Limits...
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.

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

Exaggerated Tfh responses to OVA antigen. (a and b) 0.5 × 106 CD4+ enriched SP thymocytes from OTII Tg or OTII Tg Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before i.p. immunization with OVA + alum (a) or OVA-NP16 + alum (b). (a) Unimmunized controls are shown at day 3 after transfer in the first column, and immunized mice are shown at days 3 and 7 in the second and third columns. Summary data were compiled from two separate experiments, each with four to five mice per group, and statistical analyses are shown on the right. FACS analysis shows staining of gated donor cells in the spleen for PD1 and CXCR5 (top two rows), Batf (middle two rows), Bcl6 (bottom two rows). Numbers above panels in the top two rows represent absolute numbers of donor cells recovered in the recipient spleens (mean ± SEM), with the percentage of PD1hiCXCR5hi cells indicated in the top right quadrant of each dot plot. In the bottom four rows, numbers represent mean fluorescence intensity (MFI), with shaded gray histograms representing background staining. (b) Comparative levels of Batf and Bcl6 proteins (expressed as OTII Cul3cKO/WT MFI ratio) in CD4+ SP thymocytes before (day 0) and after parking for 3 and 7 d (in vivo transfer) in individual unimmunized mice. Data are combined from two independent experiments with four to eight mice in each group. (c) Serum IgG1 antibodies against BSA-NP41 (left) and BSA-NP4 (right) at days 0 and 21 after immunization with OVA-NP16 + alum. Data are a compilation of two independent experiments, with a total of eight immunized mice in each group. Horizontal bars indicate mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Exaggerated Tfh responses to OVA antigen. (a and b) 0.5 × 106 CD4+ enriched SP thymocytes from OTII Tg or OTII Tg Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before i.p. immunization with OVA + alum (a) or OVA-NP16 + alum (b). (a) Unimmunized controls are shown at day 3 after transfer in the first column, and immunized mice are shown at days 3 and 7 in the second and third columns. Summary data were compiled from two separate experiments, each with four to five mice per group, and statistical analyses are shown on the right. FACS analysis shows staining of gated donor cells in the spleen for PD1 and CXCR5 (top two rows), Batf (middle two rows), Bcl6 (bottom two rows). Numbers above panels in the top two rows represent absolute numbers of donor cells recovered in the recipient spleens (mean ± SEM), with the percentage of PD1hiCXCR5hi cells indicated in the top right quadrant of each dot plot. In the bottom four rows, numbers represent mean fluorescence intensity (MFI), with shaded gray histograms representing background staining. (b) Comparative levels of Batf and Bcl6 proteins (expressed as OTII Cul3cKO/WT MFI ratio) in CD4+ SP thymocytes before (day 0) and after parking for 3 and 7 d (in vivo transfer) in individual unimmunized mice. Data are combined from two independent experiments with four to eight mice in each group. (c) Serum IgG1 antibodies against BSA-NP41 (left) and BSA-NP4 (right) at days 0 and 21 after immunization with OVA-NP16 + alum. Data are a compilation of two independent experiments, with a total of eight immunized mice in each group. Horizontal bars indicate mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: We transferred 0.5 × 106 WT or Cul3cKO CD4+ enriched SP OTII thymocytes i.v. into unirradiated CD45 congenic recipients to study their response after immunization with cognate antigen OVA in alum. First, we followed unimmunized recipients. Surprisingly, we noted that, although both CD4+ SP thymocytes and 24-h RTEs in Cul3cKO mice failed to express CXCR5 (chemokine [C-X-C motif] receptor 5) and other surface Tfh markers (Fig. 1 a), the transferred cells expressed conspicuously higher amounts of basal Batf (67%) and Bcl6 (41%) on average than their WT counterparts 3 d after transfer, as measured by intracellular flow cytometry (Fig. 3, a [left column] and b). These increased basal levels were maintained without further increase at day 7 after transfer (Fig. 3 b). However, these changes were not sufficient to induce the acquisition of the stereotypical Tfh phenotype because levels of CXCR5, PD1 (programmed cell death 1), and ICOS were not detectably increased in the absence of immunization. Thus, Cul3 ablation at the DP stage resulted in delayed but stable increases of basal Batf and Bcl6 proteins, without overt Tfh transformation, suggesting that some degree of Tfh dysregulation was already present before antigen exposure.


