The proto-oncogene MYC is required for selection in the germinal center and cyclic reentry

Journal name:
Nature Immunology
Volume:
13,
Pages:
1083–1091
Year published:
DOI:
doi:10.1038/ni.2428
Received
Accepted
Published online

Abstract

After antigenic challenge, B cells enter the dark zone (DZ) of germinal centers (GCs) to proliferate and hypermutate their immunoglobulin genes. Mutants with greater affinity for the antigen are positively selected in the light zone (LZ) to either differentiate into plasma and memory cells or reenter the DZ. The molecular circuits that govern positive selection in the GC are not known. We show here that the GC reaction required biphasic regulation of expression of the cell-cycle regulator c-Myc that involved its transient induction during early GC commitment, its repression by Bcl-6 in DZ B cells and its reinduction in B cells selected for reentry into the DZ. Inhibition of c-Myc in vivo led to GC collapse, which indicated an essential role for c-Myc in GCs. Our results have implications for the mechanism of GC selection and the role of c-Myc in lymphomagenesis.

At a glance

Figures

  1. A subset of LZ GC B cells express c-Myc under physiological conditions.
    Figure 1: A subset of LZ GC B cells express c-Myc under physiological conditions.

    (a) Immunofluorescence microscopy of paraffin-embedded sections of reactive human lymph nodes. Staining of CD23 (left), expressed in FDCs51, indicates the boundaries of the LZ; AID (right) serves as a DZ marker4, 52; the DNA-intercalating dye DAPI identifies nuclei. Scale bars, 200 μm. (b) Sorting profiles of LZ and DZ GC B cell subpopulations in human tonsils (Supplementary Fig. 1). (c) Immunoblot analysis of the populations in b. Na, naive B cells (control); Bulk, bulk CD77+ GC B cells, isolated as described9; *, nonspecific band. Actin serves as loading control. (d) Quantitative RT-PCR analysis of c-Myc and Bcl-6 mRNA in LZ and DZ B cell pools; results are presented relative to expression of the control gene ACTB. (e) Immunofluorescence microscopy of paraffin-embedded isolated from human tonsils as in b sections of a mouse lymph node 12 d after immunization with SRBCs. Staining for AID identifies the GC (DZ); IgG identifies the FDC network (LZ); PC, plasma cell; SCS, subcapsular sinus. Scale bar, 50 μm. (f) Quantification of GFP–c-Myc+ B cells in LZ and DZ GC B cell subsets from GFP–c-Myc mice 12 d after immunization with SRBCs, assessed by flow cytometry. Each symbol represents an individual mouse; small horizontal lines and numbers above indicate the mean. (g) Distribution of GFP–c-Myc+ GC B cells among LZ and DZ subsets, assessed by flow cytometry (left), and fluorescence intensity of GFP–c-Myc in those subsets (right). Numbers adjacent to outlined areas (left) indicate percent cells in each throughout. Data are representative of one experiment with three specimens (a), three experiments (be average and s.d. of three independent cell pools per population in d), four independent experiments with 13 mice (f) or four experiments (g).

  2. Alternating peaks of c-Myc expression and Bcl-6 expression during T cell-dependent antigen responses and GC formation.
    Figure 2: Alternating peaks of c-Myc expression and Bcl-6 expression during T cell–dependent antigen responses and GC formation.

    (a) Experimental approach. B, B cell. (b) Distribution of the expression of GFP–c-Myc (top) and Bcl-6 (bottom) in CD45.2+ host B cells, primed CD45.1+ Igλ+ B cells and unprimed CD45.1+B1-8hi Igλ+ B cells (key) on days 1–8 after immunization with NP-OVA (left; additional information, Supplementary Fig. 2); far right, concatenation of results at left (dashed lines indicate background signals). (c) Temporal evolution of antigen-primed B cells (black line) and unprimed B cells (gray line) among donor CD45.1+ B1-8hi Igλ+ cells (left vertical axis) and frequency of GFP–c-Myc+ and Bcl-6+ cells (right axis) in the antigen-primed B1-8hi B cell population during GC formation. (d) Topographic distribution of GFP–c-Myc+ primed B cells in each GC compartment (LZ, DZ) at days 5 and 8 after immunization, assessed by flow cytometry (left), and GFP–c-Myc+ cells in the LZ and DZ (right). Data are representative of three experiments (two mice per time point in c; error bars (c,d), s.d.).

