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T145 0-148 Sentence denotes Interleukin-10 Production by Th1 Cells Requires Interleukin-12-Induced STAT4 Transcription Factor and ERK MAP Kinase Activation by High Antigen Dose
T1 0-148 Sentence denotes Interleukin-10 Production by Th1 Cells Requires Interleukin-12-Induced STAT4 Transcription Factor and ERK MAP Kinase Activation by High Antigen Dose
T2 151-159 Sentence denotes Abstract
T146 160-167 Sentence denotes Summary
T3 160-167 Sentence denotes Summary
T147 168-274 Sentence denotes CD4+ T cells producing interleukin-10 (IL-10) and interferon-γ (IFN-γ) are reported in chronic infections.
T4 168-274 Sentence denotes CD4+ T cells producing interleukin-10 (IL-10) and interferon-γ (IFN-γ) are reported in chronic infections.
T148 275-380 Sentence denotes However, the signals that direct the development of IL-10-producing T helper 1 (Th1) cells are undefined.
T5 275-380 Sentence denotes However, the signals that direct the development of IL-10-producing T helper 1 (Th1) cells are undefined.
T149 381-592 Sentence denotes We showed that development of IL-10-producing Th1 cells required high T cell receptor (TCR) ligation, sustained ERK1 and ERK2 MAP kinases phosphorylation, and IL-12-induced STAT4 transcription factor activation.
T6 381-592 Sentence denotes We showed that development of IL-10-producing Th1 cells required high T cell receptor (TCR) ligation, sustained ERK1 and ERK2 MAP kinases phosphorylation, and IL-12-induced STAT4 transcription factor activation.
T150 593-798 Sentence denotes Repeated TCR triggering led to enhanced IL-10 production by Th1 cells, and continued IL-12 action and high-dose TCR signaling were required for the development and maintenance of IL-10-producing Th1 cells.
T7 593-798 Sentence denotes Repeated TCR triggering led to enhanced IL-10 production by Th1 cells, and continued IL-12 action and high-dose TCR signaling were required for the development and maintenance of IL-10-producing Th1 cells.
T151 799-1001 Sentence denotes Although Th1, Th2, and Th17 cells require the activation of distinct STATs for their differentiation, activation of ERK1 and ERK2 was a common requirement for production of IL-10 by all Th cell subsets.
T8 799-1001 Sentence denotes Although Th1, Th2, and Th17 cells require the activation of distinct STATs for their differentiation, activation of ERK1 and ERK2 was a common requirement for production of IL-10 by all Th cell subsets.
T152 1002-1057 Sentence denotes IL-10 expression also correlated with c-maf expression.
T9 1002-1057 Sentence denotes IL-10 expression also correlated with c-maf expression.
T153 1058-1238 Sentence denotes Despite having distinct functions in protection against pathogens, all Th cells share the important task of controlling overexuberant immune responses by means of IL-10 production.
T10 1058-1238 Sentence denotes Despite having distinct functions in protection against pathogens, all Th cells share the important task of controlling overexuberant immune responses by means of IL-10 production.
T11 1240-1252 Sentence denotes Introduction
T1116 1253-1409 Sentence denotes Interleukin-10 (IL-10) is a cytokine with broad anti-inflammatory properties that inhibits macrophage and dendritic cell (DC) function (Moore et al., 2001).
T12 1253-1409 Sentence denotes Interleukin-10 (IL-10) is a cytokine with broad anti-inflammatory properties that inhibits macrophage and dendritic cell (DC) function (Moore et al., 2001).
T1117 1410-1686 Sentence denotes IL-10 limits the immune and inflammatory responses to pathogens and gut flora and prevents damage to the host (Moore et al., 2001; O'Garra and Vieira, 2004), but when dysregulated may result in chronic infection (Brooks et al., 2006; Ejrnaes et al., 2006; Moore et al., 2001).
T13 1410-1686 Sentence denotes IL-10 limits the immune and inflammatory responses to pathogens and gut flora and prevents damage to the host (Moore et al., 2001; O'Garra and Vieira, 2004), but when dysregulated may result in chronic infection (Brooks et al., 2006; Ejrnaes et al., 2006; Moore et al., 2001).
T1118 1687-2228 Sentence denotes IL-10 is expressed by T helper 2 (Th2) cells, B cells, DCs, and macrophages (Moore et al., 2001), and also by Th1 cells (Anderson et al., 2007; Assenmacher et al., 1994; Del Prete et al., 1993; Gerosa et al., 1996; Jankovic et al., 2007; Pohl-Koppe et al., 1998) and (reviewed in O'Garra and Vieira, 2007; Trinchieri, 2007), certain regulatory (Treg) T cells (Moore et al., 2001; O'Garra and Vieira, 2004; Roncarolo et al., 2006), and Th17 cells (Awasthi et al., 2007; Fitzgerald et al., 2007; McGeachy et al., 2007; Stumhofer et al., 2007).
T14 1687-2228 Sentence denotes IL-10 is expressed by T helper 2 (Th2) cells, B cells, DCs, and macrophages (Moore et al., 2001), and also by Th1 cells (Anderson et al., 2007; Assenmacher et al., 1994; Del Prete et al., 1993; Gerosa et al., 1996; Jankovic et al., 2007; Pohl-Koppe et al., 1998) and (reviewed in O'Garra and Vieira, 2007; Trinchieri, 2007), certain regulatory (Treg) T cells (Moore et al., 2001; O'Garra and Vieira, 2004; Roncarolo et al., 2006), and Th17 cells (Awasthi et al., 2007; Fitzgerald et al., 2007; McGeachy et al., 2007; Stumhofer et al., 2007).
T1119 2229-2539 Sentence denotes In vitro human CD4+ and CD8+ T cell clones, or mouse CD4+ T cells that produce both interferon-γ (IFN-γ) and IL-10, can be differentiated by T cell receptor (TCR)-stimulation in the presence of IL-12 (Chang et al., 2007; Gerosa et al., 1996; Jeannin et al., 1996; Meyaard et al., 1996; Windhagen et al., 1996).
T15 2229-2539 Sentence denotes In vitro human CD4+ and CD8+ T cell clones, or mouse CD4+ T cells that produce both interferon-γ (IFN-γ) and IL-10, can be differentiated by T cell receptor (TCR)-stimulation in the presence of IL-12 (Chang et al., 2007; Gerosa et al., 1996; Jeannin et al., 1996; Meyaard et al., 1996; Windhagen et al., 1996).
T1120 2540-2719 Sentence denotes Furthermore, Th1 cell clones coproducing IFN-γ and IL-10 have been isolated from bronchoalveolar lavage (BAL) of active pulmonary tuberculosis (TB) patients (Gerosa et al., 1999).
T16 2540-2719 Sentence denotes Furthermore, Th1 cell clones coproducing IFN-γ and IL-10 have been isolated from bronchoalveolar lavage (BAL) of active pulmonary tuberculosis (TB) patients (Gerosa et al., 1999).
T1121 2720-3033 Sentence denotes IL-10 production by Th1 cells was also reported in animals infected with Toxoplasma gondii (Jankovic et al., 2002; Shaw et al., 2006) or with Leishmania major (Anderson et al., 2007) and shown to be required for regulation of the immune response in these infections (Anderson et al., 2007; Jankovic et al., 2007).
T17 2720-3033 Sentence denotes IL-10 production by Th1 cells was also reported in animals infected with Toxoplasma gondii (Jankovic et al., 2002; Shaw et al., 2006) or with Leishmania major (Anderson et al., 2007) and shown to be required for regulation of the immune response in these infections (Anderson et al., 2007; Jankovic et al., 2007).
T1122 3034-3297 Sentence denotes The relative amounts of IL-10 and IFN-γ produced by Th1 cells may influence the balance between clearance and persistent infection with certain pathogens (Moore et al., 2001; Trinchieri, 2007), thus determining whether chronic infection or immunopathology ensues.
T18 3034-3297 Sentence denotes The relative amounts of IL-10 and IFN-γ produced by Th1 cells may influence the balance between clearance and persistent infection with certain pathogens (Moore et al., 2001; Trinchieri, 2007), thus determining whether chronic infection or immunopathology ensues.
T1123 3298-3460 Sentence denotes Th1, Th2, and Th17 cell responses differentiate along distinct signaling pathways (Glimcher and Murphy, 2000; Ivanov et al., 2007; Stockinger and Veldhoen, 2007).
T19 3298-3460 Sentence denotes Th1, Th2, and Th17 cell responses differentiate along distinct signaling pathways (Glimcher and Murphy, 2000; Ivanov et al., 2007; Stockinger and Veldhoen, 2007).
T1124 3461-3852 Sentence denotes Th1 cell development requires signal transducer and activator of transcription (STAT)1 activation, induced by type I IFN or IFN-γ, the transcription factor T-box 21 (T-bet), and IL-12-induced STAT4 signaling, which can couple with IL-18-induced IRAK and NF-κB transcription factors to drive the high amounts of IFN-γ required to eradicate intracellular pathogens (Glimcher and Murphy, 2000).
T20 3461-3852 Sentence denotes Th1 cell development requires signal transducer and activator of transcription (STAT)1 activation, induced by type I IFN or IFN-γ, the transcription factor T-box 21 (T-bet), and IL-12-induced STAT4 signaling, which can couple with IL-18-induced IRAK and NF-κB transcription factors to drive the high amounts of IFN-γ required to eradicate intracellular pathogens (Glimcher and Murphy, 2000).
T1125 3853-4026 Sentence denotes Th2 cell development, with expression of IL-4, IL-5, and IL-13, requires IL-4, STAT6, and the transcription factor GATA binding protein (GATA)-3 (Glimcher and Murphy, 2000).
T21 3853-4026 Sentence denotes Th2 cell development, with expression of IL-4, IL-5, and IL-13, requires IL-4, STAT6, and the transcription factor GATA binding protein (GATA)-3 (Glimcher and Murphy, 2000).
T1126 4027-4201 Sentence denotes The development of Th17 cells requires IL-6, TGF-β, and the STAT3-dependent expression of the transcription factor RORγt (Ivanov et al., 2007; Stockinger and Veldhoen, 2007).
T22 4027-4201 Sentence denotes The development of Th17 cells requires IL-6, TGF-β, and the STAT3-dependent expression of the transcription factor RORγt (Ivanov et al., 2007; Stockinger and Veldhoen, 2007).
T1127 4202-4347 Sentence denotes Th1 and Th2 cell responses can also be induced by varying the dose of antigen presented to the naive T cell by the antigen-presenting cell (APC).
