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. Author manuscript; available in PMC: 2015 Sep 11.
Published in final edited form as: Cell Rep. 2014 Aug 28;8(5):1497–1508. doi: 10.1016/j.celrep.2014.07.059

The BCL6 RD2 domain governs commitment of activated B-cells to form germinal centers

Chuanxin Huang 1, David G Gonzalez 2, Christine M Cote 2, Yanwen Jiang 1, Katerina Hatzi 1, Matt Teater 1, Kezhi Dai 3, Timothy Hla 3, Ann M Haberman 2,*, Ari Melnick 1,*
PMCID: PMC4163070  NIHMSID: NIHMS620022  PMID: 25176650

Summary

To understand how the Bcl6 transcriptional repressor functions in the immune system we disrupted its RD2 repression domain in mice. Bcl6RD2MUT mice exhibit a complete loss of GC formation but retain normal extrafollicular responses. Bcl6RD2MUT antigen-engaged B-cells migrate to the interfollicular zone and interact with cognate T helper cells. However, these cells fail to complete early GC-commitment differentiation and coalesce as nascent GC aggregates. Bcl6 directly binds and represses trafficking receptors S1pr1 and Gpr183 by recruiting Hdac2 through the RD2 domain. Deregulation of these genes impairs B-cell migration and may contribute to GC failure in Bcl6RD2MUT mice. The development of functional GC-TFH cells was partially impaired in Bcl6RD2MUT mice. In contrast to Bcl6−/− mice, Bcl6RD2MUT animals experience no inflammatory disease or macrophage deregulation. These results reveal an essential role for RD2 repression in early GC commitment and striking biochemical specificity in Bcl6 control of humoral and innate immune-cell phenotypes.

Introduction

In response to T-cell dependent (TD) antigen stimulation, antigen-specific B-cells migrate to the periphery of B-cell follicles and interfollicular zones of secondary lymphoid organs, where they interact with cognate T-helper cells within 1-3 days (Kerfoot et al., 2011; Okada et al., 2005; Qi et al., 2008). After then, B-cells can follow one of three alternative fates by differentiating into extrafollicular plasma cells, follicular germinal center (GC) B-cells or recirculating early memory B-cells (McHeyzer-Williams et al., 2012). GC B-cells undergo massive clonal expansion, somatic hypermutation and class switch recombination, after which selected clones undergo differentiation into memory B-cells and long-live plasma cells (Allen et al., 2007; Victora and Nussenzweig, 2012). T follicular helper (TFH) cells express the chemokine receptor CXCR5 and costimulatory molecule PD-1 at high levels and specialize in providing help to B-cells during the humoral immune response (Crotty, 2011). Antigen-presenting dendritic cells mediate the initiation of TFH-cell differentiation at early time-points after immunization (Choi et al., 2011; Goenka et al., 2011). Interactions with cognate B-cells, especially GC B-cells within GCs, are critical for further polarization, maintenance and function of TFH-cells at later stages of the immune response (Choi et al., 2011; Kerfoot et al., 2011; Kitano et al., 2011). Thus, the reciprocal development of GC B-cells and TFH-cells is crucial for establishment of the GC reaction, including formation of high-affinity antibody and generation of long-live plasma cells.

The Bcl6 transcriptional repressor is a master regulator of the GC reaction required for development of both GC B-cells and TFH-cells respectively (Dent et al., 1997; Fukuda et al., 1997; Johnston et al., 2009; Nurieva et al., 2009; Ye et al., 1997; Yu et al., 2009). In addition Bcl6 plays key roles in suppressing inflammatory cytokine expression in macrophages (Toney et al., 2000). Bcl6 deficient (Bcl6−/−) mice in addition to failing to form GCs are sickly and die within weeks from a lethal inflammatory syndrome primarily driven by macrophages with secondary contributions from Th2 and Th17-cells (Mondal et al., 2010; Toney et al., 2000). However, from the mechanistic standpoint the function of Bcl6 is poorly understood outside of the context of already established GC B-cells. Bcl6 enables proliferation and tolerance of DNA damage by silencing DNA-damage sensing and cell cycle checkpoint genes, and also delays plasma cell differentiation by repression of critical GC exit and plasma cell differentiation genes (Bunting and Melnick, 2013; Klein and Dalla-Favera, 2008). However, Bcl6−/− mice display complete loss of GC formation with no evidence of capacity to establish nascent GC clusters (Dent et al., 1997; Fukuda et al., 1997; Ye et al., 1997), suggesting that Bcl6 might have biological functions prior to GC formation. Indeed Bcl6 protein is up regulated in early GC-committed B-cells (i.e. “pre-GC B-cells”) outside GCs 3-5 days after immunization (Kerfoot et al., 2011; Kitano et al., 2011) and plays essential roles in maintaining interactions with TFH cell as well as subsequent migration and clustering into GC structures, at least in part through repressing the expression of Gpr183, encoding G protein-coupled receptor Ebi2 (Kitano et al., 2011; Shaffer et al., 2000).

Bcl6 functions as a transcription repressor via its N-terminal BTB domain and middle “second repression”, or “RD2” domain (Chang et al., 1996; Seyfert et al., 1996). Loss of function of the BCL6 BTB domain markedly impairs survival and proliferation of mature GC B-cells in B-cell intrinsic manner with no effects on T-cells or macrophages (Huang et al., 2013). Notably, unlike Bcl6−/− B cells, BTB-deficient B-cells still form GCs although their numbers and sizes are markedly reduced. This discrepancy illustrates our incomplete understanding of the molecular underpinnings of Bcl6-mediated GC B-cell development and suggests that other functions of Bcl6 are dominantly involved in modulating early pre-GC B cell fate.