  3. Bcl-6 represses c-Myc protein expression in DZ GC B cells.
    Figure 3: Bcl-6 represses c-Myc protein expression in DZ GC B cells.

    (a) Immunofluorescence microscopy of c-Myc and Bcl-6 costaining in a secondary follicle of a human lymph node; staining of Bcl-6 indicates the boundaries of the GC. Scale bars, 200 μm. (b) Binding of Bcl-6 to chromatin at the MYC locus in human CD77+ GC B cells (Bcl-6 CB), assessed by ChIP plus microarray (top; raw data, Supplementary Table 1), and organization of the MYC locus (below) in the region around exon 1 and the transcription start site (+1), including the quantitative ChIP (qChIP) amplicons used in c (C1 and B1–B3) and the B6BS and M0 potential Bcl-6-binding sites (based on a previously defined consensus14). Gray shading indicates regions enriched for CpG islands. (c) Quantitative PCR analysis of DNA (obtained by ChIP of Bcl-6) isolated from a sample of CD77+ GC B cells independent of that in b; results for 'normalized' binding at each region (enrichment in Bcl-6 immunoprecipitates over background of species- or isotype-matched irrelevant antibody) are presented relative to that at C1, set as 1. (d) Dual-luciferase reporter assay of HEK293T cells transfected to express a wild-type c-Myc luciferase reporter (c-Myc–luc) and hemagglutinin-tagged wild-type Bcl-6 (HA–Bcl-6) or Bcl-6 truncation mutants with deletion of the entire zinc-finger domain (HA–Bcl-6(ΔZF)) or the amino-terminal transrepression domain (HA–Bcl-6(ZF)), showing the effects on the 1.2-kilobase upstream MYC regulatory region with two potential Bcl-6-binding sites (top); results are presented relative to those of renilla luiferase. (e) Dual-luciferase reporter assay (as in d) of HEK293T cells transfected to express hemagglutinin-tagged wild-type Bcl-6 and a luciferase reporter with wild-type c-Myc or c-Myc with point mutation of either (c-Myc–luc(mutB1) or c-Myc–luc(mutB2)) or both (c-Myc–luc(mutB1,B2)) putative consensus Bcl-6-binding sites (B1 and B2). Data are representative of three experiments (a), two experiments (b) or one experiment (ce; average and s.d. of three (c) or two (d,e) technical replicates).

  4. Coordinated upregulation of genes of the 'immune activation' signature and those encoding molecules involved in entry into the cell cycle in GFP-c-Myc+ GC B cells.
    Figure 4: Coordinated upregulation of genes of the 'immune activation' signature and those encoding molecules involved in entry into the cell cycle in GFP–c-Myc+ GC B cells.