T23 4202-4347 Sentence denotes Th1 and Th2 cell responses can also be induced by varying the dose of antigen presented to the naive T cell by the antigen-presenting cell (APC).
T1128 4348-4770 Sentence denotes Whereas high doses of antigen, with sustained TCR signaling and extracellular-signal regulated (ERK) mitogen-activated protein kinase (MAPK) phosphorylation, result in Th1 cells producing IFN-γ via an IL-12-independent mechanism, low doses of antigen, with transient ERK1 and ERK2 activation, favor Th2 responses and IL-4 secretion (Constant et al., 1995; Hosken et al., 1995; Jorritsma et al., 2003; Yamane et al., 2005).
T24 4348-4770 Sentence denotes Whereas high doses of antigen, with sustained TCR signaling and extracellular-signal regulated (ERK) mitogen-activated protein kinase (MAPK) phosphorylation, result in Th1 cells producing IFN-γ via an IL-12-independent mechanism, low doses of antigen, with transient ERK1 and ERK2 activation, favor Th2 responses and IL-4 secretion (Constant et al., 1995; Hosken et al., 1995; Jorritsma et al., 2003; Yamane et al., 2005).
T1129 4771-5179 Sentence denotes Because Th1 and Th2 cells cross regulate each other's development and function and can suppress Th17 cell responses, and all differentiate along distinct signaling pathways (Glimcher and Murphy, 2000; Stockinger and Veldhoen, 2007), IL-10 produced by all these Th cells may thus act as a feedback regulator to control the pathology associated with an overexuberant, albeit efficacious, inflammatory response.
T25 4771-5179 Sentence denotes Because Th1 and Th2 cells cross regulate each other's development and function and can suppress Th17 cell responses, and all differentiate along distinct signaling pathways (Glimcher and Murphy, 2000; Stockinger and Veldhoen, 2007), IL-10 produced by all these Th cells may thus act as a feedback regulator to control the pathology associated with an overexuberant, albeit efficacious, inflammatory response.
T1130 5180-5302 Sentence denotes Whether IL-10 production by these different Th cell subsets is induced by independent and/or common mechanisms is unknown.
T26 5180-5302 Sentence denotes Whether IL-10 production by these different Th cell subsets is induced by independent and/or common mechanisms is unknown.
T1131 5303-5529 Sentence denotes Here, we showed that in vitro differentiation of IL-10-producing Th1 cells from naive CD4+ T cells required IL-12-induced STAT4 signaling, strong TCR activation (high antigen dose), and sustained ERK1 and ERK2 phosphorylation.
T27 5303-5529 Sentence denotes Here, we showed that in vitro differentiation of IL-10-producing Th1 cells from naive CD4+ T cells required IL-12-induced STAT4 signaling, strong TCR activation (high antigen dose), and sustained ERK1 and ERK2 phosphorylation.
T1132 5530-5662 Sentence denotes Furthermore, we showed that activation of ERK1 and ERK2 is a requirement for production of IL-10 by Th1, Th2, and Th17 cell subsets.
T28 5530-5662 Sentence denotes Furthermore, we showed that activation of ERK1 and ERK2 is a requirement for production of IL-10 by Th1, Th2, and Th17 cell subsets.
T1133 5663-5889 Sentence denotes This common but highly regulated pathway for IL-10 induction and maintenance ensures its function as a feedback loop to control damage to the host and also allows a protective response to ensue as opposed to chronic infection.
T29 5663-5889 Sentence denotes This common but highly regulated pathway for IL-10 induction and maintenance ensures its function as a feedback loop to control damage to the host and also allows a protective response to ensue as opposed to chronic infection.
T30 5891-5898 Sentence denotes Results
T2963 5900-5983 Sentence denotes IL-12 and High Doses of Antigen Induce the Development of Th1 Cells Producing IL-10
T31 5900-5983 Sentence denotes IL-12 and High Doses of Antigen Induce the Development of Th1 Cells Producing IL-10
T2964 5984-6200 Sentence denotes To study the differentiation of Th1 cells coproducing IFN-γ and IL-10, we cultured purified TCR-transgenic DO11.10 naive CD4+ T cells with purified DCs as APCs and increasing doses of ovalbumin peptide 323-339 (OVA).
T32 5984-6200 Sentence denotes To study the differentiation of Th1 cells coproducing IFN-γ and IL-10, we cultured purified TCR-transgenic DO11.10 naive CD4+ T cells with purified DCs as APCs and increasing doses of ovalbumin peptide 323-339 (OVA).
T2965 6201-6396 Sentence denotes Culture with high doses of antigen for 7 days gave rise to Th1 cells expressing IFN-γ upon restimulation (Constant et al., 1995; Hosken et al., 1995), but not IL-10 (Figure S1A available online).
T33 6201-6396 Sentence denotes Culture with high doses of antigen for 7 days gave rise to Th1 cells expressing IFN-γ upon restimulation (Constant et al., 1995; Hosken et al., 1995), but not IL-10 (Figure S1A available online).
T2966 6397-6563 Sentence denotes Culture with low antigen doses under the same conditions led to the differentiation of Th2 cells, which expressed both IL-4 and IL-10 upon restimulation (Figure S1A).
T34 6397-6563 Sentence denotes Culture with low antigen doses under the same conditions led to the differentiation of Th2 cells, which expressed both IL-4 and IL-10 upon restimulation (Figure S1A).
T2967 6564-6835 Sentence denotes Culture of naive CD4+ T cells in an APC-free system by stimulation with anti-CD3 and anti-CD28 antibodies in the presence of IL-12 resulted in IFN-γ-producing Th1 cells, a proportion of which coproduced IL-10, as did Th2 cells resulting from culture in IL-4 (Figure S1B).
T35 6564-6835 Sentence denotes Culture of naive CD4+ T cells in an APC-free system by stimulation with anti-CD3 and anti-CD28 antibodies in the presence of IL-12 resulted in IFN-γ-producing Th1 cells, a proportion of which coproduced IL-10, as did Th2 cells resulting from culture in IL-4 (Figure S1B).
T2968 6836-7111 Sentence denotes To investigate whether the lack of IL-10 produced by Th1 cells resulted from inhibition of IL-10 production by DCs and high antigen dose or alternatively required IL-12, we cultured naive CD4+ T cells with increasing doses of antigen presented by DC in the presence of IL-12.
T36 6836-7111 Sentence denotes To investigate whether the lack of IL-10 produced by Th1 cells resulted from inhibition of IL-10 production by DCs and high antigen dose or alternatively required IL-12, we cultured naive CD4+ T cells with increasing doses of antigen presented by DC in the presence of IL-12.
T2969 7112-7364 Sentence denotes At low doses of antigen, IL-12 abrogated the development of Th2 cells and induced IFN-γ expression but only low levels of IL-10 expression, suggesting that IL-12 per se was not sufficient to induce significant IL-10 production in Th1 cells (Figure 1A).
T37 7112-7364 Sentence denotes At low doses of antigen, IL-12 abrogated the development of Th2 cells and induced IFN-γ expression but only low levels of IL-10 expression, suggesting that IL-12 per se was not sufficient to induce significant IL-10 production in Th1 cells (Figure 1A).
T2970 7365-7583 Sentence denotes Strikingly, as the antigen dose was increased, Th1 populations driven with IL-12 now contained higher numbers of IL-10-producing cells (Figure 1A) and produced more IL-10 protein upon restimulation (Figures 1A and 1B).
T38 7365-7583 Sentence denotes Strikingly, as the antigen dose was increased, Th1 populations driven with IL-12 now contained higher numbers of IL-10-producing cells (Figure 1A) and produced more IL-10 protein upon restimulation (Figures 1A and 1B).
T2971 7584-7681 Sentence denotes Thus, the development of Th1 cells producing IL-10 required both IL-12 and high doses of antigen.
T39 7584-7681 Sentence denotes Thus, the development of Th1 cells producing IL-10 required both IL-12 and high doses of antigen.
T2972 7682-7803 Sentence denotes Th1 cells differentiated to produce large amounts of IL-10 and IFN-γ and lost their capacity to produce IL-2 (Figure 1B).
T40 7682-7803 Sentence denotes Th1 cells differentiated to produce large amounts of IL-10 and IFN-γ and lost their capacity to produce IL-2 (Figure 1B).
T2973 7804-8057 Sentence denotes Because the presence of IL-12 reduced the proliferation of CD4+ T cells at both high and low antigen doses (Table S1), and only the former showed IL-10 production, the development of high IL-10-producing cells is most likely not related to limited IL-2.
T41 7804-8057 Sentence denotes Because the presence of IL-12 reduced the proliferation of CD4+ T cells at both high and low antigen doses (Table S1), and only the former showed IL-10 production, the development of high IL-10-producing cells is most likely not related to limited IL-2.
T2974 8058-8377 Sentence denotes Naive CD4+ T cells from DO11.10/recombination-activating gene 1 (Rag1)-deficient animals, cultured with high doses of antigen in the presence of IL-12, also resulted in IL-10 expression by Th1 cells, showing that this expression was not dependent on the presence of effector or memory T cells or Treg cells (Figure S2).
T42 8058-8377 Sentence denotes Naive CD4+ T cells from DO11.10/recombination-activating gene 1 (Rag1)-deficient animals, cultured with high doses of antigen in the presence of IL-12, also resulted in IL-10 expression by Th1 cells, showing that this expression was not dependent on the presence of effector or memory T cells or Treg cells (Figure S2).
T2975 8378-8583 Sentence denotes Although it has been suggested that TGF-β can induce IL-10 in CD4+ T cells (Kitani et al., 2003; Schiott et al., 2000), we found that in developing Th1 and Th2 cells this was not the case (data not shown).
T43 8378-8583 Sentence denotes Although it has been suggested that TGF-β can induce IL-10 in CD4+ T cells (Kitani et al., 2003; Schiott et al., 2000), we found that in developing Th1 and Th2 cells this was not the case (data not shown).
T2976 8584-8686 Sentence denotes In fact, neutralization of TGF-β led to increased IL-10 production by both T cell subsets (Figure S3).
T44 8584-8686 Sentence denotes In fact, neutralization of TGF-β led to increased IL-10 production by both T cell subsets (Figure S3).
T2977 8687-8905 Sentence denotes Thus, the development of IL-10-producing Th1 cells only depended on the presence of IL-12 together with high antigen dose and not on other soluble factors such as IL-2 or TGF-β or on the presence of other T cell types.
