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of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation\nT cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25−, or CD4+CD44loCD25− to \u003e98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009). In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained. Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture. 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. 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). Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006). 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). When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3β inhibitor), or a similar amount of DMSO were present in the culture. More details of specific culture conditions are provided in Figures S6 and S7."}

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of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation\nT cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25−, or CD4+CD44loCD25− to \u003e98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009). In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained. Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture. 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. 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). Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006). 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). When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3β inhibitor), or a similar amount of DMSO were present in the culture. More details of specific culture conditions are provided in Figures S6 and S7."}

{"project":"GO-BP","denotations":[{"id":"T19807","span":{"begin":1398,"end":1401},"obj":"http://purl.obolibrary.org/obo/GO_0004707"}],"text":"Isolation of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation\nT cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25−, or CD4+CD44loCD25− to \u003e98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009). In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained. Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture. 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. 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). Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006). 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). When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3β inhibitor), or a similar amount of DMSO were present in the culture. More details of specific culture conditions are provided in Figures S6 and S7."}

{"project":"GO-MF","denotations":[{"id":"T19808","span":{"begin":750,"end":755},"obj":"http://purl.obolibrary.org/obo/GO_0005143"},{"id":"T19809","span":{"begin":1398,"end":1401},"obj":"http://purl.obolibrary.org/obo/GO_0004707"}],"text":"Isolation of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation\nT cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25−, or CD4+CD44loCD25− to \u003e98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009). In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained. Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture. 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. 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). Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006). 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). When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3β inhibitor), or a similar amount of DMSO were present in the culture. More details of specific culture conditions are provided in Figures S6 and S7."}

{"project":"GO-CC","denotations":[{"id":"T19810","span":{"begin":48,"end":52},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19811","span":{"begin":67,"end":71},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19812","span":{"begin":100,"end":105},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19813","span":{"begin":484,"end":489},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19814","span":{"begin":548,"end":553},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19815","span":{"begin":563,"end":568},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19816","span":{"begin":705,"end":710},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19817","span":{"begin":805,"end":810},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19818","span":{"begin":828,"end":833},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19819","span":{"begin":940,"end":945},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19820","span":{"begin":1055,"end":1060},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19821","span":{"begin":1136,"end":1141},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19822","span":{"begin":1262,"end":1267},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T19823","span":{"begin":757,"end":760},"obj":"http://purl.obolibrary.org/obo/GO_0005680"}],"text":"Isolation of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation\nT cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25−, or CD4+CD44loCD25− to \u003e98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009). In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained. Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture. 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. 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). Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006). 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). When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3β inhibitor), or a similar amount of DMSO were present in the culture. More details of specific culture conditions are provided in Figures S6 and S7."}

{"project":"sentences","denotations":[{"id":"T19130","span":{"begin":0,"end":97},"obj":"Sentence"},{"id":"T19131","span":{"begin":98,"end":272},"obj":"Sentence"},{"id":"T19132","span":{"begin":273,"end":396},"obj":"Sentence"},{"id":"T19133","span":{"begin":397,"end":523},"obj":"Sentence"},{"id":"T19134","span":{"begin":524,"end":756},"obj":"Sentence"},{"id":"T19135","span":{"begin":757,"end":1026},"obj":"Sentence"},{"id":"T19136","span":{"begin":1027,"end":1110},"obj":"Sentence"},{"id":"T19137","span":{"begin":1111,"end":1335},"obj":"Sentence"},{"id":"T19138","span":{"begin":1336,"end":1497},"obj":"Sentence"},{"id":"T19139","span":{"begin":1498,"end":1576},"obj":"Sentence"},{"id":"T164","span":{"begin":0,"end":97},"obj":"Sentence"},{"id":"T165","span":{"begin":98,"end":272},"obj":"Sentence"},{"id":"T166","span":{"begin":273,"end":396},"obj":"Sentence"},{"id":"T167","span":{"begin":397,"end":523},"obj":"Sentence"},{"id":"T168","span":{"begin":524,"end":756},"obj":"Sentence"},{"id":"T169","span":{"begin":757,"end":1026},"obj":"Sentence"},{"id":"T170","span":{"begin":1027,"end":1110},"obj":"Sentence"},{"id":"T171","span":{"begin":1111,"end":1335},"obj":"Sentence"},{"id":"T172","span":{"begin":1336,"end":1497},"obj":"Sentence"},{"id":"T173","span":{"begin":1498,"end":1576},"obj":"Sentence"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/ontology/tao.owl#"}],"text":"Isolation of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation\nT cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25−, or CD4+CD44loCD25− to \u003e98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009). In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained. Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture. 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. 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). Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006). 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). When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3β inhibitor), or a similar amount of DMSO were present in the culture. More details of specific culture conditions are provided in Figures S6 and S7."}

