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Supporting Information Desnues et al. 10.1073/pnas.1314121111 SI Materials and Methods Mice. Toll-like receptor (TLR)8 / and TLR9 / mice were generated as described previously (1, 2). TLR9 / mice were obtained from S. Akira (Osaka University, Osaka, Japan). Double TLR8/9 / mice were generated by intercrossing TLR8 / with TLR9 / mice. All three TLR-deficient mouse genotypes were backcrossed on the C57BL/6 background for more than 10 generations. C57BL/6 mice were purchased from the Charles River Laboratories. Unless otherwise specified, age- and sex-matched mice were used for each experiment. Mice were housed under specific pathogen-free conditions at the Centre d Immunologie de Marseille- Luminy and experiments were conducted in accordance with institutional guidelines for animal care and use and European directives. Reagents. R848, LPS from Escherichia coli 0111-B4, Pam 3 CSK 4, CpG ODN 1826, and poly I:C were purchased from Invivogen. Recombinant mouse IFN-α was from Calbiochem. Preparation of Cells and Quantification of Cytokines. Bone marrow cells from mice were extracted from femur and tibias and cultured with GM-CSF or M-CSF for the production of bone marrow dendritic cells (BM-DCs) or BM-macrophages, respectively as previously described (1). For the production of bone marrowplasmacytoid DCs (pdc), bone marrow cells depleted of erythrocytes were cultured in RPMI, 10% (vol/vol) FBS, 2 mm GlutaMAX (Gibco), 1 mm sodium pyruvate, 1% (vol/vol) MEM Non-Essential Amino Acids (Gibco), 50 μm β-mercaptoethanol, and 2.5% (vol/vol) B16-Flt3L cell line supernatant (source of Flt3 ligand) at 2 10 6 cells/ml in six-well plates for 9 d (3). B cells were purified from spleen by negative selection using the B-cell isolation kit (Miltenyi). Purity was assessed by FACS and above 96%. Cytokines levels in culture supernatants were measured by IL-6 and TNF (ebioscience) ELISA kits. For intracellular cytokine staining splenocytes were treated with the indicated stimuli in the presence of Brefeldin A (e-biosciences) for 4 h. RNA Isolation and Quantitative PCR. Total RNA was isolated with TRIzol reagent (Gibco, Invitrogen) or RNA easy kit (Qiagen). RNA was reversed-transcribed with Supercsript II reverse transcriptase (Invivogen) and quantitative PCR for TLR7, IFN-β, and β-actin was performed as described previously (1). Serological Analysis. Evaluation of IgM and IgG2a, and IgG autoantibodies against DNA, RNA, ribonucleoprotein (RNP) and Smith ribonucleoprotein (smrnp) on serum samples were performed as described previously (1). Flow Cytometric Analysis. Cell suspensions were incubated with 24G2 hybridoma supernatant and then stained using immunofluorescence-labeled antibodies against the following antigens: B220, CD3, CD4, CD8, NK1.1, CD19, CD11b, CD11c, Ly6G, Ly6C, CD21, CD23, CD5, CD44,, CD38, CD138, CD24, and CD172a (SIRPα) from BD Biosciences, IA/IE (MHC class II), GL7 from ebioscience, and SiglecH from Biolegend. For intracellular cytokine staining cells were fixed with Cytofix/Cytoperm (BD Biosciences) for 20 min on ice and stained using immunofluorescence-labeled antibodies for TNF and IL12p40/p70 (BD Biosciences). Flow cytometry was conducted using an LSR2 (BD Biosciences) and data were analyzed with FlowJo (Tree Star). Histology, Immunofluorescence, and Antinuclear Antibody Staining. For histopathology studies, kidneys were fixed in formalin and embedded in paraffin. For light microscopy, 3- to 4-μm-thick tissue sections were stained with H&E and periodic acid-schiff. To determine the extent of renal damage, all renal biopsies were analyzed in a blinded fashion by a pathologist. Typical glomerular active lesions of lupus nephritis were evaluated: glomerular cellularity (mesangial cell proliferation, endocapillary proliferation, and inflammatory cells), glomerular deposits, extracapillary proliferation, as well as, tubulointerstitial chronic lesions: tubular atrophy, interstitial fibrosis, and interstitial infiltrates. Lesions were graded semiquantitatively using a scoring system from 0 to 4 (0, no changes; 1, mild; 2, moderate; 3, high; 4, severe). The total histological score for each specimen was derived from the sum of all of the described parameters. Immunofluorescence IgG and IgM staining on kidney sections was performed as described previously (1). Antinuclear antibodies (ANA) in mouse sera were tested with Hep-2 cells fixed on slides (Biomedical Diagnostics). Sera were diluted in PBS 1:60 and an Alexa Fluor 488 anti-mouse IgG antibody (Invitrogen) was used to detect mouse antibodies. Statistical Analysis. Statistics were calculated using Prism 5 (GraphPad Software) by Mann Whitney U test with P values indicated throughout as P < 0.05, P < 0.01, and P < 0.001. 1. Demaria O, et al. (2010) TLR8 deficiency leads to autoimmunity in mice. J Clin Invest 120(10):3651 3662. 2. Hemmi H, et al. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408(6813): 740 745. 3. Gilliet M, et al. (2002) The development of murine plasmacytoid dendritic cell precursors is differentially regulated by FLT3-ligand and granulocyte/macrophage colony-stimulating factor. J Exp Med 195(7):953 958. 1of6

