In brief, cells were harvested and proteins were cross-linked to DNA by adding

Transcription

1 Immunity, Volume 31 Supplemental Data The Transcriptional Repressor Bcl-6 Directs T Follicular Helper Cell Lineage Commitment Di Yu, Sudha Rao, Louis M. Tsai, Sau K. Lee, Yiqing He, Elissa L. Sutcliffe, Monika Srivastava, Michelle Linterman, Lei Zheng, Nicholas Simpson, Julia I. Ellyard, Ian A. Parish, Cindy Ma, Qi- Jing Li, Christopher R. Parish, Charles R. Mackay, and Carola G. Vinuesa SUPPLEMENTAL EXPERIMENTAL PROECEDURES Detailed ChIP Assay In brief, cells were harvested and proteins were cross-linked to DNA by adding formaldehyde to a final concentration of 1% for 1 min at room temperature followed by the addition of.25m glycine. Cells were washed three times with phosphatebuffered saline and resuspended in sodium dodecyl sulfate lysis buffer with the addition of complete EDTA-free protease inhibitor cocktail tablets (Roche Molecular Biochemicals). Formaldehyde-fixed cells were incubated on ice for 1 min and sonicated to shear chromatin by using an Utrasonic processor (Cole/Parmer) under optimized conditions that gave a range of DNA fragments from 1 to 5 bp, as determined by 2% agarose gel electrophoresis. Before the addition of antibody, samples were precleared with salmon sperm DNA-protein A-agarose for 3 min at 4 C with rotation. The soluble chromatin fraction was incubated overnight with rotation at 4 C with 5 µg of anti-bcl-6 antibody (SC-858, Santa Cruz) or without antibodies as a control. Immune complexes were collected with salmon sperm DNAprotein A-agarose, washed with 1 ml of low-salt buffer, high-salt buffer, LiCl wash buffer, and Tris-EDTA buffer (2x) and eluted with 4 µl of elution buffer with rotation at room temperature. Protein-DNA cross-links were reversed at 65 C overnight. Samples were recovered by phenol-chloroform extraction and ethanol precipitated. DNA pellets were washed in 7% ethanol, resuspended in 5 µl of Tris, ph 8., and 1

2 subsequently used for SYBR real-time PCR amplification (Applied Biosystems) (Figure S4). Antibody Used in Experiments For cell stimulation and differentiation: anti-cd3 (Clone 5A2, BD Pharmingen), anti-cd28 (Clone 37.51, BD Pharminggen), anti-ifn-γ (Clone XMG1.2, Biolegend), anti-il-4 (Clone 11B11, Biolegend), anti-il-12/il-23 p4 (Clone C17.8, Biolegend). For flow cytometry, antibodies were purchased from BD Pharmingen, ebiosciences and Biolegend except where otherwise indicated: Anti-mouse: CCR7 (Clone 4B7), CD25 (Clone Jo2), CD44 (Clone IM7), (Clone A2), (Clone 14), CD62L (Clone MEL-14), CXCR4 (Clone 2B11), CXCR5 (Clone 2G8), Fas (Clone PC61.5), GATA-3 (Clone L5-823), GL-7 (Clone GL7), ICOS (Clone 7E.17G9), IFN-γ (Clone XMG1.2), IL-17 (Clone TC11-18H1), IL-2 (Clone JES6-5H4), PD-1 (Clone J43), T-bet (Clone ebio4b1), Thy1.1 (Clone HIS51); Anti-human: CCR7 (Clone 1553, R&D Systems), CD45RA (Clone HI1), CD45RO (Clone UCHL1), CXCR4 (Clone 12G5), CXCR5 (Clone RF8B2), ICOS (Clone ISA-3), IFN-γ (Clone 4S.B3), IL- 17 (Clone ebio64dec17), IL-2 (Clone MQ1-17H12), IL-4 (Clone 8D4-8), PD-1 (Clone MIH4). For immunofluorescence: anti-mouse biotin (Clone 14, ebioscience) and biotinylated Peanut Agglutinin (PNA) (Vector Laboratories) conjugated to Alexa Fluor 555-Streptavidin (Invitrogen), CD4 FITC (Clone RM4-5, BD Bioscience) and IgD Alexa Fluor 647 (Clone 11-26c.2a, Biolegend). 2

