T follicular helper cell (T_FH) differentiation is defective in the absence of Bcl6 BTB repressor domain function

Abstract

Follicular helper T cells (T_FH) are essential for germinal centers and most long term humoral immunity. Differentiation of T_FH cells depends on the transcriptional repressor, Bcl6. Bcl6 mediates gene repression via the recruitment of co-repressors. Currently, it is unknown how Bcl6 recruits corepressors to regulate gene expression of T_FH cells. Here we demonstrate, using a mutant form of Bcl6 with two BTB (bric-a-brac, tramtrack, broad-complex) mutations that abrogate corepressor binding, that the Bcl6 BTB domain is required for proper differentiation of T_FH and GC T_FH cells in vivo. Importantly, we also observe a significant defect in germinal center B cell development. These results are consistent in multiple contexts, including a novel LCMV NP-specific TCR transgenic mouse model. Taken together, these data suggest that the Bcl6 BTB domain is a key mediator of the differentiation of T_FH cells.

INTRODUCTION

B cell CLL/lymphoma 6 (Bcl6) is a transcriptional repressor that is essential for the differentiation of T follicular helper cells (T_FH) and germinal center B cells. T_FH cells are CD4 T cells specialized in providing help for B cells (1). The absence of T_FH cells results in the loss of germinal centers (GC) and, consequently, abrogated memory B cell, plasma cell, and neutralizing antibody responses. Thus, T_FH cells have critical roles in protective immune responses against pathogens, as well as deleterious roles in numerous autoimmune diseases (1, 2).

Bcl6 consists of a bric-a-brac, tramtrack, broad-complex (BTB/POZ) domain, a middle domain (also known as RDII), and a zinc finger domain consisting of six krueppel-like zinc fingers (1). BTB domains are evolutionarily conserved protein interaction domains that are widely present in transcription factors (3, 4). The BTB domain forms the interface of the obligate homodimer and the corepressors BCOR, SMRT, and NCOR bind at the cleft formed by this interface (5-8). While Bcl6 is required for T_FH differentiation (9-12), the contributions of its functional domains in CD4⁺ T cells are not well understood. In this study, we sought to examine to role of the Bcl6 BTB domain in T_FH differentiation and function.

MATERIALS AND METHODS

Mice and vectors

C57BL/6J (B6) and Cre^CD4 mice were purchased from the Jackson Laboratory. Bcl6^fl/fl (13), CD45.1 congenic and Smarta TCR transgenic (‘SM’, specific for LCMV GP (glycoprotein) 66-77 on I-A^b) (14) mice were on a full B6 background and were bred at LJI. Bcl6^BTBMUT mice, engineered to express the Bcl6 BTBmut from the endogenous Bcl6 locus, were generously provided by Dr. Ari Melnick (15), which were crossed to homozygosity at LJI for use in all experiments. NIP TCR transgenic mice were generated as described below and in Supplementary Figure 1. TCR hybridomas were generated (Janice White and Philippa Marrack, unpublished data) and TCR sequences were cloned and sequenced using cDNA isolated from LCMV-reactive clones. TCR sequences were expressed in 58 αβ⁻ T cell hybridomas and tested for reactivity against LCMV-infected dendritic cells. The TCRαβ pair showing the strongest reactivity (Vα1-Jα8 and Vβ6-Dβ1-Jβ2.3 rearrangements) was chosen and cloned into genomic TCR expression cassette vectors. Linearized DNA fragments were injected into fertilized C57BL/6 eggs at the UCSD transgenic mouse facility. Pups were genotyped (Fig. S1). A single α/β TCR transgenic founder mouse (“NIP”) was selected and crossed to B6.SJL mice to generate CD45.1⁺ NIP mice. All animal experiments were conducted in accordance with approved animal protocols. The green fluorescent protein (GFP)-expressing retroviral (RV) expression vector pMIG was used. BTB domain mutant Bcl6 RV (BTBmut) was generated by inducing two point mutations in the protein interaction domain that do not affect dimerization (16). RV particles were produced as previously described (9).

Infections and immunizations

Lymphocytic choriomeningitis virus (LCMV) Armstrong stocks were prepared and quantified as previously described (9). Infections were performed by intraperitoneal injection of 0.5-2 × 10⁵ plaque-forming units of LCMV Armstrong per mouse. GP61-keyhole limpet hemocyanin (KLH) was prepared in alum and injected as described previously (17). A total of 20 μg GP61-KLH was resuspended in alum for bilateral footpad injections.

