the DNA-binding domain in human primary CD4+CD45RA+ T cells using a TAT-fused, recombinantly expressed GATA3.

T9843

20895-21003

Sentence

denotes

After transduction, the cells were activated with soluble anti-CD3/CD28 in the presence or absence of TGF-β.

T155

20895-21003

Sentence

denotes

After transduction, the cells were activated with soluble anti-CD3/CD28 in the presence or absence of TGF-β.

T9844

21004-21134

Sentence

denotes

TAT-GATA3 was successfully transduced in a homogeneous and dose-dependent manner into human CD4+ T cells (Figure 7A, upper panel).

T156

21004-21134

Sentence

denotes

TAT-GATA3 was successfully transduced in a homogeneous and dose-dependent manner into human CD4+ T cells (Figure 7A, upper panel).

T9845

21135-21356

Sentence

denotes

TAT-GATA3 reduced FOXP3 expression in a dose-dependent manner, whereas a DNA-binding domain truncated version (TAT-ΔDBD-GATA3) did not affect FOXP3 expression as compared with expression in untransduced cells (Figure 7A).

T157

21135-21356

Sentence

denotes

TAT-GATA3 reduced FOXP3 expression in a dose-dependent manner, whereas a DNA-binding domain truncated version (TAT-ΔDBD-GATA3) did not affect FOXP3 expression as compared with expression in untransduced cells (Figure 7A).

T9846

21357-21556

Sentence

denotes

In addition, we analyzed the inhibitory effect of GATA3 on FOXP3 in transgenic DO11.10 mice, constitutively overexpressing GATA3 under the control of the CD2 locus control region (DO11.10xCD2-GATA3).

T158

21357-21556

Sentence

denotes

In addition, we analyzed the inhibitory effect of GATA3 on FOXP3 in transgenic DO11.10 mice, constitutively overexpressing GATA3 under the control of the CD2 locus control region (DO11.10xCD2-GATA3).

T9847

21557-21670

Sentence

denotes

The thymic selection into the CD4 lineage is largely intact in DO11.10xCD2-GATA3 (RW Hendriks, unpublished data).

T159

21557-21670

Sentence

denotes

The thymic selection into the CD4 lineage is largely intact in DO11.10xCD2-GATA3 (RW Hendriks, unpublished data).

T9848

21671-21773

Sentence

denotes

These mice develop lymphomas at an older age, but signs of autoimmune disease were not described [22].

T160

21671-21773

Sentence

denotes

These mice develop lymphomas at an older age, but signs of autoimmune disease were not described [22].

T9849

21774-21956

Sentence

denotes

To investigate the effect of GATA3 on iTreg, CD4+CD62L+CD25– cells were isolated, activated with OVA in the presence or absence of TGF-β, and Foxp3 expression was analyzed after 4 d.

T161

21774-21956

Sentence

denotes

To investigate the effect of GATA3 on iTreg, CD4+CD62L+CD25– cells were isolated, activated with OVA in the presence or absence of TGF-β, and Foxp3 expression was analyzed after 4 d.

T9850

21957-22014

Sentence

denotes

The naive CD4+CD25– cells were Foxp3– (unpublished data).

T162

21957-22014

Sentence

denotes

The naive CD4+CD25– cells were Foxp3– (unpublished data).

T9851

22015-22126

Sentence

denotes

As described for the human cells, TGF-β dramatically up-regulated Foxp3 in the DO11.10 littermate control mice.

T163

22015-22126

Sentence

denotes

As described for the human cells, TGF-β dramatically up-regulated Foxp3 in the DO11.10 littermate control mice.

T9852

22127-22269

Sentence

denotes

In contrast, cells from the CD2-GATA3xDO11.10 mice showed dramatically reduced Foxp3 expression when activated with TGF-β and OVA (Figure 7B).

T164

22127-22269

Sentence

denotes

In contrast, cells from the CD2-GATA3xDO11.10 mice showed dramatically reduced Foxp3 expression when activated with TGF-β and OVA (Figure 7B).

T9853

22270-22439

Sentence

denotes

All mice produced similar amounts of TGF-β; in addition, Smad7 was equally expressed [23] in T cells of both mice strains (Figure 7C), indicating intact TGF-β signaling.

T165

22270-22439

Sentence

denotes

All mice produced similar amounts of TGF-β; in addition, Smad7 was equally expressed [23] in T cells of both mice strains (Figure 7C), indicating intact TGF-β signaling.

T166

22440-22504

Sentence

denotes

Figure 7 GATA3 Acts as a Negative Regulator of FOXP3 Expression

T26337

22450-22504

Sentence

denotes

GATA3 Acts as a Negative Regulator of FOXP3 Expression

T26338

22505-22713

Sentence

denotes

(A) Human naive CD4+CDRA+ T cells were transduced with 0, 20, 100, and 500 nM of TAT-GATA3 protein, and intracellular presence of GATA3 was analyzed using FACS following anti-CD3/CD28 activation of the cells.

T167

22505-22713

Sentence

denotes

(A) Human naive CD4+CDRA+ T cells were transduced with 0, 20, 100, and 500 nM of TAT-GATA3 protein, and intracellular presence of GATA3 was analyzed using FACS following anti-CD3/CD28 activation of the cells.

T26339

22714-22840

Sentence

denotes

GFP-positive cells were gated and analyzed for intracellular FOXP3 expression following a 2-d incubation period (lower panel).

T168

22714-22840

Sentence

denotes

GFP-positive cells were gated and analyzed for intracellular FOXP3 expression following a 2-d incubation period (lower panel).

T26340

22841-22897

Sentence

denotes

Data are representative of four independent experiments.

T169

22841-22897

Sentence

denotes

Data are representative of four independent experiments.

T26341

22898-23014

Sentence

denotes

(B) CD4+CD25− T cells were isolated from D011.10 and D011.10xCD2-GATA3 mice and treated with OVA and TGF-β for 96 h.

T170

22898-23014

Sentence

denotes

(B) CD4+CD25− T cells were isolated from D011.10 and D011.10xCD2-GATA3 mice and treated with OVA and TGF-β for 96 h.

T26342

23015-23073

Sentence

denotes

Surface CD4 and intracellular FOXP3 were measured by FACS.

T171

23015-23073

Sentence

denotes

Surface CD4 and intracellular FOXP3 were measured by FACS.

T26343

23074-23137

Sentence

denotes

These data are representative of three independent experiments.

T172

23074-23137

Sentence

denotes

These data are representative of three independent experiments.

T26344

23138-23257

Sentence

denotes

(C) The cells treated as in (B) were harvested and mRNA was quantified by real-time PCR for SMAD7 and TGF-β expression.

T173

23138-23257

Sentence

denotes

(C) The cells treated as in (B) were harvested and mRNA was quantified by real-time PCR for SMAD7 and TGF-β expression.

T26345

23258-23315

Sentence

denotes

Bars show the mean ± SD of three independent experiments.

T174

23258-23315

Sentence

denotes

Bars show the mean ± SD of three independent experiments.

T9854

23318-23455

Sentence

denotes

Taken together, these results demonstrated a repressive role of IL-4-induced GATA3 transcription factor in the generation of iTreg cells.

T175

23318-23455

Sentence

denotes

Taken together, these results demonstrated a repressive role of IL-4-induced GATA3 transcription factor in the generation of iTreg cells.

T11568

23457-23491

Sentence

denotes

GATA3 Represses the FOXP3 Promoter

T176

23457-23491

Sentence

denotes

GATA3 Represses the FOXP3 Promoter

T11569

23492-23669

Sentence

denotes

To investigate the molecular mechanism of GATA3-mediated repression of human FOXP3, the human FOXP3 promoter was studied and a palindromic binding site for GATA3 was discovered.

T177

23492-23669

Sentence

denotes

To investigate the molecular mechanism of GATA3-mediated repression of human FOXP3, the human FOXP3 promoter was studied and a palindromic binding site for GATA3 was discovered.

T11570

23670-23764

Sentence

denotes

The GATA-binding site is located 303 bp upstream from the transcription start site (TSS) [24].

T178

23670-23764

Sentence

denotes

The GATA-binding site is located 303 bp upstream from the transcription start site (TSS) [24].

T11571

23765-23899

Sentence

denotes

This site is highly conserved between humans, mice, and rats (Figure S4) and may therefore play an important role in FOXP3 regulation.

T179

23765-23899

Sentence

denotes

This site is highly conserved between humans, mice, and rats (Figure S4) and may therefore play an important role in FOXP3 regulation.

T11572

23900-23988

Sentence

denotes

The functional relevance of this site was studied using a FOXP3-promoter construct [24].

T180

23900-23988

Sentence

denotes

The functional relevance of this site was studied using a FOXP3-promoter construct [24].

T11573

23989-24190

Sentence

denotes

We transfected human primary CD4+ T cells, in vitro differentiated Th2 cells, and Jurkat cells (Jurkat cells are known to constitutively express GATA3 [25,26]), and we measured FOXP3 promoter activity.

T181

23989-24190

Sentence

denotes

We transfected human primary CD4+ T cells, in vitro differentiated Th2 cells, and Jurkat cells (Jurkat cells are known to constitutively express GATA3 [25,26]), and we measured FOXP3 promoter activity.

