Murine germinal center B cells require functional Fms-like tyrosine kinase 3 signaling for IgG1 class-switch recombination Mattias N. D. Svenssona,1, Karin M. E. Anderssona, Caroline Waséna, Malin C. Erlandssona, Merja Nurkkala-Karlssona, Ing-Marie Jonssona, Mikael Brisslerta, Mats Bemarkb, and Maria I. Bokarewaa aDepartment of Rheumatology and Inflammation Research at Sahlgrenska Academy, University of Gothenburg, Gothenburg 41346, Sweden; and bDepartment of Microbiology and Immunology at Sahlgrenska Academy, University of Gothenburg, Gothenburg 41390, Sweden Edited by Tak W. Mak, The Campbell Family Institute for Breast Cancer Research at Princess Margaret Cancer Centre, University Health Network, Toronto, Canada, and approved October 23, 2015 (received for review July 21, 2015) Switched antibody classes are important for efficient immune receptor signals, primary and secondary stimuli control CSR in B responses. Aberrant antibody production to otherwise harmless cells. Whereas T-cell–dependent (i.e., CD40L) or T-cell–independent antigens may result in autoimmunity. The protein kinase fms-like (i.e., TLR) primary stimuli induce expression of AID, secondary tyrosine kinase 3 receptor (Flt3) has an important role during early stimuli such as IL-4 (IgG1, IgE), IFN-γ (IgG2c), and TGF-β (IgA) B-cell development, but the role of Flt3 in peripheral B cells has not are needed for directing the class switch to a specific antibody been assessed before. Herein we describe a previously unappre- isotype through the induction of GLT (5). ciated role for Flt3 in IgG1 class-switch recombination (CSR) and – + During T-cell dependent responses, CSR mainly occurs within production. We show that Flt3 is reexpressed on B-cell lymphoma 6 germinal centers (GCs) (6). IgG1 production is dependent on GC germinal center B cells in vivo and following LPS activation of formation and the type I cytokine IL-4 (7). Binding of IL-4 to the . peripheral B cells in vitro Absence of Flt3 signaling in Flt3 ligand- IL-4 receptor (IL-4R) leads to phosphorylation of signal trans- deficient mice results in impaired IgG1 CSR and accumulation of ducer and activator of transcription (Stat) 6 by Janus kinase (8). IgM-secreting plasma cells. On activated B cells, Flt3 is coexpressed Phosphorylated Stat6 binds the promoter region of γ1, inducing and functions in synergy with the common-gamma chain receptor GLT and subsequent CSR to IgG1 (8). IL-4 is produced by fol- family. B cells from Flt3 ligand-deficient mice have impaired IL-4R licular T cells (T ) that are specialized B-helper T cells involved signaling, with reduced phosphorylation of signal transducer and FH activator of transcription (Stat) 6, and demonstrate a failure to ini- in GC establishment and function (9). tiate CSR to IgG1 with low expression of γ1 germ-line transcripts, The protein kinase fms-like tyrosinekinase3receptor(Flt3)isa resulting in impaired IgG1 production. Thus, functional synergy be- tyrosine kinase receptor expressed on early hematopoietic and tween Flt3 and IL-4R signaling is critical for Stat-mediated regulation lymphoid progenitors in the bone marrow (BM) (10). Flt3 is acti- of sterile γ1 germ-line transcripts and CSR to IgG1. vated by Flt3-ligand (FL) binding, promoting survival and differ- entiation (11–13). FL is expressed in multiple cell types including class-switch recombination | germinal center B cells | Flt3 | IgG1 | IL-4R BM stroma cells and activated T cells, either in a membrane-bound form or as a soluble protein (14, 15). Generally, FL has a weak ctivation of B cells by foreign antigens and the subsequent stimulatory effect on its own and acts in combination with other Aformation of antibody-producing plasma cells are crucial cytokines (16). For example, Flt3 induces responsiveness to IL-7 in steps in protective humoral immunity. The immune system re- sponds to different invading pathogens by production of anti- Significance bodies with unique effector functions. This is accomplished by class-switch recombination (CSR), where the rearranged variable The protein kinase fms-like tyrosine kinase 3 receptor (Flt3) has region of an antibody heavy chain is joined with different an important role during early B-cell development. Recent re- constant regions (CH) (1). Impaired CSR can cause serious com- ports have shown that Flt3 is reexpressed on peripheral B cells plications, such as hyper-IgM syndromes with increased susceptibil- during activation, however the function for Flt3 during B-cell ity to bacterial infections (2), but also systemic or organ-specific activation is poorly understood. The present study describes a autoimmunity (3). role for Flt3 during B-cell activation and antibody production. During CSR, the Ig heavy chain CH exons coding for IgM (Cμ) We show that Flt3 is reexpressed on activated germinal center are deleted and replaced with CH exons coding for either IgG B cells and that