T45 8687-8905 Sentence denotes Thus, the development of IL-10-producing Th1 cells only depended on the presence of IL-12 together with high antigen dose and not on other soluble factors such as IL-2 or TGF-β or on the presence of other T cell types.
T5019 8907-9001 Sentence denotes IL-10 Production by Th1 Cells Is Dependent on STAT4 but Not on STAT6, IFN-γ, or IL-4 Signaling
T46 8907-9001 Sentence denotes IL-10 Production by Th1 Cells Is Dependent on STAT4 but Not on STAT6, IFN-γ, or IL-4 Signaling
T5020 9002-9203 Sentence denotes To further elucidate the mechanisms required for the development of Th1 cells producing IL-10, we investigated the role of STAT4, one of the signaling pathways activated by IL-12 (Murphy et al., 2000).
T47 9002-9203 Sentence denotes To further elucidate the mechanisms required for the development of Th1 cells producing IL-10, we investigated the role of STAT4, one of the signaling pathways activated by IL-12 (Murphy et al., 2000).
T5021 9204-9319 Sentence denotes Naive CD4+ D011.10 T cells deficient in STAT4 (Ouyang et al., 1998) were cultured in the presence of IL-12 and OVA.
T48 9204-9319 Sentence denotes Naive CD4+ D011.10 T cells deficient in STAT4 (Ouyang et al., 1998) were cultured in the presence of IL-12 and OVA.
T5022 9320-9454 Sentence denotes Again, IL-10-producing Th1 cells were differentiated at the high antigen dose in the presence of IL-12 in DO11.10 T cells (Figure 2A).
T49 9320-9454 Sentence denotes Again, IL-10-producing Th1 cells were differentiated at the high antigen dose in the presence of IL-12 in DO11.10 T cells (Figure 2A).
T5023 9455-9740 Sentence denotes In contrast, in the absence of STAT4, the percentage of cells expressing IFN-γ was dramatically diminished as expected and resulted in an increase in the percentage of cells expressing IL-4, but not IL-10 (Figure 2A), suggesting that STAT4 contributes to IL-10 expression by Th1 cells.
T50 9455-9740 Sentence denotes In contrast, in the absence of STAT4, the percentage of cells expressing IFN-γ was dramatically diminished as expected and resulted in an increase in the percentage of cells expressing IL-4, but not IL-10 (Figure 2A), suggesting that STAT4 contributes to IL-10 expression by Th1 cells.
T5024 9741-10016 Sentence denotes Because IL-10 expression is associated with an IL-4-induced Th2 cell phenotype, we investigated whether the differentiation of the IL-10-producing Th1 cells depended on signaling through the IL-4 receptor via STAT6 activation (Glimcher and Murphy, 2000; Murphy et al., 2000).
T51 9741-10016 Sentence denotes Because IL-10 expression is associated with an IL-4-induced Th2 cell phenotype, we investigated whether the differentiation of the IL-10-producing Th1 cells depended on signaling through the IL-4 receptor via STAT6 activation (Glimcher and Murphy, 2000; Murphy et al., 2000).
T5025 10017-10147 Sentence denotes The absence of STAT6 did not impair the differentiation of IL-10-producing Th1 cells in the presence of IL-12 and OVA (Figure 2A).
T52 10017-10147 Sentence denotes The absence of STAT6 did not impair the differentiation of IL-10-producing Th1 cells in the presence of IL-12 and OVA (Figure 2A).
T5026 10148-10349 Sentence denotes In fact, a higher percentage of STAT6-deficient cells compared with WT cells produced both IL-10 and IFN-γ (Figure 2A), which may be the result of the loss of Th2 cell control over a Th1 cell response.
T53 10148-10349 Sentence denotes In fact, a higher percentage of STAT6-deficient cells compared with WT cells produced both IL-10 and IFN-γ (Figure 2A), which may be the result of the loss of Th2 cell control over a Th1 cell response.
T5027 10350-10494 Sentence denotes As expected, lack of STAT6 abrogated both IL-4 and IL-10 production by T cells developed with IL-4 or with low antigen dose (Figures 2B and 2C).
T54 10350-10494 Sentence denotes As expected, lack of STAT6 abrogated both IL-4 and IL-10 production by T cells developed with IL-4 or with low antigen dose (Figures 2B and 2C).
T5028 10495-10624 Sentence denotes However, in the absence of STAT4 signaling, IL-10 and IL-4 production by Th2 cells was if anything increased (Figures 2B and 2C).
T55 10495-10624 Sentence denotes However, in the absence of STAT4 signaling, IL-10 and IL-4 production by Th2 cells was if anything increased (Figures 2B and 2C).
T5029 10625-10783 Sentence denotes Thus, in contrast to what was observed under Th1 conditions, IL-10 expression by Th2 cells depended on STAT6, but not on STAT4, signaling (Figures 2B and 2C).
T56 10625-10783 Sentence denotes Thus, in contrast to what was observed under Th1 conditions, IL-10 expression by Th2 cells depended on STAT6, but not on STAT4, signaling (Figures 2B and 2C).
T5030 10784-11069 Sentence denotes To investigate whether the inability of STAT4-deficient T cells to produce IL-10 might be due to the absence of IFN-γ, as suggested before (Shaw et al., 2006), we differentiated DO11.10 or DO11.10 IFN-γ-deficient naive CD4+ T cells in the presence of IL-12 and increasing doses of OVA.
T57 10784-11069 Sentence denotes To investigate whether the inability of STAT4-deficient T cells to produce IL-10 might be due to the absence of IFN-γ, as suggested before (Shaw et al., 2006), we differentiated DO11.10 or DO11.10 IFN-γ-deficient naive CD4+ T cells in the presence of IL-12 and increasing doses of OVA.
T5031 11070-11263 Sentence denotes The secretion of IL-10 as induced by high antigen dose, and IL-12 was not affected by an absence of IFN-γ (Figure 2D), showing that the expression of IL-10 by Th1 cells is independent of IFN-γ.
T58 11070-11263 Sentence denotes The secretion of IL-10 as induced by high antigen dose, and IL-12 was not affected by an absence of IFN-γ (Figure 2D), showing that the expression of IL-10 by Th1 cells is independent of IFN-γ.
T5032 11264-11363 Sentence denotes In the absence of IFN-γ, we observed an increase in the secreted IL-4 as expected (data not shown).
T59 11264-11363 Sentence denotes In the absence of IFN-γ, we observed an increase in the secreted IL-4 as expected (data not shown).
T5033 11364-11567 Sentence denotes We also tested for any potential role of IL-4 in the development of Th1 cells producing IL-10 by culturing DO11.10 or DO11.10 IL-4-deficient naive CD4+ T cells with IL-12 and increasing doses of antigen.
T60 11364-11567 Sentence denotes We also tested for any potential role of IL-4 in the development of Th1 cells producing IL-10 by culturing DO11.10 or DO11.10 IL-4-deficient naive CD4+ T cells with IL-12 and increasing doses of antigen.
T5034 11568-11781 Sentence denotes As observed in the absence of STAT6 (Figure 2A), IL-4 deficiency had no effect on the development of Th1 cells producing IL-10 (Figure 2E), but compromised the development of Th2 cells producing IL-10 (Figure 2F).
T61 11568-11781 Sentence denotes As observed in the absence of STAT6 (Figure 2A), IL-4 deficiency had no effect on the development of Th1 cells producing IL-10 (Figure 2E), but compromised the development of Th2 cells producing IL-10 (Figure 2F).
T5035 11782-12038 Sentence denotes Thus, our data suggested that IL-10 production by Th1 or Th2 cells was dependent on the specific signaling pathways required for their differentiation, given that STAT4 is required for the induction of IL-10 production by Th1 cells and STAT6 for Th2 cells.
T62 11782-12038 Sentence denotes Thus, our data suggested that IL-10 production by Th1 or Th2 cells was dependent on the specific signaling pathways required for their differentiation, given that STAT4 is required for the induction of IL-10 production by Th1 cells and STAT6 for Th2 cells.
T7035 12040-12137 Sentence denotes High Antigen Doses and STAT4 Are Required for the In Vivo Generation of IL-10-Producing Th1 Cells
T63 12040-12137 Sentence denotes High Antigen Doses and STAT4 Are Required for the In Vivo Generation of IL-10-Producing Th1 Cells
T7036 12138-12381 Sentence denotes To address the mechanisms regulating IL-10 production by Th1 cells in vivo, we transferred DO11.10 cells into BALB/c recipient mice and immunized the recipients with very high doses of OVA-protein with or without added lipopolysacharide (LPS).
T64 12138-12381 Sentence denotes To address the mechanisms regulating IL-10 production by Th1 cells in vivo, we transferred DO11.10 cells into BALB/c recipient mice and immunized the recipients with very high doses of OVA-protein with or without added lipopolysacharide (LPS).
T7037 12382-12509 Sentence denotes T cells were recovered from the inguinal lymph nodes 3 days after priming and restimulated in vitro with OVA peptide for 48 hr.
T65 12382-12509 Sentence denotes T cells were recovered from the inguinal lymph nodes 3 days after priming and restimulated in vitro with OVA peptide for 48 hr.
T7038 12510-12735 Sentence denotes This in vivo immunization induced IL-10 and IFN-γ production, and the amount of IL-10 production was enhanced by addition of LPS in the immunization (Figure 3A) and with higher doses of OVA (3 μM versus 1 μM, data not shown).
T66 12510-12735 Sentence denotes This in vivo immunization induced IL-10 and IFN-γ production, and the amount of IL-10 production was enhanced by addition of LPS in the immunization (Figure 3A) and with higher doses of OVA (3 μM versus 1 μM, data not shown).
T7039 12736-12977 Sentence denotes To test the role of STAT4 and STAT6 signaling in the in vivo development of IL-10-producing Th1 cells, we transferred STAT4- or STAT6-deficient or WT DO11.10 cells into recipient BALB/c mice and immunized with OVA-protein plus LPS as before.
T67 12736-12977 Sentence denotes To test the role of STAT4 and STAT6 signaling in the in vivo development of IL-10-producing Th1 cells, we transferred STAT4- or STAT6-deficient or WT DO11.10 cells into recipient BALB/c mice and immunized with OVA-protein plus LPS as before.
T7040 12978-13218 Sentence denotes In vivo expression of both IL-10 and IFN-γ was markedly reduced but not completely abrogated in the absence of STAT4 signaling (Figures 3B and 3C), suggesting the existence of compensatory mechanisms that were absent in the in vitro system.
