The cell-cycle regulator c-myc is essential for the formation and maintenance of germinal centers
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1 SUPPLEMENTARY INFORMATION The cell-cycle regulator c-myc is essential for the formation and maintenance of germinal centers Dinis Pedro Calado 1,2, Yoshiteru Sasaki 3, Susana A Godinho 4, Alex Pellerin 1, Karl Köchert 2, Barry P Sleckman 5, Ignacio Moreno de Alborán 6, Martin Janz 2,7, Scott Rodig 8 & Klaus Rajewsky 1,2 1 Program of Cellular and Molecular Medicine, Children's Hospital, and Immune Disease Institute, Harvard Medical School, Boston, Massachusetts, USA. 2 Max Delbrück Center for Molecular Medicine, Berlin, Germany. 3 Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto, Japan. 4 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA. 5 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA. 6 Department of Immunology and Oncology, National Centre for Biotechnology, Madrid, Spain. 7 Hematology, Oncology and Tumor Immunology, Charité, University Medical School, Berlin, Germany. 8 Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts, USA. Correspondence should be addressed to D.P.C. (dinis.calado@mdc-berlin.de) or K.R. (klaus.rajewsky@mdc-berlin.de). 1
2 Supplementary Figure 1: c-myc target gene expression analysis suggests increased c- Myc transcriptional activity in GC B cells. (a) Left panel, GSEA of FO and GC B cell gene expression profiles for the c-myc target gene set V$MYC_Q2. Right panel, GSEA of FO and GC B cell gene expression profiles for the c-myc target gene set V$MYC_Q2 upon removal of genes, from the gene set, associated with the GO terms proliferation, cell cycle and growth, MINUSPROLGENES. (b) Left panel, GSEA of FO and GC B cell gene expression profiles for the c-myc target gene set V$MYCMAX_B. Right panel, GSEA of FO and GC B cell profiles for the c-myc target gene set V$MYCMAX_B upon removal of genes, from the gene set, associated with the GO terms proliferation, cell cycle and growth, MINUSPROLGENES. (c) GSEA of FO and GC B cell gene expression profiles for a gene set of c-myc up-regulated genes obtained through the intersection of c-myc ChIP-Seq and MYC sirna knockdown data in GC B cell derived Burkitt lymphoma cell lines. See Methods for GSEA analysis details. V$MYC_Q2 and V$MYCMAX_B gene sets were obtained from the Broad Institute molecular signatures database (MSigDB). 2
3 Supplementary Figure 2: Time course of GC formation. Flow cytometry analysis of splenic GC B cells (B220 pos CD38 low CD95 high ) of 2, 4, 5, 7, and 10 day SRBC immunized C57BL/6 wild-type mice. Data are representative of 2 independent experiments. 3
4 Supplementary Figure 3: Expression of c-myc in GC and non-gc B cell subsets. (a) Flow cytometry analysis of splenic MZ (Marginal Zone, B220 pos CD38 high AA4.1 neg CD21 high CD23 low ) and FO (Follicular, B220 pos CD38 high AA4.1 neg CD21 pos CD23 high ) B cells for c-myc-egfp expression of 10 day SRBC immunized (D10 SRBCi) C57BL/6 wild-type and Myc-eGFP mice (b) Flow cytometry analysis of splenic GC B cells (B220 pos CD38 low CD95 high ) for c-myc-egfp expression of 4 and 10 day SRBC immunized (D4 SRBCi and D10 SRBCi, respectively) C57BL/6 wild-type and Myc-eGFP mice. (c) Graphical representation of the frequency of c-myc-egfp positive cells within MZ, FO and GC B cells of Myc-eGFP mice at D4 SRBCi (D4P) and D10 SRBCi (D10P). (d) Graphical representation of c-myc-egfp fluorescence of MZ, FO and GC B cells of Myc-eGFP mice at D4P and D10P. gmfi, geometrical mean fluorescence intensity, black bar represents the mean. Symbols represent data from individual mice (c,d). ns, not significant. * p 0.05, ** p 0.01, *** p
