Supplementary data: Control DC Blimp-1 ko DC 8 6 4 2-2 IL-1β p=.5 medium 8 6 4 2 IL-2 Medium p=.16 8 6 4 2 IL-6 medium p=.3 5 4 3 2 1-1 medium IL-1 n.s. 25 2 15 1 5 IL-12(p7) p=.15 5 IFNγ p=.65 4 3 2 1 4 3 2 1 TNFα p=.31 Medium medium medium 1 8 6 4 KC n.s 6 4 2 TIMP-1 P=.26 2 Medium Medium Supplementary figure 1. Quantification of proinflammatory cytokines. DCs were purified from spleens of (ο) or DCBlimp-1 ko ( ) mice and treated with or without 1 µg of overnight. 5 µl of undiluted supernatant was applied to multicytokine precoated well for 2 h at rt. Each dot represents an individual mouse and bar is the mean ± SEM.
1 5 1 4 1 3 1 2 1.78 1 2 1 3 1 4 1 5 MHCII A. 2.5 2. 1.5 1..5. TLR3 n.s. Blimp-1 ko TLR4 6 p=.26 4 2 Blimp-1 ko 2. 1.5 1..5. TLR7 p=.27 Blimp-1 ko 1.5 1..5. TLR9 n.s. Blimp-1 ko B. CD4 B7-H4 CD86 CD273 CD275 15 25 12 12 12 DC # Cells 1 2 15 1 9 6 9 6 9 6 5 5 3 3 3 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 25 15 12 12 15 2 Blimp-1 ko DC # Cells 1 5 15 1 5 1 5 9 6 3 9 6 3 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 Supplementary figure 2. Surface expression of co-stimulatory molecules on DCs by flow cytometry. Spleens were collected from 6-8 week old and DCBlimp-1 ko mice. Total spleen cells were stained with anti- and anti-mhcii for cdcs (left top corner). From the gated population, the level of TLRs measured by qpcr in A (Mean ± SEM, n=6) and various molecules were analyzed by flow cytometry and MFI was compared in B. A representative flow image of 3 independent experiments.
4 3 p=.29 p=.29 RLU 2 1 Blimp-1 3 UTR mirna - + + + - - Let-7c Supplementary figure 3. Blimp-1 regulation by mirna measured by luciferase assay. 1 µg of Blimp-1 3 UTR/luciferase vector was transfected alone or with 1.4 µg of either mirna or Let-7c mirna vector into the 293T cell line. Luciferase activity was measured from 3x1 5 cells 48 h post transfection. Renilla luciferase activity was measured to compare transfection efficiency. Mean ± SEM of 4 independent experiments.
A. 1.5 SOCS1 Control DC Blimp-1 ko DC /HPRT 1..5. Transfection - - Blimp-1 B. 1 5 1 5 1 4 1 4 1 3 1 3 1 2 37.8.274 1 2 47.2 16.7 1 2 1 3 1 4 1 5 GFP 1 2 1 3 1 4 1 5 GFP # Cells 15 1 5 % of Max 1 8 6 4 2 % of Max 1 8 6 4 2 Blimp-1 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 1 2 1 3 1 4 1 5 SOCS1 SOCS1 SOCS1 Supplementary figure 4. Expression of SOCS-1 was measured from BM-DCs. Blimp- 1 ko BM-DCs were transduced with lentivirus expressing either gene or Blimp-1 gene with a GFP reporter. (A) To measure SOCS-1 mrna, GFP positive BM-DCs were sorted and SOCS-1 was measured by qpcr. Mean ± SEM, n=3. (B) SOCS-1 protein was measured by flow cytometry. To induce SOCS-1, BM-DCs were stimulated with 1 µg of for 24 h. On the left panel, SOCS-1 expression from untransduced DCs. On the right panel, SOCS-1 expression from lentivirus transduced Blimp-1 ko DCs. SOCS-1 expression was analyzed from + GFP + population or + GFP - population. Flow cytometry figure is a representative image of 3 independent experiments.
Blimp-1 ko DC transduced with Control DC Blimp-1 ko DC Control vector Blimp-1 vector DAPI//SOCS-1 DAPI//GFP/SOCS-1 SOCS-1 SOCS-1 DAPI/ GFP Supplementary figure 5. Immunofluorescence of SOCS-1 expression in DCs. BM-DCs were differentiated from either or DCBlimp-1 ko mice in vitro. Lentivirus carrying either gene or Blimp-1 was infected into Blimp-1 ko DCs and 24 h post infection, BM-DCs were counted and transferred to a chamber slide well for 2 additional days. (1 µg) was added for overnight before staining to induce SOCS-1. Images were taken at 1x magnification and the bar represents 15 µm. Representative images are from 3 independent experiments.