3 SUPPLEMENTARY FIGURE LEGENDS Figure S1. GC and Tfh Profiles in Control Unimmunized Chimeric Mice. (A-B) Representative profiles showing GC cells (A) and Tfh cells (B) in unimmunized : (Group A) or : Bcl6 -/- (Group B) fetal liver chimeric mice. Figure S2. Development of Effector CD4 + T Cells from Bcl6 -/- T cells. (A) Flow cytometric profiles showing CD25 vs CD44 expression on CD4+ cells. in 5%:5% : (Group A) or : Bcl6 -/- (right) fetal liver chimeric mice one week after SRBC immunization. Gates identify effector (CD44 hi CD25 - ) CD4 + T cells and the percentages of cells in this gate are indicated. (B) Bar graphs showing the percentage of effector cells gated in (A) amongst and compartments from the same mice. (C) Ratio of CD4 + T effector cells between and compartments in each mouse. Each symbol represents one mouse. The lines are drawn through the median values in each group. P value was calculated by Student's t-test. Figure S3. Effects of Bcl-6 Deficiency on IL-17 and IFN-γ ssecretion by Mouse CD4 + cells (A, B) IFNγ (A) and IL-17 (B) production by total CD4 + T cells from : (Group A) or : Bcl6 -/- (Group B) fetal liver chimeras 8 days after SRBC immunization. Left panel shows representative flow cytometric profiles of IFN-γ vs CD44 expression and gates used to enumerate cytokine-producing cells quantified in the bar graphs (middle panel). The right panel shows ratios calculated by dividing the percentage of cytokine-secreting cells among CD4 + T cells ( or Bcl6 -/- ) by the percentage of cytokine-secreting cells amongst CD4 + T cells ( ) in each individual mouse. 3

4 (C, D) IFN-γ (C) and IL-17 (D) production by CD44 hi CD25 - CD62 lo effector cells sorted from the same mice as in (A-B). The ratios shown are calculated as in (A-B). (E, F) IFN-γ (E) and IL-17 (F) production by naïve CD44 lo CD25 - CD62L hi CD4 + cells sorted from the same mice as the panels above and differentiated for 3 days under Th1 (E) or Th17 (F) conditions. The ratios shown are calculated as in (A-B). In A-F, each symbol represents one mouse and the lines are drawn through the median values in each group. P values were calculated by Student's t-test. Figure S4. FACS Sorting Strategies (A) Strategy used to sort naïve CD4 + T cells (CD4 + B22-7AAD - CD44 lo CD62L hi CD25 - ) and activated CD4 + T cells (CD4 + B22-7AAD - CD44 hi CD62L low CD25 - ) from mouse spleens and lymph nodes. Percentages indicate the purity of populations. (B) Strategy used to sort human tonsil naïve CD4+ T cells (CD4 + 7AAD - CD45RO - CXCR5 lo ); Tfh cells (CD4 + 7AAD - CD45RO + CXCR5 hi PD-1 hi ); non-tfh effector cells (CD4 + 7AAD - CD45RO + CXCR5 int/lo PD-1 int/lo ); and a B cell-enriched population (CD4-7AAD - CXCR5 hi ). (C) Strategy to indentify Tfh cells and non-tfh effectors in mice. Figure S5. Bcl-6 Deficiency Does Not Influence Treg Development. (A) Representative flow cytometric profiles showing CD25 vs FoxP3 gated on CD4 + cells that are either Bcl-6 +/+ or Bcl-6 -/- from the fetal liver chimeras in the previous figure. (B) Numbers indicate the percentage of T regulatory cells (Tregs, FoxP3 + CD4 + ) amongst total CD4 + cells of the indicated genotypes. Each symbol represents one mouse and the lines are drawn through the mean values in each group. Figure S6. Increased Bcl-6 Expression Suppresses Th1 and Th17 cytokine secretion. 4

