Upon recognition of antigen, B cells undertake a bifurcated response in which some cells rapidly differentiate into plasmablasts while others undergo affinity maturation in germinal centers (GCs). Here we identified a double-negative feedback loop between the transcription factors IRF4 and IRF8 that regulated the initial developmental bifurcation of activated B cells as well as the GC response. IRF8 dampened signaling via the B cell antigen receptor (BCR), facilitated antigen-specific interaction with helper T cells, and promoted antibody affinity maturation while antagonizing IRF4-driven differentiation of plasmablasts. Genomic analysis revealed concentration-dependent actions of IRF4 and IRF8 in regulating distinct gene-expression programs. Stochastic modeling suggested that the double-negative feedback was sufficient to initiate bifurcation of the B cell developmental trajectories.
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Jacob, J., Kassir, R. & Kelsoe, G. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173, 1165–1175 (1991).
MacLennan, I.C. et al. Extrafollicular antibody responses. Immunol. Rev. 194, 8–18 (2003).
Victora, G.D. & Nussenzweig, M.C. Germinal centers. Annu. Rev. Immunol. 30, 429–457 (2012).
Sciammas, R. et al. Graded expression of interferon regulatory factor-4 coordinates isotype switching with plasma cell differentiation. Immunity 25, 225–236 (2006).
Sciammas, R. et al. An incoherent regulatory network architecture that orchestrates B cell diversification in response to antigen signaling. Mol. Syst. Biol. 7, 495 (2011).
Ochiai, K. et al. Transcriptional regulation of germinal center B and plasma cell fates by dynamical control of IRF4. Immunity 38, 918–929 (2013).
Singh, H., Glasmacher, E., Chang, A.B. & Vander Lugt, B. The molecular choreography of IRF4 and IRF8 with immune system partners. Cold Spring Harb. Symp. Quant. Biol. 78, 101–104 (2014).
Brüstle, A. et al. The development of inflammatory TH-17 cells requires interferon-regulatory factor 4. Nat. Immunol. 8, 958–966 (2007).
Ouyang, X. et al. Transcription factor IRF8 directs a silencing programme for TH17 cell differentiation. Nat. Commun. 2, 314 (2011).
Vander Lugt, B. et al. Transcriptional programming of dendritic cells for enhanced MHC class II antigen presentation. Nat. Immunol. 15, 161–167 (2014).
Carotta, S. et al. The transcription factors IRF8 and PU.1 negatively regulate plasma cell differentiation. J. Exp. Med. 211, 2169–2181 (2014).
Pathak, S., Ma, S., Shukla, V. & Lu, R. A role for IRF8 in B cell anergy. J. Immunol. 191, 6222–6230 (2013).
Lee, C.H. et al. Regulation of the germinal center gene program by interferon (IFN) regulatory factor 8/IFN consensus sequence-binding protein. J. Exp. Med. 203, 63–72 (2006).
Feng, J. et al. IFN regulatory factor 8 restricts the size of the marginal zone and follicular B cell pools. J. Immunol. 186, 1458–1466 (2011).
Kitamura, D., Roes, J., Kuhn, R. & Rajewsky, K.A. B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin μ chain gene. Nature 350, 423–426 (1991).
Burne-Taney, M.J. et al. B cell deficiency confers protection from renal ischemia reperfusion injury. J. Immunol. 171, 3210–3215 (2003).
Bajénoff, M. & Germain, R.N. B-cell follicle development remodels the conduit system and allows soluble antigen delivery to follicular dendritic cells. Blood 114, 4989–4997 (2009).
Pillai, S., Cariappa, A. & Moran, S.T. Marginal zone B cells. Annu. Rev. Immunol. 23, 161–196 (2005).
Liu, D. et al. T-B-cell entanglement and ICOSL-driven feed-forward regulation of germinal centre reaction. Nature 517, 214–218 (2015).
Allen, D., Simon, T., Sablitzky, F., Rajewsky, K. & Cumano, A. Antibody engineering for the analysis of affinity maturation of an anti-hapten response. EMBO J. 7, 1995–2001 (1988).
Cattoretti, G. et al. Nuclear and cytoplasmic AID in extrafollicular and germinal center B cells. Blood 107, 3967–3975 (2006).
Glasmacher, E. et al. A genomic regulatory element that directs assembly and function of immune-specific AP-1-IRF complexes. Science 338, 975–980 (2012).
Xu, Y., Harder, K.W., Huntington, N.D., Hibbs, M.L. & Tarlinton, D.M. Lyn tyrosine kinase: accentuating the positive and the negative. Immunity 22, 9–18 (2005).
