© 2006 Nature Publishing Group http://www.nature.com/natureimmunology
ARTICLES
Transcription factor IRF4 controls plasma cell differentiation and class-switch recombination Ulf Klein1, Stefano Casola2, Giorgio Cattoretti1, Qiong Shen1, Marie Lia1, Tongwei Mo1, Thomas Ludwig1, Klaus Rajewsky2 & Riccardo Dalla-Favera1 B cells producing high-affinity antibodies are destined to differentiate into memory B cells and plasma cells, but the mechanisms leading to those differentiation pathways are mostly unknown. Here we report that the transcription factor IRF4 is required for the generation of plasma cells. Transgenic mice with conditional deletion of Irf4 in germinal center B cells lacked post–germinal center plasma cells and were unable to differentiate memory B cells into plasma cells. Plasma cell differentiation required IRF4 as well as the transcriptional repressor Blimp-1, which both acted ‘upstream’ of the transcription factor XBP-1. In addition, IRF4-deficient B cells had impaired expression of activation-induced deaminase and lacked class-switch recombination, suggesting an independent function for IRF4 in this process. These results identify IRF4 as a crucial transcriptional ‘switch’ in the generation of functionally competent plasma cells.
The hallmark of antibody-mediated adaptive immunity is the generation of plasma cells producing high titers of antigen-specific antibodies as well as memory B cells destined to become plasma cells after reencounter with antigen. However, the mechanisms that control the differentiation of germinal center (GC) B cells toward the plasma cell or memory B cell pathway are not known. Both cell types derive from antigen-activated B cells that have undergone the ‘GC reaction’, in which they specifically modify their immunoglobulin through somatic hypermutation and class-switch recombination (CSR)1,2. The commitment to plasma or memory cell differentiation is thought to occur in a specific area in the GC structure, the light zone, where GC centrocytes seem to undergo specific changes in their transcriptional program. Specifically, centrocytes downregulate the transcription factor Bcl-6, which is required for GC formation and maintenance3,4, and activate the transcription factors Blimp-1 (ref. 5), IRF4 (ref. 6), BMI-1 (ref. 7), NF-kB8, STAT3 (ref. 9) and STAT5 (ref. 10). Although Blimp-1 has been shown to be required for the generation of plasma cells11 and STAT3, for those of a specific isotype9, the overall transcriptional programs necessary for plasma cell versus memory B cell differentiation have not been identified. Because of its expression in GC centrocytes destined to become plasma cells or memory B cells, we have investigated the function of IRF4 in post-GC B cell differentiation. IRF4 (also called Pip12, LSIRF13, ICSAT14 and MUM1 (ref. 15)) is a member of the interferon-regulatory factor family of transcription factors characterized by a specific DNA-binding domain and the ability to bind to regulatory elements in promoters of interferon-inducible genes16.
Expression of IRF4 is restricted to cells of the immune system, including lymphocytes, dendritic cells and macrophages17,18, in which it has been linked to a variety of functions, including proliferation, apoptosis and differentiation17,18. That heterogeneous and often ‘opposite’ mode of action may be explained by alternative interaction with cofactors, including Pu.1 (ref. 12), E47 (ref. 19), STAT-6 (ref. 20) and IRF8 (ref. 21), which can influence IRF4 transcriptional activity. In the B lineage, IRF4 has a biphasic expression pattern: it is expressed in immature B cells in the bone marrow21 and is absent from proliferating GC centroblasts6 and then is reexpressed in a subpopulation of centrocytes in the GC and in plasma cells6. Consistent with involvement of IRF4 in early B cell development, mice with germline deletion of IRF4 have a differentiation block at the transition from the immature to the mature, follicular B cell22 and, as a consequence, lack progeny of the latter cell type, including GC as well as memory B cells and plasma cells. However, because of the developmental block at the immature B cell stage, possible involvement of IRF4 reexpression in post-GC differentiation and in particular in plasma cell–versus–memory B cell commitment cannot be investigated in those mice22. Elucidation of the function of IRF4 in late B cell differentiation is likely to be relevant to understanding normal mature B cell development as well as the genesis of B cell lymphomas. Indeed, IRF4, originally identified also as the product of a proto-oncogene involved in chromosomal translocations in multiple myeloma, is capable of transforming cells in vitro15 and is often abnormally expressed in B cell lymphomas6,23,24. To investigate the involvement of IRF4 in post-GC B cell development, we have generated mice in which Irf4 can be conditionally
1Institute 2CBR
for Cancer Genetics, Department of Pathology and Herbert Irving Comprehensive Cancer Center, Columbia University, New York, New York 10032, USA. Institute for Biomedical Research, Harvard Medical School, Boston, Massachusetts 02115, USA. Correspondence should be to R.D.-F. (
[email protected]).