T11574

24191-24406

Sentence

denotes

The promoter was not active in the GATA3-expressing cell line Jurkat or in the in vitro-differentiated Th2 cells, whereas the construct was active in the CD4 cells, which express a lower amount of GATA3 (Figure 8A).

T182

24191-24406

Sentence

denotes

The promoter was not active in the GATA3-expressing cell line Jurkat or in the in vitro-differentiated Th2 cells, whereas the construct was active in the CD4 cells, which express a lower amount of GATA3 (Figure 8A).

T11575

24407-24546

Sentence

denotes

Overexpression of GATA3 in naive T cells diminished luciferase activity of the FOXP3 promoter compared with the control vector (Figure 8B).

T183

24407-24546

Sentence

denotes

Overexpression of GATA3 in naive T cells diminished luciferase activity of the FOXP3 promoter compared with the control vector (Figure 8B).

T11576

24547-24667

Sentence

denotes

To further address the function of the GATA3 site, we inserted a site-specific mutation deleting the GATA3-binding site.

T184

24547-24667

Sentence

denotes

To further address the function of the GATA3 site, we inserted a site-specific mutation deleting the GATA3-binding site.

T11577

24668-24901

Sentence

denotes

This mutation increased luciferase activity by 3-fold in memory CD4+CD45RO+ T cells, whereas no difference was observed in naive (GATA3–) CD4+CD45RA+ T cells, revealing a repressor activity of GATA3 on the FOXP3 promoter (Figure 8C).

T185

24668-24901

Sentence

denotes

This mutation increased luciferase activity by 3-fold in memory CD4+CD45RO+ T cells, whereas no difference was observed in naive (GATA3–) CD4+CD45RA+ T cells, revealing a repressor activity of GATA3 on the FOXP3 promoter (Figure 8C).

T11578

24902-24992

Sentence

denotes

Furthermore GATA3 binds directly to the FOXP3 promoter as investigated by pull-down assay.

T186

24902-24992

Sentence

denotes

Furthermore GATA3 binds directly to the FOXP3 promoter as investigated by pull-down assay.

T11579

24993-25246

Sentence

denotes

HEK cells were transiently transfected with GATA3 or a control vector, and increasing amounts of lysates were incubated with oligonucleotides containing the GATA3 site of the FOXP3 promoter or a control oligonucleotide with a mutated GATA3-binding site.

T187

24993-25246

Sentence

denotes

HEK cells were transiently transfected with GATA3 or a control vector, and increasing amounts of lysates were incubated with oligonucleotides containing the GATA3 site of the FOXP3 promoter or a control oligonucleotide with a mutated GATA3-binding site.

T11580

25247-25311

Sentence

denotes

After the pull-down, GATA3 binding was detected by Western blot.

T188

25247-25311

Sentence

denotes

After the pull-down, GATA3 binding was detected by Western blot.

T11581

25312-25398

Sentence

denotes

Similarly, GATA3-expressing Th2 cells and iTreg cells were subjected to this approach.

T189

25312-25398

Sentence

denotes

Similarly, GATA3-expressing Th2 cells and iTreg cells were subjected to this approach.

T11582

25399-25498

Sentence

denotes

Only HEK cells overexpressing GATA3 and Th2 cells showed GATA3-binding activity (Figure 8D and 8E).

T190

25399-25498

Sentence

denotes

Only HEK cells overexpressing GATA3 and Th2 cells showed GATA3-binding activity (Figure 8D and 8E).

T11583

25499-25578

Sentence

denotes

These experiments demonstrated that GATA3 binds the palindromic FOXP3 promoter.

T191

25499-25578

Sentence

denotes

These experiments demonstrated that GATA3 binds the palindromic FOXP3 promoter.

T11584

25579-25811

Sentence

denotes

To gain insights into the in vivo situation, we performed a chromatin immunoprecipitation (ChIP) using an anti-GATA3 antibody and showed that GATA3 binds to the FOXP3 promoter region in Th2 cells, but not in iTreg cells (Figure 7F).

T192

25579-25811

Sentence

denotes

To gain insights into the in vivo situation, we performed a chromatin immunoprecipitation (ChIP) using an anti-GATA3 antibody and showed that GATA3 binds to the FOXP3 promoter region in Th2 cells, but not in iTreg cells (Figure 7F).

T11585

25812-25926

Sentence

denotes

Taken together these data demonstrate that the GATA3-binding to the FOXP3 promoter is repressing FOXP3 expression.

T193

25812-25926

Sentence

denotes

Taken together these data demonstrate that the GATA3-binding to the FOXP3 promoter is repressing FOXP3 expression.

T194

25927-25977

Sentence

denotes

Figure 8 GATA3 Represses the Human FOXP3 Promoter

T26927

25937-25977

Sentence

denotes

GATA3 Represses the Human FOXP3 Promoter

T26928

25978-26176

Sentence

denotes

(A) Jurkat, Th2 cells, and human primary CD4 cells were transfected with an empty vector (pGL3 basic) or a vector containing the putative FOXP3 promoter region fused to the luciferase reporter gene.

T195

25978-26176

Sentence

denotes

(A) Jurkat, Th2 cells, and human primary CD4 cells were transfected with an empty vector (pGL3 basic) or a vector containing the putative FOXP3 promoter region fused to the luciferase reporter gene.

T26929

26177-26330

Sentence

denotes

Bars show the mean ± SD of arbitrary light units normalized for renilla luciferase of four independent experiments; samples were measured in triplicates.

T196

26177-26330

Sentence

denotes

Bars show the mean ± SD of arbitrary light units normalized for renilla luciferase of four independent experiments; samples were measured in triplicates.

T26930

26331-26472

Sentence

denotes

(B) Naive CD4 T cells were transfected with the FOXP3 promoter reporter construct together with a GATA3 expression vector or an empty vector.

T197

26331-26472

Sentence

denotes

(B) Naive CD4 T cells were transfected with the FOXP3 promoter reporter construct together with a GATA3 expression vector or an empty vector.

T26931

26473-26530

Sentence

denotes

Bars show the mean ± SD of three independent experiments.

T198

26473-26530

Sentence

denotes

Bars show the mean ± SD of three independent experiments.

T26932

26531-26716

Sentence

denotes

(C) Naive (left panel) or memory (right panel) CD4 T cells were transfected with wild-type or a GATA3 mutated 511-FOXP3 promoter reporter construct and activated with PMA and ionomycin.

T199

26531-26716

Sentence

denotes

(C) Naive (left panel) or memory (right panel) CD4 T cells were transfected with wild-type or a GATA3 mutated 511-FOXP3 promoter reporter construct and activated with PMA and ionomycin.

T26933

26717-26871

Sentence

denotes

Bars show the mean ± SD of arbitrary light units normalized for renilla luciferase of eight independent experiments; samples were measured in triplicates.

T200

26717-26871

Sentence

denotes

Bars show the mean ± SD of arbitrary light units normalized for renilla luciferase of eight independent experiments; samples were measured in triplicates.

T26934

26872-27063

Sentence

denotes

(D) Nuclear extracts were prepared from HEK cells transfected with GATA3 or an empty vector, (E) Th1, Th2, or iTreg cells and binding factors precipitated using biotinylated oligonucleotides.

T201

26872-27063

Sentence

denotes

(D) Nuclear extracts were prepared from HEK cells transfected with GATA3 or an empty vector, (E) Th1, Th2, or iTreg cells and binding factors precipitated using biotinylated oligonucleotides.

T26935

27064-27149

Sentence

denotes

The oligonucleotides–transcription factor complexes were separated on a SDS-PAGE gel.

T202

27064-27149

Sentence

denotes

The oligonucleotides–transcription factor complexes were separated on a SDS-PAGE gel.

T26936

27150-27251

Sentence

denotes

The amounts of GATA3 protein in the precipitates were assessed by immunoblotting with anti-GATA3 mAb.

T203

27150-27251

Sentence

denotes

The amounts of GATA3 protein in the precipitates were assessed by immunoblotting with anti-GATA3 mAb.

T26937

27252-27301

Sentence

denotes

Total nuclear extracts were also run as controls.

T204

27252-27301

Sentence

denotes

Total nuclear extracts were also run as controls.

T26938

27302-27357

Sentence

denotes

Data are representative of three different experiments.

T205

27302-27357

Sentence

denotes

Data are representative of three different experiments.

T26939

27358-27443

Sentence

denotes

(F) iTreg or Th2 cells were analyzed by ChIP for GATA3 binding to the FOXP3 promoter.

T206

27358-27443

Sentence

denotes

(F) iTreg or Th2 cells were analyzed by ChIP for GATA3 binding to the FOXP3 promoter.

T26940

27444-27551

Sentence

denotes

The “input” represents PCR amplification of the total sample, which was not subjected to any precipitation.

T207

27444-27551

Sentence

denotes

The “input” represents PCR amplification of the total sample, which was not subjected to any precipitation.

T26941

27552-27612

Sentence

denotes

Results are representative of three independent experiments.

T208

27552-27612

Sentence

denotes

Results are representative of three independent experiments.

