ARTICLES Lineage-specific functions of Bcl-6 in immunity and inflammation are mediated by distinct biochemical mechanisms Chuanxin Huang, Katerina Hatzi & Ari Melnick The transcription factor Bcl-6 orchestrates germinal center (GC) reactions through its actions in B cells and T cells and regulates inflammatory signaling in macrophages. Here we found that genetic replacement with mutated Bcl6 encoding Bcl-6 that cannot bind corepressors to its BTB domain resulted in disruption of the formation of GCs and affinity maturation of immunoglobulins due to a defect in the proliferation and survival of B cells. In contrast, loss of function of the BTB domain had no effect on the differentiation and function of follicular helper T cells or that of other helper T cell subsets. Bcl6-null mice had a lethal inflammatory phenotype, whereas mice with a mutant BTB domain had normal healthy lives with no inflammation. The repression of inflammatory responses by Bcl-6 in macrophages was accordingly independent of the repressor function of the BTB domain. Bcl-6 thus mediates its actions through lineage-specific biochemical functions. Bcl-6 is a transcriptional repressor originally identified as being Bcl-6 is a member of the BTB–zinc finger family of proteins. Its BTB encoded by a locus frequently translocated in diffuse large B cell lym- domain forms an obligate homodimer and it contains C2H2 zinc fin- phomas (DLBCLs)1. During normal development, Bcl-6 has critical gers that bind to DNA. The interface between monomers of the Bcl-6 functions in various cell types of the adaptive and innate compart- BTB domain creates two symmetrical extended lateral grooves that ments of the immune system. Bcl-6 expression undergoes substantial form docking sites for the corepressors SMRT, NCOR and BCOR20–22. upregulation in B cells after challenge with T cell–dependent antigens2, Those three corepressors bind to Bcl-6 via an unstructured 18–amino and Bcl-6 is required for the formation of germinal centers (GCs) acid Bcl-6-binding domain23. The Bcl-6-binding domains of NCOR in which B cells undergo affinity maturation of immunoglobulins. and SMRT are identical, whereas that of BCOR is completely differ- © 2013 Nature America, Inc. All rights reserved. America, Inc. © 2013 Nature Bcl-6-deficient (Bcl6–/–) mice do not form GCs and thus are unable ent, yet all three bind to the lateral groove of the Bcl-6 BTB domain to generate high-affinity antibodies3–5. The proposed biological func- in perfectly overlapping configurations23,24. Substitution of lysine tion of Bcl-6 in B cells in the GC is to facilitate the simultaneous rapid for asparagine at position 21 (N21K) and of alanine for histamine at npg proliferation and tolerance to the genomic damage that occurs during position 116 (H116A) in the Bcl-6 BTB domain completely abrogates clonal expansion and somatic hypermutation by directly repressing the binding of Bcl-6 to NCOR, SMRT and BCOR without impairing genes encoding molecules involved in DNA-damage sensing and folding or dimerization23. Mutant Bcl-6 with those N21K and H116A checkpoints, such as ATR6, CHEK1 (ref. 7), EP300 (ref. 8), TP53 (ref. 9) substitutions in its BTB domain is completely inactive, which indicates 10 and CDKN1A . Follicular helper T cells (TFH cells), specifically that the lateral groove–Bcl-6-binding domain interface is responsible those in GCs, are specialized CD4+ helper T cells that provide help to for the repressor activity of the Bcl-6 BTB domain23. However, Bcl-6 B cells during the GC reaction11,12. Bcl-6 expression is upregulated also has a middle autonomous repression region often called ‘repres- –/– 25 during the differentiation of TFH cells, and Bcl6 T cells do not dif- sion domain 2’ (RD2) , which may recruit other corepressors, such as 13–15 26,27 ferentiate into TFH cells in vivo . Constitutive expression of Bcl-6 NuRD and CTBP . The sequence of the reported Bcl-6 consensus 14–16 enhances the differentiation of TFH cells . The requirement for binding site (TTCCT(A/C)GAA, where A/C indicates either adeno- Bcl-6 in both B cells and TFH cells in GCs is cell autonomous, and loss sine or cytosine) overlaps that of STAT transcription factor–binding of Bcl-6 in either cell type leads to abrogation of the GC reaction17. sites25,28,29, and several lines of evidence indicate that Bcl-6 may Bcl-6 also has an important role in macrophages, in which it mediates antagonize STAT signaling, with potential relevance for inflamma- a dampening effect on inflammatory signaling through repression tory and innate immunological functions3,30,31. of chemokine expression and target genes of the transcription fac- Collectively, the data suggest complex biological roles for Bcl-6 in tor NF-κB18,19. Bcl6–/– mice develop a lethal inflammatory