T68 12978-13218 Sentence denotes In vivo expression of both IL-10 and IFN-γ was markedly reduced but not completely abrogated in the absence of STAT4 signaling (Figures 3B and 3C), suggesting the existence of compensatory mechanisms that were absent in the in vitro system.
T7041 13219-13406 Sentence denotes Signaling through STAT6 had no effect on IL-10 production by Th1 cells as shown by intracellular cytokine staining (ICS) and by immunoassay in STAT6-deficient T cells (Figures 3B and 3C).
T69 13219-13406 Sentence denotes Signaling through STAT6 had no effect on IL-10 production by Th1 cells as shown by intracellular cytokine staining (ICS) and by immunoassay in STAT6-deficient T cells (Figures 3B and 3C).
T7927 13408-13476 Sentence denotes IL-10 Production Is Maintained by High TCR Signal Strength and IL-12
T70 13408-13476 Sentence denotes IL-10 Production Is Maintained by High TCR Signal Strength and IL-12
T7928 13477-13635 Sentence denotes We next investigated whether repeated strong TCR activation is a compensatory signal for IL-12-induced STAT4 signaling in the induction of IL-10 in Th1 cells.
T71 13477-13635 Sentence denotes We next investigated whether repeated strong TCR activation is a compensatory signal for IL-12-induced STAT4 signaling in the induction of IL-10 in Th1 cells.
T7929 13636-13790 Sentence denotes For this, CD4+ T cells were differentiated for 2 consecutive weeks with high antigen doses in the presence or absence of IL-12 throughout (Figures 4A–4D).
T72 13636-13790 Sentence denotes For this, CD4+ T cells were differentiated for 2 consecutive weeks with high antigen doses in the presence or absence of IL-12 throughout (Figures 4A–4D).
T7930 13791-13975 Sentence denotes High antigen dose and IL-12 cooperated to induce maximal IL-10 production (Figures 4A and 4B), given that this combination resulted in the highest numbers of IL-10-producing Th1 cells.
T73 13791-13975 Sentence denotes High antigen dose and IL-12 cooperated to induce maximal IL-10 production (Figures 4A and 4B), given that this combination resulted in the highest numbers of IL-10-producing Th1 cells.
T7931 13976-14226 Sentence denotes Repeated high antigen dose stimulation in the absence of exogenously added IL-12 resulted in the production of IL-10 by Th1 cells, suggesting that repeated strong TCR triggering may overcome the need for IL-12 for IL-10 induction (Figures 4C and 4D).
T74 13976-14226 Sentence denotes Repeated high antigen dose stimulation in the absence of exogenously added IL-12 resulted in the production of IL-10 by Th1 cells, suggesting that repeated strong TCR triggering may overcome the need for IL-12 for IL-10 induction (Figures 4C and 4D).
T7932 14227-14347 Sentence denotes However, IL-10 induction under these conditions was abrogated when IL-12p40-deficient DCs were used as APCs (Figure 4E).
T75 14227-14347 Sentence denotes However, IL-10 induction under these conditions was abrogated when IL-12p40-deficient DCs were used as APCs (Figure 4E).
T7933 14348-14466 Sentence denotes Thus, IL-12 is essential during both primary and secondary antigenic stimulation for production of IL-10 by Th1 cells.
T76 14348-14466 Sentence denotes Thus, IL-12 is essential during both primary and secondary antigenic stimulation for production of IL-10 by Th1 cells.
T7934 14467-14790 Sentence denotes To determine the requirements for stability of the IL-10-producing Th1 cells, we differentiated CD4+ T cells for 1 week with high antigen doses with or without IL-12 (Figures 4A and 4C), washed them, and then restimulated them for an additional week with a low antigen dose, in the absence or presence of IL-12 (Figure 4F).
T77 14467-14790 Sentence denotes To determine the requirements for stability of the IL-10-producing Th1 cells, we differentiated CD4+ T cells for 1 week with high antigen doses with or without IL-12 (Figures 4A and 4C), washed them, and then restimulated them for an additional week with a low antigen dose, in the absence or presence of IL-12 (Figure 4F).
T7935 14791-15162 Sentence denotes Th1 cells induced in the first week to produce IL-10 by culture with high antigen doses and IL-12 lost their ability to express IL-10 when recultured with low doses of OVA, which could be compensated for, to some extent, by addition of IL-12 to the secondary cultures (Figure 4F), again suggesting that antigen dose and IL-12 signals cooperate for the induction of IL-10.
T78 14791-15162 Sentence denotes Th1 cells induced in the first week to produce IL-10 by culture with high antigen doses and IL-12 lost their ability to express IL-10 when recultured with low doses of OVA, which could be compensated for, to some extent, by addition of IL-12 to the secondary cultures (Figure 4F), again suggesting that antigen dose and IL-12 signals cooperate for the induction of IL-10.
T7936 15163-15463 Sentence denotes Finally, DO11.10 CD4+ cells that were exposed to low doses of antigen and IL-12 during the primary differentiation phase produced high amounts of IFN-γ but little IL-10, but they could be induced to produce IL-10 when both high antigen dose and IL-12 were present during the recall phase (Figure S4).
T79 15163-15463 Sentence denotes Finally, DO11.10 CD4+ cells that were exposed to low doses of antigen and IL-12 during the primary differentiation phase produced high amounts of IFN-γ but little IL-10, but they could be induced to produce IL-10 when both high antigen dose and IL-12 were present during the recall phase (Figure S4).
T7937 15464-15573 Sentence denotes Thus, high antigen dose and IL-12 are required for sustaining the induction of IL-10 production by Th1 cells.
T80 15464-15573 Sentence denotes Thus, high antigen dose and IL-12 are required for sustaining the induction of IL-10 production by Th1 cells.
T9380 15575-15638 Sentence denotes IL-10 Production by Th1 Cells Requires ERK1 and ERK2 Activation
T81 15575-15638 Sentence denotes IL-10 Production by Th1 Cells Requires ERK1 and ERK2 Activation
T9381 15639-15824 Sentence denotes Our data showed that the maintenance of IL-10 induction in Th1 cells required stimulation with high antigen dose, which to some extent could be compensated for by the addition of IL-12.
T82 15639-15824 Sentence denotes Our data showed that the maintenance of IL-10 induction in Th1 cells required stimulation with high antigen dose, which to some extent could be compensated for by the addition of IL-12.
T9382 15825-16150 Sentence denotes Signaling through the TCR with high doses of antigen induced stronger ERK1 and ERK2 activation than that induced by low antigen dose, not only in naive CD4+ T cells (data not shown) as previously demonstrated (Jorritsma et al., 2003) but also in CD4+ T cells restimulated with the same high and low antigen doses (Figure 5A).
T83 15825-16150 Sentence denotes Signaling through the TCR with high doses of antigen induced stronger ERK1 and ERK2 activation than that induced by low antigen dose, not only in naive CD4+ T cells (data not shown) as previously demonstrated (Jorritsma et al., 2003) but also in CD4+ T cells restimulated with the same high and low antigen doses (Figure 5A).
T9383 16151-16502 Sentence denotes Although the apparent peak and amount of ERK1 and ERK2 activation varied slightly between experiments, a consistent finding was that high antigen dose differentiated Th1 cells always showed enhanced and prolonged ERK1 and ERK2 activation in the presence of IL-12, regardless of whether they were restimulated with high or low antigen dose (Figure 5B).
T84 16151-16502 Sentence denotes Although the apparent peak and amount of ERK1 and ERK2 activation varied slightly between experiments, a consistent finding was that high antigen dose differentiated Th1 cells always showed enhanced and prolonged ERK1 and ERK2 activation in the presence of IL-12, regardless of whether they were restimulated with high or low antigen dose (Figure 5B).
T9384 16503-16687 Sentence denotes We then investigated whether ERK1 and ERK2 activation was required for the induction of IL-10 in Th1 cells by using U0126 (Figure 5C), a compound that blocks downstream ERK activation.
T85 16503-16687 Sentence denotes We then investigated whether ERK1 and ERK2 activation was required for the induction of IL-10 in Th1 cells by using U0126 (Figure 5C), a compound that blocks downstream ERK activation.
T9385 16688-16901 Sentence denotes To ensure that only T cell signaling was being affected by U0126, we used an APC-free system in which the T cells were differentiated in the presence of increasing doses of anti-CD3 and a constant amount of IL-12.
T86 16688-16901 Sentence denotes To ensure that only T cell signaling was being affected by U0126, we used an APC-free system in which the T cells were differentiated in the presence of increasing doses of anti-CD3 and a constant amount of IL-12.
T9386 16902-17080 Sentence denotes As in the APC-driven cultures, stronger TCR stimulation together with IL-12 led to higher percentages of cells producing both IL-10 and IFN-γ after 1 week of culture (Figure 5C).
T87 16902-17080 Sentence denotes As in the APC-driven cultures, stronger TCR stimulation together with IL-12 led to higher percentages of cells producing both IL-10 and IFN-γ after 1 week of culture (Figure 5C).
T9387 17081-17186 Sentence denotes Addition of U0126 to the cultures abrogated the production of IL-10 at all doses of anti-CD3 (Figure 5C).
T88 17081-17186 Sentence denotes Addition of U0126 to the cultures abrogated the production of IL-10 at all doses of anti-CD3 (Figure 5C).
T9388 17187-17550 Sentence denotes Because U0126 inhibits the MEK5-catalyzed activation of ERK5, as well as the MEK1- and MEK2-catalyzed activation of ERK1 and ERK2 (Bain et al., 2007; Mody et al., 2001), we also used the more specific, structurally unrelated MEK1 and MEK2 inhibitor PD184352 at concentrations in which it inhibits MEK1 and MEK2 but not MEK5 (Bain et al., 2007; Mody et al., 2001).
T89 17187-17550 Sentence denotes Because U0126 inhibits the MEK5-catalyzed activation of ERK5, as well as the MEK1- and MEK2-catalyzed activation of ERK1 and ERK2 (Bain et al., 2007; Mody et al., 2001), we also used the more specific, structurally unrelated MEK1 and MEK2 inhibitor PD184352 at concentrations in which it inhibits MEK1 and MEK2 but not MEK5 (Bain et al., 2007; Mody et al., 2001).
T9389 17551-17676 Sentence denotes PD184352 caused a similar inhibition of IL-10 production by Th1 cells in a dose-dependent fashion (Figure 5D and Figure S5A).