5 Intracellular IFN-γ and IL-17 production measured by flow cytometric staining of naïve CD4 + T cells sorted from C57BL/6 mice, stimulated with plate-bound anti-cd3ε and CD28 under Th1 or Th17 differentiation conditions in the presence of IL-2 for 2 days, followed by retroviral transduction with a Bcl-6 or empty vector control. After transduction, cells were cultured under Th1 or Th17 differentiation conditions for the indicated times before cytokine production was measured. For analysis, transduced cells were divided into three groups -, Intermediate (int), High (hi) - according to levels of GFP (Bcl-6) transduction as shown in Figure 3C and 3D. Figure S7 Bcl-6 Expression Does Not Suppress IL-2 Secretion. IL-2 production by naïve CD4 + T cells transduced with either a Bcl-6-expressing retrovirus or a vector control and stimulated under Th17 differentiation conditions for the times indicated. Transduced cells were divided into three groups: -, Intermediate (int), High (hi), according to the levels of GFP (Bcl-6). Day 5 representative FACS plots for each of the three groups are shown. Data shown represent mean values ± S.D., with n = 3. Figure S8. Location and Sequences of the Primer Sets Used for ChIP Analysis Figure S9 Bcl-6 Overexpression Suppresses GATA-3 Expression (A) Intracellular GATA-3 expression in sorted mouse naïve CD4 + T cells transduced with either a Bcl-6-expressing retrovirus or a vector control and cultured in Th2 differentiation conditions for 2 days (left panels). Bar-graphs show Geometric Mean Fluorescent Intensity (Geom MFI) and positive percentages in transduced (GFP + ) and non-transduced (GFP - ) cells (right panels). Data shown represent mean values ± S.D., with n = 3. P values were calculated by Student's t-test. (B) Representative flow cytometric profiles showing CD4 vs CD44 expression and CXCR5 vs PD-1 from Bcl6+/+ : Bcl-6-/- mixed chimeric mice 5

6 (described in Fig. 1), used to gate T naive (T N ), Tfh and non-tfh effectors (left panel). The histograms (middle panel) show GATA-3 expression in Bcl-6 +/+ (top) and Bcl-6 -/- (bottom panel) T cells from the chimeric mice; numbers indicate mean fluorescence intensity in each population. The graphs (right panel) show GATA-3 MFI in Bcl-6 +/+ Tnaive, Tfh and non-tfh effector cells from 4 individual mice. Figure S1. Bcl-6 Promotes the Tfh Gene Expression Signature. Flow cytometric analysis of PD-1, CXCR5, CXCR4 and CCR7 expression on naïve CD4 + T cells sorted from C57BL/6 mice, stimulated with plate-bound anti-cd3ε and anti-cd28 in the presence of IL-2, transduced by either a Bcl-6-expressing retrovirus or a vector control and cultured under ThN, Th1 or Th2 differentiation conditions for 2 days. Red, green and blue cells represent cells expressing no, intermediate or high levels of GFP (Bcl-6) respectively, with Geom MFI values of PD-1 or positive percentages of chemokine receptors shown. Data are representative of two experiments. Figure S11. mirna Expression by Non-transduced Cells (A) Naïve CD4 + T cells were sorted from C57BL/6 mice, stimulated with plate-bound anti-cd3ε and CD28 in the presence of IL-2 and transduced with either a Bcl-6- expressing retrovirus or a vector control. After transduction, cells were cultured under Th conditions for 2 days. Cells expressing intermediate to high levels of GFP were sorted to >98% purity to compare mirna gene expression changes in cells transduced with Bcl-6 vs empty vector. These results are shown in Fig. 6. Untransduced cells expressing no GFP (GFP - ) were sorted as an additional control. (B) MiRNA expression in GFP - cells from (A). No mirnas expressed at levels >1 were up- or down-regulated by more than 2-fold. 6