T209

27614-27624

Sentence

denotes

Discussion

T13433

27625-27796

Sentence

denotes

The current study reveals that FOXP3 induction, an important step in iTreg commitment, is inhibited by GATA3, which is the key regulator for polarization toward Th2 cells.

T210

27625-27796

Sentence

denotes

The current study reveals that FOXP3 induction, an important step in iTreg commitment, is inhibited by GATA3, which is the key regulator for polarization toward Th2 cells.

T13434

27797-27899

Sentence

denotes

After differentiation, the effector Th2 cells become refractory to conversion into a FOXP3+ phenotype.

T211

27797-27899

Sentence

denotes

After differentiation, the effector Th2 cells become refractory to conversion into a FOXP3+ phenotype.

T13435

27900-28003

Sentence

denotes

In accordance with other studies, we found that CD4+CD25– cells were able to up-regulate FOXP3 [12,27].

T212

27900-28003

Sentence

denotes

In accordance with other studies, we found that CD4+CD25– cells were able to up-regulate FOXP3 [12,27].

T13436

28004-28200

Sentence

denotes

Already-committed cells such as memory T cells and Th1 cells showed only moderate and transient FOXP3 induction, which is not sufficient to change the phenotype toward a regulatory T cell profile.

T213

28004-28200

Sentence

denotes

Already-committed cells such as memory T cells and Th1 cells showed only moderate and transient FOXP3 induction, which is not sufficient to change the phenotype toward a regulatory T cell profile.

T13437

28201-28497

Sentence

denotes

In contrast, naive T cells could efficiently up-regulate FOXP3 when treated with TGF-β to induce iTreg cells [10,28–34], suggesting that FOXP3 plays an important role in the early differentiation process and may act in a way similar to that known for the Th1/Th2 decision factors T-bet and GATA3.

T214

28201-28497

Sentence

denotes

In contrast, naive T cells could efficiently up-regulate FOXP3 when treated with TGF-β to induce iTreg cells [10,28–34], suggesting that FOXP3 plays an important role in the early differentiation process and may act in a way similar to that known for the Th1/Th2 decision factors T-bet and GATA3.

T13438

28498-28754

Sentence

denotes

This commitment is characterized by competitive expression of these factors [35,36], which we also observed in differentiating FOXP3+ iTreg cells including a phase of co-expression, which turns into nonoverlapping expression upon completed differentiation.

T215

28498-28754

Sentence

denotes

This commitment is characterized by competitive expression of these factors [35,36], which we also observed in differentiating FOXP3+ iTreg cells including a phase of co-expression, which turns into nonoverlapping expression upon completed differentiation.

T13439

28755-28908

Sentence

denotes

In this competitive process TGF-β appeared to be mandatory for the induction of FOXP3, possibly by keeping the expression of GATA3 and T-bet low [37,38].

T216

28755-28908

Sentence

denotes

In this competitive process TGF-β appeared to be mandatory for the induction of FOXP3, possibly by keeping the expression of GATA3 and T-bet low [37,38].

T13440

28909-29188

Sentence

denotes

In contrast, differentiating naive T cells in the absence of polarization factors (Th0) such as IL-4, IL-12, or TGF-β showed only a transient FOXP3 expression and failed to generate a population of FOXP3-expressing cells, but GATA3 and T-bet were up-regulated (unpublished data).

T217

28909-29188

Sentence

denotes

In contrast, differentiating naive T cells in the absence of polarization factors (Th0) such as IL-4, IL-12, or TGF-β showed only a transient FOXP3 expression and failed to generate a population of FOXP3-expressing cells, but GATA3 and T-bet were up-regulated (unpublished data).

T13441

29189-29386

Sentence

denotes

Interestingly, as we and others previously described, FOXP3-promoting factors, such as dexamethasone [39], CTLA-4 [40], and estrogens [41], are also known as inhibitors of GATA3 expression [42–45].

T218

29189-29386

Sentence

denotes

Interestingly, as we and others previously described, FOXP3-promoting factors, such as dexamethasone [39], CTLA-4 [40], and estrogens [41], are also known as inhibitors of GATA3 expression [42–45].

T13442

29387-29518

Sentence

denotes

Therefore GATA3 not only induces differentiation into Th2 cells but also inhibits FOXP3 expression and commitment into iTreg cells.

T219

29387-29518

Sentence

denotes

Therefore GATA3 not only induces differentiation into Th2 cells but also inhibits FOXP3 expression and commitment into iTreg cells.

T13443

29519-29690

Sentence

denotes

The Th2 cytokine IL-4 but not IL-13 (unpublished data) was able to inhibit TGF-β–mediated FOXP3 induction and therefore prevented conversion into the regulatory phenotype.

T220

29519-29690

Sentence

denotes

The Th2 cytokine IL-4 but not IL-13 (unpublished data) was able to inhibit TGF-β–mediated FOXP3 induction and therefore prevented conversion into the regulatory phenotype.

T13444

29691-29819

Sentence

denotes

To prove the inhibitory effect of IL-4 on inducible or natural Treg commitment in vivo, we treated mice with IL-4 and anti-IL-4.

T221

29691-29819

Sentence

denotes

To prove the inhibitory effect of IL-4 on inducible or natural Treg commitment in vivo, we treated mice with IL-4 and anti-IL-4.

T13445

29820-29892

Sentence

denotes

This has been shown to increase the effect of the cytokine in vivo [19].

T222

29820-29892

Sentence

denotes

This has been shown to increase the effect of the cytokine in vivo [19].

T13446

29893-30035

Sentence

denotes

Only the IL-4/IL-4 mAb complex resulted in a decrease of the amount of natural or inducible Treg (CD25+ and Foxp3+) cells 7 d after treatment.

T223

29893-30035

Sentence

denotes

Only the IL-4/IL-4 mAb complex resulted in a decrease of the amount of natural or inducible Treg (CD25+ and Foxp3+) cells 7 d after treatment.

T13447

30036-30145

Sentence

denotes

Our results suggest that IL-4 is only interfering with the differentiation of naive T cells into iTreg cells.

T224

30036-30145

Sentence

denotes

Our results suggest that IL-4 is only interfering with the differentiation of naive T cells into iTreg cells.

T13448

30146-30458

Sentence

denotes

But since a distinction of nTreg and iTreg cells is currently not possible, because iTreg cells also transiently express CD25 after activation, we cannot exclude that IL-4 may also inhibit Foxp3 expression in nTreg cells in vivo or that additional effects may contribute to the observed drop in Foxp3 expression.

T225

30146-30458

Sentence

denotes

But since a distinction of nTreg and iTreg cells is currently not possible, because iTreg cells also transiently express CD25 after activation, we cannot exclude that IL-4 may also inhibit Foxp3 expression in nTreg cells in vivo or that additional effects may contribute to the observed drop in Foxp3 expression.

T13449

30459-30598

Sentence

denotes

IL-4 has already been shown to negatively regulate the development of naive T cells into Th1 or the IL-17–producing T cells (Th17) [46,47].

T226

30459-30598

Sentence

denotes

IL-4 has already been shown to negatively regulate the development of naive T cells into Th1 or the IL-17–producing T cells (Th17) [46,47].

T13450

30599-30801

Sentence

denotes

Similar effects have been recently described for IL-6, which, combined with TGF-β, inhibits the generation of iTreg cells and induces differentiation into the Th17 cells by an unknown mechanism [48,49].

T227

30599-30801

Sentence

denotes

Similar effects have been recently described for IL-6, which, combined with TGF-β, inhibits the generation of iTreg cells and induces differentiation into the Th17 cells by an unknown mechanism [48,49].

T13451

30802-30905

Sentence

denotes

Thus the polarization into iTreg cells is negatively regulated by the effector cytokines IL-4 and IL-6.

T228

30802-30905

Sentence

denotes

Thus the polarization into iTreg cells is negatively regulated by the effector cytokines IL-4 and IL-6.

T13452

30906-31005

Sentence

denotes

IL-4 has been previously shown to induce the generation of FOXP3+ Treg cells out of CD4+CD25– [50].

T229

30906-31005

Sentence

denotes

IL-4 has been previously shown to induce the generation of FOXP3+ Treg cells out of CD4+CD25– [50].

T13453

31006-31156

Sentence

denotes

In those experiments, the concentrations of IL-4 used were low, and as we also observed, IL-4 at low concentration slightly enhanced FOXP3 expression.

T230

31006-31156

Sentence

denotes

In those experiments, the concentrations of IL-4 used were low, and as we also observed, IL-4 at low concentration slightly enhanced FOXP3 expression.

T13454

31157-31238

Sentence

denotes

Importantly, these concentrations were not sufficient to induce GATA3 expression.

T231

31157-31238

Sentence

denotes

Importantly, these concentrations were not sufficient to induce GATA3 expression.

T13455

31239-31359

Sentence

denotes

IL-4 may favor proliferation of nTreg cells [47] or directly regulate FOXP3 expression in a STAT-dependent fashion [51].

T232

31239-31359

Sentence

denotes

IL-4 may favor proliferation of nTreg cells [47] or directly regulate FOXP3 expression in a STAT-dependent fashion [51].