Chew, V. & Lam, K.P. Leupaxin negatively regulates B cell receptor signaling. J. Biol. Chem. 282, 27181–27191 (2007).
Khalil, A.M., Cambier, J.C. & Shlomchik, M.J. B cell receptor signal transduction in the GC is short-circuited by high phosphatase activity. Science 336, 1178–1181 (2012).
Chu, C. et al. SAP-regulated T Cell-APC adhesion and ligation-dependent and -independent Ly108–CD3ζ interactions. J. Immunol. 193, 3860–3871 (2014).
Choe, J. & Choi, Y.S. IL-10 interrupts memory B cell expansion in the germinal center by inducing differentiation into plasma cells. Eur. J. Immunol. 28, 508–515 (1998).
Todd, D.J., Lee, A.H. & Glimcher, L.H. The endoplasmic reticulum stress response in immunity and autoimmunity. Nat. Rev. Immunol. 8, 663–674 (2008).
Gillespie, D.T. The chemical Langevin equation. J. Chem. Phys. 113, 297–306 (2000).
Kaern, M., Elston, T.C., Blake, W.J. & Collins, J.J. Stochasticity in gene expression: from theories to phenotypes. Nat. Rev. Genet. 6, 451–464 (2005).
Ettinger, R. et al. IL-21 induces differentiation of human naive and memory B cells into antibody-secreting plasma cells. J. Immunol. 175, 7867–7879 (2005).
We thank core facilities of the Cincinnati Children's Hospital Medical Center for help with mouse colony management, cell sorting and high-throughput DNA sequencing. Supported by the Cincinnati Children's Research Foundation (H.S.) and the Leukemia & Lymphoma Society Career Development Program (5442-16 for H.X.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Expression of Bcl-6, IRF4 and IRF8 in B cells after immunization with NP-KLH plus alum and LPS.
(a) Gating strategies for resting B cells (Grey), GC B cells (Blue) and plasmablasts (Red).(b) Expression of Bcl-6, IRF4 and IRF8 on dpi 7 is plotted individually. Grey curves indicate the corresponding background with control antibodies.(c) Expression of Bcl-6, IRF4 and IRF8 was analyzed on dpi 13. GC B cells and plasma cells are indicated with blue and red colors, respectively.
Supplementary Figure 2 Analysis of IRF8’s function in GC B cells and plasmablasts trough the use of μMT chimeric mice.
(a) Diagrammatic representation of the experimental protocol.(b) Gating strategies for plasmablasts (left) and GC B cells (right) in μMT mice.(c) Equations for calculating competitive competencies of CD45.2 (wild-type or IRF8 knockout) cells. The normalized ratio controls for the variation in follicular reconstitution efficiencies among different mice and reflects the relative competency of IRF8 knockout B cells in relation to wild-type counterparts in the generation of GC B cells or plasmablasts.
Supplementary Figure 3 B cell and T cell responses in Irf8fl/flCd19+/cre and Irf8+/+Cd19+/cre mice after immunization.
(a) Percentage of IgMloIgG1+ cells in GC B cell (B220+CD38–GL7hi) population on day 7 post immunization with NP-KLH plus alum and LPS. Each circle represents an individual mouse. Data were pooled from two independent experiments (mean±SEM, **P < 0.01).(b) Percentage of GC B cells on day 13 post immunization as described in a. Each circle represents an individual mouse. Data were pooled from four independent experiments (mean±SEM, **P < 0.01).(c) IRF8 expression in Irf8fl/flCd19+/cre and Irf8+/+Cd19+/cre GC B cells on day 13 post immunization as described in a. The cells were gated on B220+FashiGL7hi. Data are representative of analysis of six mice of each genotype from two independent experiments.(d) Percentage of GC-Tfh cells (CXCR5hiPD-1hi) in activated CD4 T cell compartment (B220–CD4+CD44hi) on day 13 post immunization as described in a. Each circle represents an individual mouse. Data were pooled from five independent experiments (mean±SEM, *P < 0.05).(e) Percentages of plasmablasts (B220loCD138+) in NP positive splenocytes on day 3 post immunization with NP-FICOLL. Each circle represents an individual mouse. Data were pooled from two independent experiments (mean±SEM, ***P < 0.001).
Supplementary Figure 4 IRF4hi and IRF8hi activated B cells manifest distinct gene-expression states.