7 Figure S12. Predicted Binding Sites within 3 UTRs of Tfh Signature Molecules for mirnas Downregulated upon forced Bcl-6 Expression Schematic representation of predicted mirna binding sites within the 3 untranslated region (3 UTR) of mouse CXCR5, CXCR4, PD-1 and IL-21 as predicted by miranda and Targetscan. Figure S13 mir-11 and mir-13 Are Downregulated in Cells Expressing Bcl-6 and in Tfh Cells. Relative mrna levels of mir-11 and mir13 in (A) naïve CD4 + T cells transduced with either a Bcl-6-expressing retrovirus or a vector control, cultured under Th conditions for 2 days and sorted for high GFP expression; (B) mouse naïve and Tfh cells. Figure S14 Overexpression of mir Does Not Repress CXCR4 Expression (A) Histograms showing CXCR4 fluorescence on mouse B22 + cells transduced with retrovirus expressing empty vector (filled histogram) or mir (empty histogram). (B) The bar graphs show CXCR4 geometric mean fluorescence intensity in the same cells as in (A). Data shown represent mean values ± S.D., with n = 3. 7

8 Figure S1 A GL-7 B CXCR5 B Bcl6 -/-.3.2 Fas CD Bcl6 -/ Group A Group B Group A Group B PD-1

9 Figure S2 A Group A Group B Bcl6 -/- CD % 14.17% 25.16% 9.35% CD25 CD44 B Teffectors (CD44 hi CD25 - ) in CD4 + (%) Bcl6 CD45. Group A Group B +/+ +/+ +/+ -/ C Ratio of Teffector cells P<.1 Bcl6 -/-

10 Figure S3 A B C IFN-γ IL-17 IFN-γ Bcl6 -/- CD CD Bcl6 -/ Ratio of IFN-γ + cells 1.1 Bcl6 -/- CD CD Group A Group B Group A Group B P <.1 IFN-γ + in CD4 + cells (%) IL-17 + in CD4 + cells (%) Bcl6 -/ Group A Group A D IL-17 Bcl6 -/- Bcl6 -/ Bcl6 -/ Group B Group B Ratio of IL-17 + cells Ratio of IFN-γ + cells Ratio of IL-17 + cells P =.3 Group A 1.. P =.5 Bcl6 -/ Bcl6 -/- Group B Group A Group B.5 P =.2 Bcl6 -/- E IFN-γ Bcl6 -/ Ratio of IFN-γ + cells P <.1 Bcl6 -/- F IL Bcl6 -/ Ratio of IL-17 + cells P <.1. Bcl6 -/-

11 Figure S4 A 7AAD - CD4 + B22-7AAD - CD4 + B22 - CD25 - Effector CD25 CD62L 97.1% 97.6% Naïve CD44 CD44 B 7AAD - 7AAD - CD4 + 7AAD - CD4 + CD45RO + Tfh 92.% CXCR5 B cells CXCR5 CXCR5 PD % Naïve non-tfh 93.4% 92.8% CD4 CD45RO PD-1 C Tnaive CD4 CXCR5 Tfh non-tfh CD44 PD1

12 Figure S5 A Bcl6+/+ Bcl6-/- + CD Foxp3 CD25 B Treg (Foxp3 + ) in CD4 + (%) Bcl6 -/-

13 Figure S6 INF-γ + cells (%) GFP Bcl-6 - GFP Bcl-6 int GFP Bcl-6 hi Vector Bcl Time (days) IL-17 + cells (%) GFP Bcl-6 nil Vector Bcl-6 GFP Bcl-6 int GFP Bcl-6 hi Time (days)

14 Figure S7 8 GFP Bcl-6 nil GFP Bcl-6 int GFP Bcl-6 hi Vector Bcl-6 IL-2 + cells (%) Time (days) IL-2 Vector Bcl-6 GFP Bcl-6