T13456

31360-31520

Sentence

denotes

Since IL-13 does not effectively reduce FOXP3 and fails to induce GATA3, we hypothesized that the IL-4–dependent inhibition of FOXP3 could be mediated by GATA3.

T233

31360-31520

Sentence

denotes

Since IL-13 does not effectively reduce FOXP3 and fails to induce GATA3, we hypothesized that the IL-4–dependent inhibition of FOXP3 could be mediated by GATA3.

T13457

31521-31660

Sentence

denotes

In fact, GATA3-inducing IL-4 concentrations repressed TGF-β–mediated FOXP3 expression, whereas IL-4 as well as TGF-β signaling were intact.

T234

31521-31660

Sentence

denotes

In fact, GATA3-inducing IL-4 concentrations repressed TGF-β–mediated FOXP3 expression, whereas IL-4 as well as TGF-β signaling were intact.

T13458

31661-31829

Sentence

denotes

This result suggested a competitive mechanism between GATA3 and FOXP3 transcription factors in determining lineage commitment during the early phase of differentiation.

T235

31661-31829

Sentence

denotes

This result suggested a competitive mechanism between GATA3 and FOXP3 transcription factors in determining lineage commitment during the early phase of differentiation.

T13459

31830-31932

Sentence

denotes

Accordingly, we investigated naturally high GATA3-expressing cells and confirmed the absence of FOXP3.

T236

31830-31932

Sentence

denotes

Accordingly, we investigated naturally high GATA3-expressing cells and confirmed the absence of FOXP3.

T13460

31933-32049

Sentence

denotes

Protein transduction of GATA3 into naive T cells inhibited FOXP3 induction in human, differentiating, naive T cells.

T237

31933-32049

Sentence

denotes

Protein transduction of GATA3 into naive T cells inhibited FOXP3 induction in human, differentiating, naive T cells.

T13461

32050-32195

Sentence

denotes

This inhibitory effect of GATA3 was further confirmed in BALB/c transgenic mice, expressing GATA3 in T cells (DO11.10:CD2-GATA3 transgenic mice).

T238

32050-32195

Sentence

denotes

This inhibitory effect of GATA3 was further confirmed in BALB/c transgenic mice, expressing GATA3 in T cells (DO11.10:CD2-GATA3 transgenic mice).

T13462

32196-32371

Sentence

denotes

In line with the transient overexpression of GATA3 in human T cells, cells of these mice failed to induce FOXP3 expression upon exposure with antigen in the presence of TGF-β.

T239

32196-32371

Sentence

denotes

In line with the transient overexpression of GATA3 in human T cells, cells of these mice failed to induce FOXP3 expression upon exposure with antigen in the presence of TGF-β.

T13463

32372-32529

Sentence

denotes

Strikingly, the DO11.10:CD2-GATA3 mice do have peripheral FOXP3+ cells, which however displayed a 10%–25% lower frequency compared to wild-type DO11.10 mice.

T240

32372-32529

Sentence

denotes

Strikingly, the DO11.10:CD2-GATA3 mice do have peripheral FOXP3+ cells, which however displayed a 10%–25% lower frequency compared to wild-type DO11.10 mice.

T13464

32530-32671

Sentence

denotes

Thus GATA3 restrains the development of certain Treg subsets, presumably the inducible, peripheral population and not those of thymic origin.

T241

32530-32671

Sentence

denotes

Thus GATA3 restrains the development of certain Treg subsets, presumably the inducible, peripheral population and not those of thymic origin.

T13465

32672-32801

Sentence

denotes

Thymic T cells undergo a different maturation process, which may explain the insensitivity of nTreg to GATA3 overexpression [52].

T242

32672-32801

Sentence

denotes

Thymic T cells undergo a different maturation process, which may explain the insensitivity of nTreg to GATA3 overexpression [52].

T13466

32802-33172

Sentence

denotes

In contrast to the Th2-differentiating and iTreg-inhibiting function of GATA3 in peripheral T cells, GATA3 acts in the thymus together with other transcription factors such as the Repressor of GATA3 (ROG) in the differentiation process toward CD8 cells [53,54] or participates in complex transcriptional feedback network to regulate sympathoadrenal differentiation [55].

T243

32802-33172

Sentence

denotes

In contrast to the Th2-differentiating and iTreg-inhibiting function of GATA3 in peripheral T cells, GATA3 acts in the thymus together with other transcription factors such as the Repressor of GATA3 (ROG) in the differentiation process toward CD8 cells [53,54] or participates in complex transcriptional feedback network to regulate sympathoadrenal differentiation [55].

T13467

33173-33258

Sentence

denotes

Therefore, the role of GATA3 appears to be tissue specific and cannot be generalized.

T244

33173-33258

Sentence

denotes

Therefore, the role of GATA3 appears to be tissue specific and cannot be generalized.

T13468

33259-33369

Sentence

denotes

Our study demonstrates that GATA3 repressed FOXP3 expression directly by binding to the FOXP3 promoter region.

T245

33259-33369

Sentence

denotes

Our study demonstrates that GATA3 repressed FOXP3 expression directly by binding to the FOXP3 promoter region.

T13469

33370-33525

Sentence

denotes

A palindromic GATA-site is located 303 bp upstream of the TSS in a highly conserved region, which we have previously identified as the FOXP3 promoter [24].

T246

33370-33525

Sentence

denotes

A palindromic GATA-site is located 303 bp upstream of the TSS in a highly conserved region, which we have previously identified as the FOXP3 promoter [24].

T13470

33526-33797

Sentence

denotes

Site-specific mutation of this site increased the activity of the promoter constructs, thus revealing the repressive nature of this GATA element in memory T cells, which naturally express GATA3, whereas no difference was seen in naive T cells, which do not express GATA3.

T247

33526-33797

Sentence

denotes

Site-specific mutation of this site increased the activity of the promoter constructs, thus revealing the repressive nature of this GATA element in memory T cells, which naturally express GATA3, whereas no difference was seen in naive T cells, which do not express GATA3.

T13471

33798-33891

Sentence

denotes

This palindromic GATA element is bound by GATA3 protein as proven with pull-down experiments.

T248

33798-33891

Sentence

denotes

This palindromic GATA element is bound by GATA3 protein as proven with pull-down experiments.

T13472

33892-33976

Sentence

denotes

Furthermore, it is shown by ChIP that GATA3 binds this element also in intact cells.

T249

33892-33976

Sentence

denotes

Furthermore, it is shown by ChIP that GATA3 binds this element also in intact cells.

T13473

33977-34184

Sentence

denotes

It is known that GATA3 can induce transcription by chromatin remodeling [56], by directly transactivating promoters [36], or, as shown in the current study, acts as a repressor of gene expression [21,57–59].

T250

33977-34184

Sentence

denotes

It is known that GATA3 can induce transcription by chromatin remodeling [56], by directly transactivating promoters [36], or, as shown in the current study, acts as a repressor of gene expression [21,57–59].

T13474

34185-34291

Sentence

denotes

Therefore keeping GATA3 expression low might be required to induce efficient FOXP3+ iTreg cell generation.

T251

34185-34291

Sentence

denotes

Therefore keeping GATA3 expression low might be required to induce efficient FOXP3+ iTreg cell generation.

T13475

34292-34581

Sentence

denotes

The molecular interactions enabling GATA3 to inhibit FOXP3 are not identified yet, but the GATA-binding site is located adjacent to positive, inducing sites, composed of AP-1-NFATc2 sites [24], and GATA3 may compete with the binding of AP-1/NFAT to the promoter (unpublished observations).

T252

34292-34581

Sentence

denotes

The molecular interactions enabling GATA3 to inhibit FOXP3 are not identified yet, but the GATA-binding site is located adjacent to positive, inducing sites, composed of AP-1-NFATc2 sites [24], and GATA3 may compete with the binding of AP-1/NFAT to the promoter (unpublished observations).

T13476

34582-34671

Sentence

denotes

In summary, we demonstrated that FOXP3 is negatively regulated by cytokines such as IL-4.

T253

34582-34671

Sentence

denotes

In summary, we demonstrated that FOXP3 is negatively regulated by cytokines such as IL-4.

T13477

34672-34845

Sentence

denotes

GATA3 acts as an inhibitor of FOXP3 expression in early T cell differentiation, as well as in differentiated Th2 cells by directly binding and repressing the FOXP3 promoter.

T254

34672-34845

Sentence

denotes

GATA3 acts as an inhibitor of FOXP3 expression in early T cell differentiation, as well as in differentiated Th2 cells by directly binding and repressing the FOXP3 promoter.

T13478

34846-34954

Sentence

denotes

We therefore describe a new mechanism of how Il-4 avoids tolerance induction by repressing FOXP3 expression.

T255

34846-34954

Sentence

denotes

We therefore describe a new mechanism of how Il-4 avoids tolerance induction by repressing FOXP3 expression.

T13479

34955-35146

Sentence

denotes

These findings will give new perspectives toward understanding molecular mechanisms of iTreg induction and thus pathways of peripheral tolerance induction, particularly in allergy and asthma.

T256

34955-35146

Sentence

denotes

These findings will give new perspectives toward understanding molecular mechanisms of iTreg induction and thus pathways of peripheral tolerance induction, particularly in allergy and asthma.