{"project":"events-check-again","denotations":[{"id":"T19870","span":{"begin":13,"end":16},"obj":"Protein"},{"id":"T19871","span":{"begin":122,"end":125},"obj":"Protein"},{"id":"T19872","span":{"begin":126,"end":131},"obj":"Protein"},{"id":"T19873","span":{"begin":135,"end":138},"obj":"Protein"},{"id":"T19874","span":{"begin":139,"end":144},"obj":"Protein"},{"id":"T19875","span":{"begin":146,"end":150},"obj":"Protein"},{"id":"T19876","span":{"begin":156,"end":159},"obj":"Protein"},{"id":"T19877","span":{"begin":160,"end":164},"obj":"Protein"},{"id":"T19878","span":{"begin":166,"end":170},"obj":"Protein"},{"id":"T19879","span":{"begin":343,"end":346},"obj":"Protein"},{"id":"T19880","span":{"begin":477,"end":482},"obj":"Protein"},{"id":"T19881","span":{"begin":541,"end":544},"obj":"Protein"},{"id":"T19882","span":{"begin":739,"end":742},"obj":"Protein"},{"id":"T19883","span":{"begin":750,"end":755},"obj":"Protein"},{"id":"T19884","span":{"begin":798,"end":801},"obj":"Protein"},{"id":"T19885","span":{"begin":861,"end":864},"obj":"Protein"},{"id":"T19886","span":{"begin":874,"end":878},"obj":"Protein"},{"id":"T19887","span":{"begin":1371,"end":1374},"obj":"Protein"},{"id":"T19888","span":{"begin":1375,"end":1385},"obj":"Negative_regulation"},{"id":"T19889","span":{"begin":1398,"end":1401},"obj":"Protein"},{"id":"T19890","span":{"begin":1402,"end":1411},"obj":"Negative_regulation"},{"id":"T19891","span":{"begin":1423,"end":1428},"obj":"Protein"},{"id":"T19892","span":{"begin":1429,"end":1438},"obj":"Negative_regulation"}],"relations":[{"id":"R15758","pred":"themeOf","subj":"T19887","obj":"T19888"},{"id":"R15759","pred":"themeOf","subj":"T19889","obj":"T19890"},{"id":"R15760","pred":"themeOf","subj":"T19891","obj":"T19892"}],"text":"Isolation of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation\nT cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25−, or CD4+CD44loCD25− to \u003e98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009). In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained. Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture. 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. 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). Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006). 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). When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3β inhibitor), or a similar amount of DMSO were present in the culture. More details of specific culture conditions are provided in Figures S6 and S7."}

{"project":"bionlp-st-ge-2016-reference-tees","denotations":[{"id":"T19893","span":{"begin":13,"end":17},"obj":"Protein"},{"id":"T19894","span":{"begin":33,"end":43},"obj":"Protein"},{"id":"T19895","span":{"begin":0,"end":9},"obj":"Localization"},{"id":"T19896","span":{"begin":122,"end":125},"obj":"Protein"},{"id":"T19897","span":{"begin":126,"end":133},"obj":"Protein"},{"id":"T19898","span":{"begin":135,"end":138},"obj":"Protein"},{"id":"T19899","span":{"begin":139,"end":150},"obj":"Protein"},{"id":"T19900","span":{"begin":156,"end":159},"obj":"Protein"},{"id":"T19901","span":{"begin":160,"end":170},"obj":"Protein"},{"id":"T19902","span":{"begin":343,"end":347},"obj":"Protein"},{"id":"T19903","span":{"begin":477,"end":483},"obj":"Protein"},{"id":"T19904","span":{"begin":750,"end":755},"obj":"Protein"},{"id":"T19905","span":{"begin":798,"end":802},"obj":"Protein"},{"id":"T19906","span":{"begin":856,"end":864},"obj":"Protein"},{"id":"T19907","span":{"begin":869,"end":878},"obj":"Protein"},{"id":"T19908","span":{"begin":1124,"end":1127},"obj":"Protein"},{"id":"T19909","span":{"begin":1361,"end":1369},"obj":"Protein"},{"id":"T19910","span":{"begin":1371,"end":1374},"obj":"Protein"},{"id":"T19911","span":{"begin":1388,"end":1396},"obj":"Protein"},{"id":"T19912","span":{"begin":1398,"end":1401},"obj":"Protein"},{"id":"T19913","span":{"begin":1414,"end":1421},"obj":"Protein"},{"id":"T19914","span":{"begin":1423,"end":1428},"obj":"Protein"},{"id":"T19915","span":{"begin":1375,"end":1385},"obj":"Negative_regulation"},{"id":"T19916","span":{"begin":1402,"end":1411},"obj":"Negative_regulation"},{"id":"T19917","span":{"begin":1402,"end":1411},"obj":"Negative_regulation"},{"id":"T19918","span":{"begin":1429,"end":1438},"obj":"Negative_regulation"},{"id":"T19919","span":{"begin":1429,"end":1438},"obj":"Negative_regulation"},{"id":"T19920","span":{"begin":1474,"end":1481},"obj":"Gene_expression"},{"id":"T19921","span":{"begin":1474,"end":1481},"obj":"Gene_expression"},{"id":"T19922","span":{"begin":1474,"end":1481},"obj":"Gene_expression"},{"id":"T19923","span":{"begin":1573,"end":1575},"obj":"Protein"}],"relations":[{"id":"R15761","pred":"themeOf","subj":"T19893","obj":"T19895"},{"id":"R15762","pred":"themeOf","subj":"T19909","obj":"T19920"},{"id":"R15763","pred":"themeOf","subj":"T19910","obj":"T19915"},{"id":"R15764","pred":"themeOf","subj":"T19911","obj":"T19916"},{"id":"R15765","pred":"themeOf","subj":"T19911","obj":"T19921"},{"id":"R15766","pred":"themeOf","subj":"T19912","obj":"T19917"},{"id":"R15767","pred":"themeOf","subj":"T19913","obj":"T19918"},{"id":"R15768","pred":"themeOf","subj":"T19913","obj":"T19922"},{"id":"R15769","pred":"themeOf","subj":"T19914","obj":"T19919"}],"text":"Isolation of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation\nT cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25−, or CD4+CD44loCD25− to \u003e98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009). In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained. Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture. 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. 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). Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006). 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). When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3β inhibitor), or a similar amount of DMSO were present in the culture. More details of specific culture conditions are provided in Figures S6 and S7."}