(a) Naïve splenic B cells were isolated and stimulated with LPS or anti-CD40 plus IL-2, IL-5 and IL-4 as described in Methods. B cells were harvested and analyzed for IgG3 or IgG1 CSR at 72 h post activation.(b) Flow sorting gates use to enrich IRF4hi (IgMhiCD138hi, indicated as IRF4hi) or IRF8hi (IgMloCD138lo, indicated as IRF8hi) cells (left panel). IRF4 and IRF8 protein amounts in IRF4hi and IRF8hi B cells analyzed by western blotting (right panel).(c) Irf4 and Irf8 transcripts in indicated cells were measured by Q-PCR. Pou2f1 transcripts were used for normalization. Data are from three independent experiments (mean±SEM, *P < 0.01, **P < 0.001).(d) Selected GO functional categories for differentially expressed genes in IRF4hi and IRF8hi cells are displayed with their odds ratios.
(a) Naïve splenic B cells were isolated from wild-type mice and activated with LPS. Cells were cross-linked and processed for IRF4 and IRF8 ChIP-seq on day 2 and 3 after stimulation. Binding peaks were called using MACS and motif enrichment analysis performed using Homer. The fold-enrichment and P value of EICE (GSE21512), AICE (GSE39756) and ISRE (GSE43036) motifs are indicated.(b) IRF4 (red) and IRF8 (blue) ChIP-seq tracks for selected genes shown in Fig. 5f. The DNaseI-seq tracks are from ENCODE (GSM1014190). Tag counts for peaks are plotted on y-axis.(c) Numbers of IRF4 only, IRF8 only and shared ChIP-seq peaks are displayed using Venn diagram. Peaks with maxima within 100 bp of each other were considered as shared peaks.(d) Differentially enriched transcription factor motifs (>2-fold, P < 0.001) within IRF4 only or IRF8 only peaks displayed in histogram. They are Bach1 (GSE31477), Nfr1 (GSE37589), Bach2 (GSE31477), Jun (GSE31477), Oct2 (GSE21512), PBX1 (GSE28007), NF-E2 (GSE31477), ATF3 (GSE33912), BATF (GSE39756) and AP-1 (GSE21512) for IRF4 only peaks (red); T1ISRE and RARγ (GSE30538) for IRF8 only peaks (blue).
(a) ChIP-seq tracks for IRF4 (red) and IRF8 (blue) at Irf4 gene. IRF8 targeted region in Irf4 gene is highlighted with box. The DNaseI-seq track is from ENCODE (GSM1014190).(b) Expression of Irf4 gene was analyzed by Q-PCR at indicated time points in Irf8+/+ and Irf8–/– FO B cells stimulated with 0.4 μg/ml LPS. Pou2f1 transcripts were used for normalization. The relative expression of indicated gene in Irf4–/– cells is in relation to its expression in wild-type cells at 24 h. Data is from two independent experiments (mean±SEM, *P < 0.001).(c) Proposed gene regulatory network with two double negative feedback loops that orchestrates activated B cell responses. Schematic is generated using BioTapestry. Arrowheads represent gene activation inputs and barred lines signify repression; dashed lines indicate that a gene regulatory relationship could be indirect; thin red line represents a low level IRF4 gene output whereas thick red line represents a higher level IRF4 gene output.
Supplementary Figures 1–6 and Supplementary Note (PDF 1447 kb)
Homer motif enrichment analysis for IRF8 targeted regions in GC B cells are listed in the first sheet. GO analysis of genes associated with IRF8 targeted regions in GC B cells are listed in the second sheet. (XLSX 155 kb)
Transcriptional abundance (FPKM) of functionally important genes in GC B cells (Fig. 3c). (XLSX 45 kb)
Differentially expressed genes within IRF4++ and IRF8++ B cells, termed IRF4++ and Irf8++ genes, are listed in the first sheet. Average log2 transformed FPKM values from two independent experiments are indicated in column B and C in the first sheet, respectively. GO analysis results for the differentially expressed genes are listed in the second sheet. (XLSX 311 kb)
Transcriptional abundance of functionally important GC B cell genes (FPKM) in IRF4++ and IRF8++ B cells (Fig. 5e). (XLSX 42 kb)
Antibodies and reagents used for flow cytometry. (XLSX 10 kb)
Primers used for gene expression and VH186.2 mutation analysis. (XLSX 10 kb)
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Xu, H., Chaudhri, V., Wu, Z. et al. Regulation of bifurcating B cell trajectories by mutual antagonism between transcription factors IRF4 and IRF8. Nat Immunol 16, 1274–1281 (2015) doi:10.1038/ni.3287
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