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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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Altered Tfh gene expression in Cul3-deficient thymocytes. (a) qRT-PCR analysis of Batf in large DP, small DP, and CD4+ SP thymocytes shown as ratio of Cul3cKO/littermate (LM) control. (b) qRT-PCR analysis of Batf and Bcl6 in CD4+ SP thymocytes shown as ratio of Cul3cKO/littermate control. Bar graphs represent mean ± SEM from 5–10 pairs of KO and controls from five independent experiments. (c) Gene resolution fold changes of CD4+ SP thymocyte microarrays in Cul3cKO versus littermate controls, with biological replicates plotted as x- and y-axis coordinates. The Tfh gene set, indicated as large black scatter, is significantly up-regulated relative to other genome-wide changes in expression, as shown by comparative SSMD analysis of Monte Carlo–generated sets (P = 2 × 10−6). In contrast, the Th1 and Th2 gene sets (not indicated in the figure) were not significantly altered (P = 0.3). (d) CD69-MACS–depleted OTII Tg thymocytes were stimulated with T cell–depleted CD45 congenic splenic APCs at different concentrations of OVA peptide for 20 h before FACS analysis of CD4+ SP cells for surface CD69 and intracellular Batf. Mean ± SEM of two independent experiments with three WT and three Cul3cKO is shown. (e) MHC II–deficient hosts were lethally irradiated and reconstituted with bone marrow cells from OTII Tg in a Cul3cKO or WT background as indicated. CD4/CD8 dot plots show absence of SP thymocytes at 5–6 wk after reconstitution, as expected. Bar graph shows Batf expression measured by qRT-PCR as a ratio of Cul3cKO/WT purified small DP thymocytes (mean ± SEM). Data are compiled from three WT and six KO from two independent experiments. (f) Same experiment as in panel e for MHC I/II double-deficient hosts reconstituted with Cul3cKO or WT bone marrow cells as indicated. Data are compiled from three WT and three KO from one experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig5: Altered Tfh gene expression in Cul3-deficient thymocytes. (a) qRT-PCR analysis of Batf in large DP, small DP, and CD4+ SP thymocytes shown as ratio of Cul3cKO/littermate (LM) control. (b) qRT-PCR analysis of Batf and Bcl6 in CD4+ SP thymocytes shown as ratio of Cul3cKO/littermate control. Bar graphs represent mean ± SEM from 5–10 pairs of KO and controls from five independent experiments. (c) Gene resolution fold changes of CD4+ SP thymocyte microarrays in Cul3cKO versus littermate controls, with biological replicates plotted as x- and y-axis coordinates. The Tfh gene set, indicated as large black scatter, is significantly up-regulated relative to other genome-wide changes in expression, as shown by comparative SSMD analysis of Monte Carlo–generated sets (P = 2 × 10−6). In contrast, the Th1 and Th2 gene sets (not indicated in the figure) were not significantly altered (P = 0.3). (d) CD69-MACS–depleted OTII Tg thymocytes were stimulated with T cell–depleted CD45 congenic splenic APCs at different concentrations of OVA peptide for 20 h before FACS analysis of CD4+ SP cells for surface CD69 and intracellular Batf. Mean ± SEM of two independent experiments with three WT and three Cul3cKO is shown. (e) MHC II–deficient hosts were lethally irradiated and reconstituted with bone marrow cells from OTII Tg in a Cul3cKO or WT background as indicated. CD4/CD8 dot plots show absence of SP thymocytes at 5–6 wk after reconstitution, as expected. Bar graph shows Batf expression measured by qRT-PCR as a ratio of Cul3cKO/WT purified small DP thymocytes (mean ± SEM). Data are compiled from three WT and six KO from two independent experiments. (f) Same experiment as in panel e for MHC I/II double-deficient hosts reconstituted with Cul3cKO or WT bone marrow cells as indicated. Data are compiled from three WT and three KO from one experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: The stable baseline increase of Batf and Bcl6 proteins observed shortly after thymic maturation of Cul3cKO CD4+ T cells suggested that fresh thymocytes might already express detectable changes at the transcriptional level. Using quantitative real-time PCR (qRT-PCR), we measured the Batf transcript levels in large and small DP thymocytes, as well as in CD4+ SP thymocytes. Fig. 5 a shows that although Batf transcripts were not quantitatively altered at the early, large-DP stage in Cul3cKO mice, they were already significantly increased at the small-DP stage compared with littermate controls and further elevated at the SP stage. This kinetics was consistent with the genetic ablation of Cul3 at the early DP stage by the Cd4-Cre transgene. Thus, although increased Batf protein was detected soon after thymic emigration at the single cell level, transcriptional changes were already evident at the DP stage immediately after Cul3 ablation. A similar picture was noted for Bcl6, which was expressed at a higher level in Cul3cKO than in WT CD4+ SP thymocytes (Fig. 5 b). Importantly, these alterations were cell intrinsic, as the analysis included several mixed (1:1) bone marrow chimeras in which Cul3cKO and WT CD4+ SP thymocytes could be compared side by side.