In this study we generated mice in which the native Bcl6 locus was engineered to produce a form of Bcl6 containing a mutant RD2 domain that disrupts its repressor function. We found that RD2 domain is essential for pre-GC B-cell differentiation and clustering into nascent GC within follicles, in part through repressing key trafficking receptors S1pr1 and Gpr183 by recruiting Hdac2. Thus, these findings implicate the Bcl6 RD2 domain as defining the fate of activated B-cells towards the GC phenotype, which is mechanistically distinct from its role in enabling proliferation and survival upon induction of the proliferative burst within GCs. The activities of bcl6 can thus be parsed out into unique biochemical elements, each contributing different actions to the functionality of immune system.

Results

Generation and characterization of Bcl6 RD2 mutant knockin mice

To more precisely localize the repressor function of the Bcl6 RD2 domain, we first mapped the minimal region sufficient for its transcription repression activity. The full-length RD2, which spans the region from BTB domain to zinc finger domain (amino acids 129 to 519), mediated strong transcriptional repression similar to the Bcl6 BTB domain (Figure S1A-B). Progressive truncation yielded a 45-amino acid region (amino acids 350 to 595) as the minimal domain containing a similar repressive effect as full-length RD2 (Figure S1B). This 45-animo acid domain encompassed a known, functionally important KKYK motif (amino acids 387 to 390). Introduction of K387Q, K388Q and K390Q substitutions (lysine at 387, 388 and 390 to glutamine), mimics of acetylation of the KKYK, completely abrogated transcriptional repression mediated by either the 45-amino acid region or full-length RD2 domain (Figure S1B).

It was reported that the bcl6 middle region containing the RD2 domain binds to histone deacetylase 2 (HDAC2), MTA3/NuRD complex and CtBP (Bereshchenko et al., 2002; Fujita et al., 2004; Mendez et al., 2008). We found that wild-type minimal 45-animo acid RD2 domain, but not its QQYQ mutant form, interacts with HDAC2 as well as NuRD subunits Mi-2 and MTA3, but not CtBP, in co-immunoprecipitation assays (Figure S1C). Reporter assays showed that both HDAC2 and MTA3 contribute to the repressor function of the minimal RD2 domain (Figure S1D). Collectively, these results show that a 45-amino acid domain mediates the autonomous repressor function of the Bcl6 RD2 domain and its activity can be disrupted through mutations of key lysine residues.

We next utilized a homologous recombination strategy to introduce K387Q, K388Q and K390Q point mutations into the native Bcl6 locus in mice (Figure S1E-F). This enabled us to generate a Bcl6 RD2 mutant knockin mouse strain (called “Bcl6RD2MUT” from hereon) (Figure S1G). In contrast to Bcl6−/− mice, Bcl6RD2MUT mice were born at the expected Mendelian ratios, and were grossly normal in growth and survival. Expression of mutant Bcl6 gene product was confirmed by immunoblotting and sequencing (Figure S1H-I). Quantitative chromatin immunoprecipitation (QChIP) assays demonstrated that endogenous Bcl6RD2MUT protein binds to target genes in primary murine macrophages to a similar extent as WT Bcl6 protein, and retains the ability to recruit SMRT corepressor through the BTB domain (Figure S1J)

Bcl6RD2MUT mice display normal extrafollicular response, but complete abrogation of GC reaction

Bcl6−/− mice display a reduction of immature B-cells in bone marrow and mature B-cells in spleen (Duy et al., 2010). However, Bcl6RD2MUT display normal B-cell development prior to the GC stage and form (Figure S1K-L) and form normal splenic primary lymphoid follicles (Figure S1M). We next studied the role of the Bcl6 RD2 domain in the TD-antigen immune response. TD-antigen stimulation first induces an extrafollicular response to give rise to an early wave of low-affinity antibodies. We immunized a cohort of age-matched WT and Bcl6RD2MUT mice, with sheep red blood cells (SRBCs), a T-cell dependent antigen. Bcl6RD2MUT and WT mice produced similar amount of splenic NP-specific plasmablast/plasma cells (NP+CD138+B220lo-neg) and splenic IgM and IgG NP-specific antibody secreting cells (ASCs) at day 7 after immunization with 4-hydroxy-3-nitrophenylacetyl conjugated to chicken gamma globulin (NP-CGG), a well defined TD antigen (Figure S2A-B).

At later timepoints TD-antigen immunization induces the GC (follicular) response to produce high-affinity antibodies. Remarkably, Bcl6RD2MUT exhibited complete absence of any GCs in B-cell follicles of spleens at day 5,10 and 14 after SRBC immunization, revealed by immunochemistry (Figure 1A), similar to Bcl6−/− mice. Complete loss of GCs was also observed in Peyer's patches (Figure 1A). Accordingly, flow cytometry analysis showed that CD38lo-negFas+ GC B-cells were virtually undetectable in immunized Bcl6RD2MUT mice at day 10 after SRBC immunization (<0.1%; Figure 1B), identical to Bcl6−/− mice. Both CXCR5hiPD1hi GC-T and CXCR5intPD1int FH TFH were profoundly reduced in immunized Bcl6RD2MUT mice (0.7±0.2% and 2.7±0.4% respectively; P<0.01 versus WT; Figure 1C). As expected, GC-TFH and TFH cells were virtually absent in immunized Bcl6−/− mice (<0.1 and <1.0 respectively; Figure 1C). Similar defects were detected in Bcl6RD2MUT mice challenged with NP-CGG (Figure S2C). Bcl6RD2MUT mice displayed progressively lower levels of total NP-specific immunoglobulins after day 7 after NP-CGG immunization, most strikingly at day 32 (Figure S2D). The titers of all high-affinity NP-specific immunoglobulin isotypes, captured by NP4, were significantly lower in Bcl6RD2MUT than WT mice at day 32 (P<0.01; Figure S2E). The ratio of titers of high-affinity IgG1 to those of total IgG1, which indicates antibody maturation, was also lower in Bcl6RD2MUT than WT mice (P<0.01; Figure S2F). Collectively these data demonstrate that transcriptional repression through the RD2 domain of Bcl6 is required for the GC response and high affinity immunoglobulin maturation, but dispensable for the extrafollicular response.