15 Figure S8 A Predicted Bcl-6 binding sites: Human Primer: TSS TSS Tbx21 Rorc B Gene Rorc Tbx21 Primer Sets F: GTA GAA GCA TCA GGG GGT CA R: GAG ATG GAG AGG GCA GGT TT F: CCT CTG AGT GCC AGG AGA AT R: CTT GCT TTC TGG GAG TGT CA

16 Figure S9 A Cell number GFP + cells positive GFP - cells positive GATA-3 GATA-3 (Geom MFI) GFP + cells GFP - cells ** Bcl-6 Vector Bcl-6 Vector GATA-3 + cells (%) 1 75 GFP + cells GFP - cells ** 5 25 Bcl-6 Vector Bcl-6 Vector B CXCR5 CD4 Tnaive CD44 Tfh non-tfh PD1 Bcl6 -/- Gata-3 Tnaive = 35 non-tfh= 74 Tfh= 94 Tnaive = 39 Non-Tfh=9 Gata-3 MFI Tnaive Non-Tfh Tfh

17 % 1.59% 2.27% 5.36% 1.99% 1.39% 1.81% 4.17% 2.2% 1.68% 3.19% 6.4% Vector Bcl-6 GFP 22.7% 24.6% 25.2% 24.7% 3.7% 59.1% 21.1% 24.9% 21.9% 21.2% 24.6% 23.2% 15.8% 16.8% 17.9% 27.6% 35.4% 68.9% CCR7 22.4% 25.5% 22.3% 19.1% 21.4% 24.% 32.5% 32.2% 53.1% 69.5% 91.7% 5.2% 51.7% 57.5% 14.3% 22.% 35.4% Vector 29.8% CXCR4 2.35% 1.69% 1.97% 2.36% 1.78% Bcl-6 2.% CXCR Vector PD-1 Figure S1 Bcl-6 Vector Bcl-6 Thn Th1 Th2

18 Figure S11 A GFP 98.7% Figure 6 FSC 99.9% B Bcl-6 (probe signal) Vector (probe signal)

19 Figure S12 Mouse CXCR5 3 UTR (length: 1446) mir-18a, 18b mir-93, 17/2a, 2b, 16a, 16b Mouse CXCR4 3 UTR (length: 642) mir-132, mir-212 mir-23a Mouse PD-1 3 UTR (length: 956) mir-17-3p let-7d, let-7f, let-7a, let-7i, let-7g Mouse IL-21 3 UTR (length: 2578) mir-27a, mir-27b mir-21 mir-25 mir-21 mir-3e mir-142-5p mir-142-5p Predicted mirna target site

20 Figure S13 A 2.5 mir-11 5 mir-13 Relative to U6 (x1-4 ) Relative to U6 (x1-2 ) Vector Bcl-6 Vector Bcl-6 B 12 mir-11 5 mir-13 Relative to U6 (x1-4 ) Relative to U6 (x1-2 ) Tnaïve Tfh Tnaïve Tfh

21 Figure S14 A GFP + cells Cell number GFP - cells CXCR4 B 25 GFP - cells GFP + cells GMFI (x1 3 ) Vector mir Vector mir-17-92

22 Table S1 Details of the putative Bcl-6 binding sites generated by MatInspector Gene (Human) Location of Bcl-6 binding sites From To Optimised Core Similarity Matrix Similarity Strand Sequence (capitals: core sequence) Stat (+) atatacctagaaaagca Stat (+) ggactcctagagtcttc (+) gactttctggagaaaag (+) ggagtcctggcaagagc RORγt (+) tgcatcctagagaagag (+) tcttaggtagaaatcac (-) gccttccgagatggccc T-bet (+) aatttccccgagggtcc (+) acactcccagaaagcaa (Mouse) Gene Location of Bcl-6 binding sites From To Optimised Core Similarity Matrix Similarity Strand Sequence (capitals: core sequence) Stat (-) gtattccttgatatgga Stat (+) cttttcctccaagggac RORgt (+) ctcttccttgaggttgc T-bet (+) ttcctcctagacagggc

