Bcl6 middle domain repressor function is required for T follicular helper cell differentiation and utilizes the corepressor MTA3

Abstract

T follicular helper (Tfh) cells are essential providers of help to B cells. The transcription factor B-cell CLL/lymphoma 6 (Bcl6) is a lineage-defining regulator of Tfh cells and germinal center B cells. In B cells, Bcl6 has the potential to recruit distinct transcriptional corepressors through its BTB domain or its poorly characterized middle domain (also known as RDII), but in Tfh cells the roles of the Bcl6 middle domain have yet to be clarified. Mimicked acetylation of the Bcl6 middle domain (K379Q) in CD4 T cells results in significant reductions in Tfh differentiation in vivo. Blimp1 (Prdm1) is a potent inhibitor of Tfh cell differentiation. Although Bcl6 K379Q still bound to the Prdm1 cis-regulatory elements in Tfh cells, Prdm1 expression was derepressed. This was a result of the failure of Bcl6 K379Q to recruit metastasis-associated protein 3 (MTA3). The loss of Bcl6 function in Bcl6 K379Q-expressing CD4 T cells could be partially rescued by abrogating Prdm1 expression. In addition to Prdm1, we found that Bcl6 recruits MTA3 to multiple genes involved in Tfh cell biology, including genes important for cell migration, cell survival, and alternative differentiation pathways. Thus, Bcl6 middle domain mediated repression is a major mechanism of action by which Bcl6 controls CD4 T-cell fate and function.

Keywords: B-cell help; T follicular helper cells; germinal centers.

Fig. 1.

Acetylation of Bcl6 middle domain inhibits Tfh cell development. (A) Bcl6-null CD4 T cells do not develop into Tfh cells. Bcl6^fl/fl and Bcl6^fl/fl Cre^CD4 mice were infected with LCMV. Tfh cell development was analyzed 7 d following infection. CD44^hi CD4⁺ T cells are shown. (B) Schematic of Bcl6 domains and acetylation motif KKYK in the middle domain. (C–E and G) Bcl6^fl/fl Cre^CD4 CD45.1⁺ SMARTA (SM) cells were retrovirally transduced with empty GFP vector, Bcl6 WT, Bcl6 K379Q, or Bcl6 3Q, then transferred to Bcl6^fl/fl Cre^CD4 mice and analyzed 7 d following acute LCMV infection. (C) CD45.1⁺GFP⁺ SMARTA cell frequencies. (D) LCMV-specific SMARTA Tfh cells (CXCR5^hiSLAM^lo). (E) LCMV-specific SMARTA GC Tfh cells (CXCR5^hi PSGL1^lo). (F) Early Tfh cells (CXCR5^hiSLAM^lo) among transduced Bcl6^fl/fl Cre^CD4 SMARTA cells at 3 d following LCMV infection. (G) CD45.1⁺GFP⁺ SMARTA cell frequencies. Data shown are representative of at least three independent experiments. G is representative of more than six independent experiments (*P< 0.05, **P< 0.01, and ***P< 0.001).

Fig. 2.

Acetylation of middle domain diminishes the inhibition of Blimp-1 by Bcl6. (A) Transduced Bcl6^fl/fl Cre^CD4 CD45.1⁺ SMARTA cells were transferred to B6 mice. At 7 d following LCMV infection, RNA was isolated from transduced cells and analyzed for Prdm1 transcript levels. (B and C) Bcl6^fl/fl Cre^CD4 Blimp1-YFP⁺ SMARTA cells were transduced with GFP, Bcl6, or K379Q RV, and total SMARTA CD4⁺ T cells (B) or SMARTA Th1 (SLAM⁺CXCR5⁻) and Tfh (CXCR5⁺SLAM^lo) cells (C) were analyzed for Blimp1-YFP expression 7 d following acute LCMV infection. Dotted line in the bar graph represents mean fluorescence intensity (MFI) of the YFP-negative control. (D–F) Bcl6^fl/fl Cre^CD4 CD45.1⁺ SMARTA cells were transduced with GFP, Bcl6, or K379Q RV (GFP) with or without Prdm1shRNA-RV (Ametrine), then transferred to B6 mice and analyzed 7 d following LCMV infection. (E) Tfh cell differentiation (CXCR5^hiSLAM^lo). (F) GC Tfh cell differentiation (CXCR5^hi PSGL1^lo). Data shown are representative of at least two independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 3.