T257

35148-35169

Sentence

denotes

Materials and Methods

T17772

35171-35176

Sentence

denotes

Mice.

T258

35171-35176

Sentence

denotes

Mice.

T17773

35177-35257

Sentence

denotes

Normal B6 mice were purchased from the Jackson Laboratories (Bar Harbor, Maine).

T259

35177-35257

Sentence

denotes

Normal B6 mice were purchased from the Jackson Laboratories (Bar Harbor, Maine).

T17774

35258-35504

Sentence

denotes

Transgenic DO11.10 mice, expressing a T cell receptor for OVA323–339 peptide in the context of H-2d, were backcrossed with mice expressing GATA-3, driven by the human CD2 locus control region (CD2-GATA3) [22], resulting in DO11.10xCD2-GATA3 mice.

T260

35258-35504

Sentence

denotes

Transgenic DO11.10 mice, expressing a T cell receptor for OVA323–339 peptide in the context of H-2d, were backcrossed with mice expressing GATA-3, driven by the human CD2 locus control region (CD2-GATA3) [22], resulting in DO11.10xCD2-GATA3 mice.

T17775

35505-35636

Sentence

denotes

Mice used for experiments were backcrossed on a BALB/c background for a minimum of eight generations and used at an age of 8–12 wk.

T261

35505-35636

Sentence

denotes

Mice used for experiments were backcrossed on a BALB/c background for a minimum of eight generations and used at an age of 8–12 wk.

T17776

35637-35780

Sentence

denotes

Mice were housed under specific pathogen-free conditions and all animal studies were performed according to institutional and state guidelines.

T262

35637-35780

Sentence

denotes

Mice were housed under specific pathogen-free conditions and all animal studies were performed according to institutional and state guidelines.

T18073

35782-35808

Sentence

denotes

Isolation of CD4+ T cells.

T263

35782-35808

Sentence

denotes

Isolation of CD4+ T cells.

T18074

35809-35964

Sentence

denotes

CD4+ T cells were isolated from blood of healthy human volunteers using the anti-CD4 magnetic beads (Dynal, Hamburg, Germany) as previously described [60].

T264

35809-35964

Sentence

denotes

CD4+ T cells were isolated from blood of healthy human volunteers using the anti-CD4 magnetic beads (Dynal, Hamburg, Germany) as previously described [60].

T18075

35965-36035

Sentence

denotes

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

T265

35965-36035

Sentence

denotes

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

T18076

36036-36143

Sentence

denotes

Monoclonality of T cell clones was confirmed by TCR-chain mapping and was identified to be Vbeta8 positive.

T266

36036-36143

Sentence

denotes

Monoclonality of T cell clones was confirmed by TCR-chain mapping and was identified to be Vbeta8 positive.

T18077

36144-36197

Sentence

denotes

The clones were characterized by high IL-4 secretion.

T267

36144-36197

Sentence

denotes

The clones were characterized by high IL-4 secretion.

T18328

36199-36226

Sentence

denotes

Quantitative real-time PCR.

T268

36199-36226

Sentence

denotes

Quantitative real-time PCR.

T18329

36227-36655

Sentence

denotes

The 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′-GAA ACA GCA CAT TCC CAG AGT TC-3′; FOXP3 reverse primer 5′-ATG GCC CAG CGG ATG AG-3′; EF-1α forward primer and reverse primer as described [61]; GATA3 forward primer 5′-GCG GGC TCT ATC ACA AAA TGA-3′ and rwd 5′-GCT CTC CTG GCT GCA GAC AGC-3′.

T269

36227-36655

Sentence

denotes

The 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′-GAA ACA GCA CAT TCC CAG AGT TC-3′; FOXP3 reverse primer 5′-ATG GCC CAG CGG ATG AG-3′; EF-1α forward primer and reverse primer as described [61]; GATA3 forward primer 5′-GCG GGC TCT ATC ACA AAA TGA-3′ and rwd 5′-GCT CTC CTG GCT GCA GAC AGC-3′.

T18330

36656-36845

Sentence

denotes

The prepared cDNAs were amplified using SYBR-PCR mastermix (Biorad) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

T270

36656-36845

Sentence

denotes

The prepared cDNAs were amplified using SYBR-PCR mastermix (Biorad) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).

T18331

36846-36976

Sentence

denotes

Quantitative PCR of murine samples was performed with Brilliant SYBR Green QPCR master mix (Stratagene) and the following primers:

T271

36846-36976

Sentence

denotes

Quantitative PCR of murine samples was performed with Brilliant SYBR Green QPCR master mix (Stratagene) and the following primers:

T18332

36977-37223

Sentence

denotes

Ubiquitin C, 5′- AGG TCA AAC AGG AAG ACA GAC GTA-3′ and 5′-TCACACCCAAGAACAAGCACA-3′; Smad-7, 5′-GAA ACC GGG GGA ACG AAT TAT-3′ and 5′-CGC GAG TCT TCT CCT CCC A-3′; TGF-ß1, 5′-TGA CGT CAC TGG AGT TGT ACG G-3′ and 5′-GGT TCA TGT CAT GGA TGG TGC-3′.

T272

36977-37223

Sentence

denotes

Ubiquitin C, 5′- AGG TCA AAC AGG AAG ACA GAC GTA-3′ and 5′-TCACACCCAAGAACAAGCACA-3′; Smad-7, 5′-GAA ACC GGG GGA ACG AAT TAT-3′ and 5′-CGC GAG TCT TCT CCT CCC A-3′; TGF-ß1, 5′-TGA CGT CAC TGG AGT TGT ACG G-3′ and 5′-GGT TCA TGT CAT GGA TGG TGC-3′.

T18333

37224-37362

Sentence

denotes

Primer pairs were evaluated for integrity by analysis of the amplification plot, dissociation curves, and efficiency of PCR amplification.

T273

37224-37362

Sentence

denotes

Primer pairs were evaluated for integrity by analysis of the amplification plot, dissociation curves, and efficiency of PCR amplification.

T18334

37363-37515

Sentence

denotes

PCR conditions were 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 60 °C for 1 min using an 7300 real-time PCR system (Applied Biosystems).

T274

37363-37515

Sentence

denotes

PCR conditions were 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 60 °C for 1 min using an 7300 real-time PCR system (Applied Biosystems).

T18335

37516-37665

Sentence

denotes

PCR amplification of the housekeeping gene encoding ubiquitin C was performed during each run for each sample to allow normalization between samples.

T275

37516-37665

Sentence

denotes

PCR amplification of the housekeeping gene encoding ubiquitin C was performed during each run for each sample to allow normalization between samples.

T18336

37666-37781

Sentence

denotes

Relative quantification and calculation of the range of confidence was performed using the comparative ΔΔCT method.

T276

37666-37781

Sentence

denotes

Relative quantification and calculation of the range of confidence was performed using the comparative ΔΔCT method.

T18787

37783-37813

Sentence

denotes

Inducible murine Treg culture.

T277

37783-37813

Sentence

denotes

Inducible murine Treg culture.

T18788

37814-37945

Sentence

denotes

Naive CD4+ T cells (CD4+, CD62L+, and CD25–) were isolated from pooled lymph nodes and spleens by FACS (FACS Aria, BD Biosciences).

T278

37814-37945

Sentence

denotes

Naive CD4+ T cells (CD4+, CD62L+, and CD25–) were isolated from pooled lymph nodes and spleens by FACS (FACS Aria, BD Biosciences).

T18789

37946-38157

Sentence

denotes

5 × 105 T cells were co-cultured with 2.5 × 104 bone marrow–derived dendritic cells [62] and 0.01 μg/ml OVA323–339 peptide (Ansynth) in the presence or absence of 20-ng/μl rhTGF-ß1 (Peprotech) in 48-well plates.

T279

37946-38157

Sentence

denotes

5 × 105 T cells were co-cultured with 2.5 × 104 bone marrow–derived dendritic cells [62] and 0.01 μg/ml OVA323–339 peptide (Ansynth) in the presence or absence of 20-ng/μl rhTGF-ß1 (Peprotech) in 48-well plates.

T18790

38158-38288

Sentence

denotes

After 4 d, cells were harvested and analyzed for intracellular FOXP3 expression by FACS or gene expression by quantitative RT-PCR.

T280

38158-38288

Sentence

denotes

After 4 d, cells were harvested and analyzed for intracellular FOXP3 expression by FACS or gene expression by quantitative RT-PCR.

T19106

38290-38322

Sentence

denotes

In vitro T cell differentiation.

T281

38290-38322

Sentence

denotes

In vitro T cell differentiation.