5 Supplementary Figure 4: Early GC B cells express both c-myc and Bcl-6. (a) Representative image of histological immunofluorescence analysis of spleen of 4 day SRBC immunized (D4 SRBCi) C57BL/6 wild-type mice. T cells (CD3, green), FDCs (CD35, Blue), Bcl-6 (grey), c-myc (red), cut-off of 20 Bcl-6 positive cells per FDC area. (b) Representative image of histological immunofluorescence analysis of spleen of D4 SRBCi C57BL/6 wild-type mice as in (a), cut-off of >20 Bcl-6 positive cells per FDC area. Data are representative of 3 independent experiments, with at least 10 FDC areas analyzed per experiment per mouse (a,b). Scale bar 25µm. 5
6 Supplementary Figure 5: Generation of conditional alleles in the Rosa26 locus. (a) Schematic representation of truncated human CD2 (hcd2) targeting strategy in the Rosa26 locus. Cre-mediated deletion of the STOP cassette (neo r -stop) induces expression of hcd2 under transcriptional control of the endogenous Rosa26 promoter. Probes A and B used for Southern Blot analysis are represented by black bars. (b) Southern blot analysis of targeted ES clones. Genomic DNA from wild-type (lane 1) and one targeted ES clones (lane 2) was digested with EcoRI (left panel) or with PacI (right panel) and probed respectively with the external probe A (left panel) and the internal probe B (right panel). Ex1-3: Rosa26 exons 1-3; SA: splice acceptor; DT: diphtheria toxin. (c) Schematic representation of human MYC targeting strategy into the Rosa26 locus. Cre-mediated deletion of the STOP cassette (neo r- stop) induces expression of MYC under transcriptional control of a CAG promoter. A frtflanked IRES-hCD2 sequence is inserted between the MYC cdna and the polya sequence 6
7 (pa) in order to monitor expression from the CAG promoter through hcd2 expression. (d) Southern blot analysis of targeted ES clones. Genomic DNA from wild-type (lane 1) and two targeted ES clones (lane 2 and 3) was digested with EcoRI (left panel) or with PacI (right panel) and probed respectively with the external probe A (left panel) and the internal probe B (right panel). Ex1-3: Rosa26 exons 1-3; SA: splice acceptor; DT: diphtheria toxin. 7
8 8
9 Supplementary Figure 6: Both Bcl-6 and Bcl-2 fail to rescue GC formation upon Myc ablation. (a) Graphical representation of absolute cell numbers of hcd2 positive splenic GC B cells of D10 SRBCi mice of the indicated genotypes hcd2, Cg1-cre hcd2 stopfl (Myc intact); FF hcd2, Cg1-cre Myc FF hcd2 stopfl (Myc ablated), as shown in Fig. 4c and FF MYC, Cg1-cre Myc FF MYC stopfl (Myc ablated + MYC); BCL-6, Cg1-cre IµBCL-6 hcd2 stopfl (Myc intact + Bcl6 TG); FF BCL-6, Cg1-cre Myc FF IµBCL-6 hcd2 stopfl (Myc ablated + Bcl6 TG); BCL-2, Cg1-cre EmuBCL-2 hcd2 stopfl (Myc intact + Bcl2 TG); FF BCL-2, Cg1-cre Myc FF EmuBCL-2 hcd2 stopfl (Myc ablated + Bcl2 TG). (b) Graphical representation of the frequency of hcd2 positive peyer s patch GC B cells of mice of the indicated genotypes as in (a). (c) Flow cytometry analysis of peyer s patch GC B cells of mice of the indicated genotypes, lower panel, analysis of hcd2 reporter positive GC B cells. (d) Flow cytometry analysis of splenic GC B cells 10 days after SRBC immunization (D10 SRBCi) of mice of the indicated genotypes, lower panel, analysis of hcd2 reporter GC B cells. (e) Flow cytometry analysis of peyer s patch GC B cells of mice of the indicated genotypes, lower panel, analysis of hcd2 9