Acetylation of middle domain inhibits generation of Tfh cells following immunization. Bcl6^fl/fl Cre^CD4 CD45.1⁺ SMARTA cells were transduced with the indicated RV, transferred into Bcl6^fl/fl Cre^CD4 mice, and analyzed 10 d after immunization with KLH-GP61 in alum. (A) CXCR5⁺ SMARTA cells. (B) GC Tfh SMARTA cells (CXCR5^hiPD-1^hi). (C) GC B-cell frequency (Fas^hiPNA^hi of CD19⁺). Data are pooled from four experiments (n = 17–20 per group), normalized to the GFP condition (GFP = 1). Data shown are representative of at least three independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. S1.

Bcl6 middle-domain function in Tfh cells is necessary to support GC formation. Bcl6^fl/fl Cre^CD4 CD45.1⁺ SMARTA cells were transduced with the indicated RV, transferred into Bcl6^fl/fl Cre^CD4 mice, and analyzed by histology 10 d after immunization with KLH-GP61 in alum. Draining lymph node sections were stained with B220 (blue) and GL-7 (green) antibodies. Representative sections of the lymph nodes are shown. Data are representative of three or four mice per group.

Fig. 4.

Constitutive deacetylation does not augment Tfh cell development. (A–C) Bcl6^fl/fl Cre^CD4 CD45.1⁺ SMARTA cells were retrovirally transduced with empty GFP vector, WT-Bcl6, or K379R-Bcl6 (constitutive deacetylation), then transferred to B6 mice and analyzed 3 d (A) or 7 d (B and C) following acute LCMV infection. (A) Early Tfh cells (CXCR5^hiSLAM^lo). (B) Tfh cells (CXCR5^hiSLAM^lo). (C) GC Tfh cells (CXCR5^hiPSGL-1^lo). (D) BCL6 was immunoprecipitated from total tonsil cells or PD-1⁺ GC Tfh cells isolated from tonsil followed by immunoblot analysis with anti-BCL6 and anti-acetylated lysine antibodies. Data shown are representative of two (A) or three (B–D) independent experiments (*P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 5.

Acetylation of Bcl6 prevents association with MTA3. Bcl6 represses Blimp-1 through recruitment of MTA3 and the Mi-2/NURD histone deacetylase complex. (A–F) Bcl6^fl/fl Cre^CD4 SMARTA cells were retrovirally transduced with Bcl6-WT and Bcl6-K379Q, transferred in B6 mice, and isolated by FACS 7 d after LCMV infection. (A–D) Chromatin was prepared and ChIP analyses were performed for Bcl6, MTA3, STAT3, and STAT5 at the Prdm1 promoter. (E–H) (Left) ChIP for MTA3 at Bcl6-binding sites for Ccr7, Ifngr1, Il7r, and Runx3. (Right) qPCR for Ccr7, Ifngr1, Il7r, and Runx3. Data are shown as fold induction of K379Q to WT transduced cells. (I) Model: acetylation of Bcl6 by p300 prevents association with the corepressor MTA3. If Bcl6 is deacetylated, MTA3 is able to bind to the middle domain and recruit the Mi-2/NURD complex. This complex mediates the repression of target genes such as Prdm1 through an HDAC-dependent mechanism. Although MTA3 has been shown to directly bind Bcl6 in B cells, it remains formally possible that the association between MTA3 and Bcl6 in Tfh cells is mediated by an intermediary protein, not represented in the figure. Data shown are representative of at least two independent experiments.

Fig. S2.

Mutant Bcl6 RV expression. (A) Plat-E cells were transfected with GFP-only, Bcl6-WT, BTB middle domain mutant (BTB-mut), or RDII middle domain mutant (RDII-mut) plasmid DNA. Bcl6 expression was measured by intracellular staining 48 h after transfection. Histogram overlay shows relative Bcl6 expression. Quantification shows Bcl6 MFI. (B) SMARTA CD4⁺ T cells were transduced with GFP-only, Bcl6-WT, BTB-mut, or RDII-mut RV. Transduced cells were adoptively transferred into C57BL/6J mice that were subsequently infected with LCMV. Splenocytes were analyzed 8 d after infection. Bcl6 expression in SMARTA TCR transgenic CD4⁺ T cells was measured by intracellular staining. Quantification shows Bcl6 MFI. Error bars in all graphs depict SEM.