Figure 1. Complete loss of GCs in Bcl6RD2MUT mice.

Figure 1

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(A-C) WT and Bcl6RD2MUT mice were immunized i.p. with SRBCs. (A) Representative immunohistochemical staining for PNA, B220 and Bcl6 on paraffin-embedded serial spleen sections and Peyer's patches. Scale bars: 200 μm. ND, not detected; n=3 each time-point per group. (B-C) Flow cytometry of murine splenic cells 10 days post-immunization. The adjacent dot plots indicate the percentage of Fas+ CD38lo-neg GC B-cells among total B-cells (B), as well as (C): CXCR5hiPD1hi GC-TFH and CXCR5intPD1int TFH cells among total CD4+ T-cells. N.S., not significant * P<0.05 and **P<0.01 (two-tailed t-test).

The Bcl6 RD2 domain is essential for GC B-cell but not GC-TFH cell formation

The requirement of Bcl6 for GC formation is due to its dual and cell autonomous role in development of both GC B- and TFH cells. Moreover, the development of these two cell types is interdependent. Thus, loss of GC B- and TFH cells in Bcl6RD2MUT mice could be caused by B- and/or T-cell deficiency. To determine the potential contribution of these cell types to the Bcl6RD2MUT phenotype, we generated mixed bone marrow chimeras by transferring 50% of congenic CD45.1+ WT bone marrow cells together with 50% of CD45.2+ bone marrow cells from WT, Bcl6−/− or Bcl6RD2MUT mice into sub-lethally irradiated Rag1−/− mice, which were then immunized after reconstitution (Figure 2A). As expected, CD45.2+ Bcl6−/− donor cell-derived GC B-cells, GC-TFH and TFH cells were essentially absent from CD45.1+ WT/CD45.2+Bcl6−/− mixed chimeras (P<0.01; Figure 2B-C). Analysis of CD45.2+ Bcl6RD2MUT chimeras revealed complete absence of CD45.2+ GC B-cells (Figure 2B), but yet still formed a normal number of CD45.2+ TFH (P>0.05) and approximately 60% CD45.2+ GC-TFH cells compared to WT donor T-cells (P<0.05; Figure 2C), which was similar to a hypomorphic defect observed in Bcl6+/− T cells (Yu et al., 2009). Together, these results indicate that RD2-dependent repression is essential for the generation of GC B-cells, but were more dispensable for GC-TFH cell differentiation.

Figure 2. Intrinsic defects in GC B but not TFH cells in Bcl6RD2MUT mice.

Figure 2

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(A) Schematic outline of generation of mixed chimeras and immunization. (B) Flow cytometry and adjacent dot plots showing percent of Fas+ CD38lo-neg GC B-cells among live B220+ cells, and (C), percent of CXCR5hiPDhi GC-TFH and CXCR5intPD1int TFH cells among live CD4+ cells from CD45.1 or CD45.2 donors. The ratio was calculated by dividing percent GC B-cells or TFH cells from CD45.2 donor by that from CD45.1 donor. Each symbol represents an individual mouse and small horizontal lines indicate means. Data are from three independent experiments with four mice per genotype. N.S., not significant; *P<0.05 and *P<0.01 (two-tailed t-test).

RD2-deficient B-cells are unable to support a functional GC reaction

We next examined whether the functional defects in GC formation in Bcl6RD2MUT mice were cell intrinsic to either GC B-cells and/or GC-TFH cells. For this we reconstructed chimeras by transferring µMT bone marrow (80%) along with bone marrow cells (20%) from WT, Bcl6RD2MUT or Bcl6−/− mice into sub-lethally irradiated Rag1−/− recipients (Figure 3A). μMT bone marrow cells provide a source of normal T-cells but not B-cells, thus all B-cells in these chimeras originate from tested donor bone marrow. GC B-cells were virtually absent in Bcl6RD2MUT mixed chimeras 10 days after immunization with SRBCs (Figure 3B). Accordingly titers of total NP specific antibodies (binding NP26) were reduced by 88% and the titers of high-affinity IgG1 NP-specific antibodies (binding NP4) were reduced by 95% in Bcl6RD2MUT versus WT mixed chimeras (P<0.05 versus WT) 21 days after NP-CGG immunization (Figure 3C). Notably, the impairment in GC B-cell numbers and titers of high-affinity antibodies were indistinguishable between Bcl6RD2MUT and Bcl6−/− mixed chimeras (Figure 3C), demonstrating that B-cells absolutely require the Bcl6 RD2 domain for GC formation and functionality.

Figure 3. Severely impaired function of RD2-deficient GC B- cells and modest defect in T-cells.

Figure 3

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(A) Schematic outline of generation of μMT chimeras and immunization. (B) Flow cytometry of FAS+ CD38lo-neg GC B-cells among live splenic B220+ cells from SRBC-immunized μMT chimeras. (C) ELISA of NP-specific IgG1 in sera from NP26-CGG-immunized μMT chimeras. (D) Schematic outline of generation of Tcrb−/−Tcrd−/− chimeras and immunization. (E) Flow cytometry of FAS+CD38lo-neg GC B-cells among live splenic B220+ cells from SRBC-immunized Tcrb−/−Tcrd−/− chimeras. (F) ELISA of NP-specific IgG1 in sera from NP-CGG-immunized Tcrb−/−Tcrd−/− chimeras. Each symbol represents an individual mouse and small horizontal lines indicate means (B-C and E-F). Data are from two independent experiments with four mice per genotype. RU, relative units; N.S., not significant; *P<0.05 and **P<0.01 (two-tailed t-test).

