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A negative feedback loop mediated by the Bcl6-cullin 3 complex limits Tfh cell differentiation.

Mathew R, Mao AP, Chiang AH, Bertozzi-Villa C, Bunker JJ, Scanlon ST, McDonald BD, Constantinides MG, Hollister K, Singer JD, Dent AL, Dinner AR, Bendelac A - J. Exp. Med. (2014)

Bottom Line: Intriguingly, we found that Bcl6 was also highly and transiently expressed during the CD4(+)CD8(+) (double positive [DP]) stage of T cell development, in association with the E3 ligase cullin 3 (Cul3), a novel binding partner of Bcl6 which ubiquitinates histone proteins.Although they maintained an apparently normal phenotype after emigration, they expressed increased amounts of Batf and Bcl6 at basal state and produced explosive and prolonged Tfh responses upon subsequent antigen encounter.Ablation of Cul3 in mature CD4(+) splenocytes also resulted in dramatically exaggerated Tfh responses.

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Affiliation: Committee on Immunology, Department of Pathology, Howard Hughes Medical Institute, and Department of Chemistry, University of Chicago, Chicago, IL 60637Committee on Immunology, Department of Pathology, Howard Hughes Medical Institute, and Department of Chemistry, University of Chicago, Chicago, IL 60637Committee on Immunology, Department of Pathology, Howard Hughes Medical Institute, and Department of Chemistry, University of Chicago, Chicago, IL 60637.

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Bcl6 interaction with Cul3 in thymocytes. (a) Intracellular staining of Bcl6 in unimmunized CD4+8+ (DP) and CD4+ SP thymocytes and in splenic CD4+ OTII Tg cells transferred into naive recipients and immunized with OVA 3 d before analysis. Data are representative of two to three independent experiments with two to three mice per group. (b) Immunoprecipitation and Western blot analysis of lysates from fresh DP or CD4+ SP thymocyte (20 × 106 per immunoprecipitation), as indicated. Data are representative of two independent experiments. Molecular mass is indicated in kilodaltons. IN, input lysate. (c) Confocal microscopic analysis of Bcl6 and Cul3 in transfected HeLa cells, as indicated. Cul3mut has the double mutation (L52AE55A) abolishing binding to BTB domains. Data are representative of two independent experiments. Bars, 1 µm.
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fig6: Bcl6 interaction with Cul3 in thymocytes. (a) Intracellular staining of Bcl6 in unimmunized CD4+8+ (DP) and CD4+ SP thymocytes and in splenic CD4+ OTII Tg cells transferred into naive recipients and immunized with OVA 3 d before analysis. Data are representative of two to three independent experiments with two to three mice per group. (b) Immunoprecipitation and Western blot analysis of lysates from fresh DP or CD4+ SP thymocyte (20 × 106 per immunoprecipitation), as indicated. Data are representative of two independent experiments. Molecular mass is indicated in kilodaltons. IN, input lysate. (c) Confocal microscopic analysis of Bcl6 and Cul3 in transfected HeLa cells, as indicated. Cul3mut has the double mutation (L52AE55A) abolishing binding to BTB domains. Data are representative of two independent experiments. Bars, 1 µm.

Mentions: Human cortical thymocytes were previously shown to express the Bcl6 protein (Hyjek et al., 2001), and their mouse counterparts transiently express Bcl6 mRNA at the DP stage according to a public database (ImmGen). Flow cytometric analysis of WT mouse thymocytes revealed high amounts of Bcl6 in DP thymocytes and rapid down-regulation in SP cells (Fig. 6 a). Notably, the thymic expression levels were comparable or superior to those elicited after immunization in peripheral Tfh cells.


A negative feedback loop mediated by the Bcl6-cullin 3 complex limits Tfh cell differentiation.

Mathew R, Mao AP, Chiang AH, Bertozzi-Villa C, Bunker JJ, Scanlon ST, McDonald BD, Constantinides MG, Hollister K, Singer JD, Dent AL, Dinner AR, Bendelac A - J. Exp. Med. (2014)

Bcl6 interaction with Cul3 in thymocytes. (a) Intracellular staining of Bcl6 in unimmunized CD4+8+ (DP) and CD4+ SP thymocytes and in splenic CD4+ OTII Tg cells transferred into naive recipients and immunized with OVA 3 d before analysis. Data are representative of two to three independent experiments with two to three mice per group. (b) Immunoprecipitation and Western blot analysis of lysates from fresh DP or CD4+ SP thymocyte (20 × 106 per immunoprecipitation), as indicated. Data are representative of two independent experiments. Molecular mass is indicated in kilodaltons. IN, input lysate. (c) Confocal microscopic analysis of Bcl6 and Cul3 in transfected HeLa cells, as indicated. Cul3mut has the double mutation (L52AE55A) abolishing binding to BTB domains. Data are representative of two independent experiments. Bars, 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4042651&req=5

fig6: Bcl6 interaction with Cul3 in thymocytes. (a) Intracellular staining of Bcl6 in unimmunized CD4+8+ (DP) and CD4+ SP thymocytes and in splenic CD4+ OTII Tg cells transferred into naive recipients and immunized with OVA 3 d before analysis. Data are representative of two to three independent experiments with two to three mice per group. (b) Immunoprecipitation and Western blot analysis of lysates from fresh DP or CD4+ SP thymocyte (20 × 106 per immunoprecipitation), as indicated. Data are representative of two independent experiments. Molecular mass is indicated in kilodaltons. IN, input lysate. (c) Confocal microscopic analysis of Bcl6 and Cul3 in transfected HeLa cells, as indicated. Cul3mut has the double mutation (L52AE55A) abolishing binding to BTB domains. Data are representative of two independent experiments. Bars, 1 µm.
Mentions: Human cortical thymocytes were previously shown to express the Bcl6 protein (Hyjek et al., 2001), and their mouse counterparts transiently express Bcl6 mRNA at the DP stage according to a public database (ImmGen). Flow cytometric analysis of WT mouse thymocytes revealed high amounts of Bcl6 in DP thymocytes and rapid down-regulation in SP cells (Fig. 6 a). Notably, the thymic expression levels were comparable or superior to those elicited after immunization in peripheral Tfh cells.

Bottom Line: Intriguingly, we found that Bcl6 was also highly and transiently expressed during the CD4(+)CD8(+) (double positive [DP]) stage of T cell development, in association with the E3 ligase cullin 3 (Cul3), a novel binding partner of Bcl6 which ubiquitinates histone proteins.Although they maintained an apparently normal phenotype after emigration, they expressed increased amounts of Batf and Bcl6 at basal state and produced explosive and prolonged Tfh responses upon subsequent antigen encounter.Ablation of Cul3 in mature CD4(+) splenocytes also resulted in dramatically exaggerated Tfh responses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Committee on Immunology, Department of Pathology, Howard Hughes Medical Institute, and Department of Chemistry, University of Chicago, Chicago, IL 60637Committee on Immunology, Department of Pathology, Howard Hughes Medical Institute, and Department of Chemistry, University of Chicago, Chicago, IL 60637Committee on Immunology, Department of Pathology, Howard Hughes Medical Institute, and Department of Chemistry, University of Chicago, Chicago, IL 60637.

Show MeSH
Related in: MedlinePlus