T90 17551-17676 Sentence denotes PD184352 caused a similar inhibition of IL-10 production by Th1 cells in a dose-dependent fashion (Figure 5D and Figure S5A).
T9390 17677-17885 Sentence denotes Upon addition of inhibitors to other signaling pathways, including a p38 MAPK inhibitor, SB203580, or the GSK3β inhibitor, CT99021 (Bain et al., 2007), no effect on IL-10 production was observed (Figure S5B).
T91 17677-17885 Sentence denotes Upon addition of inhibitors to other signaling pathways, including a p38 MAPK inhibitor, SB203580, or the GSK3β inhibitor, CT99021 (Bain et al., 2007), no effect on IL-10 production was observed (Figure S5B).
T9391 17886-18078 Sentence denotes Our data thus suggested that IL-10 production by Th1 cells in response to high antigen dose and IL-12 requires ERK1 and ERK2 signaling, but not the activation of the p38 or the GSK3β pathways.
T92 17886-18078 Sentence denotes Our data thus suggested that IL-10 production by Th1 cells in response to high antigen dose and IL-12 requires ERK1 and ERK2 signaling, but not the activation of the p38 or the GSK3β pathways.
T11022 18080-18157 Sentence denotes IL-10 Production by Th2 and Th17 Cells Also Requires ERK1 and ERK2 Activation
T93 18080-18157 Sentence denotes IL-10 Production by Th2 and Th17 Cells Also Requires ERK1 and ERK2 Activation
T11023 18158-18476 Sentence denotes To address whether IL-10 production by Th2 and Th17 cells was also dependent on ERK1 and ERK2 activation, we differentiated these cells with anti-CD3 and anti-CD28 in the absence of APCs (Shoemaker et al., 2006; Veldhoen et al., 2009; Veldhoen et al., 2006), in the presence or absence of the MEK inhibitor (PD184352).
T94 18158-18476 Sentence denotes To address whether IL-10 production by Th2 and Th17 cells was also dependent on ERK1 and ERK2 activation, we differentiated these cells with anti-CD3 and anti-CD28 in the absence of APCs (Shoemaker et al., 2006; Veldhoen et al., 2009; Veldhoen et al., 2006), in the presence or absence of the MEK inhibitor (PD184352).
T11024 18477-18748 Sentence denotes We showed that ERK1 and ERK2 activation is a common pathway required for induction of IL-10 in different Th cell subsets because IL-10 production by both Th2 and Th17 cells was markedly inhibited in the presence of the MEK inhibitor (PD184352) (Figure 5D and Figure S5B).
T95 18477-18748 Sentence denotes We showed that ERK1 and ERK2 activation is a common pathway required for induction of IL-10 in different Th cell subsets because IL-10 production by both Th2 and Th17 cells was markedly inhibited in the presence of the MEK inhibitor (PD184352) (Figure 5D and Figure S5B).
T11025 18749-18878 Sentence denotes In contrast, inhibitors of p38 MAPK or of GSK-3β activation did not affect the expression of IL-10 by these subsets (Figure S5B).
T96 18749-18878 Sentence denotes In contrast, inhibitors of p38 MAPK or of GSK-3β activation did not affect the expression of IL-10 by these subsets (Figure S5B).
T11026 18879-19029 Sentence denotes Activation of the ERK1 and ERK2 signaling pathway is therefore a common requirement for the induction of IL-10 production by Th1, Th2, and Th17 cells.
T97 18879-19029 Sentence denotes Activation of the ERK1 and ERK2 signaling pathway is therefore a common requirement for the induction of IL-10 production by Th1, Th2, and Th17 cells.
T11943 19031-19108 Sentence denotes c-maf Expression Correlates with IL-10 Production in Th1, Th2, and Th17 Cells
T98 19031-19108 Sentence denotes c-maf Expression Correlates with IL-10 Production in Th1, Th2, and Th17 Cells
T11944 19109-19391 Sentence denotes To investigate further the downstream factors involved in regulating IL-10 production, we differentiated DO11.10 CD4+ T cells with increasing doses of OVA, in the presence or absence of IL-12, and quantified the expression of cytokines and transcription factors by real-time RT-PCR.
T99 19109-19391 Sentence denotes To investigate further the downstream factors involved in regulating IL-10 production, we differentiated DO11.10 CD4+ T cells with increasing doses of OVA, in the presence or absence of IL-12, and quantified the expression of cytokines and transcription factors by real-time RT-PCR.
T11945 19392-19514 Sentence denotes Low-dose antigen resulted in transcription of Il4 and this was abrogated by both high antigen doses and IL-12 (Figure 6A).
T100 19392-19514 Sentence denotes Low-dose antigen resulted in transcription of Il4 and this was abrogated by both high antigen doses and IL-12 (Figure 6A).
T11946 19515-19723 Sentence denotes A low amount of transcription of Ifnγ was induced by IL-12 when cells were differentiated with low antigen dose, but this effect of IL-12 was markedly upregulated with increasing doses of antigen (Figure 6A).
T101 19515-19723 Sentence denotes A low amount of transcription of Ifnγ was induced by IL-12 when cells were differentiated with low antigen dose, but this effect of IL-12 was markedly upregulated with increasing doses of antigen (Figure 6A).
T11947 19724-19932 Sentence denotes A low amount of Il10 transcription was observed at low doses of antigen accompanying Il4 expression (Th2 cell response), and this was abrogated by increased doses of antigen as was Il4 expression (Figure 6A).
T102 19724-19932 Sentence denotes A low amount of Il10 transcription was observed at low doses of antigen accompanying Il4 expression (Th2 cell response), and this was abrogated by increased doses of antigen as was Il4 expression (Figure 6A).
T11948 19933-20073 Sentence denotes At low doses of antigen, IL-12 had little effect to increase IL-10 mRNA expression (Figure 6A) in keeping with the protein data (Figure 1A).
T103 19933-20073 Sentence denotes At low doses of antigen, IL-12 had little effect to increase IL-10 mRNA expression (Figure 6A) in keeping with the protein data (Figure 1A).
T11949 20074-20253 Sentence denotes However, IL-12 induced a high amount of Il10 transcription as well as Ifnγ expression with increased antigen doses (Figure 6A), again in keeping with the protein data (Figure 1A).
T104 20074-20253 Sentence denotes However, IL-12 induced a high amount of Il10 transcription as well as Ifnγ expression with increased antigen doses (Figure 6A), again in keeping with the protein data (Figure 1A).
T11950 20254-20419 Sentence denotes CD4+ T cells differentiated with increasing doses of antigen did not express high amounts of Tbx-21 (T-bet) mRNA, unless they were cocultured with IL-12 (Figure 6B).
T105 20254-20419 Sentence denotes CD4+ T cells differentiated with increasing doses of antigen did not express high amounts of Tbx-21 (T-bet) mRNA, unless they were cocultured with IL-12 (Figure 6B).
T11951 20420-20679 Sentence denotes In contrast, high amounts of GATA-3 mRNA expression were only observed under Th2 cell differentiation conditions (low-dose antigen) (Figure 6B), and this expression was markedly downregulated by both increasing antigen dose and coculture in IL-12 (Figure 6B).
T106 20420-20679 Sentence denotes In contrast, high amounts of GATA-3 mRNA expression were only observed under Th2 cell differentiation conditions (low-dose antigen) (Figure 6B), and this expression was markedly downregulated by both increasing antigen dose and coculture in IL-12 (Figure 6B).
T11952 20680-20895 Sentence denotes Differentiation of T cells under low antigen dose led to expression of c-maf, in keeping with the Th2 cell profile (Ho et al., 1996), which was almost completely abrogated by increasing doses of antigen (Figure 6B).
T107 20680-20895 Sentence denotes Differentiation of T cells under low antigen dose led to expression of c-maf, in keeping with the Th2 cell profile (Ho et al., 1996), which was almost completely abrogated by increasing doses of antigen (Figure 6B).
T11953 20896-21006 Sentence denotes Interestingly, IL-12 sustained the high expression of c-Maf mRNA even at the highest antigen dose (Figure 6B).
T108 20896-21006 Sentence denotes Interestingly, IL-12 sustained the high expression of c-Maf mRNA even at the highest antigen dose (Figure 6B).
T11954 21007-21098 Sentence denotes Moreover, IL-12 maintenance of c-maf expression required STAT4 activation (data not shown).
T109 21007-21098 Sentence denotes Moreover, IL-12 maintenance of c-maf expression required STAT4 activation (data not shown).
T11955 21099-21294 Sentence denotes In Th17 cells that expressed IL-17a as well as IL-10 mRNA (Figure 6C), T-bet and GATA-3 mRNA were undetectable (data not shown), whereas that of ROR-γt was high (Figure 6C) (Ivanov et al., 2007).
T110 21099-21294 Sentence denotes In Th17 cells that expressed IL-17a as well as IL-10 mRNA (Figure 6C), T-bet and GATA-3 mRNA were undetectable (data not shown), whereas that of ROR-γt was high (Figure 6C) (Ivanov et al., 2007).
T11956 21295-21607 Sentence denotes Th17 cells also expressed high amounts of c-maf (Figure 6C), confirming a recent report (Bauquet et al., 2009). c-Maf is therefore expressed in all IL-10-expressing T cell populations tested (Figures 6B and 6C) and may not be just a Th2 cell-specific transcription factor as originally thought (Ho et al., 1996).
T111 21295-21607 Sentence denotes Th17 cells also expressed high amounts of c-maf (Figure 6C), confirming a recent report (Bauquet et al., 2009). c-Maf is therefore expressed in all IL-10-expressing T cell populations tested (Figures 6B and 6C) and may not be just a Th2 cell-specific transcription factor as originally thought (Ho et al., 1996).
T11957 21608-21829 Sentence denotes We showed also that like Il10 expression, c-maf expression was inhibited in Th1 and Th17 cells in the presence of the MEK1 and MEK2 inhibitor (PD184352), whereas T-bet and RORγt expression was hardly affected (Figure 6D).
T112 21608-21829 Sentence denotes We showed also that like Il10 expression, c-maf expression was inhibited in Th1 and Th17 cells in the presence of the MEK1 and MEK2 inhibitor (PD184352), whereas T-bet and RORγt expression was hardly affected (Figure 6D).
T113 21831-21841 Sentence denotes Discussion
T14192 21842-22038 Sentence denotes IL-10 expression by cells of the innate and adaptive immune systems reflects the importance of this cytokine in the tight regulation of the immune response, to minimize pathology during infection.