    (a) Overlap (12 genes) between the consensus activation signature (Activation; integrated by 76 genes common to two or more published immunological activation signatures; details, Supplementary Table 4) and genes upregulated in GFP–c-Myc+ GC B cells (Myc+ upreg), determined by hypergeometric distribution (P = 0.000000126). (b) Expression profile of the 12 consensus 'immune activation' genes with enrichment in GFP–c-Myc+ cells relative to their expression in GFP–c-Myc cells. Top, sample identifiers (color bars group samples); numbers on right margin indicate euclidean distance. (c) Expression of mRNA of the subset of 12 'immune activation' genes with enrichment in GFP–c-Myc+ cells; results are presented relative expression of to the control gene Actb. (d) Immunofluorescence microscopy of the coexpression of AID (red) and c-Myc (green) in GC B cells from a mouse lymph node. M, mantle zone. Scale bar, 20 μm. (e) Expression of Aicda mRNA in GC B cell subsets isolated from GFP–c-Myc mice 12 d after immunization with SRBCs; results are presented relative to those of GFP–c-Myc cells, set as 1. (f) Quantitative RT-PCR analysis of Ccnd2 and Myc mRNA in GFP–c-Myc+ and GFP–c-Myc populations; results are presented relative to expression of the control gene Actb. (g) Cell-cycle profile analysis of LZ and DZ GC B cells (defined by surface expression of CXCR4-CD86)6, with cell-cycle phases defined by DNA content, adjusted to the Watson pragmatic model (left), and cell-cycle distribution of bulk GFP–c-Myc+ GC B cells and all GC B cells. Data are from one experiment (a,b), three experiments (c,f; average and s.d. of three independent cell pools with two mice per pool), two experiments (d), two independent experiments (e; average and s.d.) or one experiment with one mouse, representative of three (g).

  5. The BCR repertoire of GFP-c-Myc+ GC B cells shows enrichment for high-affinity variants.
    Figure 5: The BCR repertoire of GFP–c-Myc+ GC B cells shows enrichment for high-affinity variants.

    (a) Three populations (1–3) purified from GC B cell pools of GFP–c-Myc mice 9 d after immunization with NP-KLH. (b) Distribution of GFP–c-Myc+ and GFP–c-Myc subpopulations in the DZ and LZ compartments (gating strategy, Supplementary Fig. 1). (c) Fraction of variable heavy-chain region 186.2 segments (complementarity-determining region 1) with the W33L substitution (W33L+), among all sequenced segments (W33L mutants/total segments; additional details, Supplementary Table 5) in populations 1–3 identified in a; W33L, segments without the W33L substitution. *P = 0.211, **P = 0.001 and ***P < 0.0001 (Fisher's exact test). Data are representative of two experiments (b) or are pooled from two independent experiments with two to three mice each (with ~45–50 clones per sample in each; c).

  6. Access to T cell help triggers c-Myc expression before reentry into the DZ.
    Figure 6: Access to T cell help triggers c-Myc expression before reentry into the DZ.

    (a) Experimental strategy: NP haptens are detected by B cells; OVA peptide conjugates are detected by T cells. Black, unprimed cells; Green (TOV), OVA-primed T cells; red, NP-primed B cells; surface triangles, DEC-205+ (CD205+Ly75+) cells; WT, wild-type; DEC-205-KO, DEC-205-deficient; α-DEC-OVA, OVA–anti-DEC-205. DEC-205 wild-type cells express a photoactivatable variant of GFP (PAGFP) that allows specific identification of these cells. (b) Expected dynamics and topographical distribution of DEC-205+ (PAGFP+) cells before (Untreated) and 12 and 40 h after injection of OVA–anti-DEC-205 (top), and immunofluorescence microscopy (below) of paraffin-embedded sections of popliteal lymph nodes 12 and 40 h after injection of OVA–anti-DEC-205, to detect PAGFP+ (DEC-205+) B cells in B220+ GC compartments (outlined). Scale bars, 50 μm. (c) Gating strategy used to isolate DEC-205+ and DEC-205 GC B cells from the LZ of mice 12 h after injection of OVA–anti-DEC-205. (d) Quantitative analysis of Myc, Cd70 and Ccnd2 mRNA in DEC-205+ and DEC-205 populations isolated as in c (additional details, Supplementary Fig. 5); results are presented relative to the control gene Actb. Data are representative of three experiments (b) or two experiments (c) or are from one experiment representative of two (d; average and s.d. of three technical replicates).

  7. The biological activity of c-Myc is required for normal GC maintenance.
    Figure 7: The biological activity of c-Myc is required for normal GC maintenance.