RD2-deficient T-cells are modestly impaired in supporting a functional GC reaction

The Bcl6 RD2 domain appears to play a relatively limited role in instructing GC-TFH cell differentiation (Figure 2C). To determine whether these cells were functionally impaired in driving humoral immunity, chimeras were generated by transferring a 4:1 mixture of Tcrb−/−Tcrd−/− and WT, Tcrb−/−Tcrd−/− and Bcl6RD2MUT, or Tcrb−/−Tcrd−/− and Bcl6−/− bone marrow cells into sub-lethally irradiated Rag1−/− recipients (Figure 3D). In these chimeras the Tcrb−/−Tcrd−/− strain contributes normal B-cells, whereas T-cells are derived from tested WT, Bcl6RD2MUT or Bcl6−/− marrow cells. Eight weeks after reconstitution, animals were immunized with SRBCs or NP-CGG. The frequency of GC B-cells among splenic B-cells in Tcrb−/−Tcrd−/− and WT mixed chimeras was 3.1±0.3% 10 days after SRBC immunization, whereas GC B-cells were essentially undetectable in Bcl6−/− mixed chimeras (<0.1%; Figure 3E). Notably, Bcl6RD2MUT mixed chimeras displayed a mild, albeit significant, reduction of GC B-cells, with a frequency of 1.8±0.6% (P<0.05 versus WT; Figure 3E). The titers of total NP-specific IgG1 antibodies in NP-CGG immunized Bcl6RD2MUT mixed chimeras were indistinguishable to WT (Figure 3F). In contrast, the titers of high-affinity NP-specific IgG1 antibodies in Bcl6RD2MUT chimers were significantly reduced compared to WT chimeras (P<0.05; one-tailed t test, Figure 3F). However, antibody titers were still significantly higher in Bcl6RD2MUT mixed chimeras as compared Bcl6−/− chimeras (P<0.05; Figure 3F). Taken together these data indicate that inactivation of the RD2 domain leads to a TFH functional defect, which, although significant, is much less severe than that caused by Bcl6 knockout.

To gain some insight into the basis of this modest defect, we isolated CD45.2+ Bcl6RD2MUT GC-TFH and CD45.1+ WT counterparts from SRBC-immunized mixed CD45.1+ WT / CD45.2+ WT chimeras (Figure S3A) and examined the expression of several key TFH cell-associated genes including Il-21 and Blimp1. IL-21 is expressed by TFH cells and signals to B-cells to regulate GC responses (Linterman et al., 2010; Zotos et al., 2010). Blimp1 is an important Bcl6 direct target and antagonistic regulator of TFH cell differentiation (Johnston et al., 2009). Notably, Bcl6RD2MUT GC-TFH expressed lower abundance of Il-21 and higher levels of Blimp1 compared to WT counterparts (Figure S3B). The Bcl6 RD2 domain has been reported to repress Blimp1 transcription in B cells (Fujita et al., 2004). We next analyzed whether RD2-deficiency might directly impair Bcl6-dependent repression of Blimp1 transcription in T cells using a luciferase reporter driven by the Blimp1β promoter, which contains the consensus Bcl6 binding site (Figure S3C) which is the major binding site for Bcl6 at Blimp1 locus (Hatzi et al., 2013). Indeed, we observed that RD2-deficiency significantly impaired Bcl6-mediated repression of the Blimp1β promoter in T-cells (Figure S3D).

The early phase of Bcl6RD2MUT B-cell response appears normal

The striking and complete defect in GC formation observed in Bcl6RD2MUT mice is similar to Bcl6−/− mice and different than the Bcl6 BTB mutant mouse strain, in which nascent GCs are still observed at late time points (day 10) (Huang et al., 2013), suggesting that the functional defect of Bcl6RD2MUT B-cells occurs at an early time-point of GC development (day 4) before the onset of rapid population expansion of GC B-cells (Calado et al., 2012). Indeed, SRBC-immunized BTB mutant mice formed robust early GC clusters in spleen and Peyer's patches at day 4, whereas in Bcl6RD2MUT mice the ability to develop early GC clusters was completely abrogated (Figure S4A).

We next examined the early stages of Bcl6RD2MUT B-cell development in response to TD immunization. To this end, Bcl6RD2MUT mice were bred with a green fluorescent protein (GFP) expressing strain and their offspring further crossed to MD4 mice, which bear a transgenic B-cell receptor that specifically binds the hen egg lysosome (HEL), to finally obtain GFP-Bcl6RD2MUT-MD4 mice. Red fluorescent protein (RFP) mice were crossed to the WT MD4 background to generate RFP-MD4 mice. Naïve B-cells from GFP-Bcl6RD2MUT-MD4 and RFP-WT-MD4 mice (at 1:1 ratio) were transferred together with cyan fluorescent protein (CFP) expressing ovalbumin (OVA)-specific T cells to recipients, which were then immunized at the footpad with HEL-conjugated ovalbumin (HEL-OVA) in complete Freund's adjuvant (CFA) two days later (summarized in Figure 4A). This protocol ensured the specific recruitment of MD4 B-cells to the GC formation pathway at the expense of the recipient B-cell pool. SMARTA TCR-transgenic mice were used as recipients to eliminate the endogenous T cell response to OVA. One day post-immunization, time resolved intravital imaging of adoptively transferred fluorescent cells within popliteal LNs was performed. Like WT B-cells, Bcl6RD2MUT B-cells relocated to the periphery of B-cell follicles and interfollicular zones (Figure 4B and Video S1-2). Moreover, analysis of T-B cell contact times in vivo indicated that the duration of such interactions by Bcl6RD2MUT B-cells are comparable to that observed among WT B-cells, typically lasting longer than 30 minutes and often for the duration of the imaging time period (Figure 4B-C and Video S1-2).