T114 21842-22038 Sentence denotes IL-10 expression by cells of the innate and adaptive immune systems reflects the importance of this cytokine in the tight regulation of the immune response, to minimize pathology during infection.
T14193 22039-22154 Sentence denotes IL-10 expression by Th1 cells has been reported to regulate the immune response in leishmaniasis and toxoplasmosis.
T115 22039-22154 Sentence denotes IL-10 expression by Th1 cells has been reported to regulate the immune response in leishmaniasis and toxoplasmosis.
T14194 22155-22256 Sentence denotes However, in many situations, IL-10 is not produced by Th1 cells in response to antigenic stimulation.
T116 22155-22256 Sentence denotes However, in many situations, IL-10 is not produced by Th1 cells in response to antigenic stimulation.
T14195 22257-22380 Sentence denotes Our goal was to address the molecular signals that determine whether Th1 cells develop to produce IL-10 (Trinchieri, 2007).
T117 22257-22380 Sentence denotes Our goal was to address the molecular signals that determine whether Th1 cells develop to produce IL-10 (Trinchieri, 2007).
T14196 22381-22530 Sentence denotes Here, we showed that Th1 cells required high-antigen-dose-induced ERK1 and ERK2 phosphorylation and IL-12-induced STAT-4 activation to produce IL-10.
T118 22381-22530 Sentence denotes Here, we showed that Th1 cells required high-antigen-dose-induced ERK1 and ERK2 phosphorylation and IL-12-induced STAT-4 activation to produce IL-10.
T14197 22531-22899 Sentence denotes Our findings that ERK1 and ERK2 activation was a common pathway required for the production of IL-10 by Th1, Th2, and Th17 cell subsets, which differentiate along distinct pathways, such that IL-10 provides a highly regulated feedback loop to avoid the extremes of excessive inflammation or chronic infections and also allow a protective response to diverse pathogens.
T119 22531-22899 Sentence denotes Our findings that ERK1 and ERK2 activation was a common pathway required for the production of IL-10 by Th1, Th2, and Th17 cell subsets, which differentiate along distinct pathways, such that IL-10 provides a highly regulated feedback loop to avoid the extremes of excessive inflammation or chronic infections and also allow a protective response to diverse pathogens.
T14198 22900-23113 Sentence denotes In certain viral or parasitic infections (Anderson et al., 2007; Brooks et al., 2006; Ejrnaes et al., 2006), high amounts of stimulation may lead to the chronic nonhealing infection shown to be regulated by IL-10.
T120 22900-23113 Sentence denotes In certain viral or parasitic infections (Anderson et al., 2007; Brooks et al., 2006; Ejrnaes et al., 2006), high amounts of stimulation may lead to the chronic nonhealing infection shown to be regulated by IL-10.
T14199 23114-23301 Sentence denotes During the course of infection, after initial triggering with antigen, T cells migrate to the tissue encountering high doses of antigen and factors produced by the innate immune response.
T121 23114-23301 Sentence denotes During the course of infection, after initial triggering with antigen, T cells migrate to the tissue encountering high doses of antigen and factors produced by the innate immune response.
T14200 23302-23554 Sentence denotes Under these conditions, we speculate that Th1 cells will be induced to express high amounts of IL-10, in keeping with reports that IL-10-producing Th1 cells were found in CD4+ clones isolated from BAL but not blood of TB patients (Gerosa et al., 1999).
T122 23302-23554 Sentence denotes Under these conditions, we speculate that Th1 cells will be induced to express high amounts of IL-10, in keeping with reports that IL-10-producing Th1 cells were found in CD4+ clones isolated from BAL but not blood of TB patients (Gerosa et al., 1999).
T14201 23555-23920 Sentence denotes Similarly, the immune response to a clinical isolate of L. major, which produces heavily infected nonhealing lesions, was found to be regulated by IL-10 derived from Foxp3− Th1 cells that coproduce IL-10 and IFN-γ (Anderson et al., 2007), and the immune response during T. gondii infection was found also to be regulated by Foxp3− Th1 cells (Jankovic et al., 2007).
T123 23555-23920 Sentence denotes Similarly, the immune response to a clinical isolate of L. major, which produces heavily infected nonhealing lesions, was found to be regulated by IL-10 derived from Foxp3− Th1 cells that coproduce IL-10 and IFN-γ (Anderson et al., 2007), and the immune response during T. gondii infection was found also to be regulated by Foxp3− Th1 cells (Jankovic et al., 2007).
T14202 23921-24298 Sentence denotes It is likely that IL-10 production by Th1 cells is evoked under conditions of high inflammation and antigenic stimulation, whereas regulatory CD4+ T cells producing IL-10 may operate to regulate the immune response under conditions in which the pathogen is clinically controlled, such as in infection with L. major (Friedlin strain) (Belkaid et al., 2002; Suffia et al., 2006).
T124 23921-24298 Sentence denotes It is likely that IL-10 production by Th1 cells is evoked under conditions of high inflammation and antigenic stimulation, whereas regulatory CD4+ T cells producing IL-10 may operate to regulate the immune response under conditions in which the pathogen is clinically controlled, such as in infection with L. major (Friedlin strain) (Belkaid et al., 2002; Suffia et al., 2006).
T14203 24299-24603 Sentence denotes We now also reported that CD4+ T cells cultured with high antigen dose and IL-12 differentiate into canonical Th1 effector cells, which, in addition to expressing large amounts of IFN-γ and IL-10, lose their IL-2 expression as described before in certain chronic infection models (Sallusto et al., 2004).
T125 24299-24603 Sentence denotes We now also reported that CD4+ T cells cultured with high antigen dose and IL-12 differentiate into canonical Th1 effector cells, which, in addition to expressing large amounts of IFN-γ and IL-10, lose their IL-2 expression as described before in certain chronic infection models (Sallusto et al., 2004).
T14204 24604-24790 Sentence denotes Our demonstration that loss of IL-2 is accompanied by production of IL-10 offers potential additional mechanisms whereby effector T cell responses may be dampened during chronic disease.
T126 24604-24790 Sentence denotes Our demonstration that loss of IL-2 is accompanied by production of IL-10 offers potential additional mechanisms whereby effector T cell responses may be dampened during chronic disease.
T14205 24791-24992 Sentence denotes Using an in vivo transfer model of DO11.10 TCR transgenic cells (Castro et al., 2000), we showed that IL-10-producing Th1 cells were differentiated in the presence of high doses of OVA protein and LPS.
T127 24791-24992 Sentence denotes Using an in vivo transfer model of DO11.10 TCR transgenic cells (Castro et al., 2000), we showed that IL-10-producing Th1 cells were differentiated in the presence of high doses of OVA protein and LPS.
T14206 24993-25187 Sentence denotes We showed here that this induction of IL-10 in Th1 cells in vivo was markedly, but not totally, reduced in STAT4-deficient T cells as observed during T. gondii infection (Jankovic et al., 2002).
T128 24993-25187 Sentence denotes We showed here that this induction of IL-10 in Th1 cells in vivo was markedly, but not totally, reduced in STAT4-deficient T cells as observed during T. gondii infection (Jankovic et al., 2002).
T14207 25188-25414 Sentence denotes A high antigenic activation during T. gondii infection or high antigen doses delivered in the presence of LPS, as seen in our system, may compensate for an absolute requirement for IL-12 in the induction of IL-10 by Th1 cells.
T129 25188-25414 Sentence denotes A high antigenic activation during T. gondii infection or high antigen doses delivered in the presence of LPS, as seen in our system, may compensate for an absolute requirement for IL-12 in the induction of IL-10 by Th1 cells.
T14208 25415-25579 Sentence denotes In our in vitro system, repeated stimulation of Th1 cells with high antigen doses allowed the development of Th1 cells producing IL-10 in an IL-12-dependent manner.
T130 25415-25579 Sentence denotes In our in vitro system, repeated stimulation of Th1 cells with high antigen doses allowed the development of Th1 cells producing IL-10 in an IL-12-dependent manner.
T14209 25580-25733 Sentence denotes IL-10 production by Th1 cells induced by high antigen dose and IL-12 was independent of IFN-γ, in keeping with previous findings (Jankovic et al., 2002).
T131 25580-25733 Sentence denotes IL-10 production by Th1 cells induced by high antigen dose and IL-12 was independent of IFN-γ, in keeping with previous findings (Jankovic et al., 2002).
T14210 25734-25886 Sentence denotes However, a role for IFN-γ in mediating IL-10 reactivation by Th1 cells during secondary infection with T. gondii has been suggested (Shaw et al., 2006).
T132 25734-25886 Sentence denotes However, a role for IFN-γ in mediating IL-10 reactivation by Th1 cells during secondary infection with T. gondii has been suggested (Shaw et al., 2006).
T14211 25887-26123 Sentence denotes We have found that CD4+ T cells exposed to a high dose of antigen do not express IL-10 upon restimulation, but can be induced to produce IL-10 upon re-exposure to a high dose of antigen in the recall phase in the absence of added IL-12.
T133 25887-26123 Sentence denotes We have found that CD4+ T cells exposed to a high dose of antigen do not express IL-10 upon restimulation, but can be induced to produce IL-10 upon re-exposure to a high dose of antigen in the recall phase in the absence of added IL-12.
T14212 26124-26203 Sentence denotes However, this is dependent on the induction of IL-12 by antigen-presenting DCs.
T134 26124-26203 Sentence denotes However, this is dependent on the induction of IL-12 by antigen-presenting DCs.
T14213 26204-26371 Sentence denotes The combination of both high antigen dose and IL-12 resulted in the highest levels of IL-10 production and correlated with the high levels of ERK1 and ERK2 activation.
T135 26204-26371 Sentence denotes The combination of both high antigen dose and IL-12 resulted in the highest levels of IL-10 production and correlated with the high levels of ERK1 and ERK2 activation.
T14214 26372-26589 Sentence denotes The increased expression of IFN-γ observed during the secondary phase will induce increased IL-12 production by DCs and suggests that repeated high-level TCR activation feeds back to upregulate IL-12 production by DC.
T136 26372-26589 Sentence denotes The increased expression of IFN-γ observed during the secondary phase will induce increased IL-12 production by DCs and suggests that repeated high-level TCR activation feeds back to upregulate IL-12 production by DC.
T14215 26590-26800 Sentence denotes It is thus likely that in T. gondii infection in vivo (Shaw et al., 2006), the requirement for IFN-γ to induce IL-10, was for feedback upregulation of IL-12 by DCs, which in turn induced IL-10 in the Th1 cells.