Flow cytometry

Flow cytometry was done with monoclonal antibodies against SLAM (CD150, Biolegend) and CD4, CD8, CD44, CD62L, CD25, B220, Fas, and GL7 (eBioscience). Stains were done for 30 minutes at 4 °C in PBS supplemented with 0.5% bovine serum albumin and 0.1% sodium azide, unless specified otherwise. CXCR5 staining was done as described (9, 18). Intracellular staining for Bcl6 was performed with an Alexa 647-conjugated monoclonal antibody to Bcl6 (clone K112-91) (BD Pharmingen) using the FoxP3 ICS kit buffers and protocol (eBioscience).

Statistical Analysis

Statistical tests were performed using Prism 5.0 (GraphPad). P values were calculated by two-tailed unpaired Student’s t tests with a 95% confidence interval. Error bars depict the standard error of the mean. *, p < 0.05. **, p < 0.01, ***, p < 0.001.

RESULTS AND DISCUSSION

The Bcl6 BTB domain regulates T_FH differentiation

Given that the Bcl6 BTB domain is known to interact with corepressors (such as BCOR) in B cells that are also expressed in CD4 T cells, we sought to determine the role of the Bcl6 BTB domain in T_FH differentiation. To this end, we generated N21K and H116A mutations in the Bcl6 BTB domain. These point mutations together have been shown to prevent co-repressor binding to the BTB domain without affecting the ability of Bcl6 to dimerize (16). Similar levels of Bcl6 protein expression were observed in CD4 T cells transduced with an RV expressing either WT-Bcl6 or the mutant form (BTBmut), and the amount of Bcl6 expression was comparable to that of GC T_FH cells (Fig. 1A). To examine the role of the Bcl6 BTB domain in vivo, we determined if expression of the Bcl6 BTBmut protein in Bcl6-deficient cells could rescue Tfh development in response to acute LCMV infection. LCMV glycoprotein-specific Bcl6^fl/fl Cre^CD4 Smarta (SM) TCR transgenic CD45.1 cells were transduced with Bcl6 WT, BTBmut, or an empty GFP vector (GFP) and transferred to Bcl6^fl/fl Cre^CD4 hosts. We first examined early T_FH development 3 days following an acute LCMV infection. Cells transduced with the empty vector (GFP⁺) did not differentiate into T_FH cells, confirming the requirement of Bcl6 for T_FH differentiation. Ectopic expression of WT Bcl6 was sufficient to rescue T_FH development (Fig. 1B). We observed a defect in early T_FH differentiation in BTBmut⁺ SM cells compared with Bcl6-WT⁺ SM (p < 0.0001, Fig. 1B). We then examined T_FH and GC B cell development at 7 days following acute LCMV infection. Again, there was a significant impairment in T_FH differentiation in BTBmut⁺ cells (p < 0.0001, Fig. 1C) compared with WT. This was coupled with an even larger reduction in GC T_FH cells (p = 0.006, Fig. 1D). GC T_FH cells are the more polarized T_FH cells that are present in the germinal centers and are identifiable as CXCR5^hiPD1^hiPSGL1^loBcl6^hi cells (1). In parallel we observed a significant defect in GC B cell development (p = 0.005, Fig. 1E. Thus, Bcl6 BTB domain functions appeared to be necessary for the majority of T_FH differentiation and B cell help.

Figure 1. The Bcl6 BTB domain is necessary for proper T_FH cell differentiation.

(A) Similar levels of Bcl6 expression in Bcl6-RV, BTBmut-RV and GC T_FH. Bcl6 MFI of Bcl6^fl/fl Cre^CD4 CD4 T cells transduced with either BTBmut-RV or Bcl6-RV compared to ex vivo GC T_FH isolated from mice at 7 days following LCMV infection. (B-E) Bcl6^fl/fl Cre^CD4 SM cells were retrovirally transduced with empty GFP vector, Bcl6 WT, or BTBmut, then transferred to Bcl6^fl/fl Cre^CD4 mice and analyzed at day 3 or 7 following an acute LCMV infection. (B) Early Tfh differentiation (CXCR5^hi SLAM^lo) by transduced Bcl6^fl/fl Cre^CD4 SM cells at day 3. (C) Tfh differentiation (CXCR5^hi SLAM^lo) by transduced Bcl6^fl/fl Cre^CD4 SM cells at day 7. (D) GC T_FH (CXCR5^hi PD1^hi) by transduced Bcl6^fl/fl Cre^CD4 SM cells at day 7. (E) GC B cells (PNA^hi Fas^hi) at day 7. At least 3 mice were used for each condition. Experiments were repeated at least 3 times. * p < 0.05, ** p < 0.01, *** p < 0.001.