Figure 4. Bcl6RD2MUT B cells exhibit normal contacts with cognate T helper cells, but fail to cluster into nascent GCs.

Figure 4

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(A) Schematic outline of experimental approach. (B) Time lapse images of CFP-OT-II T cells interacting with WT RFP-MD4 B cells and GFP-Bcl6RD2MUT-MD4 B cells showing the tracks of motile T and B cells in conjugate pairs (See also Video S1 and S2). Time is shown in min:s. (C) Distribution of T-B cell contact durations of a representative experiment described in Experimental Procedures. (D) Bcl6 staining in sections of draining popliteal lymph nodes 5 days after immunization. Nascent GC is identified as the Bcl6hi cell cluster (blue or white) and highlighted by open circle. A representative region close to the GC is boxed and shown in bottom panel. Open and filled arrows indicate Bcl6hi WT B-cells and Bcl6RD2MUT B-cells respectively. Scale bars, 50 μm

Bcl6RD2MUT B-cells failed to form nascent GC clusters

After immunization and initial cognate contact with T-helper cells some antigen-specific B-cells proceed toward pre-GC B-cell differentiation, characterized by up-regulation of Bcl6 protein in the outer follicle, and finally coalesce and form small GC clusters in the follicle center four to five days after immunization (Kerfoot et al., 2011; Kitano et al., 2011). We next examined the ability of transferred antigen-specific Bcl6RD2MUT B-cells to form Bcl6hi pre-GC B-cells and GC structures in SMARTA TCR-transgenic mice at day 5 post-immunization (Figure 4A). As expected, WT RFP-MD4 B-cells formed PNA+ GC clusters within follicles on day 5 after immunization (Figure S4B). WT Bcl6hi RFP-MD4 B-cells were predominantly found within GC and also in the region around GC (Figure 4D). In contrast Bcl6hi GFP-Bcl6RD2MUT-MD4 B-cells failed to self-aggregate or join GC structures, although they remained Ki67+, and instead appeared randomly distributed (Figure 4D, Figure S4C). Similar to WT B-cells, Ki67+ Bcl6RD2MUT-B-cells were also observed within the medullary cords and the periphery of follicles (data not shown), including the area adjacent to the sub-capsular sinus, previously shown to support the extrafollicular formation of plasmablasts (Kerfoot et al., 2011).

The Bcl6 RD2 domain specifically controls genes involve in B-cell trafficking

Formation of GCs requires coordinated differential expression of specific lymphocyte trafficking factors. In murine B-cells, down-regulation of Gpr183 (also known as Ebi2) is important for the clustering of pre-GC B-cells within follicle centers (Gatto et al., 2009; Pereira et al., 2009). Up-regulation of S1pr2 confines GC B-cells within GCs (Green et al., 2011). S1pr1 enables B-cell trafficking (Cinamon et al., 2004), and low S1pr1 levels in GC B-cells was suggested to enable their retention within follicles (Green et al., 2011). Differential expression of these factors between murine GC and follicular B-cells has been reported (Green et al., 2011). We also observed a similar expression pattern in human GC and follicular B-cells (Figure 5A). Thus we asked whether the BCL6 RD2 domain controls expression of these factors.

Figure 5. The BCL6 RD2 domain recruits HDAC2 to repress S1PR1 and GPR183.

Figure 5

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(A) Relative transcript abundance of S1PR1, S1PR2 and GPR183 in human tonsillar GC B-cells (GCB) compared to naïve B cells (NB) after normalization of HPRT. (B) ChIP-seq tags (y axis) are shown as a histogram for the enrichment of BCL6, H3K4me2, H3K27ac in GC B-cells and naïve B-cells for S1PR1 and GPR183 loci. (C) Enrichment of indicated proteins at S1PR1 and GPR183 loci in human tonsillar GC B-cells. (D-F) OCI-LY1 cells stably expressing a control vector, siRNA-resistant BCL6 or BCL6RD2MUT were transfected with BCL6 siRNA#1 for two days. Relative transcript abundance of indicated genes (D) and fold change of enrichment of indicated proteins or histone marks at the S1PR1 locus (E) were calculated compared to vector. Data are representative of three independent experiments. *P<0.05 and **P<0.01 (two-tailed t-test). (F) GSEA of the gene-expression profiles of BCL6 and BCL6RD2MUT-expressing OCI-LY1 cells for BCL6_repressesd and GC_B_cell_upregulated gene sets. FDR: false-discovery rate (q value); NES: normalized enrichment score. The cartoon illustrates that that RD2 mutant BCL6 is deficient in repressing many BCL6 target genes and deficient in enabling the GC B-cell transcriptional program as compared to WT BCL6.

Analysis of ChIP-seq data (Beguelin et al., 2013; Huang et al., 2013) showed that BCL6 binds to distal regulatory sites associated with the GPR183 and S1PR1 genes respectively (Figure 5B). Both these binding sites carry classical enhancer marks: histone 3 lysine 27 acetylation (H3K27ac) and histone 3 lysine 4 dimethylation (H3K4me2) in naïve B-cells. In contrast the active enhancer mark H3K27ac (Creyghton et al., 2010), was clearly reduced in the presence of BCL6 in GC B-cells (Figure 5B), consistent with the reduced abundance of GPR183 and S1PR1 mRNA. QChIP assays for BCL6 repression complexes at the S1PR1 and GPR183 loci in primary human GC B-cells showed that HDAC2 was specifically bound along with BCL6 at both the S1PR1 and GPR183 enhancer regions (Figure 5C). In contrast the core NuRD component Mi-2 and CtBP were absent from these sites, although they still occupied their known binding sites with BCL6 at intron 3 of PRDM1 for Mi-2, and the BCL6 promoter for CtBP (Figure S5A).