T137 26590-26800 Sentence denotes It is thus likely that in T. gondii infection in vivo (Shaw et al., 2006), the requirement for IFN-γ to induce IL-10, was for feedback upregulation of IL-12 by DCs, which in turn induced IL-10 in the Th1 cells.
T14216 26801-27038 Sentence denotes Although IL-10 may be differentially regulated in Th1 and Th2 cells as has been reported (Chang et al., 2007; Wang et al., 2005), some studies suggest the existence of common pathways, but the molecular basis for these is as yet unclear.
T138 26801-27038 Sentence denotes Although IL-10 may be differentially regulated in Th1 and Th2 cells as has been reported (Chang et al., 2007; Wang et al., 2005), some studies suggest the existence of common pathways, but the molecular basis for these is as yet unclear.
T14217 27039-27293 Sentence denotes Costimulatory OX-40 signals have been shown to negatively regulate IL-10 production (Ito et al., 2005) both in Th1 and Th2 cells, whereas ICOS signaling has been suggested to induce IL-10 (Ito et al., 2007; Witsch et al., 2002) in both Th1 and Th2 cells.
T139 27039-27293 Sentence denotes Costimulatory OX-40 signals have been shown to negatively regulate IL-10 production (Ito et al., 2005) both in Th1 and Th2 cells, whereas ICOS signaling has been suggested to induce IL-10 (Ito et al., 2007; Witsch et al., 2002) in both Th1 and Th2 cells.
T14218 27294-27407 Sentence denotes However, in some cases, ICOS signaling also regulates IL-4 production and Th2 responses (Greenwald et al., 2005).
T140 27294-27407 Sentence denotes However, in some cases, ICOS signaling also regulates IL-4 production and Th2 responses (Greenwald et al., 2005).
T14219 27408-27635 Sentence denotes We now provide a common mechanism of ERK1 and ERK2 activation for the regulation of IL-10 production in Th1, Th2, and Th17 cells, although each subset differentiates along a distinct and subset-specific transcriptional pathway.
T141 27408-27635 Sentence denotes We now provide a common mechanism of ERK1 and ERK2 activation for the regulation of IL-10 production in Th1, Th2, and Th17 cells, although each subset differentiates along a distinct and subset-specific transcriptional pathway.
T14220 27636-27806 Sentence denotes This reinforces the fact that IL-10 is not a Th cell-subset-specific cytokine, but instead is produced in a tightly regulated fashion during each differentiation pathway.
T142 27636-27806 Sentence denotes This reinforces the fact that IL-10 is not a Th cell-subset-specific cytokine, but instead is produced in a tightly regulated fashion during each differentiation pathway.
T14221 27807-27983 Sentence denotes Of note, a role for ERK1 and ERK2 activation in the induction of IL-10 production has already been described for macrophages and DC (Agrawal et al., 2006; Hacker et al., 1999).
T143 27807-27983 Sentence denotes Of note, a role for ERK1 and ERK2 activation in the induction of IL-10 production has already been described for macrophages and DC (Agrawal et al., 2006; Hacker et al., 1999).
T14222 27984-28249 Sentence denotes Differential transcriptional regulation of IL-10 in Th1 and Th2 cells has been suggested (Chang et al., 2007; Wang et al., 2005), and extensive histone acetylation of the IL-10 gene is detectable in fully polarized Th2 cells, but not Th1 cells (Chang et al., 2007).
T144 27984-28249 Sentence denotes Differential transcriptional regulation of IL-10 in Th1 and Th2 cells has been suggested (Chang et al., 2007; Wang et al., 2005), and extensive histone acetylation of the IL-10 gene is detectable in fully polarized Th2 cells, but not Th1 cells (Chang et al., 2007).
T14223 28250-28546 Sentence denotes We provide evidence that IL-10 is produced in canonical Th1 cells and that its expression correlates with the expression of T-bet and the highest IFN-γ production, in keeping with our observations that high-dose antigen stimulation and IL-12 signaling are required for IL-10 and IFN-γ expression.
T145 28250-28546 Sentence denotes We provide evidence that IL-10 is produced in canonical Th1 cells and that its expression correlates with the expression of T-bet and the highest IFN-γ production, in keeping with our observations that high-dose antigen stimulation and IL-12 signaling are required for IL-10 and IFN-γ expression.
T14224 28547-28853 Sentence denotes It has also been shown that maintenance of IL-10 expression is conditional on IL-12 or IL-4 unless the IL-10 gene is imprinted by GATA-3 (Chang et al., 2007), which can remodel the IL-10 locus, thus explaining the highest amounts of IL-10 produced by Th2 cells (Chang et al., 2007; Shoemaker et al., 2006).
T146 28547-28853 Sentence denotes It has also been shown that maintenance of IL-10 expression is conditional on IL-12 or IL-4 unless the IL-10 gene is imprinted by GATA-3 (Chang et al., 2007), which can remodel the IL-10 locus, thus explaining the highest amounts of IL-10 produced by Th2 cells (Chang et al., 2007; Shoemaker et al., 2006).
T14225 28854-29052 Sentence denotes We show here that high antigen dose and IL-12 drastically downregulate Gata-3 expression, suggesting that additional factors are in place to induce IL-10 expression in Th1 cells, albeit transiently.
T147 28854-29052 Sentence denotes We show here that high antigen dose and IL-12 drastically downregulate Gata-3 expression, suggesting that additional factors are in place to induce IL-10 expression in Th1 cells, albeit transiently.
T14226 29053-29200 Sentence denotes Expression of c-maf was greatly diminished by high antigen doses in T cells and yet was unexpectedly maintained by IL-12 and present in Th17 cells.
T148 29053-29200 Sentence denotes Expression of c-maf was greatly diminished by high antigen doses in T cells and yet was unexpectedly maintained by IL-12 and present in Th17 cells.
T14227 29201-29505 Sentence denotes That c-maf expression is common to IL-10-producing Th1, Th2, and Th17 cells and, like IL-10, is dependent on ERK activation in Th1 and Th17 cells for its expression is of interest because c-Maf has been shown to be an essential transcription factor for IL-10 expression in macrophages (Cao et al., 2005).
T149 29201-29505 Sentence denotes That c-maf expression is common to IL-10-producing Th1, Th2, and Th17 cells and, like IL-10, is dependent on ERK activation in Th1 and Th17 cells for its expression is of interest because c-Maf has been shown to be an essential transcription factor for IL-10 expression in macrophages (Cao et al., 2005).
T14228 29506-29686 Sentence denotes In summary, we show that although Th1, Th2, and Th17 CD4+ T cell subsets differentiate along distinct signaling and transcriptional pathways, they can all be induced to make IL-10.
T150 29506-29686 Sentence denotes In summary, we show that although Th1, Th2, and Th17 CD4+ T cell subsets differentiate along distinct signaling and transcriptional pathways, they can all be induced to make IL-10.
T14229 29687-29774 Sentence denotes ERK1 and ERK2 activation is required for IL-10 production by all these Th cell subsets.
T151 29687-29774 Sentence denotes ERK1 and ERK2 activation is required for IL-10 production by all these Th cell subsets.
T14230 29775-29992 Sentence denotes With regard to the expression of IL-10 by Th1 cells, our data provide a mechanism for how IL-10 expression is induced and then amplified and regulated by the levels of antigen and IL-12 encountered in the environment.
T152 29775-29992 Sentence denotes With regard to the expression of IL-10 by Th1 cells, our data provide a mechanism for how IL-10 expression is induced and then amplified and regulated by the levels of antigen and IL-12 encountered in the environment.
T14231 29993-30290 Sentence denotes This provides a mechanism whereby a Th1 cell responds to extrinsic signals, reflecting increased inflammation in the tissue, to tightly regulate the production of IL-10 so as to allow a protective response to eradicate a pathogen with minimal damage to the host and also prevent chronic infection.
T153 29993-30290 Sentence denotes This provides a mechanism whereby a Th1 cell responds to extrinsic signals, reflecting increased inflammation in the tissue, to tightly regulate the production of IL-10 so as to allow a protective response to eradicate a pathogen with minimal damage to the host and also prevent chronic infection.
T14232 30291-30540 Sentence denotes Moreover, our findings have important implications for the regulation of IL-10 production during an inflammatory Th1 response in infection and may be of relevance for the design of vaccines and for strategies in immunotherapy in infectious diseases.
T154 30291-30540 Sentence denotes Moreover, our findings have important implications for the regulation of IL-10 production during an inflammatory Th1 response in infection and may be of relevance for the design of vaccines and for strategies in immunotherapy in infectious diseases.
T155 30542-30565 Sentence denotes Experimental Procedures
T18692 30567-30614 Sentence denotes Mice, Cytokines, Antibodies, and Other Reagents
T156 30567-30614 Sentence denotes Mice, Cytokines, Antibodies, and Other Reagents
T157 30615-31043 Sentence denotes BALB/c DO11.10 mice transgenic for OVA-specific TCR WT or crossed back with Rag1-, IL-4-, IFN-γ-, STAT4-, and STAT6-deficient mice were used as a source of antigen-specific T cells (Murphy et al., 1990; Ouyang et al., 1998; Shoemaker et al., 2006) and were bred and maintained under SPF conditions at the NIMR, London, Home Office, UK, Animals (Scientific Procedures) Act 1986 or at the Washington University School of Medicine.
T18693 30615-31195 Sentence denotes BALB/c DO11.10 mice transgenic for OVA-specific TCR WT or crossed back with Rag1-, IL-4-, IFN-γ-, STAT4-, and STAT6-deficient mice were used as a source of antigen-specific T cells (Murphy et al., 1990; Ouyang et al., 1998; Shoemaker et al., 2006) and were bred and maintained under SPF conditions at the NIMR, London, Home Office, UK, Animals (Scientific Procedures) Act 1986 or at the Washington University School of Medicine. Female mice were used at 8–12 weeks old, and animal protocols were approved according to the Animals (Scientific Procedures) Act 1986, Home Office, UK.
T158 31044-31195 Sentence denotes Female mice were used at 8–12 weeks old, and animal protocols were approved according to the Animals (Scientific Procedures) Act 1986, Home Office, UK.
T18694 31196-31432 Sentence denotes Reagents, including antibodies for T cell and DC preparation, purification and culture, media, cytokines, and cytokine mAbs have been described (Hosken et al., 1995; Shoemaker et al., 2006; Veldhoen et al., 2009; Veldhoen et al., 2006).