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)

Exaggerated Tfh responses to OVA antigen. (a and b) 0.5 × 106 CD4+ enriched SP thymocytes from OTII Tg or OTII Tg Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before i.p. immunization with OVA + alum (a) or OVA-NP16 + alum (b). (a) Unimmunized controls are shown at day 3 after transfer in the first column, and immunized mice are shown at days 3 and 7 in the second and third columns. Summary data were compiled from two separate experiments, each with four to five mice per group, and statistical analyses are shown on the right. FACS analysis shows staining of gated donor cells in the spleen for PD1 and CXCR5 (top two rows), Batf (middle two rows), Bcl6 (bottom two rows). Numbers above panels in the top two rows represent absolute numbers of donor cells recovered in the recipient spleens (mean ± SEM), with the percentage of PD1hiCXCR5hi cells indicated in the top right quadrant of each dot plot. In the bottom four rows, numbers represent mean fluorescence intensity (MFI), with shaded gray histograms representing background staining. (b) Comparative levels of Batf and Bcl6 proteins (expressed as OTII Cul3cKO/WT MFI ratio) in CD4+ SP thymocytes before (day 0) and after parking for 3 and 7 d (in vivo transfer) in individual unimmunized mice. Data are combined from two independent experiments with four to eight mice in each group. (c) Serum IgG1 antibodies against BSA-NP41 (left) and BSA-NP4 (right) at days 0 and 21 after immunization with OVA-NP16 + alum. Data are a compilation of two independent experiments, with a total of eight immunized mice in each group. Horizontal bars indicate mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Exaggerated Tfh responses to OVA antigen. (a and b) 0.5 × 106 CD4+ enriched SP thymocytes from OTII Tg or OTII Tg Cul3cKO donors were injected i.v. into CD45 congenic recipients 24 h before i.p. immunization with OVA + alum (a) or OVA-NP16 + alum (b). (a) Unimmunized controls are shown at day 3 after transfer in the first column, and immunized mice are shown at days 3 and 7 in the second and third columns. Summary data were compiled from two separate experiments, each with four to five mice per group, and statistical analyses are shown on the right. FACS analysis shows staining of gated donor cells in the spleen for PD1 and CXCR5 (top two rows), Batf (middle two rows), Bcl6 (bottom two rows). Numbers above panels in the top two rows represent absolute numbers of donor cells recovered in the recipient spleens (mean ± SEM), with the percentage of PD1hiCXCR5hi cells indicated in the top right quadrant of each dot plot. In the bottom four rows, numbers represent mean fluorescence intensity (MFI), with shaded gray histograms representing background staining. (b) Comparative levels of Batf and Bcl6 proteins (expressed as OTII Cul3cKO/WT MFI ratio) in CD4+ SP thymocytes before (day 0) and after parking for 3 and 7 d (in vivo transfer) in individual unimmunized mice. Data are combined from two independent experiments with four to eight mice in each group. (c) Serum IgG1 antibodies against BSA-NP41 (left) and BSA-NP4 (right) at days 0 and 21 after immunization with OVA-NP16 + alum. Data are a compilation of two independent experiments, with a total of eight immunized mice in each group. Horizontal bars indicate mean. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: We transferred 0.5 × 106 WT or Cul3cKO CD4+ enriched SP OTII thymocytes i.v. into unirradiated CD45 congenic recipients to study their response after immunization with cognate antigen OVA in alum. First, we followed unimmunized recipients. Surprisingly, we noted that, although both CD4+ SP thymocytes and 24-h RTEs in Cul3cKO mice failed to express CXCR5 (chemokine [C-X-C motif] receptor 5) and other surface Tfh markers (Fig. 1 a), the transferred cells expressed conspicuously higher amounts of basal Batf (67%) and Bcl6 (41%) on average than their WT counterparts 3 d after transfer, as measured by intracellular flow cytometry (Fig. 3, a [left column] and b). These increased basal levels were maintained without further increase at day 7 after transfer (Fig. 3 b). However, these changes were not sufficient to induce the acquisition of the stereotypical Tfh phenotype because levels of CXCR5, PD1 (programmed cell death 1), and ICOS were not detectably increased in the absence of immunization. Thus, Cul3 ablation at the DP stage resulted in delayed but stable increases of basal Batf and Bcl6 proteins, without overt Tfh transformation, suggesting that some degree of Tfh dysregulation was already present before antigen exposure.

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