We next performed functional assays in GC-derived lymphoma cells to study the transcriptional mechanism of the BCL6 RD2 domain. BCL6 knockdown in BCL6-dependent OCI-Ly1 lymphoma cells de-repressed both GPR183 and S1PR1 (P<0.05, Figure S5B) and resulted in upregulation of S1PR2 (possibly an indirect effect since BCL6 is not known to activate genes). Ectopic expression of WT, but not RD2 mutant BCL6, recruited HDAC2 to the S1PR1 locus in BCL6-null MutuIII cells (Figure S5C). BCL6 knockdown in OCI-LY1 cells impaired recruitment of HDAC2 to S1PR1 and GPR183 loci, with consequent increase of H3K27 acetylation of both loci. (Figure S5D) HDAC2 knockdown also induced H3K27 acetylation at both of these loci in these cells (Figure S5D).

To confirm whether repression of S1PR1 and GPR183 is specifically dependent on the BCL6 RD2 we performed rescue experiments in which we introduced WT or RD2 mutant BCL6 using expression constructs insensitive to siRNA into OCI-LY1 cells in which endogenous BCL6 was depleted by siRNA knockdown (Figure S5E). RD2 mutant BCL6 failed to repress S1PR1 and GPR183 expression (Figure 5D). Moreover, RD2 mutant BCL6 was unable to recruit HDAC2 and deacetylate H3K27 at the S1PR1 locus as compared to WT BCL6 (Figure 5E). Similar results were observed in an additional GC-derived lymphoma cell line (Figure S5F). Finally, migration assays in GC derived lymphoma cell revealed that S1PR1 inhibits S1PR2-induced migration in presence or absence of chemotactic cytokine CXCL12 (Figure S5G), consistent with antagonistic functions of S1PR1 and S1PR2 in B-cell trafficking. Overall the data suggest that BCL6 RD2 domain-dependent recruitment of HDAC2 mediates silencing of S1PR1 and GPR183 and indirectly induces upregulation of S1PR2, contributing to clustering of B-cells within follicles,.

The BCL6 RD2 domain regulates gene sets linked to the GC phenotype

To examine whether the BCL6 RD2 domain might regulate additional genes linked to the GC phenotype, we performed RNA-seq to compare the gene expression profiles of endogenous BCL6-depleted OCI-LY1 cells rescued with either WT or RD2 mutant BCL6. Unsupervised hierarchical clustering showed that the WT BCL6 and RD2 mutant gene expression profiles where clearly distinct and distributed to distinct clusters (Figure S5H). Gene-set enrichment analysis (GSEA) revealed that a canonical set of genes, known to be downregulated by BCL6 in B-cell lymphoma cells (Shaffer et al., 2000), was significantly repressed in WT BCL6 vs. RD2 mutant BCL6 (NES = -1.50, FDR = 0.02, Figure 5F). Conversely, a canonical GC B-cell gene set, consisting of genes that are upregulated in GC B-cells compared to other B-cell types (Shaffer et al., 2001), was significantly enriched (NES = 2.17, FDR = 0.00) in WT BCL6 as compared to RD2-mutant BCL6 expressing B-cells (Figure 5F). This result is consistent with the notion that the RD2 domain functions of BCL6 are required for B-cells to acquire the GC phenotype.

The RD2 domain is dispensable for the anti-inflammatory function of Bcl6

Bcl6−/− mice are born at sub-Mendelian frequency. The pups exhibit developmental delays and runted size (Dent et al., 1997; Fukuda et al., 1997; Ye et al., 1997). Within weeks they develop a fatal inflammatory syndrome driven by macrophage and Th2 cells (Mondal et al., 2010; Toney et al., 2000). In marked contrast Bcl6RD2MUT mice were born at the expected ratios, exhibited normal body weights (Figure 6A) and lived normal healthy lifespans. Bcl6RD2MUT animals did not exhibit any evidence of tissue damage or cellular infiltrates upon histopathology exam of various target organs of the Bcl6−/− inflammatory disease including lungs (Figure 6B) and other organs (data not shown). Bcl6 represses expression of inflammatory chemokines such as Ccl2, Ccl3, Ccl6, Ccl7 and Il-1a in macrophages. Loss of Bcl6 results in marked up-regulation of these genes in macrophages and splenic B-cells, which is believed to contribute to the lethal Bcl6−/− phenotype (Shaffer et al., 2000; Toney et al., 2000). These inflammatory response genes were only minimally increased in resting or lipopolysaccharide (LPS)-stimulated macrophages from Bcl6RD2MUT mice as compared to their WT counterparts (Figure 6C). Moreover, the abundance of these inflammatory cytokines in Bcl6RD2MUT was comparable to WT B-cells in either resting or LPS-stimulated status (Shaffer et al., 2000)(Figure 6D). Finally, Bcl6−/− mice display a high proportion of Th2 and Th17 cells, which play a critical role in the manifestation of inflammatory disease (Mondal et al., 2010; Toney et al., 2000). This phenotype can be further induced and aggravated by immunization. However, Bcl6RD2MUT mice displayed similar numbers of Th2 (1.6±0.2% versus 2.0±0.3%) and Th17 cells (0.5±0.1% versus 0.6±0.2%) to WT mice after immunization (Figure 6E), further demonstrating that Bcl6 RD2 domain functions are dispensable to the actions of BCL6 in repressing the inflammatory phenotype.

Figure 6. Bcl6RD2MUT mice fail to develop macrophage/TH2 driven inflammatory disease.

