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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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Excessive Tfh formation in Cul3fl/fl Cd4-cre mice. (a) FACS analysis of thymic CD4+ SP, RTEs, and splenic CD4+ in 10–15-wk-old Cul3cKO and littermate (LM) controls. Numbers indicate percentage (mean ± SEM) of CXCR5+PD1+ Tfh cells among two to eight mice/group from two to four independent experiments. (b) Tfh cell numbers in 16-wk-old Cul3cKO and littermate control splenic, mesenteric, cervical, and pancreatic lymph nodes, inguinal/axillary lymph nodes, and Peyer’s patches. Data are a compilation of two to six independent experiments with a total of 3–11 mice. (c) Mixed bone marrow chimeras reconstituted with various KO/WT bone marrow cell ratios analyzed for Tfh frequency 10 wk after reconstitution. Data are representative of two to three mixed chimeras per group from two independent experiments. (d) FACS analysis of splenic CD4+ Tfh, T reg, and Tfr cells from 9-wk-old Cul3cKO and littermate control mice. The leftmost column shows the gating strategy for CD4+Foxp3− and CD4+Foxp3+ cells. Data represent two independent experiments with a total of three mice each examined. Values represent the percentage of cells (mean ± SEM). (e) qRT-PCR analysis of cMaf and Il21 in splenic CD4+ T cells (sorted as CD4+CD25−CD1d-PBS57-tetramer−) from unimmunized 6–8-wk-old mice shown as the ratio of Cul3cKO/littermate control (n = 5 for each group from three independent experiments; mean ± SEM). (f) Hep-2 antinuclear antibodies quantified as mean nuclear fluorescence intensity at 1:40 dilution of sera from 30–50-wk-old Cul3cKO (seven females and one male) and WT littermates (four females), along with a positive control serum from MRLlpr/lpr and a no-serum negative control. (b and f) Horizontal bars indicate mean. (g) Survival curves of cohorts of Cul3cKO mice (n = 21) and WT littermates (n = 19; mean ± SEM). *, P < 0.05; ***, P < 0.001.
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fig1: Excessive Tfh formation in Cul3fl/fl Cd4-cre mice. (a) FACS analysis of thymic CD4+ SP, RTEs, and splenic CD4+ in 10–15-wk-old Cul3cKO and littermate (LM) controls. Numbers indicate percentage (mean ± SEM) of CXCR5+PD1+ Tfh cells among two to eight mice/group from two to four independent experiments. (b) Tfh cell numbers in 16-wk-old Cul3cKO and littermate control splenic, mesenteric, cervical, and pancreatic lymph nodes, inguinal/axillary lymph nodes, and Peyer’s patches. Data are a compilation of two to six independent experiments with a total of 3–11 mice. (c) Mixed bone marrow chimeras reconstituted with various KO/WT bone marrow cell ratios analyzed for Tfh frequency 10 wk after reconstitution. Data are representative of two to three mixed chimeras per group from two independent experiments. (d) FACS analysis of splenic CD4+ Tfh, T reg, and Tfr cells from 9-wk-old Cul3cKO and littermate control mice. The leftmost column shows the gating strategy for CD4+Foxp3− and CD4+Foxp3+ cells. Data represent two independent experiments with a total of three mice each examined. Values represent the percentage of cells (mean ± SEM). (e) qRT-PCR analysis of cMaf and Il21 in splenic CD4+ T cells (sorted as CD4+CD25−CD1d-PBS57-tetramer−) from unimmunized 6–8-wk-old mice shown as the ratio of Cul3cKO/littermate control (n = 5 for each group from three independent experiments; mean ± SEM). (f) Hep-2 antinuclear antibodies quantified as mean nuclear fluorescence intensity at 1:40 dilution of sera from 30–50-wk-old Cul3cKO (seven females and one male) and WT littermates (four females), along with a positive control serum from MRLlpr/lpr and a no-serum negative control. (b and f) Horizontal bars indicate mean. (g) Survival curves of cohorts of Cul3cKO mice (n = 21) and WT littermates (n = 19; mean ± SEM). *, P < 0.05; ***, P < 0.001.

Mentions: Cul3cKO mice show spontaneous enlargement of secondary lymphoid tissues because of the selective accumulation of Tfh cells and the resulting expansion of GC B cells, whereas other T helper responses are unaffected (Mathew et al., 2012). We found that the Tfh phenotype was not observed in thymic CD4+ single-positive (SP) cells or in recent thymic emigrants (RTEs) but developed progressively in the spleen and in mesenteric, cervical, and pancreatic lymph nodes, whereas axillary/inguinal lymph nodes and Peyer’s patches were much less affected (Fig. 1, a and b). Interestingly, although the Tfh expansion was mostly made of conventional CD4+ T cells, it also included Foxp3+ T follicular regulatory (Tfr) cells (Fig. 1 d). In lethally irradiated mice reconstituted with CD45 congenic mixtures of WT and KO bone marrows, the T cell defect was clearly cell intrinsic, but a modest Tfh conversion of WT cells could also be observed at high KO/WT ratio (Fig. 1 c), suggesting some bystander effect, perhaps caused by increased expression of the Tfh-promoting factors cMaf and Il21 (Fig. 1 e).