Impaired T_FH differentiation by germline Bcl6^BTBMUT CD4 T cells

These results are different from a recent report using mixed bone marrow chimeric mice with Bcl6^BTBMUT and Tcrb^−/− donor cells, in which no difference in total Tfh frequencies were observed upon immunization with sheep red blood cells (SRBC), though antigen-specific cells were not directly assessed in that study (19). Therefore, in light of those data, it was incumbent upon us to explore the role of the Bcl6 BTB domain in T_FH differentiation in multiple contexts, directly examining antigen-specific Tfh cells in each case. Thus, we obtained the germline “knock-in” Bcl6^BTBMUT mice for further studies. CD4 T cells were isolated from Bcl6^BTBMUT SM CD45.1⁺ or WT SM CD45.1⁺ mice and transferred to Bcl6^fl/fl Cre^CD4 hosts. Antigen-specific T_FH and GC T_FH differentiation was examined at 7 days following acute LCMV infection. We observed a significant defect in both T_FH (p = 0.017, Fig. 2A) and GC T_FH differentiation (p = 0.014, Fig. 2B), confirming the requirement for the Bcl6 BTB domain in the generation of T_FH cells.

Figure 2. Impaired T_FH differentiation by Bcl6^BTBMUT CD4 T cells.

Bcl6^BTBMUT SM or WT SM cells were transferred to Bcl6^fl/fl Cre^CD4 mice and analyzed 7 days following acute LCMV infection. (A) Tfh (CXCR5^hi SLAM^lo) Bcl6^BTBMUT SM cells. (B) GC T_FH (CXCR5^hi PSGL1^lo) Bcl6^BTBMUT SM cells. At least 3 mice were used for each condition. Experiments were repeated at least 3 times. * p < 0.05, ** p < 0.01, *** p < 0.001.

BTB dependent T_FH differentiation in NIP-transgenic Bcl6^BTBMUT CD4 T cells

One possibility is that this observed phenotype may be selective to the SM CD4 T cell response, though SM CD4 T cells have been very informative in numerous contexts (9, 14, 17, 20). To this end, we developed a new LCMV-specific TCR transgenic mouse. The LCMV nucleoprotein (NP) is the major target of the antibody response during LMCV infection, and the currently used GP-specific SM CD4 T cells do not allow for the study of the immunodominant antibody response because GP-specific CD4 T cells do not provide help to NP-specific B cells (14). Epitope mapping defined a specific peptide (NP 311-325) of the NP protein efficiently recognized by CD4 T cells during LCMV infection (21, 22). For this reason, NP 311-325 was chosen as a target to generate a novel TCR transgenic mouse model (Fig. S1). NIP-transgenic CD4 T cells transferred into B6 mice exhibited robust expansion and differentiated into T_FH and GC T_FH cells following LCMV infection (Fig. S1B-E). NIP CD4 T cells transferred into B6 hosts were able to induce robust B cell responses, including the generation of GC B cells (Fig. S1F), plasma cells (Fig. S1G), and LCMV-specific antibodies (Fig. S1H).

We examined the role of the Bcl6 BTB domain in the context of a retrogenic mouse model where bone marrow (BM) from Bcl6^BTBMUT or B6 (WT) mice was transduced with an NP-specific TCRα and TCRβ retroviral construct (NIP-RV) and the transduced bone marrow was transferred into irradiated WT recipients (23). WT or Bcl6^BTBMUT NP-specific naive CD4 T cells were isolated from these mice after reconstitution and transferred to Bcl6^fl/fl Cre^CD4 hosts. The NIP⁺ WT and NIP⁺ Bcl6^BTBMUT CD4 T cells expanded robustly in response to an acute LCMV infection. At 7 days following LCMV infection, we observed a significant defect in T_FH differentiation (Fig. S2) and GC T_FH differentiation (Fig. 3A, Fig. S2) for Bcl6^BTBMUT NIP-RV⁺ compared to WT NIP-RV⁺ mice (p = 0.01 and p = 0.0014). We then made use of germline NIP TCR transgenic mice crossed with Bcl6^BTBMUT mice. Bcl6^BTBMUT NIP or WT NIP CD4 T cells were transferred into Bcl6^fl/fl Cre^CD4 recipients followed by LCMV infection. We observed severe defects in T_FH (p = 0.0088, Fig. 3B) and GC T_FH (p = 0.0006, Fig. 3C) differentiation in Bcl6^BTBMUT NIP CD4 T cells. The consequence of the substantial loss of GC Tfh was a significant loss of GC B cell development (Fig. 3D) and reduced anti-LCMV IgG response (Fig. 3E). Taken together, these data demonstrate that the Bcl6 BTB domain is important for T_FH differentiation and function in response to at least two different antigens.

Figure 3. Bcl6^BTBMUT LCMV-specific NIP transgenic CD4 T cells are defective in T_FH cell differentiation and function.

