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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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Cul3 regulates Tfh responses in mature CD4+ splenocytes. CD4+ splenocytes from OTII Tg Cul3fl/fl mice were transduced with MIGR1 retrovirus expressing Cre and GFP, or GFP alone, as indicated, and injected into CD45 congenic recipients 24 h before immunization with OVA + alum, as described in Fig. 3. Mice were analyzed 5 and 7 d after immunization, as indicated. The first column shows the fraction of GFP+ cells among donor cells (CD45.2+) at time of recovery. The second and third columns show staining of gated CD4+ donor cells separated according to GFP expression. The fourth column shows summaries of individual data. Numbers represent the percentage of PD1hiCXCR5hi Tfh cells based on three separate experiments for day 5 (n = 5) and day 7 (n = 6). Horizontal bars indicate mean. ***, P < 0.001.
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fig8: Cul3 regulates Tfh responses in mature CD4+ splenocytes. CD4+ splenocytes from OTII Tg Cul3fl/fl mice were transduced with MIGR1 retrovirus expressing Cre and GFP, or GFP alone, as indicated, and injected into CD45 congenic recipients 24 h before immunization with OVA + alum, as described in Fig. 3. Mice were analyzed 5 and 7 d after immunization, as indicated. The first column shows the fraction of GFP+ cells among donor cells (CD45.2+) at time of recovery. The second and third columns show staining of gated CD4+ donor cells separated according to GFP expression. The fourth column shows summaries of individual data. Numbers represent the percentage of PD1hiCXCR5hi Tfh cells based on three separate experiments for day 5 (n = 5) and day 7 (n = 6). Horizontal bars indicate mean. ***, P < 0.001.

Mentions: In Cul3cKO mice, the early dysregulation of Tfh-inducing genes in thymocytes preempted the assessment of an additional role of Cul3 during antigen stimulation of mature CD4+ T cells. To test whether a similar autoregulatory feedback existed in mature CD4+ T cells undergoing Tfh cell differentiation, we deleted Cul3 by retroviral expression of Cre recombinase in Cul3fl/fl OTII Tg CD4+ splenocytes. After transduction, cells were reinjected into naive hosts, which were subsequently immunized with OVA, following the same protocol as in Fig. 3. Fig. 8 shows that the GFP-expressing cells derived after transduction with MIGR1-Cre, but not control MIGR1, also exhibited a highly significant exaggeration of Tfh cell differentiation at various times after immunization (P < 0.001). Thus, we conclude that the Cul3-mediated negative feedback of Tfh cell differentiation also operates in mature CD4+ T cells undergoing antigen stimulation.


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)

Cul3 regulates Tfh responses in mature CD4+ splenocytes. CD4+ splenocytes from OTII Tg Cul3fl/fl mice were transduced with MIGR1 retrovirus expressing Cre and GFP, or GFP alone, as indicated, and injected into CD45 congenic recipients 24 h before immunization with OVA + alum, as described in Fig. 3. Mice were analyzed 5 and 7 d after immunization, as indicated. The first column shows the fraction of GFP+ cells among donor cells (CD45.2+) at time of recovery. The second and third columns show staining of gated CD4+ donor cells separated according to GFP expression. The fourth column shows summaries of individual data. Numbers represent the percentage of PD1hiCXCR5hi Tfh cells based on three separate experiments for day 5 (n = 5) and day 7 (n = 6). Horizontal bars indicate mean. ***, P < 0.001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig8: Cul3 regulates Tfh responses in mature CD4+ splenocytes. CD4+ splenocytes from OTII Tg Cul3fl/fl mice were transduced with MIGR1 retrovirus expressing Cre and GFP, or GFP alone, as indicated, and injected into CD45 congenic recipients 24 h before immunization with OVA + alum, as described in Fig. 3. Mice were analyzed 5 and 7 d after immunization, as indicated. The first column shows the fraction of GFP+ cells among donor cells (CD45.2+) at time of recovery. The second and third columns show staining of gated CD4+ donor cells separated according to GFP expression. The fourth column shows summaries of individual data. Numbers represent the percentage of PD1hiCXCR5hi Tfh cells based on three separate experiments for day 5 (n = 5) and day 7 (n = 6). Horizontal bars indicate mean. ***, P < 0.001.
Mentions: In Cul3cKO mice, the early dysregulation of Tfh-inducing genes in thymocytes preempted the assessment of an additional role of Cul3 during antigen stimulation of mature CD4+ T cells. To test whether a similar autoregulatory feedback existed in mature CD4+ T cells undergoing Tfh cell differentiation, we deleted Cul3 by retroviral expression of Cre recombinase in Cul3fl/fl OTII Tg CD4+ splenocytes. After transduction, cells were reinjected into naive hosts, which were subsequently immunized with OVA, following the same protocol as in Fig. 3. Fig. 8 shows that the GFP-expressing cells derived after transduction with MIGR1-Cre, but not control MIGR1, also exhibited a highly significant exaggeration of Tfh cell differentiation at various times after immunization (P < 0.001). Thus, we conclude that the Cul3-mediated negative feedback of Tfh cell differentiation also operates in mature CD4+ T cells undergoing antigen stimulation.

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