LPS CD40 + IL-4. Vorinostat (24 Hours) Vorinostat (24 Hours) Panobinostat (24 Hours) Panobinostat (24 Hours) Romidepsin (48 Hours)

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1 A) CD + IL- B) LPS ( Hours) ( Hours) Cell number (x1-3 ) M 1. M. M.1 M Cell number (x1 - ) M 1. M.7 M.38 M Cell number (x1-3 ) Cell number (x1-3 ) ( Hours) 7.nM.nM 1.7nM.nM Romidepsin (8 Hours) 3.nM 1.8nM 1.8nM.nM.39nM.3nM Cell number (x1 - ) Cell number (x1 - ) ( Hours) 1 1.7nM.nM.8nM Romidepsin (8 Hours).nM.3nM.1nM.8nM.nM Supplementary Figure 1 HDACi modulate B cell survival in response to T-dependent and T- independent stimulation in vitro: CFSE labeled B cells were stimulated with (A) αcd + IL- or (B) LPS in the presence of the indicated concentrations of HDACi and cell numbers measured at or 8 hours post stimulation. Data are mean ± SEM of triplicate samples and are representative from -3 independent experiments. Statistical significance was determined by one-way ANOVA and multiple comparisons made using Bonferroni comparison with regards to vehicle only treated cultures. * = P., ** = P.1, *** = P.1, **** = P.1.

2 Pan HDACi Class I Specific Romidepsin Mean Division Number.nM.8nM.nM Mean Division Number 3. M 1. M.7 M.38 M.19 M. M Mean Division Number.1nM.8nM.nM.nM Supplementary Figure Effect of HDACi on mean division number of CFSE profiles. The mean division number of CFSE profiles from LPS stimulated B cells treated with vorinostat, panobinostat and romidepsin cultures was calculated as previously described 1.

3 A B C Cell number (x1 - ) Cell number (x1 - ) Hours 8 Hours *** *. M.3nM 1. M 1.nM. M.nM Cell number (x1 - ) Cell number (x1 - ) **** **. M.3nM 1. M 1.nM. M.nM. M 1. M. M Division.nM 1.nM.8nM Cell Number (x1 - ) Cell Number (x1 - ) 8. M 1. M. M 8 1 Division Division.nM 1.nM.8nM Supplementary Figure 3 Effect of HDACi on CD8 T cells in vitro: CD8 + T cells were isolated from C7BL/ mice, CFSE labeled and cultured with the indicated concentrations of vorinostat and panobinostat. (A) Total cell numbers were measured by reference to quantification beads. (B) Dilution of CFSE was used to determine the effect on cell division and number of cells in each division quantified in (C) after days in culture. Data are mean ± SEM of triplicate samples. * = P., ** = P.1, *** = P.1, **** = P.1.

4 (DW) Weight (Grams) 3 1 Weight (Grams) Days 1 3 Days Supplementary Figure Effect of panobinostat on mouse weight. Mice received vehicle or panobinostat treatment as described in materials and methods. Mice were measured throughout the dosing regime and weights recorded (n=8 mice per group).

5 1 8 dsdna Histone SMRNP * * 3 ** DW DW DW 8 TRIM1 (Ro) u1-rnp * * 3 1 DW DW 3 1 CENP-B Jo-1 PCNA DW DW DW 8 8 PmScl Ribosomes Scl DW DW DW SM SSA (Ro) SSB DW DW DW Supplementary Figure - Extractable nuclear antigen (ENA) analysis of mouse serum: Serum was isolated from mice post panobinostat therapy and the levels of auto-reactive antibody determined as described in materials and methods. (DW ()); n=8, ; n=8). Data are represented as mean ± SEM. Significance of differences were determined using two-tailed unpaired t-tests. * = P., ** = P.1.

6 A (DW) B C (DW) D Supplementary Figure H&E analysis of panobinostat treated kidney sections. Representative renal cortical glomeruli from (A) vehicle (DW) and (B) panobinostat-treated mice (H&E x). Scale Bar = 1µm. Arrows indicate areas of proliferative glomerulonephritis with early crescent formation. Sections through the medulla of kidneys from (C) DW and (D) panobinostat-treated mice. Arrows indicate areas of perivascular lymphoid infiltrate (H&E x). Scale Bar = µm.

7 A B * OD 3 1 D D9 D D9 % ANA pre/post therpy 1 1 C # CD19 + B + (x1 - )/ l 1 * 1 D Total T cell # (x1 - )/ l 3 1 Supplementary Figure 7 treatment in Lyn -/- mice reduces autoantibody titres and B cell numbers: Lyn -/- mice were treated with panobinostat for 8 days and peripheral blood analysed for (A and B) serum pooled ANA levels, and absolute numbers of (C) B cells and (D) T cells. * = P..