T159 31196-31432 Sentence denotes Reagents, including antibodies for T cell and DC preparation, purification and culture, media, cytokines, and cytokine mAbs have been described (Hosken et al., 1995; Shoemaker et al., 2006; Veldhoen et al., 2009; Veldhoen et al., 2006).
T160 31433-31612 Sentence denotes LPS (S. minnesota) was from Alexis, chicken ovalbumin protein (OVA protein) was from from Sigma-Aldrich, and ovalbumin peptide323-339 (OVA) (endotoxin-free) was from Biosynthesis.
T18695 31433-31803 Sentence denotes LPS (S. minnesota) was from Alexis, chicken ovalbumin protein (OVA protein) was from from Sigma-Aldrich, and ovalbumin peptide323-339 (OVA) (endotoxin-free) was from Biosynthesis. U0126 was from BioMol International. PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), and CT99021 (GSK3β inhibitor) were kind gifts from P. Cohen and N. Shpiro, University of Dundee, UK.
T161 31613-31649 Sentence denotes U0126 was from BioMol International.
T162 31650-31756 Sentence denotes PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), and CT99021 (GSK3β inhibitor) were kind gifts from P.
T163 31757-31803 Sentence denotes Cohen and N. Shpiro, University of Dundee, UK.
T19130 31805-31902 Sentence denotes Isolation of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation
T164 31805-31902 Sentence denotes Isolation of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation
T19131 31903-32077 Sentence denotes T cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25−, or CD4+CD44loCD25− to >98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009).
T165 31903-32077 Sentence denotes T cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25−, or CD4+CD44loCD25− to >98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009).
T19132 32078-32201 Sentence denotes In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained.
T166 32078-32201 Sentence denotes In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained.
T19133 32202-32328 Sentence denotes Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture.
T167 32202-32328 Sentence denotes Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture.
T19134 32329-32561 Sentence denotes Purified DO11.10 CD4+ T cells (1 × 105 cells/ml) were cultured as before (Hosken et al., 1995), in a total volume of 1 ml cRPMI medium in a 48-well plate, with splenic DCs (2 × 104 cells/ml), and varying amounts of OVA and of IL-12.
T168 32329-32561 Sentence denotes Purified DO11.10 CD4+ T cells (1 × 105 cells/ml) were cultured as before (Hosken et al., 1995), in a total volume of 1 ml cRPMI medium in a 48-well plate, with splenic DCs (2 × 104 cells/ml), and varying amounts of OVA and of IL-12.
T19135 32562-32831 Sentence denotes APC-independent differentiation of naive CD4+ T cells into Th1 and Th2 cells used stimulation with anti-CD3 and anti-CD28 and appropriate cytokine conditions, and control Th1 and Th2 cells were cultured as described before (Hosken et al., 1995; Shoemaker et al., 2006).
T169 32562-32831 Sentence denotes APC-independent differentiation of naive CD4+ T cells into Th1 and Th2 cells used stimulation with anti-CD3 and anti-CD28 and appropriate cytokine conditions, and control Th1 and Th2 cells were cultured as described before (Hosken et al., 1995; Shoemaker et al., 2006).
T19136 32832-32915 Sentence denotes Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006).
T170 32832-32915 Sentence denotes Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006).
T19137 32916-33140 Sentence denotes Importantly, Th1 and Th2 cells could be differentiated in cRPMI or IMDM (Hosken et al., 1995; Shoemaker et al., 2006; Veldhoen et al., 2006), but Th17 cells were only differentiated optimally in IMDM (Veldhoen et al., 2009).
T171 32916-33140 Sentence denotes Importantly, Th1 and Th2 cells could be differentiated in cRPMI or IMDM (Hosken et al., 1995; Shoemaker et al., 2006; Veldhoen et al., 2006), but Th17 cells were only differentiated optimally in IMDM (Veldhoen et al., 2009).
T19138 33141-33302 Sentence denotes When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3β inhibitor), or a similar amount of DMSO were present in the culture.
T172 33141-33302 Sentence denotes When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3β inhibitor), or a similar amount of DMSO were present in the culture.
T19139 33303-33381 Sentence denotes More details of specific culture conditions are provided in Figures S6 and S7.
T173 33303-33381 Sentence denotes More details of specific culture conditions are provided in Figures S6 and S7.
T19939 33383-33418 Sentence denotes Cytokine Detection by ICS and ELISA
T174 33383-33418 Sentence denotes Cytokine Detection by ICS and ELISA
T19940 33419-33629 Sentence denotes At day 5 or 7, cells were restimulated with immobilized anti-CD3 (2 μg/ml) and anti-CD28 (2 μg/ml) (4 hr with BrefeldinA [10 μg/ml] in the last 2 hr) or with PdBU and Ionomycin (5 hr with BrefeldinA [1 μg/ml]).
T175 33419-33629 Sentence denotes At day 5 or 7, cells were restimulated with immobilized anti-CD3 (2 μg/ml) and anti-CD28 (2 μg/ml) (4 hr with BrefeldinA [10 μg/ml] in the last 2 hr) or with PdBU and Ionomycin (5 hr with BrefeldinA [1 μg/ml]).
T19941 33630-33739 Sentence denotes After ICS FACS, data were collected on a FACSCalibur (Becton Dickinson) and analyzed with FlowJo (Tree Star).
T176 33630-33739 Sentence denotes After ICS FACS, data were collected on a FACSCalibur (Becton Dickinson) and analyzed with FlowJo (Tree Star).
T19942 33740-33910 Sentence denotes For ELISA, cells were similarly restimulated for 48 hr and supernatant was collected and analyzed for IL-4, IL-10, and IFN-γ as described before (Shoemaker et al., 2006).
T177 33740-33910 Sentence denotes For ELISA, cells were similarly restimulated for 48 hr and supernatant was collected and analyzed for IL-4, IL-10, and IFN-γ as described before (Shoemaker et al., 2006).
T20243 33912-33927 Sentence denotes In Vivo Studies
T178 33912-33927 Sentence denotes In Vivo Studies
T20244 33928-34115 Sentence denotes BALB/c mice were injected intravenously (i.v.) with a red blood cell-depleted single-cell spleen suspension (2.5 × 107 cells) (Castro et al., 2000) from DO11.10 WT or STAT-deficient mice.
T179 33928-34115 Sentence denotes BALB/c mice were injected intravenously (i.v.) with a red blood cell-depleted single-cell spleen suspension (2.5 × 107 cells) (Castro et al., 2000) from DO11.10 WT or STAT-deficient mice.
T20245 34116-34215 Sentence denotes After 48 hr, they were injected subcutaneously with PBS or with OVA protein (5 mg) plus LPS (5 μg).
T180 34116-34215 Sentence denotes After 48 hr, they were injected subcutaneously with PBS or with OVA protein (5 mg) plus LPS (5 μg).
T20246 34216-34266 Sentence denotes The inguinal lymph nodes were removed 48 hr later.
T181 34216-34266 Sentence denotes The inguinal lymph nodes were removed 48 hr later.
T20247 34267-34404 Sentence denotes A single-cell suspension (1 × 106 cells) was restimulated for 24 or 48 hr with 1 μM or 3 μM of OVA and with BrefeldinA for the last 6 hr.
T182 34267-34404 Sentence denotes A single-cell suspension (1 × 106 cells) was restimulated for 24 or 48 hr with 1 μM or 3 μM of OVA and with BrefeldinA for the last 6 hr.
T20248 34405-34501 Sentence denotes Half of the supernatant was removed before the addition of BrefeldinA for use in an ELISA assay.
T183 34405-34501 Sentence denotes Half of the supernatant was removed before the addition of BrefeldinA for use in an ELISA assay.
T20249 34502-34545 Sentence denotes The cells were fixed and stained as before.
T184 34502-34545 Sentence denotes The cells were fixed and stained as before.
T20250 34546-34691 Sentence denotes Those positive for KJ1-26-Bio and for CD4-PerCP were gated, and IFN-γ and IL-10 staining was examined for this population and analyzed as before.
T185 34546-34691 Sentence denotes Those positive for KJ1-26-Bio and for CD4-PerCP were gated, and IFN-γ and IL-10 staining was examined for this population and analyzed as before.
T20630 34693-34722 Sentence denotes Real-Time Quantitative RT-PCR
T186 34693-34722 Sentence denotes Real-Time Quantitative RT-PCR
T20631 34723-34866 Sentence denotes Cells were harvested and restimulated in the presence of immobilized anti-CD3 (2 μg/ml) plus anti-CD28 (2 μg/ml) for 3 hr or immediately lysed.
T187 34723-34866 Sentence denotes Cells were harvested and restimulated in the presence of immobilized anti-CD3 (2 μg/ml) plus anti-CD28 (2 μg/ml) for 3 hr or immediately lysed.
T20632 34867-35047 Sentence denotes RNA was extracted and reverse-transcribed and cDNA was analyzed for the expression of cytokines and transcription factors by real-time PCR assay as before (Shoemaker et al., 2006).
T188 34867-35047 Sentence denotes RNA was extracted and reverse-transcribed and cDNA was analyzed for the expression of cytokines and transcription factors by real-time PCR assay as before (Shoemaker et al., 2006).
T20633 35048-35237 Sentence denotes Target gene mRNA expression was quantified either with SYBR Green (Applied Biosystems) or with Master Mix (Applied Biosystems) and normalized to ubiquitin or HPRT mRNA levels, respectively.
T189 35048-35237 Sentence denotes Target gene mRNA expression was quantified either with SYBR Green (Applied Biosystems) or with Master Mix (Applied Biosystems) and normalized to ubiquitin or HPRT mRNA levels, respectively.
T20868 35239-35253 Sentence denotes Immunoblotting
T190 35239-35253 Sentence denotes Immunoblotting
T20869 35254-35386 Sentence denotes Differentiated CD4+ T cells were rested for 5 hr in 1% FCS-containing medium and restimulated as described for specific experiments.
T191 35254-35386 Sentence denotes Differentiated CD4+ T cells were rested for 5 hr in 1% FCS-containing medium and restimulated as described for specific experiments.
T20870 35387-35566 Sentence denotes Cell lysates were prepared, equal amounts of protein were separated by SDS-PAGE, and phosphorylated or total ERK and actin were detected as described before (Beinke et al., 2004).
T192 35387-35566 Sentence denotes Cell lysates were prepared, equal amounts of protein were separated by SDS-PAGE, and phosphorylated or total ERK and actin were detected as described before (Beinke et al., 2004).