10 reporter positive GC B cells. (f) Left panel, detection of total IgG1 serum titers, right panel detection of total IgM serum titers, measured by ELISA in sera of D10 SRBCi mice of the indicated genotypes as in (a). Horizontal line represents mean. Data are representative of 3 independent experiments with at least 2 mice per genotype per experiment (c-e) and of 4 mice per genotype (f). Symbols represent data from individual mice (a,b,f). ns, not significant. * p 0.05, ** p 0.01, *** p Supplementary Figure 7: c-myc positive B cells in mature GCs localize to the light zone and display an activated phenotype. (a) Representative image of histological immunofluorescence analysis of spleen of 10 day immunized (D10 SRBCi) C57BL/6 wildtype mice. FDCs (CD35, Blue), Bcl-6 (grey), c-myc (red). (b) Graphical representation of the frequency of the c-myc-egfp positive (D10P) and negative (D10N) GC B cells localized to 10
11 the dark zone (DZ) and light zone (LZ) within GC B cells of D10 SRBCi Myc-eGFP mice. Data obtained by flow cytometry analysis. (c) Graphical representation of the frequency of c- Myc-eGFP positive (D10P) and negative (D10N) GC B cells localized to the dark zone (DZ) and light zone (LZ) within GCs of D10 SRBCi C57BL/6 wild-type mice. Data obtained by histological immunofluorescence (IF) analysis. (d) Representative flow cytometry analysis of cell surface expression of the activation markers CD86, CD83, CD40, CD69 and MHCII of non-gc B cells, and of the GC B cell subpopulations D10P, and D10N. (e) GSEA of c-mycegfp positive and c-myc-egfp negative GC B cell gene expression profiles, of D10 SRBCi Myc-eGFP mice, for a gene set of c-myc up-regulated genes obtained through the intersection of c-myc ChIP-Seq and MYC sirna knockdown data in GC B cell derived Burkitt s lymphoma lines. See Methods for GSEA analysis details. Scale bar 25µm (a). Data are representative of 3 independent experiments with at least 10 GC clusters analyzed per mouse (a), 3 independent experiments with at least 2 mice analyzed per experiment (d). Symbols represent data from individual mice (b). ns, not significant. * p 0.05, ** p 0.01, *** p Supplementary Table 1: Flow cytometry antibodies. Antigen Clone Company Mouse_CD19 ID3 BD pharmigen Mouse_CD95 Jo2 BD pharmigen Mouse_CXCR4 2B11 BD pharmigen Mouse_GL7 GL7 BD pharmigen Mouse_CD40 3/23 BD pharmigen Mouse_CD38 90 ebioscience Mouse_CD93 AA4.1 ebioscience Mouse_MHCII M5/ ebioscience Mouse_B220 RA3-6B2 ebioscience Mouse_CD69 H1.2F3 ebioscience Human_CD2 TS1/8 Biolegend Mouse_CD83 Michel-19 Biolegend Mouse_CD21 7E9 Biolegend Mouse_CD23 B3B4 Biolegend 11
12 Supplementary Table 2: Quantitative PCR primer sequences. Bcl6 5-GCCCACGTTCCCGGAGGAGA-3 5 -CGTCTGCAGCGTGTGCCTCT-3 Nfkbia 5 -TTGGTCAGGTGAAGGGAGAC-3 5 -GCTTTCAGAAGTGCCTCAGC-3 Irf4 5 -AGGTCTGCTGAAGCCTTGGC-3 5 -CTTCAGGGCTCGTCGTGGTC-3 MybL1 5 -AGCGCCTGGGAAACCGTTGG-3 5 -GCCCTCCTGTTCCACTTTTCTTCGC-3 Ccnd2 5 -TCGCAAGCTGCCCCAGCAAA-3 5 -GGCTGCTCCCACGCTTCCAG-3 Aicda 5 -TAGTGCCACCTCCTGCTCACT-3 5 -CAACAATTCCACGTGGCAGCC-3 Ccnd3 5 -CGGCGGGGTTGTGATCACTCG-3 5 -TCCTGCGATGGCTCACGGGT-3 Myc 5 -GCCAGCCCTGAGCCCCTAGT-3 5 -GGGCTGTGCGGAGGTTTGCT-3 Hprt 5 -GTCATGCCGACCCGCAGTC-3 5 -GTCCTGTCCATAATCAGTCCATGAGGAATAAAC-3 12
13 Supplementary Table 3: Immunofluorescence antibodies. Antigen Clone Company Mouse/Human_Bcl-6 sc-c19 Santa Cruz Mouse/Human_c-Myc Y69 Epitomics Mouse_Bcl-6 mgi191e ebioscience Mouse_CD C11 BD pharmigen Mouse_CD35 8C12 BD pharmigen 13
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