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)

Excessive Tfh formation in Cul3fl/fl Cd4-cre mice. (a) FACS analysis of thymic CD4+ SP, RTEs, and splenic CD4+ in 10–15-wk-old Cul3cKO and littermate (LM) controls. Numbers indicate percentage (mean ± SEM) of CXCR5+PD1+ Tfh cells among two to eight mice/group from two to four independent experiments. (b) Tfh cell numbers in 16-wk-old Cul3cKO and littermate control splenic, mesenteric, cervical, and pancreatic lymph nodes, inguinal/axillary lymph nodes, and Peyer’s patches. Data are a compilation of two to six independent experiments with a total of 3–11 mice. (c) Mixed bone marrow chimeras reconstituted with various KO/WT bone marrow cell ratios analyzed for Tfh frequency 10 wk after reconstitution. Data are representative of two to three mixed chimeras per group from two independent experiments. (d) FACS analysis of splenic CD4+ Tfh, T reg, and Tfr cells from 9-wk-old Cul3cKO and littermate control mice. The leftmost column shows the gating strategy for CD4+Foxp3− and CD4+Foxp3+ cells. Data represent two independent experiments with a total of three mice each examined. Values represent the percentage of cells (mean ± SEM). (e) qRT-PCR analysis of cMaf and Il21 in splenic CD4+ T cells (sorted as CD4+CD25−CD1d-PBS57-tetramer−) from unimmunized 6–8-wk-old mice shown as the ratio of Cul3cKO/littermate control (n = 5 for each group from three independent experiments; mean ± SEM). (f) Hep-2 antinuclear antibodies quantified as mean nuclear fluorescence intensity at 1:40 dilution of sera from 30–50-wk-old Cul3cKO (seven females and one male) and WT littermates (four females), along with a positive control serum from MRLlpr/lpr and a no-serum negative control. (b and f) Horizontal bars indicate mean. (g) Survival curves of cohorts of Cul3cKO mice (n = 21) and WT littermates (n = 19; mean ± SEM). *, P < 0.05; ***, P < 0.001.
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fig1: Excessive Tfh formation in Cul3fl/fl Cd4-cre mice. (a) FACS analysis of thymic CD4+ SP, RTEs, and splenic CD4+ in 10–15-wk-old Cul3cKO and littermate (LM) controls. Numbers indicate percentage (mean ± SEM) of CXCR5+PD1+ Tfh cells among two to eight mice/group from two to four independent experiments. (b) Tfh cell numbers in 16-wk-old Cul3cKO and littermate control splenic, mesenteric, cervical, and pancreatic lymph nodes, inguinal/axillary lymph nodes, and Peyer’s patches. Data are a compilation of two to six independent experiments with a total of 3–11 mice. (c) Mixed bone marrow chimeras reconstituted with various KO/WT bone marrow cell ratios analyzed for Tfh frequency 10 wk after reconstitution. Data are representative of two to three mixed chimeras per group from two independent experiments. (d) FACS analysis of splenic CD4+ Tfh, T reg, and Tfr cells from 9-wk-old Cul3cKO and littermate control mice. The leftmost column shows the gating strategy for CD4+Foxp3− and CD4+Foxp3+ cells. Data represent two independent experiments with a total of three mice each examined. Values represent the percentage of cells (mean ± SEM). (e) qRT-PCR analysis of cMaf and Il21 in splenic CD4+ T cells (sorted as CD4+CD25−CD1d-PBS57-tetramer−) from unimmunized 6–8-wk-old mice shown as the ratio of Cul3cKO/littermate control (n = 5 for each group from three independent experiments; mean ± SEM). (f) Hep-2 antinuclear antibodies quantified as mean nuclear fluorescence intensity at 1:40 dilution of sera from 30–50-wk-old Cul3cKO (seven females and one male) and WT littermates (four females), along with a positive control serum from MRLlpr/lpr and a no-serum negative control. (b and f) Horizontal bars indicate mean. (g) Survival curves of cohorts of Cul3cKO mice (n = 21) and WT littermates (n = 19; mean ± SEM). *, P < 0.05; ***, P < 0.001.
Mentions: Cul3cKO mice show spontaneous enlargement of secondary lymphoid tissues because of the selective accumulation of Tfh cells and the resulting expansion of GC B cells, whereas other T helper responses are unaffected (Mathew et al., 2012). We found that the Tfh phenotype was not observed in thymic CD4+ single-positive (SP) cells or in recent thymic emigrants (RTEs) but developed progressively in the spleen and in mesenteric, cervical, and pancreatic lymph nodes, whereas axillary/inguinal lymph nodes and Peyer’s patches were much less affected (Fig. 1, a and b). Interestingly, although the Tfh expansion was mostly made of conventional CD4+ T cells, it also included Foxp3+ T follicular regulatory (Tfr) cells (Fig. 1 d). In lethally irradiated mice reconstituted with CD45 congenic mixtures of WT and KO bone marrows, the T cell defect was clearly cell intrinsic, but a modest Tfh conversion of WT cells could also be observed at high KO/WT ratio (Fig. 1 c), suggesting some bystander effect, perhaps caused by increased expression of the Tfh-promoting factors cMaf and Il21 (Fig. 1 e).

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.

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Related in: MedlinePlus