23 Table S2 MicroRNAs downregulated by Bcl-6 (by 2-fold) Clustered mirnas Bcl-6_1 Bcl-6_2 Control_1 Control_2 Fold change (mirna) Fold change (cluster) mmu-mir mmu-mir-23b mmu-mir-27b mmu-mir mmu-mir mmu-mir mmu-mir-2b mmu-mir-16a mmu-mir-19b mmu-mir-18a mmu-mir mmu-mir-19a mmu-mir-2a mmu-mir-92a mmu-mir-17* mmu-mir-31a mmu-mir-13b mmu-mir-142-5p mmu-mir-142-3p mmu-mir-23a mmu-mir-27a mmu-mir mmu-let-7g mmu-let-7i mmu-mir mmu-mir mmu-mir-16b mmu-let-7f mmu-let-7d mmu-let-7a mmu-mir mmu-mir-15b mmu-mir-3c mmu-mir-3e mmu-mir-15a mmu-mir

24 Table S3 MicroRNAs downregulated by Bcl-6 (by less than 2-fold) and upregulated by Bcl-6 Fold change (mirna) Fold change (cluster) Clustered mirnas Bcl-6_1 Bcl-6_2 Control_1 Control_2 mmu-mir-487b mmu-mir-342-3p mmu-mir-3b mmu-mir-3d mmu-mir-338-5p mmu-mir mmu-mir mmu-mir mmu-mir-22-3p mmu-mir-26a mmu-mir mmu-mir mmu-mir mmu-mir mmu-mir mmu-mir-146a mmu-mir mmu-mir-29c mmu-mir-29b mmu-mir-466c-5p mmu-mir-669c mmu-mir-669a mmu-mir mmu-mir mmu-mir-29a mmu-mir-29b mmu-mir-574-5p mmu-mir mmu-mir-466f-3p

25 Table S4 Genomic location of mirnas downregulated by BCL-6 Coordinates Gene / Intergenic Protein encoded by overlapping transcript OTTMUSG33261 No protein product 14: [+] (non-coding exon). mir-18a 14: [+] mir-17-5p/17-3p mir-19b 14: [+] mir-19a 14: [+] mir-2a 14: [+] mir-92a 14: [+] mir-93 5: mir-25 5: mir- 5: [-] 16b mir : [+] mir : [+] mir-23a 8: [+] mir-27a 8: [+] let-7d 13: [-] let-7f_1 13: [-] let-7a_1 13: [-] OTTMUSG22672 (intronic) OTTMUSG6193 (intronic) Intergenic Intergenic let-7i 1: [-] Intergenic let-7g 9: [+] ENSMUSG2257 (intronic) mir-23b 13: [+] mir-27b 13: [+] mir-24 13: [+] OTTMUSG3849 (intronic) ENSMUSG56199 (intronic) ENSMUSG21458 (intronic) mir-21 11: [-] OTTMUSG125 3'UTR (exonic) mir-3e 4: [-] OTTMUSG8852 mir-3c 4: [-] (intronic) mir-3d 15: [-] mir-3b 15: [-] mir p Intergenic 11: [+] OTTMUSG1327 (intronic) mir-15b 3: [+] OTTMUSG24475 (intronic) mir-16 14: [-] Intergenic mir-15a 14: [-] mir-2b X: [-] Intergenic mir- 16a mir- 31a mir- 13b X: [-] OTTMUSG17339 (exonic) 11: [+] OTTMUSG1218 (intronic) 16: [-] ENSMUSG49916 (intronic) Mcm7 No protein product Wdr82 WU:AC AC I1Rik-21 Tmem4 Nfyc (nuclear transcription factor-y gamma) No protein product Smc4 Kis2 locus (nonprotein coding) RP23-352L N2Rik-22 Function of protein DNA replication Gene methylation Unknown Unknown Transcription activation (cmyc, p53 ) Organisation of mitotic chromosomes unknown unknown