T19107

38323-38552

Sentence

denotes

CD4+ CD45RA+ magnetically-sorted (CD45RO depletion, MACS, according to the protocol of the manufacturer) cells were stimulated with immobilized plate-bound anti-CD3 (1 μg/ml, Okt3, IgG1) and anti-CD28 (2 μg/ml) in Th1 conditions:

T282

38323-38552

Sentence

denotes

CD4+ CD45RA+ magnetically-sorted (CD45RO depletion, MACS, according to the protocol of the manufacturer) cells were stimulated with immobilized plate-bound anti-CD3 (1 μg/ml, Okt3, IgG1) and anti-CD28 (2 μg/ml) in Th1 conditions:

T19108

38553-38620

Sentence

denotes

25 ng/ml IL-12, 5 μg/ml anti-IL-4 (R&D systems); in Th2 conditions:

T283

38553-38620

Sentence

denotes

25 ng/ml IL-12, 5 μg/ml anti-IL-4 (R&D systems); in Th2 conditions:

T19109

38621-38712

Sentence

denotes

25 ng/ml IL-4, 5 μg/ml anti-IFN-γ, 5 μg/ml anti-IL-12 (R&D systems); or in Treg conditions:

T284

38621-38712

Sentence

denotes

25 ng/ml IL-4, 5 μg/ml anti-IFN-γ, 5 μg/ml anti-IL-12 (R&D systems); or in Treg conditions:

T19110

38713-38787

Sentence

denotes

10 ng/ml TGF-β, 5 μg/ml anti-IFN-γ, 5 μg/ml anti-IL-12, 5 μg/ml anti-IL-4.

T285

38713-38787

Sentence

denotes

10 ng/ml TGF-β, 5 μg/ml anti-IFN-γ, 5 μg/ml anti-IL-12, 5 μg/ml anti-IL-4.

T19111

38788-38859

Sentence

denotes

Proliferating cells were expanded in medium containing IL-2 (30 ng/ml).

T286

38788-38859

Sentence

denotes

Proliferating cells were expanded in medium containing IL-2 (30 ng/ml).

T19516

38861-38929

Sentence

denotes

Cloning of the FOXP3 promoter and construction of mutant constructs.

T287

38861-38929

Sentence

denotes

Cloning of the FOXP3 promoter and construction of mutant constructs.

T19517

38930-39047

Sentence

denotes

The FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate the pGL3 FOXP3 −511/+176 [24].

T288

38930-39047

Sentence

denotes

The FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate the pGL3 FOXP3 −511/+176 [24].

T19518

39048-39204

Sentence

denotes

Site-directed mutagenesis in the FOXP3 promoter region were introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions.

T289

39048-39204

Sentence

denotes

Site-directed mutagenesis in the FOXP3 promoter region were introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions.

T19519

39205-39333

Sentence

denotes

The following primer and its complementary strand were used: GTT TCT CAT GAG CCC TAT TAA GTC ATT CTT ACC TCT CAC CTC TGT GGT GA.

T290

39205-39333

Sentence

denotes

The following primer and its complementary strand were used: GTT TCT CAT GAG CCC TAT TAA GTC ATT CTT ACC TCT CAC CTC TGT GGT GA.

T19761

39335-39374

Sentence

denotes

Transfections and reporter gene assays.

T291

39335-39374

Sentence

denotes

Transfections and reporter gene assays.

T19762

39375-39452

Sentence

denotes

T cells were rested in serum-free AIM-V medium (Life Technologies) overnight.

T292

39375-39452

Sentence

denotes

T cells were rested in serum-free AIM-V medium (Life Technologies) overnight.

T293

39453-39691

Sentence

denotes

3.5 μg of the FOXP3 promoter luciferase reporter vector and 0.5 μg phRL-TK were added to 3 × 106 CD4+ T cells resuspended in 100 μL of Nucleofector solution (Amaxa Biosystems) and electroporated using the U-15 program of the Nucleofector.

T19763

39453-39911

Sentence

denotes

3.5 μg of the FOXP3 promoter luciferase reporter vector and 0.5 μg phRL-TK were added to 3 × 106 CD4+ T cells resuspended in 100 μL of Nucleofector solution (Amaxa Biosystems) and electroporated using the U-15 program of the Nucleofector. 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 Biotech) according to the manufacturer's instructions.

T294

39692-39911

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 Biotech) according to the manufacturer's instructions.

T19764

39912-39971

Sentence

denotes

Data were normalized by the activity of renilla luciferase.

T295

39912-39971

Sentence

denotes

Data were normalized by the activity of renilla luciferase.

T20042

39973-40006

Sentence

denotes

RNA isolation and cDNA synthesis.

T296

39973-40006

Sentence

denotes

RNA isolation and cDNA synthesis.

T20043

40007-40100

Sentence

denotes

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

T297

40007-40100

Sentence

denotes

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

T20044

40101-40280

Sentence

denotes

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

T298

40101-40280

Sentence

denotes

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

T20171

40282-40307

Sentence

denotes

Recombinant TAT proteins.

T299

40282-40307

Sentence

denotes

Recombinant TAT proteins.

T20172

40308-40499

Sentence

denotes

The cDNAs encoding GATA3 protein or the truncated GATA3 (lacking the two zinc fingers) were cloned in frame into an expression vector along with the TAT sequence as previously described [63].

T300

40308-40499

Sentence

denotes

The cDNAs encoding GATA3 protein or the truncated GATA3 (lacking the two zinc fingers) were cloned in frame into an expression vector along with the TAT sequence as previously described [63].

T20173

40500-40625

Sentence

denotes

Proteins were expressed in BL21 Star (DE3)pLysS (Invitrogen) and lysates were purified by Ni2+-chelate column chromatography.

T301

40500-40625

Sentence

denotes

Proteins were expressed in BL21 Star (DE3)pLysS (Invitrogen) and lysates were purified by Ni2+-chelate column chromatography.

T20174

40626-40745

Sentence

denotes

Both TAT-linked proteins were more than 95% pure, based on Coomassie blue staining of sodium disulfate acrylamide gels.

T302

40626-40745

Sentence

denotes

Both TAT-linked proteins were more than 95% pure, based on Coomassie blue staining of sodium disulfate acrylamide gels.

T20439

40747-40770

Sentence

denotes

TAT-GATA3 transduction.

T303

40747-40770

Sentence

denotes

TAT-GATA3 transduction.

T20440

40771-40919

Sentence

denotes

CD4+CD45RA+ cells were cultured in AIMV medium and transduced with 20 nM, 10 nM, or 500 nM of full-length or truncated GATA3 over the course of 4 h.

T304

40771-40919

Sentence

denotes

CD4+CD45RA+ cells were cultured in AIMV medium and transduced with 20 nM, 10 nM, or 500 nM of full-length or truncated GATA3 over the course of 4 h.

T20441

40920-41024

Sentence

denotes

After 4 h, the cells were washed and activated with soluble anti-CD3 and anti-CD28 and TGF-β (10 ng/ml).

T305

40920-41024

Sentence

denotes

After 4 h, the cells were washed and activated with soluble anti-CD3 and anti-CD28 and TGF-β (10 ng/ml).

T20442

41025-41085

Sentence

denotes

Each day, the TAT proteins were freshly added to the medium.

T306

41025-41085

Sentence

denotes

Each day, the TAT proteins were freshly added to the medium.

T20443

41086-41152

Sentence

denotes

FOXP3 expression was measured after 5 d by intracellular staining.

T307

41086-41152

Sentence

denotes

FOXP3 expression was measured after 5 d by intracellular staining.

T20710

41154-41186

Sentence

denotes

Intracellular cytokine staining.

T308

41154-41186

Sentence

denotes

Intracellular cytokine staining.

T20711

41187-41282

Sentence

denotes

T cells were stimulated with 2 × 10−7 M PMA and 1 μg/ml of ionomycin (Sigma Chemicals) for 4 h.

T309

41187-41282

Sentence

denotes

T cells were stimulated with 2 × 10−7 M PMA and 1 μg/ml of ionomycin (Sigma Chemicals) for 4 h.

T20712

41283-41336

Sentence

denotes

The following mAb was used: anti-IL-4-PE (8D4–8, BD).

T310

41283-41336

Sentence

denotes

The following mAb was used: anti-IL-4-PE (8D4–8, BD).

T20713

41337-41435

Sentence

denotes

Matched isotype controls were used at the same protein concentration as the respective antibodies.

T311

41337-41435

Sentence

denotes

Matched isotype controls were used at the same protein concentration as the respective antibodies.

T20714

41436-41576

Sentence

denotes

Four-color FACS was performed using an EPICS XL-MCL (Beckman Coulter) using the software Expo32 version for data acquisition and evaluation.

T312

41436-41576

Sentence

denotes

Four-color FACS was performed using an EPICS XL-MCL (Beckman Coulter) using the software Expo32 version for data acquisition and evaluation.

T20958

41578-41593

Sentence

denotes

Flow cytometry.

T313

41578-41593

Sentence

denotes

Flow cytometry.

T20959

41594-41952

Sentence

denotes

For analysis of FOXP3 expression at the single-cell level, cells were first stained with the monoclonal antibody CD25 (Beckman Coulter), and after fixation and permeabilization, cells were incubated with PE-conjugated monoclonal antibody PCH101 (anti-human FOXP3; eBioscience) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL).

T314

41594-41952

Sentence

denotes

For analysis of FOXP3 expression at the single-cell level, cells were first stained with the monoclonal antibody CD25 (Beckman Coulter), and after fixation and permeabilization, cells were incubated with PE-conjugated monoclonal antibody PCH101 (anti-human FOXP3; eBioscience) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL).