disease the immune system. However, the link between the transcriptional caused by the interaction and crosstalk between macrophages and mechanisms of action of Bcl-6 and its biological actions in the immune helper T cells. system remains unknown. Here we generated a knock-in mouse model Division of Hematology and Oncology, Weill Cornell Medical College, New York, New York, USA. Correspondence should be addressed to A.M. ([email protected]). Received 10 September 2012; accepted 10 January 2013; published online 3 March 2013; doi:10.1038/ni.2543 380 VOLUME 14 NUMBER 4 APRIL 2013 NATURE IMMUNOLOGY ARTICLES a PNA B220 + Bcl-6 b 15 Day 7 Day 10 Day 14 d e +/+ BTBMUT –/– +/+ BTBMUT –/– ) Bcl6 Bcl6 Bcl6 Bcl6 Bcl6 Bcl6 4 5 5 15.10 8.12 8.20 2.25 1.10 0.28 10 10 10 × ** ** ** 10 4 4 +/+ m 10 10 Bcl6 µ 103 103 5 102 5.77 0.42 0.20 102 0 0 CXCR5 CD38 GC size ( 2 3 4 5 0 102 103 104 105 0 10 10 10 10 0 Fas PD-1 +/+ +/+ +/+ Bcl6BTBMUT Bcl6 BTBMUTBcl6 BTBMUTBcl6 BTBMUT Day 0 Day 10 Day 0 Day 10 Day 0 Day 10 8 15 20 Bcl6 Bcl6 Bcl6 ** ** * Bcl6+/+ 6 cells (%) 15 4 cells (%) c BTBMUT cells (%) 10 lo–hi Bcl6 hi ) 2 4 10 3 ** lo–neg PD-1 –/– ** PD-1 Bcl6 * hi 5 5 2 lo–hi 2 CD38 + 0 CXCR5 0 0 1 Fas GCs (per mm +/+ –/– +/+ –/– +/+ –/– +/+ –/– CXCR5 +/+ –/– +/+ –/– 0 Bcl6BTBMUTBcl6Bcl6 BTBMUTBcl6 Bcl6 BTBMUTBcl6Bcl6 BTBMUTBcl6 Bcl6 BTBMUTBcl6Bcl6 BTBMUTBcl6 Day 7 Day 10 Day 14 Bcl6 Bcl6 Bcl6 Bcl6 Bcl6 Bcl6 Figure 1 Impaired GC formation in Bcl6BTBMUT mice. (a) Immunohistochemistry of paraffin-embedded serial spleen sections 10 d after immunization of mice with SRBCs. Arrows (middle) indicate GCs; insets present enlargement of those GCs. PNA, peanut agglutinin. Scale bars, 200 µm (main images); original magnification (insets), ×20. (b,c) Size (b) and number (c) of GCs in spleen sections 7, 10 and 14 d after immunization of mice with SRBCs. (d) Flow cytometry (top) of GC B cells, gated on live B220+ splenic lymphocytes obtained from mice 10 d after immunization with SRBCs. Numbers in outlined areas indicate Fas+CD38lo–neg cells (GC B cells). Below, quantification of GC B cells among live B220+ cells, before and 10 d – + after immunization. (e) Flow cytometry of GC and total TFH cells, gated on live B220 CD4 splenic lymphocytes from mice 10 d after immunization hi hi lo–hi lo–hi with SRBCs. Numbers above outlined areas indicate CXCR5 PD-1 cells (right; GC TFH cells) and CXCR5 PD-1 cells (left; total TFH cells). – + Below, quantification of GC TFH and TFH cells among live B220 CD4 cells. Each symbol (b,d,e) represents an individual GC (b) or mouse (d,e); small horizontal lines indicate the mean (± s.e.m.). *P < 0.01 and **P < 0.001 (two-tailed t-test). Data are representative of five (a) or three (b–e) independent experiments (mean and s.e.m. of two to five mice). in which the endogenous Bcl6 locus encodes a mutant form of Bcl-6 chromatin-immunoprecipitation (ChIP) assays showed that Bcl-6 protein with the N21K and H116A point substitutions in the BTB protein with the mutant BTB domain retained the same ability to domain (Bcl6BTBMUT). The facts that SMRT, NCOR and BCOR are bind target genes in cultured macrophages as that of wild-type Bcl-6 coexpressed with Bcl-6 in the relevant cell types and that the mecha- protein (Supplementary Fig. 2d). The abundance of SMRT at Bcl-6 nism of the BTB domain is the only well-characterized biochemical target genes in Bcl6BTBMUT mice was identical to that in Bcl6–/– mice function of Bcl-6 favor the proposal that the biological ‘readout’ of such (Supplementary Fig. 2d), which confirmed that these substitutions in a knock-in model would be most rigorously interpretable. Notably, our the lateral groove abrogated interaction with lateral groove–binding data suggest that the transcriptional mechanisms of action of Bcl-6 are corepressors. The mutated Bcl6 alleles thus encoded a viable tran- specific to the lineage and biological function, with notable implica- scription factor able to bind to its targets but unable to recruit core- tions for the general understanding of how Bcl-6 and other transcrip- pressors through its BTB domain. © 2013 Nature America, Inc. All rights reserved. America, Inc. © 2013 Nature tion factors work, as well as for the ‘translation’ of such results into the clinical use of inhibitors of Bcl-6. Impaired GC formation in Bcl6BTBMUT mice Phenotypic analysis showed normal early development of B cells in the npg RESULTS bone marrow and spleens of Bcl6BTBMUT mice, as well as normal distri- Viability of Bcl6BTBMUT mice bution of peripheral T cells (Supplementary Fig. 3a–c). Bcl6BTBMUT To address the biological function of interactions between the Bcl- mice also formed normal splenic primary lymphoid follicles (data not 6 BTB domain and its corepressors in vivo, we introduced point shown).