Figure 6

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(A) Body weight of indicated mice at eight-weeks of age (n=6). (B) Representative hematoxylin and eosin (H&E)-stained lung sections from indicated mice. Scale bars=200 μm. (C-D) mRNA expression of indicated genes in resting or LPS-treated bone marrow-derived macrophages (C) and splenic B cells (D) from indicated mice (n=3). Data were obtained from three independent experiments and are represented as fold-upregulation relative to WT resting cells after normalization with Hprt. Numbers adjacent to error bars indicate fold induction. (E) Percentage of IFNγ, IL-4 and IL-17 secreting cells among CD4+ cells from indicated mice. Data are represented as mean+SD from three mice. N.S., not significant; *P<0.05; **P<0.01 and ***P<0.001 (two-tailed t-test).

Discussion

The development of GCs is a sequential and complex process, during which BCL6 serves as master regulator in multiple cell types (Goodnow et al., 2010; Klein and Dalla-Favera, 2008; McHeyzer-Williams et al., 2012), There are many unanswered questions regarding the mechanisms and stages at which Bcl6 controls this process. Dissection of these events is further complicated by the diverse functions of Bcl6 in B-cells, T-cells, etc.; as well as the multifaceted biochemical mechanisms of action of Bcl6 (Bunting and Melnick, 2013).

After T-cell dependent antigen stimulation, Bcl6 protein begins to accumulate in pre-GC B-cells in the outer follicle regions and is maintained at high levels within GC B-cells (Kerfoot et al., 2011; Kitano et al., 2011). Its role in mature, established GCs is fairly well defined and is linked to its silencing of cell cycle, DNA damage sensing and apoptosis checkpoint genes as well as repression of genes involved in terminal differentiation (Bunting and Melnick, 2013; Klein and Dalla-Favera, 2008). These well-known GC B-cell functions are mediated through Bcl6 BTB domain-dependent recruitment of the SMRT, NCOR and BCOR corepressors (Hatzi et al., 2013; Huang et al., 2013; Polo et al., 2004). In contrast, the significance of Bcl6 in B-cells at the pre-GC stage has not been confirmed, and the molecular basis for Bcl6-mediated pre-GC B cell differentiation remains unknown. Our current data showing that the Bcl6 RD2 domain plays an essential role in pre-GC B-cell differentiation and nascent GC formation thus significantly extends knowledge of how B-cells adopt the GC fate under the control of a specific BCL6 biochemical mechanism distinct from its known canonical function (Figure 4). The profound defect in early pre-GC differentiation in RD2-deficent B-cells best explains the complete abrogation of GC formation in Bcl6 knockout mice and emphasizes the importance of Bcl6 in early GC B-cell commitment.

The canonical BCL6 repressive mechanism of action in GC B-cells is mediated by recruitment of the SMRT, NCOR and BCOR corepressors to the BCL6 BTB domain (Hatzi and Melnick, 2014). However we find that Bcl6 BTB mutant mice are still mostly capable of forming early GC clusters. This phenotype is in contrast to the complete abrogation of GC formation observed in BCL6 RD2 mutant mice. Hence, whereby BCL6 mediates early steps in commitment to the GC fate and clustering into nascent GCs through the RD2 domain, once GCs are established, Bcl6 is then required to maintain the proliferation and survival by repressing genes through its BTB domain. These findings suggest a model of sequential and biochemically distinct biological functions of BCL6 at different GC B-cell developmental stages (Figure S6). It is also possible that the RD2 domain might also contribute to later events in established GCs such as regulation of terminal differentiation (Fujita et al., 2004; Parekh et al., 2007) although such functions cannot be assessed in the RD2 animal model developed here.

This pre-GC B-cell differentiation deficiency caused by RD2 loss of function was explained at least in part by the finding that the BCL6 RD2 domain is required to repress expression of the key migration factors GRP183 and S1PR1. Down-regulation of GRP183 is critically important for B-cell migration into the follicle center (Gatto et al., 2009; Pereira et al., 2009). S1PR1 plays a key role in enabling B-cell trafficking out of follicles (Cinamon et al., 2004). Our finding that BCL6 directly represses these genes in GC cells suggests a role for BCL6 at an early stage of the GC response whereby BCL6 can enable “capture” of B-cells within follicles to enable their clustering. At the same time, S1PR2 upregulation in GC B-cells confines them to an S1P-low niche within follicles (Green et al., 2011). We observed that S1PR1 overexpression promoted GC-type B-cell migration and antagonized S1PR2 (Figure S5G). Taken together the data suggest that dynamic regulation and equilibrium of S1PR1 and S1PR2 is a critical event in the early pre-GC phase of the humoral immune response, controlled at least in part through BCL6 direct binding and repression of S1PR1.

From the mechanistic standpoint the BCL6 RD2 domain represses the GPR183 and S1PR1 loci by recruiting HDAC2, but not MTA3-NuRD, to suppress the enhancer activation mark H3K27ac at their distal regulatory elements. Yet these data do not exclude the possibility that other as of yet unknown corepressor proteins may bind with BCL6-HDAC2 repression complexes to these key target genes. Taken together with recent findings showing that BCL6 BTB domain co-repressors distribute to different sets of genomic loci with unique functions in GC B-cells (Hatzi et al., 2013), these data suggest that transcriptional programming by BCL6 is exquisitely compartmentalized through linkage of distinct biochemical functions to genes involved in specific immunological and biological functions.

In contrast to Bcl6−/− T-cells, Bcl6RD2MUT T-cells were partially impaired in their ability to form GC-TFH cells (Figure S6), which is somewhat similar to a hypomorphic defect observed in Bcl6+/- T cells (Yu et al., 2009). This defect is partially explained by the fact that RD2 mutant GC-TFH cells exhibited reduced Il21 expression and increased Blimp1. Repression of Blimp1 by BCL6 is known to critical for GC-TFH cell differentiation and function (Johnston et al., 2009). IL-21 is expressed by GC-TFH cell to promote GC B-cell development and maintenance (Linterman et al., 2010; Zotos et al., 2010). Unlike Bcl6−/− mice, inflammatory responses and macrophage regulation were not significantly disrupted in Bcl6RD2MUT mice. Neither loss of the BTB domain lateral groove nor the RD2 domain are sufficient to elicit deregulation of inflammatory signaling, which must instead be more reliant on other functions of Bcl6. The dominant mechanism may be linked at least in part to the previously reported competition with STAT proteins for binding to promoters of genes regulating inflammatory signaling (Dent et al., 1997; Huang et al., 2013) (Figure S6).