8 A Gated on B +, IgD lo, NP +, IgG1 +, CD138 +/- 1 O Immunisation O Immunisation GC Memory CD38 B Gated on B +, IgD lo, NP +, IgG1 + Gated on B +, IgD lo, NP +, IgG1 + DW DMSO CD38 CD38 Supplementary Figure 8 Separation of germinal center and memory B-cells based on CD38 expression: (A) Histogram illustrating the differential expression of CD38 on B cells after either primary or secondary challenge with NP-KLH. (B) Overlay of B cell CD38 expression in panobinostat and vorinostat treated mice after secondary challenge with NP-KLH.

9 (DW) treated Supplementary Figure 9 Germinal centre structures are perturbed in panobinostat treated mice: Whole spleen sections were imaged using multi-position tilescan imaging. Images were compiled using post acquisition stitching. Data shown are frozen sections of spleens 1 days post immunization from treated and untreated groups stained with IgD (red) to identify B cell follicles and GL-7 (green) to identify germinal centres. Arrows indicate representative follicles. Data are representative of 3- mice per group and of independent experiments. Scale bars = µm.

10 Per Mouse A Per Group B # of GC # of GC 1 1 % B cell follicles with GC DW D 1 8 % B cell follicles with GC DW *** 8 DW E 1 C *** 1 DW DW Supplementary Figure 1 inhibits germinal centres in MRL/lpr mice: 7 week old MRL/lpr were treated with either vehicle (DW) or panobinostat for weeks and the presence of germinal centres was determined by immunohistochemistry. The total number of germinal centres in four distinct spleen sections were quantified per mouse (each column is data derived from an individual mouse) (A) and combined for each group (B). The percentage of B cell follicles with a GL-7+ germinal centre per mouse (C) and per group (D). Significance of data was determined by an unpaired T-test. (E) Representative frozen sections of spleens from treated and untreated groups stained with IgD (blue) to identify B cell follicles and GL-7 (brown) to identify germinal centres. Arrows indicate germinal centres. Scale bars = µm. n = mice per group. Data represent mean ± SEM. *** = P.1.

11 A IgM IgD Gr-1 CD138 () DMSO 1.% ±1.7 B 3.% ±.8 B C D IgG1 NP () DMSO 1.3% ±.1 3.3% ±.9 () DMSO GC CD38 89.% ±. 9.8% ±. Memory 87.3% ±. 9.9% ±. Total NP+IgG1+ cells (x1 - ) 8 () DMSO ** E () DMSO Supplementary Figure 11 inhibits primary antibody responses: Analysis of C7BL/ mice 1 days after immunization with NP-KLH in alum and treatment with vorinostat. (A) Flow cytometric identification of isotype-switched B cells (GR-1 -, CD138 -, IgM -, IgD -, B + ). (B) Gated isotype-switched B cells were analyzed for reactivity against NP antigen (NP +, IgG1 + ). NP + B cells were (C) further fractionated into germinal center (GC) and memory lineages based on CD38 expression. (D) Absolute numbers of NP + IgG1 + B cells per spleen. (E) Frozen sections of spleens 1 days post immunization stained with IgD (green) to identify B cell follicles and GL-7 (red) to identify GC. Scale bars = µm. Data are mean ± SEM of 3- mice per group and are representative of -3 independent experiments. ** = P.1.

12 A # B + CD19 + cells (x1 7 ) *** (DW) B # B + NP + IgG1 + cells (x1 ) 1 8 Pre *** Post Boost N.S Pre ** Post Boost (DW) Supplementary Figure 1 Effect of panobinostat on the humoral memory response: (A) Mice were treated for seven days with panobinostat as described in materials and methods and the number of B + CD19 + B cells in spleen quantified. (B) Mice were treated with either vehicle or panobinostat as described in materials and methods > days following primary immunization with NP-KLH. The number of NP responsive B cells in spleens of mice were then measured both prior to, and days following boosting with NP-KLH in the absence of alum. ** = P.1, *** = P.1.

13 A B C () DMSO () DMSO () DMSO.8% ±.1 1.8% ±3. 83.% ±3.9.% ±.3 IgM IgD Gr-1 IgG1.9% ±.33 1.% ±.9 8.% ±.1.% ±. B NP CD38 D Total NP+IgG1+ cells (x1 - ) 7... (DMSO) E Total B + CD19 + cells (x1-7 ) 1 8 (DMSO) Supplementary Figure 13 Memory antibody responses are not affected by vorinostat treatment: C7BL/ mice were immunized with NP-KLH, rested for days, treated with vehicle or vorinostat, rested for 1 days, and then re-challenged with NP-KLH and analyzed days later to assess memory responses. (A) Flow cytometric identification of isotype switched splenic B cells (GR-1 -, IgM -, IgD -, B + ). (B) Gated isotype switched splenic B cells were analyzed for NP reactivity (NP + IgG1 + ) and (C) memory cells identified based on CD38 expression. Absolute numbers of (D) NP+ B cells and (E) B+CD19+ B cells per spleen. Data are mean ± SEM of 3- mice per group and are representative of independent experiments.

14 # # Supplementary References 1. Turner ML, Hawkins ED, Hodgkin PD. Quantitative regulation of B cell division destiny by signal strength. Journal of immunology 8, 181(1):