T20960

41953-42278

Sentence

denotes

For cell surface marker staining, cells were incubated for 20 min at 4°C in staining buffer with the following antibodies: anti–CD152-PE (CTLA-4; BD), anti–PD-1 (eBiosciences), anti-GITR (R & D Systems), anti-CD69 (Beckman Coulter), anti-CD103 (DakoCytomation), anti-CD62L (Beckman Coulter), or anti-HLA-DR (Beckman Coulter).

T315

41953-42278

Sentence

denotes

For cell surface marker staining, cells were incubated for 20 min at 4°C in staining buffer with the following antibodies: anti–CD152-PE (CTLA-4; BD), anti–PD-1 (eBiosciences), anti-GITR (R & D Systems), anti-CD69 (Beckman Coulter), anti-CD103 (DakoCytomation), anti-CD62L (Beckman Coulter), or anti-HLA-DR (Beckman Coulter).

T20961

42279-42349

Sentence

denotes

The controls were FITC, PE, or ECD-conjugated mouse IgG1 or rat IgG2a.

T316

42279-42349

Sentence

denotes

The controls were FITC, PE, or ECD-conjugated mouse IgG1 or rat IgG2a.

T20962

42350-42512

Sentence

denotes

For staining of mouse cells, the following mAbs from BD Biosciences were used following standard techniques as described above: anti-CD3, anti-CD4, and anti-CD25.

T317

42350-42512

Sentence

denotes

For staining of mouse cells, the following mAbs from BD Biosciences were used following standard techniques as described above: anti-CD3, anti-CD4, and anti-CD25.

T20963

42513-42621

Sentence

denotes

Anti-FcγRII/III antibody (2.4G2, ATCC) was included in all stainings to reduce nonspecific antibody binding.

T318

42513-42621

Sentence

denotes

Anti-FcγRII/III antibody (2.4G2, ATCC) was included in all stainings to reduce nonspecific antibody binding.

T20964

42622-42797

Sentence

denotes

To isolate naive murine CD4 T cells from murine DO11.10 or DO11.10xCD2-GATA3 T cells, cells were stained with anti-CD25-FITC, anti-CD62L-PE, and anti-CD4-APC prior to sorting.

T319

42622-42797

Sentence

denotes

To isolate naive murine CD4 T cells from murine DO11.10 or DO11.10xCD2-GATA3 T cells, cells were stained with anti-CD25-FITC, anti-CD62L-PE, and anti-CD4-APC prior to sorting.

T20965

42798-42866

Sentence

denotes

Dead cells were excluded with 4′',6-Diamidino-2-phenylindole (DAPI).

T320

42798-42866

Sentence

denotes

Dead cells were excluded with 4′',6-Diamidino-2-phenylindole (DAPI).

T20966

42867-43140

Sentence

denotes

To analyze murine Foxp3 expression in inducible Treg cultures, cells were stained intracellularly with anti-Foxp3-PE according to manufacturer's instruction, in conjunction with anti-CD4-APC and LIVE/DEAD fixable dead cell stain kit (Invitrogen) to discriminate live cells.

T321

42867-43140

Sentence

denotes

To analyze murine Foxp3 expression in inducible Treg cultures, cells were stained intracellularly with anti-Foxp3-PE according to manufacturer's instruction, in conjunction with anti-CD4-APC and LIVE/DEAD fixable dead cell stain kit (Invitrogen) to discriminate live cells.

T20967

43141-43243

Sentence

denotes

All monoclonal antibodies for murine cell stainings were purchased from eBioscience or BD Biosciences.

T322

43141-43243

Sentence

denotes

All monoclonal antibodies for murine cell stainings were purchased from eBioscience or BD Biosciences.

T21784

43245-43262

Sentence

denotes

Western blotting.

T323

43245-43262

Sentence

denotes

Western blotting.

T21785

43263-43448

Sentence

denotes

For FOXP3 analysis on the protein level, 1 × 106 CD4+CD25– cells were lysed and loaded next to a protein-mass ladder (Magicmark, Invitrogen) on a NuPAGE 4–12% bis-tris gel (Invitrogen).

T324

43263-43448

Sentence

denotes

For FOXP3 analysis on the protein level, 1 × 106 CD4+CD25– cells were lysed and loaded next to a protein-mass ladder (Magicmark, Invitrogen) on a NuPAGE 4–12% bis-tris gel (Invitrogen).

T21786

43449-43527

Sentence

denotes

The proteins were electroblotted onto a PVDF membrane (Amersham Life Science).

T325

43449-43527

Sentence

denotes

The proteins were electroblotted onto a PVDF membrane (Amersham Life Science).

T326

43528-43702

Sentence

denotes

Unspecific binding was blocked with BSA, and the membranes were subsequently incubated with an 1:200 dilution of goat anti-FOXP3 in blocking buffer (Abcam) overnight at 4 °C.

T21787

43528-43831

Sentence

denotes

Unspecific binding was blocked with BSA, and the membranes were subsequently incubated with an 1:200 dilution of goat anti-FOXP3 in blocking buffer (Abcam) overnight at 4 °C. The blots were developed using an anti-goat HRP-labeled mAb (Amersham Biosciences) and visualized with a LAS 1000 camera (Fuji).

T327

43703-43831

Sentence

denotes

The blots were developed using an anti-goat HRP-labeled mAb (Amersham Biosciences) and visualized with a LAS 1000 camera (Fuji).

T21788

43832-43900

Sentence

denotes

Membranes were incubated in stripping buffer and re-blocked for 1 h.

T328

43832-43900

Sentence

denotes

Membranes were incubated in stripping buffer and re-blocked for 1 h.

T21789

43901-44151

Sentence

denotes

The membranes were re-probed using anti-GATA3 (HG3–31; Santa Cruz Biotechnology), anti-T-bet (4B10, Santa Cruz Biotechnology), anti-GAPDH (6C5, Ambion), anti-phospho-SMAD2 (138D4), anti-phospho-STAT6 (5A4), and anti-STAT6 (Cell Signaling Technology),

T329

43901-44151

Sentence

denotes

The membranes were re-probed using anti-GATA3 (HG3–31; Santa Cruz Biotechnology), anti-T-bet (4B10, Santa Cruz Biotechnology), anti-GAPDH (6C5, Ambion), anti-phospho-SMAD2 (138D4), anti-phospho-STAT6 (5A4), and anti-STAT6 (Cell Signaling Technology),

T22255

44153-44204

Sentence

denotes

Administration of cytokines and antibodies in vivo.

T330

44153-44204

Sentence

denotes

Administration of cytokines and antibodies in vivo.

T22256

44205-44444

Sentence

denotes

Age- and gender-matched normal B6 mice received every other day intraperitoneal (ip) injections of PBS, 1.5 μg rmIL-4, 50 μg anti-IL-4 mAb (11B11 or MAB404), or a mixture of 1.5 μg rmIL-4 plus 50 μg anti-IL-4 mAb (11B11 or MAB404) for 7 d.

T331

44205-44444

Sentence

denotes

Age- and gender-matched normal B6 mice received every other day intraperitoneal (ip) injections of PBS, 1.5 μg rmIL-4, 50 μg anti-IL-4 mAb (11B11 or MAB404), or a mixture of 1.5 μg rmIL-4 plus 50 μg anti-IL-4 mAb (11B11 or MAB404) for 7 d.

T22257

44445-44551

Sentence

denotes

Thereafter, spleen and lymph node cells were analyzed by flow cytometry for CD3, CD4, and CD25 expression.

T332

44445-44551

Sentence

denotes

Thereafter, spleen and lymph node cells were analyzed by flow cytometry for CD3, CD4, and CD25 expression.

T22258

44552-44682

Sentence

denotes

The anti-mouse IL-4 mAb MAB404 was obtained from R&D Systems, the second anti-mouse IL-4 mAb 11B11 was purchased from eBioscience.

T333

44552-44682

Sentence

denotes

The anti-mouse IL-4 mAb MAB404 was obtained from R&D Systems, the second anti-mouse IL-4 mAb 11B11 was purchased from eBioscience.

T22565

44684-44689

Sentence

denotes

ChIP.

T334

44684-44689

Sentence

denotes

ChIP.

T22566

44690-44900

Sentence

denotes

ChIP analysis was performed according to the manufacturer's protocol (Upstate Biotechnology) with the following modifications. iTreg and Th2 cells were fixed with 1% formaldehyde for 10 min at room temperature.

T335

44690-44900

Sentence

denotes

ChIP analysis was performed according to the manufacturer's protocol (Upstate Biotechnology) with the following modifications. iTreg and Th2 cells were fixed with 1% formaldehyde for 10 min at room temperature.

T22567

44901-45015

Sentence

denotes

The chromatin was sheared to 200-1000 bp of length by sonication with five pulses of 10 s at 30% power (Bandelin).

T336

44901-45015

Sentence

denotes

The chromatin was sheared to 200-1000 bp of length by sonication with five pulses of 10 s at 30% power (Bandelin).

T22568

45016-45174

Sentence

denotes

The chromatin was pre-cleared for 2 h with normal mouse IgG beads and then incubated with anti-GATA3-agarose beads (HG3–31; Santa Cruz Biotechnology) for 2 h.