B. monocytes 9.51 SSC-A HLA-DR A. 1 5 1 5 1 4 1 4 1 3 1 2 1 3 1 46.7 CD16 5K 1K15K 2K 25K FSC-A 1 2 1 5 R1 21.2 1 5 1 4 1 4 1 3 1 3 R3 59.1 1 2 1 3 1 4 1 5 6.8 1 5 1 4 1 3 93.6 R2 25K CD14 CD1a GM-CSF + IL-4 1 5 1 2 1 2 1 3 1 4 1 5 Lin CD123 CD16 1 2 1 5 1 4 1 3 1 2 71.1 13.8 1 2 1 3 1 4 1 5 CD14 R4 14.8 34.2 R5 1 2 1 3 1 4 1 5 CD141 CD14 1 2 1 5 1 4 1 3 1 2 1 2 1 3 1 4 1 5 R6 19.6 1 2 1 3 1 4 1 5 SSC-A 2K 61.8 15K 1K 5K 5K 1K 15K 2K 25K FSC-A +CD14- cells (x1 6 ) 3 2 1 CD14 1 4 9.5 1 3 1 2 1 2 1 3 1 4 1 5 CD1a number of DC Blimp-1 carrier Supplementary figure 6. Flow cytometry image of human blood DC subsets and differentiation of Mo-DCs. (A) Flow cytometry diagram of monocytes and DC subsets in human blood. (B) CD14 + monocytes were purified by Easysep, and cultured with recombinant GM-CSF and IL-4 for 7 days. After 7 days, most of monocytes were differentiated into immature DCs (CD14 - CD1a + ) (~ 9% of viable cells). The number of immature DCs was calculated and plotted. Each dot represents an individual samples and bar is mean.
Blimp-1 SLE risk allele Blimp-1 Let-7c IL-6 relative Expression (Blimp-1/β2m) 3 2 1 p=.26 p=.35 relative Expression (Let-7c/U47) 6 4 2 p=.22 p=.51 IL-6 () 3 2 1 p=.7 p=.14 PB-MC cord blood PB-MC cord blood PB-MC cord blood Supplementary figure 7. Gene expression and IL-6 production in MO-DCs derived from either adult PB-MCs or cord blood samples. CD14+ monocyte-derived DCs were prepared from either adult blood or cord blood samples with same method described in Material and Methods. Total RNA was prepared and mrna or mirna was converted into cdna. The relative expression of Blimp-1 or mirna Let-7c was measured by realtime PCR. To measure the level of IL-6, 1 6 cells/ml of Mo-DCs were stimulated with (1 µg/ml) overnight and IL-6 in the supernatants was quantified by MSD. Each dot represents an individual sample and the bar represents the mean ± SEM.
Blimp-1 Let-7c 2. 4 (Blimp-1/β2m) 1.5 1..5 (Let-7c/U47) 3 2 1. SLE carrier SLE carrier Supplementary figure 8. Gene expression of purified B cells from human blood. Total B cells were purified by negative selection kit (Stem cell technologies). Total RNA was prepared and mrna or mirna was converted into cdna to measure Blimp-1 or mirna Let-7c. Each dot represents an individual sample and the bar represents the mean ± SEM.
8 IFNγ 1 IL-1 15 TNFα 6 4 2 8 6 4 2 1 5 Agonist - ploy (I:C) Gardiquimod Agonist - ploy (I:C) Gardiquimod Agonist - ploy (I:C) Gardiquimod 4 IL-12 p7 25 IL-8 5 IL-1β 3 2 2 15 1 4 3 2 1 5 1 Agonist - ploy (I:C) Gardiquimod Agonist - ploy (I:C) Gardiquimod Agonist - ploy (I:C) Gardiquimod Supplementary figure 9. Cytokine expression from human Mo-DCs by MSD. Number of Mo-DCs differentiated from either the or Blimp-1 SLE carrier individuals was calculated at day 7 culture, and plated to 1x1 6 /ml with or without TLR agonists (TLR3 agonist: poly (I:C), 5 µg/ml, TLR4 agonist:, 1 µg/ml, and TLR7 agonist: Gardiquimod, 2 µg/ml) overnight. Supernatant was harvested and kept at -2 C until use.
Coordinate Target Coordinate Target A1, A2 PC(+) A23, A24 PC(+) B1, B2 CXCL13 B3, B4 C5a B5, B6 G-CSF B7, B8 GM-CSF B9, B1 CCL1 B11, B12 CCL11 B13, B14 sicam1 B15, B16 IFNg B17, B18 IL-1a B19, B2 IL-1b B21, B22 IL-1ra B23, B24 IL-2 C1, C2 IL-3 C3, C4 IL-4 C5, C6 IL-5 C7, C8 IL-6 C9, C1 IL-7 C11, C12 IL-1 C13, C14 IL-13 C15, C16 IL-12 p7 C17, C18 IL-16 C19, C2 IL-17 C21, C22 IL-23 C23, C24 IL-27 D1, D2 IP-1 D3, D4 I-TAC D5, D6 KC D7, D8 M-CSF D9, D1 CCL2 D11, D12 CCL12 D13, D14 MIG D15, D16 MIP-1a D17, D18 MIP-1b D19, D2 MIP-2 D21, D22 RANTES D23, D24 SDF-1 E1, E2 TARC E3, E4 TIMP-1 E5, E6 TNF-a E7, E8 TREM-1 F1, F2 PC(+) F23, F24 NC(-) Supplementary Table 1: Table for the cytokine array coordinates