Received 15 March; accepted 17 May; published online 11 June 2006; doi:10.1038/ni1357
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ARTICLES which recombine frt sites early in embryogenesis25. We identified correctly targeted CD19-enriched: embryonic stem cell lines by Southern blot Floxed Floxed (15.1 kb) 15.1 kb analysis (Supplementary Fig. 1) and tested WT (10.2 kb) WT ∆loxP (7.6 kb) 10.2 kb deletion of the floxed Irf4 promoter region ∆frt (6.0 kb) ∆loxP with simultaneous activation of eGFP tran7.6 kb scription in targeted embryonic stem cells by Nhe l, 3′ probe eGFP transient Cre expression (Fig. 1a). We conc Cγ1-Cre firmed germline transmission of the ‘condiIRES Cre 3′ UTR tional’ Irf4 allele by Southern blot analysis. CH1 H CH2 CH3 M1 M2 frt We crossed the resulting mice heterozygous Irf4 mRNA d e for a frt-flanked, floxed Irf4 allele (Irf4frtfl/+ E1 E2 Irf4 Cond. mice; called ‘Irf4fl/+ mice’ here) with Flp85.6% frt lox P lox P frt × Cγ1-Cre Irf4 Null transgenic mice to generate an Irf4-null allele frt (called ‘Irf4+/– mice’ here; Supplementary in GC and Fig. 1). Southern blot analysis of purified post-GC B cells All cells CD19+ B cells from Irf4fl/–CD19-Cre and eGFP mRNA Irf4fl/+CD19-Cre mice, in which Cre is + under transcriptional control of the B lineIrf4 Null (eGFP ) frt lox P frt × Cγ1-Cre age–restricted gene encoding CD19, showed a IgG1 eGFP Irf4 Null 7.6–kilobase (kb) band consistent with delefrt tion of the floxed region (Fig. 1b), demonFigure 1 Functional analysis of the conditionally deleted Irf4 allele. (a) Left, flow cytometry of eGFP strating the ability of the ‘conditional’ Irf4 fl/+ expression by adeno-Cre–treated Irf4 embryonic stem cells after loxP-mediated deletion of the Irf4 allele to recombine in vivo. We verified that locus. Right, Southern blot analysis of NheI-digested DNA from embryonic stem cell cultures, showing the ‘conditional’ Irf4 allele conferred an Irf4the expected 7.6-kb band after loxP-mediated deletion (DloxP) along with the 15.1-kb loxP-flanked null phenotype after homozygous deletion allele (Floxed) and the 10.2-kb wild-type allele (WT; Supplementary Fig. 1). (b) Southern blot analysis of NheI-digested DNA from purified CD19+ and CD19– B cells. CD19-enriched B cells from Irf4fl/+ (Supplementary Fig. 2 online). We conCD19-Cre mice have the wild-type allele and the allele obtained after loxP-mediated deletion (DloxP) firmed the ability of the ‘conditional’ allele and those from Irf4fl/–CD19-Cre mice have the allele obtained after loxP-mediated deletion and the to produce physiological amounts of IRF4, allele obtained after frt-mediated deletion (Dfrt); the faint 7.6-kb band in the CD19– Irf4fl/– sample is despite the insertion of several gene ‘cassettes’ due to cellular contamination of B cells. (c) ‘Knock-in’ of the internal ribosomal entry site (IRES)–Cre (Supplementary Fig. 1) into the Irf4 locus, cassette into the Cg1 locus. Black slim rectangles, Cg1 exons. UTR, untranslated region. (d) Status of fl/– by comparing the phenotype and antibody Irf4 before (top) and after Cre-mediated recombination in GC B cells of Irf4 Cg1-Cre mice. The response of Irf4fl/– mice after immunization strategy allows identification of Irf4–/– B cells by eGFP expression. Cond., conditional. (e) Flow cytometry of splenic Thy-1–IgD–IgM–F4/80–Gr-1– cells isolated from NP-KLH–immunized Irf4fl/+Cg1-Cre with a T cell–dependent antigen to that of mice to assess the fraction of eGFP+ B cells among NP-binding IgG1+ B cells, indicating deletion of Irf4+/– and/or Irf4fl/+ mice (Supplementary Irf4 (42 d after primary immunization). Right, eGFP expression of all cells (bottom) and of the gated Fig. 2 and Supplementary Table 1 online). NP+IgG1+ population (top); number above bracketed line indicates percentage of NP+IgG1+ eGFP+ We then crossed the mice with the ‘condicells. Representative result of three independent experiments. tional’ Irf4 allele with Cg1-Cre mice, which express Cre recombinase from a bicistronic deleted in GC B cells and