CD4 T cells from Bcl6^BTBMUT NIP-TCR⁺ retrogenic mice were transferred to Bcl6^fl/fl Cre^CD4 mice and analyzed at day 7 following acute LCMV infection. (A) GC Tfh cells (CXCR5^hi PSGL1^lo) among Bcl6^BTBMUT NIP-TCR⁺ cells. (B-E) Bcl6^BTBMUT NIP or WT NIP cells were transferred to Bcl6^fl/fl Cre^CD4 mice and analyzed following acute LCMV infection. (B) Tfh (CXCR5^hi SLAM^lo) Bcl6^BTBMUT NIP cells. (C) GC T_FH (CXCR5^hi PSGL1^lo) Bcl6^BTBMUT NIP cells. (D) GC B cells (PNA^hi Fas^hi). (E) LCMV-specific IgG. At least 3 mice were used for each condition. Experiments in B-D were repeated at least 3 times. * p < 0.05, ** p < 0.01, *** p < 0.001.

The Bcl6 BTB domain regulates T_FH differentiation and GC B cell development following protein immunization

To examine the role of the Bcl6 BTB domain in another experimental context we utilized a protein immunization model, keyhole limpet hemocyanin (KLH) conjugated with gp61-80 peptide in alum (17). We transferred Bcl6^BTBMUT SM or WT SM CD4 T cells into Bcl6^fl/fl Cre^CD4 hosts followed by immunization with GP61-KLH in alum. At 10 days following immunization, lymph nodes were harvested and examined for antigen-specific T_FH differentiation and function. We observed a significant reduction in CXCR5 expression by Bcl6^BTBMUT CD4 T cells (p = 0.0195, Fig. 4A) and a severe defect in GC T_FH differentiation (p < 0.0001, Fig. 4B). Bcl6^BTBMUT CD4 T cells were functionally deficient as they were unable to promote GC B cell responses (p = 0.0008, Fig. 4C). We also observed defects in T_FH and GC T_FH differentiation in Bcl6^fl/fl Cre^CD4 mice receiving BTBmut-RV⁺ SM CD4 T cells as compared with Bcl6-WT-RV⁺ SM CD4 T cells at 10 days following immunization (data not shown). Thus, the Bcl6 BTB domain is needed for optimal T_FH differentiation and function in both the context of protein immunizations and an acute viral infection.

Figure 4. Defective T_FH differentiation and function in the absence of a function Bcl6 BTB domain following protein immunization.

Bcl6^BTBMUT SM or WT SM cells were transferred to Bcl6^fl/fl Cre^CD4 mice and analyzed following GP61-KLH (alum) immunization, day 10. (A) Tfh (CXCR5^hi SLAM^lo) Bcl6^BTBMUT SM cells. (B) GC T_FH (CXCR5^hi PD1^hi) Bcl6^BTBMUT SM cells. (C) GC B cells (PNA^hi Fas^hi). At least 3 mice were used for each condition. Experiments were repeated at least 3 times. * p < 0.05, ** p < 0.01, *** p < 0.001.

Our results demonstrate the importance of the Bcl6 BTB repressor domain in CD4 T cells in the differentiation of T_FH cells and in the ability of T_FH cells to provide help to B cells. A previous study did not observe a T_FH defect in Bcl6^BTBMUT CD4 T cells (19), possibly because antigen-specific T_FH were not directly analyzed, or possibly due to a confounding negative feedback Bcl6 autoregulatory mechanism involving cullin3, whereby cullin3 is most likely recruited via the Bcl6 BTB domain (24). Alternatively, the BTB domain of Bcl6 has more prominent roles for Bcl6 activity in some in vivo contexts than others. Bcl6 functions in T_FH cells via binding to thousands of genes, demonstrating that Bcl6 is deeply integrated into T_FH cell biology (25). In this study, T_FH defects in the absence of BTB domain function were consistent when assessing antigen-specific T_FH cells, using two different antigen specificities, as well as in the context of both acute viral infection and protein immunization. The defect in T_FH differentiation and function that was observed is likely due to the inability of Bcl6 to recruit corepressors such as SMRT, NCOR, and BCOR and thus repress target genes. Indeed, it has been found that BCOR-deficient CD4 T cells are defective for GC T_FH differentiation and B cell help functions (26). Thus, BCOR may be the main co-repressor interacting with the Bcl6 BTB domain in T_FH cells. SMRT, NCOR, and BCOR are all expressed in CD4 T cells, and further investigation of the comparative roles of each of these Bcl6 BTB domain interacting transcription factors is needed. This report highlights the importance of the repressive ability of the Bcl6 BTB domain in T_FH cells for successful germinal center reactions and robust antibody responses.