In conclusion, we identify the repressor function of the Bcl6 RD2 as a critical molecular mechanism required for early stages of GC B-cell differentiation. Through recruitment of HDAC2 and perhaps additional cofactors, the RD2 domain mediates repression of genes that must be downregulated for BCL6 to commit to GC formation and form clusters within lymphoid follicles.

Experimental Procedures

Animals

Mice were housed in the SPF animal facility at the Weill Cornell Medical College and experiments performed using protocols approved by Institutional Animal Care and Use Committee. Generation of the Bcl6RD2MUT knockin mouse and other animal are described in the Supplemental Information.

Bone marrow chimera studies

8-week old sub-lethally irradiated Rag1−/− mice were used as recipients. To generate mixed chimera (Figure 2A), 4×106 bone marrow cells from a mixture of WT CD45.1 and CD45.2 (WT, Bcl6−/− or Bcl6RD2MUT) with a ratio 1:1 were injected intravenously into recipients. To generate μMT chimera (Figure 3A), 4×106 bone marrow cells from a mixture of μMT and WT, Bcl6−/− or Bcl6RD2MUT mice with a ratio 4:1 were intravenously transferred into recipients. For generation of Tcr−/− chimeras (Figure 3D), recipients received a mixture of 80% bone marrow cells of Tcrb−/−Tcrd−/− donors and 20% WT, Bcl6−/− or Bcl6RD2MUT. Eight weeks after reconstruction, the recipients were immunized i.p. with SRBCs (Cocalico Biologicals Inc.) for 10 days for analysis of GC formation. Or the recipients were immunized i.p. with NP26-CGG (Biosearch Technologies) in Alum (Thermo Sciences) at 21 days for evaluation of antibody production.

Cell isolation, Adoptive transfers and Immunizations

MD4-specific B cells were isolated from the spleens and lymph nodes of mice by immnomagnetic purification with the EasySep negative selection mouse B-Ccell enrichment kit (StemCell Technologies). T-cells were purified from the spleens and lymph nodes of OT-II mice with the EasySep negative selection CD4+ T cell enrichment kit (StemCell Technologies). For both intravital imaging and histology experiments (Figure 4), 2.5 × 106 GFP-Bcl6RD2MUT-MD4 B cells, 2.5 × 106 RFP-WT-MD4+ B cells and 1.5 × 106 T cells were transferred for all time points. Cells were injected intravenously into SMARTA recipients that were immunized with 75 μg of HEl-OVA emulsified in CFA (Thermo Sciences) at 1.5 mg/mL in the right hind footpad.

Intravital Microscopy

The popliteal lymph nodes of anesthetized mice were imaged one day post-immunization. Mice were initially anesthetized with an i.p. injection of a ketamine and xylazine mixture and were subsequently kept anesthetized with nebulized a isoflurane/O2 gas mixture. Animals were immobilized on a custom-built stage and the right popliteal lymph node was surgically prepared as previously described (Mempel et al., 2004). For imaging acquisition, an Olympus BX61WI fluorescence microscope with a 20x, 0.95NA water immersion Olympus objective and dedicated single-beam LaVision TriM laser-scanning microscope (LaVision Biotec) was controlled by Imspector Pro software. The microscope was outfitted with a Chameleon Vision II Ti:Sapphire laser (Coherent) with pulse precompensation. Emission wavelengths of 390-480 nm (blue, CFP), 500-550 nm (green, GFP), and 565-665 nm (red, RFP), were collected with an array of three photomultiplier tubes (Hamamatsu). For 4D analysis of cell migration, stacks of 24 optical sections with 3 μm z-spacing were acquired every 30 s for 60-120 min with the laser tuned to a wavelength of 880 nm. Each xy plane spanned 400-500 μm in each dimension with a resolution of 0.977 μm per pixel.

Statistical analysis

Student's t-test was performed for statistical analysis. The software GraphPad Prism 5 was used for this analysis. p-value more than 0.05 is considered to be no significance.

Supplementary Material

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Highlights.

  • RD2 mutant B-cells fail to form nascent GC clusters within follicles

  • RD2 recruits Hdac2 to transcriptionally regulate S1pr1 and Gpr183

  • RD2 deficiency partially impairs development and functionality of GC-TFH cells

  • Loss of RD2 function has minimal effect on inflammatory cytokine production

Acknowledgements

A.M. is supported by NCI R01 104348. A.M. is also supported by the Burroughs Wellcome Foundation and Chemotherapy Foundation. A.H. and D.G. are supported by NIH R01AI080850 and R21AI101704. This research was initially supported by a March of Dimes Basil O'Connor Scholar Award (A.M.). This work was facilitated by the Sackler Center for Biomedical and Physical Sciences at Weill Cornell Medical College. We thank H. Ye from the Albert Einstein College of Medicine for sharing Bcl6−/− mice and P. Wade from National Institutes of Health for providing anti-MTA3 antibodies.

Footnotes

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Author Contributions

C.H. designed the experiments and interpreted data. C.H., A.H. and A.M. conceived the study and wrote the paper. C.H., D. G., C.C., Y.J., K.H., M.T. and K.D. performed experiments. T.H. provided important reagents.

Accession Numbers

The Gene Expression omnibus (GEO) accession number for RNAseq data sets reported in this paper is GSE58365.

Additional methods are provided in supplementary materials

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Supplementary Materials

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