T337

45016-45174

Sentence

denotes

The chromatin was pre-cleared for 2 h with normal mouse IgG beads and then incubated with anti-GATA3-agarose beads (HG3–31; Santa Cruz Biotechnology) for 2 h.

T338

45175-45264

Sentence

denotes

Washing and elution buffers were used according to the protocol of Upstate Biotechnology.

T22569

45175-45409

Sentence

denotes

Washing and elution buffers were used according to the protocol of Upstate Biotechnology. Crosslinks were reversed by incubation at 65 °C for 4 h in the presence of 0.2 M NaCl, and the DNA was purified by phenol/chloroform extraction.

T339

45265-45409

Sentence

denotes

Crosslinks were reversed by incubation at 65 °C for 4 h in the presence of 0.2 M NaCl, and the DNA was purified by phenol/chloroform extraction.

T22570

45410-45463

Sentence

denotes

The amount of DNA was determined by conventional PCR.

T340

45410-45463

Sentence

denotes

The amount of DNA was determined by conventional PCR.

T22571

45464-45571

Sentence

denotes

The PCR addressed for the FOXP3 promoter region −246 to −511 and was performed using the following primers:

T341

45464-45571

Sentence

denotes

The PCR addressed for the FOXP3 promoter region −246 to −511 and was performed using the following primers:

T22572

45572-45633

Sentence

denotes

5′-gtgccctttacgagt catctg-3′ and 5′-gtgccctttacgagtcatctg-3′.

T342

45572-45633

Sentence

denotes

5′-gtgccctttacgagt catctg-3′ and 5′-gtgccctttacgagtcatctg-3′.

T22573

45634-45697

Sentence

denotes

The PCR products were visualized using an ethidium bromide gel.

T343

45634-45697

Sentence

denotes

The PCR products were visualized using an ethidium bromide gel.

T22967

45699-45715

Sentence

denotes

Pull-down assay.

T344

45699-45715

Sentence

denotes

Pull-down assay.

T345

45716-45784

Sentence

denotes

CD4+ T cells were stimulated with PMA and ionomycin for 2 h at 37°C.

T22968

45716-45980

Sentence

denotes

CD4+ T cells were stimulated with PMA and ionomycin for 2 h at 37°C. The cells were pelleted, resuspended in buffer C (20 mM HEPES [pH 7.9], 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, protease inhibitors [Sigma]. and 0.1% NP-40) and lysed on ice for 15 min.

T346

45785-45980

Sentence

denotes

The cells were pelleted, resuspended in buffer C (20 mM HEPES [pH 7.9], 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, protease inhibitors [Sigma]. and 0.1% NP-40) and lysed on ice for 15 min.

T22969

45981-46030

Sentence

denotes

Insoluble material was removed by centrifugation.

T347

45981-46030

Sentence

denotes

Insoluble material was removed by centrifugation.

T22970

46031-46109

Sentence

denotes

The supernatant was diluted 1:3 with buffer D (as buffer C, but without NaCl).

T348

46031-46109

Sentence

denotes

The supernatant was diluted 1:3 with buffer D (as buffer C, but without NaCl).

T349

46110-46284

Sentence

denotes

The lysates were incubated with 10 μg of poly(dI-dC) (Sigma) and 70 μl of streptavidin-agarose (Amersham Biosciences) carrying biotinylated oligonucleotides, for 3 h at 4 °C.

T22971

46110-46494

Sentence

denotes

The lysates were incubated with 10 μg of poly(dI-dC) (Sigma) and 70 μl of streptavidin-agarose (Amersham Biosciences) carrying biotinylated oligonucleotides, for 3 h at 4 °C. The beads were washed twice with buffer C:D (1:3) and resuspended in DTT-containing loading buffer (NuPAGE; Invitrogen), heated to 70 °C for 10 min, and the eluants on a NuPAGE 4–12% bis-tris gel (Invitrogen).

T350

46285-46494

Sentence

denotes

The beads were washed twice with buffer C:D (1:3) and resuspended in DTT-containing loading buffer (NuPAGE; Invitrogen), heated to 70 °C for 10 min, and the eluants on a NuPAGE 4–12% bis-tris gel (Invitrogen).

T22972

46495-46636

Sentence

denotes

The proteins were electroblotted onto a PVDF membrane (Amersham Biosciences) and detected using an anti-GATA3 mAb (Santa Cruz Biotechnology).

T351

46495-46636

Sentence

denotes

The proteins were electroblotted onto a PVDF membrane (Amersham Biosciences) and detected using an anti-GATA3 mAb (Santa Cruz Biotechnology).

T22973

46637-46701

Sentence

denotes

Accumulated signals were analyzed using AIDA software (Raytest).

T352

46637-46701

Sentence

denotes

Accumulated signals were analyzed using AIDA software (Raytest).

T353

46703-46725

Sentence

denotes

Supporting Information

T354

46726-46781

Sentence

denotes

Figure S1 Phenotype of In Vitro Differentiated T Cells

T355

46782-46987

Sentence

denotes

After two round of differentiation cultures, T cells were stimulated by plate-immobilized anti-CD3/CD28 and 3H-thymidine incorporation as measurement of proliferation was analyzed after 3 d of culture (A).

T356

46988-47100

Sentence

denotes

In parallel, T cells were analyzed for Treg relevant surface receptor expression as indicated on the x-axis (B).

T357

47101-47113

Sentence

denotes

(1.0 MB AI).

T358

47114-47150

Sentence

denotes

Click here for additional data file.

T359

47153-47227

Sentence

denotes

Figure S2 In Vivo Treatment of Mice with IL-4 Antibody–Cytokine Complexes

T360

47228-47456

Sentence

denotes

B6 mice were given every other day ip injections of phosphate-buffered saline (PBS), recombinant mouse IL-4 (rmIL-4), anti-IL-4 mAb (anti-IL-4 mAb, 11B11, or MAB404), or a mixture of rmIL-4 plus anti-IL-4 mAbs (11B11 or MAB404).

T361

47457-47537

Sentence

denotes

Mice were analyzed on day 7 by flow cytometry for CD3, CD4, and CD25 expression.

T362

47538-47606

Sentence

denotes

Shown is CD25 versus CD4 expression in CD3+ CD4+ spleen cells (A–F).

T363

47607-47664

Sentence

denotes

Numbers indicate percentages of CD4+ CD25high CD3+ cells.

T364

47665-47762

Sentence

denotes

Total cell counts (G) of CD4+ CD25high cells in spleen from mice in (A–F) are shown as mean ± SD.

T365

47763-47824

Sentence

denotes

The data are representative of three independent experiments.

T366

47825-47837

Sentence

denotes

(369 KB AI).

T367

47838-47874

Sentence

denotes

Click here for additional data file.

T368

47877-47954

Sentence

denotes

Figure S3 Effect of IL-4 on Already Existing Natural or Inducible Treg Cells

T369

47955-48159

Sentence

denotes

(A) CD4+CD25high nTreg cells were FACS-sorted and activated with plate-bound anti-CD3/CD28 plus IL-2 during 3 d and in the presence or absence of IL-4 (100 ng/ml) and harvested for real-time PCR analysis.

T370

48160-48235

Sentence

denotes

The results shown represent the mean ± SD of three independent experiments.

T371

48236-48274

Sentence

denotes

(B) iTreg cells were induced in vitro.

T372

48275-48523

Sentence

denotes

FOXP3 espression was assessed by real-time PCR analysis in resting cells, in cells re-stimulated with plate-bound anti-CD3/CD28, with or without TGF-β, plus IL-2 during 3 d and in the presence (black bar) or absence (white bar) of IL-4 (100 ng/ml).

T373

48524-48629

Sentence

denotes

(C) Activation dramatically increases CD4+CD25+ Treg cells suppressive capacity of CD4+CD25+ nTreg cells.

T374

48630-48744

Sentence

denotes

CD4+CD25+ nTreg cells were preactivated during 2 d in the presence or absence of an increasing IL-4 concentration.

T375

48745-48830

Sentence

denotes

After vigorous washing, their suppressive capacity on responder CD4+CD25− was tested.

T376

48831-48923

Sentence

denotes

IL-4 pretreatment did not affect the suppressive capacity of FACS-sorted CD4+CD25high cells.

T377

48924-49015

Sentence

denotes

1 × 104 CD4+CD25+ nTreg cells were added to 5 × 104 CD4+CD25– and 5 × 104 irradiated PBMCs.

T378

49016-49080

Sentence

denotes

The results are representative of three independent experiments.

T379

49081-49093

Sentence

denotes

(269 KB AI).

T380

49094-49130

Sentence

denotes

Click here for additional data file.

T381

49133-49213

Sentence

denotes

Figure S4 Schematic Structure of the FOXP3 Gene and Location of the GATA3 Sites

T382

49214-49332

Sentence

denotes

The scheme shows the location of the 11 exons spaced by a large intron (6000 bp) from the 5′untranslated region (UTR).

T383

49333-49440

Sentence

denotes

Human, murine, and rat sequences are aligned and transcription start site (TSS) is indicated with an arrow.

T384

49441-49453

Sentence

denotes

(529 KB AI).

T385

49454-49490

Sentence

denotes

Click here for additional data file.

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