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)

Altered Tfh gene expression in Cul3-deficient thymocytes. (a) qRT-PCR analysis of Batf in large DP, small DP, and CD4+ SP thymocytes shown as ratio of Cul3cKO/littermate (LM) control. (b) qRT-PCR analysis of Batf and Bcl6 in CD4+ SP thymocytes shown as ratio of Cul3cKO/littermate control. Bar graphs represent mean ± SEM from 5–10 pairs of KO and controls from five independent experiments. (c) Gene resolution fold changes of CD4+ SP thymocyte microarrays in Cul3cKO versus littermate controls, with biological replicates plotted as x- and y-axis coordinates. The Tfh gene set, indicated as large black scatter, is significantly up-regulated relative to other genome-wide changes in expression, as shown by comparative SSMD analysis of Monte Carlo–generated sets (P = 2 × 10−6). In contrast, the Th1 and Th2 gene sets (not indicated in the figure) were not significantly altered (P = 0.3). (d) CD69-MACS–depleted OTII Tg thymocytes were stimulated with T cell–depleted CD45 congenic splenic APCs at different concentrations of OVA peptide for 20 h before FACS analysis of CD4+ SP cells for surface CD69 and intracellular Batf. Mean ± SEM of two independent experiments with three WT and three Cul3cKO is shown. (e) MHC II–deficient hosts were lethally irradiated and reconstituted with bone marrow cells from OTII Tg in a Cul3cKO or WT background as indicated. CD4/CD8 dot plots show absence of SP thymocytes at 5–6 wk after reconstitution, as expected. Bar graph shows Batf expression measured by qRT-PCR as a ratio of Cul3cKO/WT purified small DP thymocytes (mean ± SEM). Data are compiled from three WT and six KO from two independent experiments. (f) Same experiment as in panel e for MHC I/II double-deficient hosts reconstituted with Cul3cKO or WT bone marrow cells as indicated. Data are compiled from three WT and three KO from one experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig5: Altered Tfh gene expression in Cul3-deficient thymocytes. (a) qRT-PCR analysis of Batf in large DP, small DP, and CD4+ SP thymocytes shown as ratio of Cul3cKO/littermate (LM) control. (b) qRT-PCR analysis of Batf and Bcl6 in CD4+ SP thymocytes shown as ratio of Cul3cKO/littermate control. Bar graphs represent mean ± SEM from 5–10 pairs of KO and controls from five independent experiments. (c) Gene resolution fold changes of CD4+ SP thymocyte microarrays in Cul3cKO versus littermate controls, with biological replicates plotted as x- and y-axis coordinates. The Tfh gene set, indicated as large black scatter, is significantly up-regulated relative to other genome-wide changes in expression, as shown by comparative SSMD analysis of Monte Carlo–generated sets (P = 2 × 10−6). In contrast, the Th1 and Th2 gene sets (not indicated in the figure) were not significantly altered (P = 0.3). (d) CD69-MACS–depleted OTII Tg thymocytes were stimulated with T cell–depleted CD45 congenic splenic APCs at different concentrations of OVA peptide for 20 h before FACS analysis of CD4+ SP cells for surface CD69 and intracellular Batf. Mean ± SEM of two independent experiments with three WT and three Cul3cKO is shown. (e) MHC II–deficient hosts were lethally irradiated and reconstituted with bone marrow cells from OTII Tg in a Cul3cKO or WT background as indicated. CD4/CD8 dot plots show absence of SP thymocytes at 5–6 wk after reconstitution, as expected. Bar graph shows Batf expression measured by qRT-PCR as a ratio of Cul3cKO/WT purified small DP thymocytes (mean ± SEM). Data are compiled from three WT and six KO from two independent experiments. (f) Same experiment as in panel e for MHC I/II double-deficient hosts reconstituted with Cul3cKO or WT bone marrow cells as indicated. Data are compiled from three WT and three KO from one experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: The stable baseline increase of Batf and Bcl6 proteins observed shortly after thymic maturation of Cul3cKO CD4+ T cells suggested that fresh thymocytes might already express detectable changes at the transcriptional level. Using quantitative real-time PCR (qRT-PCR), we measured the Batf transcript levels in large and small DP thymocytes, as well as in CD4+ SP thymocytes. Fig. 5 a shows that although Batf transcripts were not quantitatively altered at the early, large-DP stage in Cul3cKO mice, they were already significantly increased at the small-DP stage compared with littermate controls and further elevated at the SP stage. This kinetics was consistent with the genetic ablation of Cul3 at the early DP stage by the Cd4-Cre transgene. Thus, although increased Batf protein was detected soon after thymic emigration at the single cell level, transcriptional changes were already evident at the DP stage immediately after Cul3 ablation. A similar picture was noted for Bcl6, which was expressed at a higher level in Cul3cKO than in WT CD4+ SP thymocytes (Fig. 5 b). Importantly, these alterations were cell intrinsic, as the analysis included several mixed (1:1) bone marrow chimeras in which Cul3cKO and WT CD4+ SP thymocytes could be compared side by side.

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