    (a) Experimental approach and the expected dynamics in T cell–dependent antigen responses and GC formation. TRE-Omomyc rtTA-actin, mice with doxycycline (Dox)-inducible Omomyc expression; veh, vehicle. (b) Flow cytometry of B cell splenic pools from mice with doxycycline-inducible Omomyc expression, treated with vehicle or doxycycline. The CD95hiPNAhi gate (blue outline) defines the GC population; numbers adjacent to outlined areas indicate percent GC B cells. (c) Quantification of GC B cells in wild-type mice and mice with doxycycline-inducible Omomyc expression, treated with vehicle or doxycycline; results are presented relative to those of wild-type mice treated with vehicle, set as 100%.(d) Microscopy of GCs in paraffin-embedded sections of spleens from mice treated as in b; Bcl-6 staining indicates GC B cells. Scale bars, 50 μm. (e) GC size distribution (surface) in mice as in d. Each symbol represents an individual GC (~50–55 GCs per mouse); small horizontal lines indicate average and s.e.m. P < 0.00001 (two-tailed Student's t-test with unequal variance). (f) Quantitative analysis of c-Myc and Omomyc mRNA in bulk B cell pools (B (bulk)) and GC fractions (GC) from mice with doxycycline-inducible Omomyc expression (Omo) treated with doxycycline (Dx) or vehicle and from SRBC-immunized wild-type mice treated with doxycycline (reference control); results were calculated by the change-in-threshold (ΔΔCT) method after normalization to the geometric mean of CT values for three different control genes (Gapdh, Actb and Hprt1). Data are from one experiment with two mice per condition (average and s.d. of two mice, with three technical replicates per point, in f).

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Author information

  1. These authors contributed equally to this work.

    • David Dominguez-Sola &
    • Gabriel D Victora

Affiliations

  1. Institute for Cancer Genetics, Columbia University, New York, New York, USA.

    • David Dominguez-Sola,
    • Carol Y Ying,
    • Ryan T Phan,
    • Masumichi Saito &
    • Riccardo Dalla-Favera
  2. Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, USA.

    • Gabriel D Victora &
    • Michel C Nussenzweig
  3. Howard Hughes Medical Institute, New York, New York, USA.

    • Michel C Nussenzweig
  4. Herbert Irving Comprehensive Cancer Center, Columbia University, New York, New York, USA.

    • Riccardo Dalla-Favera
  5. Department of Pathology and Cell Biology, Columbia University, New York, New York, USA.

    • Riccardo Dalla-Favera
  6. Department of Genetics and Development, Columbia University, New York, New York, USA.

    • Riccardo Dalla-Favera
  7. Department of Microbiology and Immunology, Columbia University, New York, New York, USA.

    • Riccardo Dalla-Favera
  8. Present addresses: Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA (G.D.V.), Department of Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, USA (R.T.P.), and Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, Tokyo, Japan (M.S.).

    • Gabriel D Victora,
    • Ryan T Phan &
    • Masumichi Saito

Contributions

D.D.-S. and G.D.V designed the study, did the experiments, analyzed the data and wrote the manuscript; D.D.-S. did the computational analyses; C.Y.Y. did experiments; C.Y.Y., R.T.P. and M.S. identified and characterized the Bcl-6–c-Myc transcriptional interaction; R.D.-F. and M.C.N. supervised the project, provided direction in the study design and wrote the manuscript; and all authors approved the final manuscript.

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The authors declare no competing financial interests.

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Supplementary information

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  1. Supplementary Text and Figures (4M)

    Supplementary Figures 1–7 and Tables 4–6

Excel files

  1. Supplementary Table 1 (53K)

    BCL6 ChIP-on-chip binding profile across the proximal region of the MYC locus.

  2. Supplementary Table 2 (33K)

    GFPMYC signature genes.

  3. Supplementary Table 3 (25K)

    Gene Set Enrichment Analysis of GFPMYC signatures.

  4. Supplementary Table 7 (53K)

    List of primers used in real-time quantitative RT-PCR (qRT-PCR), quantitative chromatin immunoprecipitation (qChIP) and IgH-V gene sequence analysis.

Additional data