lack of functional Flt3 signaling in peripheral B (Cγ), IgE (Ce), or IgA (Cα). This process is accomplished by cells results in a specific deficiency in production of the anti- joining two DNA sequences, switch regions, which are located body subclass IgG1. Mechanistic studies indicate that Flt3 upstream of each CH gene. CSR requires the expression of activation- synergizes with the IL-4 receptor to promote proper activation induced cytidine deaminase (AID), which deaminates deox- of Stat6 and class switch to IgG1. This study provides evidence ycytosines in switch (S)-region DNA, yielding deoxyuracils. for a previously unknown function for Flt3 during peripheral During the removal of deoxyuracil bases, double-stranded B-cell activation. DNA breaks occur in the upstream (donor) and downstream (acceptor) S-regions. This activates a DNA damage response, Author contributions: M.N.D.S., K.M.E.A., M. Brisslert, M. Bemark, and M.I.B. designed research; M.N.D.S., K.M.E.A., C.W., M.C.E., M.N.-K., I.-M.J., M. Brisslert, M. Bemark, and which promotes long-range recombination. Eventually the double- M.I.B. performed research; M. Bemark and M.I.B. contributed new reagents/analytic tools; stranded DNA breaks in Sμ and the downstream target S-region M.N.D.S., K.M.E.A., C.W., M.C.E., M.N.-K., I.-M.J., M. Brisslert, M. Bemark, and M.I.B. analyzed are joined to enable expression of a new antibody isotype (1, 4). data; and M.N.D.S., K.M.E.A., M. Brisslert, M. Bemark, and M.I.B. wrote the paper. CSR is initiated through transcription from isotype-specific The authors declare no conflict of interest. intronic promoters that continues through the intronic exon, the This article is a PNAS Direct Submission. adjacent S-region, and the CH exons, creating a germ-line tran- 1To whom correspondence should be addressed. Email: [email protected]. script (GLT). GLTs are noncoding but are thought to initiate CSR This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. by rendering the S-region accessible for AID. In addition to B-cell 1073/pnas.1514191112/-/DCSupplemental. E6644–E6653 | PNAS | Published online November 16, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1514191112 Downloaded by guest on September 30, 2021 B-cell progenitors by driving expression of the IL-7R. Furthermore, Herein we investigate the role of Flt3 on mature peripheral B PNAS PLUS Flt3 signaling is suggested to potentiate IL-7–induced phosphory- cells and show that Flt3 is reexpressed on activated GC B lation of Stat5 during early B-cell differentiation (17–23). Despite cells and that signals through Flt3 are essential for proper IL- the block in early B-cell development, Flt3- and FL-targeted mice 4–induced activation of Stat6 and IgG1 production. have normal numbers of peripheral B cells, antibody levels, and responses toward T-dependent immunization (16, 24). Surface ex- Results pression of Flt3 is lost when developing B cells acquire CD19 ex- Activation of Peripheral B Cells Induces Reexpression of Surface Flt3. pression (25). The Flt3 receptor is reexpressed on mature peripheral B cells Recently, Flt3 was found to be reexpressed on splenic B cells following LPS or anti-CD40 and IL-4 in vitro stimulation (26). We after in vitro activation with LPS or anti-CD40 and IL-4 (26). + decided to further characterize B cells expressing Flt3 after in vitro Furthermore, Flt3 B cells have been described in the peripheral LPS activation and during T-cell–dependent antigen challenge blood of healthy individuals, whereas increased serum levels of FL in vivo. have been measured in patients suffering from antibody-mediated In vitro activation of naïve WT splenocytes with LPS resulted in autoimmune diseases such as rheumatoid arthritis, systemic lupus reexpression of Flt3 on B cells (Fig. 1A). Flt3 expression was ob- erythematosus, and Sjögren’s syndrome (27–29). Also, protective served on proliferating B cells, with increased cell size and granu- properties of FL are identified in mouse models of chronic asthma, − a disease associated with aberrant antibody production toward lation compared with Flt3 B cells and elevated expression of B–D otherwise harmless antigens (30–32). Stimulation of human B cells activation markers CD80, CD86, MHCII, and CD40 (Fig. 1 ). – with FL in vitro potentiate anti-IgM–induced proliferation and In vivo, immunization with three different T-cell dependent anti- survival (29). Also, FL has potent adjuvant effects in vivo, enhancing gens [methylated bovine serum albumin (mBSA), ovalbumin protective IgA immunity in streptococcal pneumonia (33). Taken (OVA), and collagen type II (CII)] resulted in a significant in- + together, these results suggest that Flt3 may participate in processes crease in Flt3 B cells in lymph nodes (LNs) (Fig. 1E). As ob- + associated with B-cell activation. served in vitro, Flt3 B cells were increased in size and granulation INFLAMMATION IMMUNOLOGY AND Fig.