the fate of the cells carrying the deletion can mRNA in which the Cre coding sequence (preceded by an internal be monitored in vivo. We show that IRF4 is critical for plasma cell ribosomal entry site) is downstream of the immunoglobulin heavychain g1 constant region (Cg1) coding region, allowing for its differentiation and CSR. independent translation26 (Fig. 1c). In these mice, Cre recombinase RESULTS is expressed after the induction of germline Cg1 transcription by Mice with conditional deletion of Irf4 in GC B cells immunization with a T cell–dependent antigen, which precedes To study the involvement of IRF4 in GC and post-GC development, switching to IgG1 (ref. 26). In unimmunized mice, less than 2% of we generated a transgenic mouse strain carrying a loxP-flanked total splenic B cells show Cre-mediated recombination; this fraction (floxed) Irf4 allele and crossed those mice with mice expressing Cre increases to include 85% of GC B cells (which are rare in unimrecombinase specifically in GC B cells. We effected conditional munized mice) after immunization26. Cre-mediated recombination deletion of Irf4 by placing loxP sites upstream of the defined promoter involves most IgG1-expressing cells and a small fraction of B cells that region of Irf4 and downstream of exon 2, containing the translational have switched to immunoglobulin classes other than IgG1 (ref. 26). As start, respectively (Supplementary Fig. 1 online). To allow tracking of the Cg1 locus is transcribed in a subset of GC B cells, these mice allow IRF4-deficient cells after Cre-mediated recombination, we placed a analysis of the function of IRF4 in GC B cells as well as in progeny gene encoding enhanced green fluorescent protein (eGFP) in an plasma cells and memory B cells, which are both derived from GC B opposite orientation upstream of the Irf4 promoter region, leading cells. Furthermore, cells with deletion of Irf4 can be specifically to its expression by the mouse phosphoglycerate kinase promoter identified in vivo by tracking of eGFP expression (Fig. 1d). We placed in intron 2 (Supplementary Fig. 1). The targeting construct analyzed the extent of the Cg1-Cre–mediated deletion of Irf4 in vivo was flanked by frt sites, which, analogous to the loxP site–Cre by determining the fraction of eGFP+ versus eGFP– IgG1+ cells in the recombinase system, are recognized specifically by Flp recombinase spleens and peripheral blood of Irf4fl/+Cg1-Cre mice in a T cell– to facilitate the generation of an Irf4-null allele lacking eGFP expres- dependent immune response (Fig. 1e). After primary immunization, sion (Supplementary Fig. 1) in mice transgenic for Flp recombinase, 87.1% ± 2.8% of splenic and 83.6% ± 11.4% of peripheral blood B Adeno-Cre: – +
P
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Irf4 fl/– Irf4 fl/+ CD19-Cre CD19-Cre – + – +
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Cell count
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ARTICLES Figure 2 Development of GC B cells in Irf4fl/–Cg1-Cre mice. Flow cytometry of spleen cells from Irf4fl/–Cg1-Cre and Irf4fl/+Cg1-Cre mice isolated 14 d after immunization with sheep red blood cells, to assess B220, PNA, eGFP and CD38. Histograms show eGFP expression of the gated PNAhiB220+ or PNAloB220+ populations (middle) and CD38 expression of the gated eGFP+PNAhi–loB220+ populations (right). Representative result of experiments using two mice each genotype on days 10, 12 and 14 after immunization.
35.1%
2.2%
2.0%
CD38
cells were eGFP+; after secondary immunization, 78.5% ± 4.4% of splenic antigen-binding IgG1+ B cells were eGFP+ (Fig. 1e). These results were similar to those obtained after crossing of mice with the Cg1-Cre–encoding allele with various reporter mice26 and demonstrate a high efficiency of Cre-mediated deletion of Irf4 in the Irf4–Cg1-Cre double-transgenic mice.
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Spleen 0.58%
0.0%
PB 0.19%
0.0%
BM 0.52%
0.0%
c Irf4
Irf4 fl/– Cγ1-Cre
fl/–
Irf4 +/– Cγ1-Cre
Cγ1-Cre
NP IgG1
100