{"project":"test2","denotations":[{"id":"T19086","span":{"begin":13,"end":16},"obj":"Protein"},{"id":"T19087","span":{"begin":122,"end":125},"obj":"Protein"},{"id":"T19088","span":{"begin":126,"end":133},"obj":"Protein"},{"id":"T19089","span":{"begin":135,"end":138},"obj":"Protein"},{"id":"T19090","span":{"begin":139,"end":150},"obj":"Protein"},{"id":"T19091","span":{"begin":156,"end":159},"obj":"Protein"},{"id":"T19092","span":{"begin":160,"end":170},"obj":"Protein"},{"id":"T19093","span":{"begin":343,"end":346},"obj":"Protein"},{"id":"T19094","span":{"begin":477,"end":482},"obj":"Protein"},{"id":"T19095","span":{"begin":541,"end":544},"obj":"Protein"},{"id":"T19096","span":{"begin":739,"end":742},"obj":"Protein"},{"id":"T19097","span":{"begin":750,"end":755},"obj":"Protein"},{"id":"T19098","span":{"begin":798,"end":801},"obj":"Protein"},{"id":"T19099","span":{"begin":861,"end":864},"obj":"Protein"},{"id":"T19100","span":{"begin":874,"end":878},"obj":"Protein"},{"id":"T19101","span":{"begin":1371,"end":1374},"obj":"Protein"},{"id":"T19102","span":{"begin":1375,"end":1385},"obj":"Negative_regulation"},{"id":"T19103","span":{"begin":1398,"end":1401},"obj":"Protein"},{"id":"T19104","span":{"begin":1402,"end":1411},"obj":"Negative_regulation"},{"id":"T19105","span":{"begin":1423,"end":1428},"obj":"Protein"},{"id":"T19106","span":{"begin":1429,"end":1438},"obj":"Negative_regulation"}],"relations":[{"id":"R15102","pred":"themeOf","subj":"T19101","obj":"T19102"},{"id":"R15103","pred":"themeOf","subj":"T19103","obj":"T19104"},{"id":"R15104","pred":"themeOf","subj":"T19105","obj":"T19106"}],"text":"Isolation of CD4+ T Cells and of Splenic DC and Cell Culture for T Cell Phenotype Differentiation\nT cells were sorted for CD4+CD62Lhi, CD4+CD62LhiCD25−, or CD4+CD44loCD25− to \u003e98% on a Moflo cytometer (Cytomation) as before (Shoemaker et al., 2006; Veldhoen et al., 2009). In most cases, experiments were reproduced with each type of purified CD4+ T cell population with similar results obtained. Splenic DCs were prepared as described (Hosken et al., 1995), and sort purified CD11c+ cells were added to the T cell culture. 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. 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). Culture conditions for Th17 cells were as described before (Veldhoen et al., 2006). 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). When indicated, U0126 or PD184352 (MEK inhibitors), SB203580 (p38 inhibitor), CT99021 (GSK3β inhibitor), or a similar amount of DMSO were present in the culture. More details of specific culture conditions are provided in Figures S6 and S7."}