A H&E B IgM Fig. S1. Increased renal pathology in TLR8/9 / mice compared with TLR8 / or TLR9 / mice. (A) H&E-stained kidney sections from 6 mo old female, TLR8 /, TLR8/9 /, and TLR9 / mice (n = 3 per group). (Left) (Scale bars, 1 mm.) (Right) Magnified details of black bordered areas. (Scale bars, 0.5 mm.) (B) Kidney sections from 5-mo-old female, TLR8 /, TLR8/9 /, or TLR9 / mice (n = 3 per group) were stained with immunofluorescence anti-igm (original magnification 100). 2of6

A TNF (ng/ml) 8 6 4 2 C vs. TLR8 -/- vs. vs. pdc B IL-6 (ng/ml) 0 2.0 1.5 1.0 0.5 0 50 100 1 10 R848 LPS (nm) (ng/ml) CD11b-like DC CD8α-like DC IL-6 (pg/ml) 0.0 0 20 50 50 R848 (nm) PolyIC (μg/ml) D DC subtype (% CD11c + cells) 50 40 30 20 10 0 pdc CD8α-like DCCD11b-like DC Fig. S2. Cytokine production and expression by, TLR8 /, TLR8/9 /, and TLR9 / DCs or macrophages. (A) BM-DCs or (B) BM-macrophages from, TLR8 /, TLR8/9 /, and TLR9 / mice left untreated or stimulated with the indicated amounts R848, LPS, or poly I:C. After 16 h, the concentration of TNF (A) or IL-6 (B) in culture supernatants was assessed by ELISA. Data are representative of two to three independent experiments (n = 3 per group). (C) Total splenocytes were left untreated or stimulated with 30 nm R848. After 16 h, the surface expression of was analyzed by FACS analysis on splenic pdcs, CD8α-like DCs, or CD11b-like DCs from (black line), TLR8 / (blue line), TLR8/9 / (red line), or TLR9 / (green line) mice (n = 3 mice per group). Untreated cells (dotted lines), R848 treated cells (full lines). n = 3 mice per group. (D) Frequencies of splenic pdcs, CD8α-like DCs, or CD11b-like DCs in the four genotypes. Data are average percentages ± SD (n = 3 mice per group). P < 0.05. 3of6

A 2.65 gated on pdc (CD5 - CD19 - CD11c + CD11b - B220 + ) 1.44 1.69 0.627 R848 (30 nm) 11.2 24 31.1 10.1 poly IC (100 μg/ml) CD11c B TNF 7.95 10.3 10.4 6.89 gated on CD11b-like DC (CD5 - CD19 - CD11c + CD11b + CD172 + CD24 - ) 1.37 1.18 1.41 0.731 R848 (100 nm) 7.82 16.4 19.3 8.54 poly IC (100 μg/ml) CD11c 2.51 2.63 3.17 3.05 C TNF gated on CD11b-like DC (CD5 - CD19 - CD11c + CD11b + CD172 + CD24 - ) 0.149 0.65 0.677 0.293 R848 (100 nm) 4.15 11.3 10.4 4.97 poly IC (100 μg/ml) CD11c IL12p40/70 0.774 1.1 1.21 0.232 Fig. S3. Analysis of TNF and IL-12p40/70 cytokine production by splenic pdcs and CD11b-like DCs. Total splenocytes were left untreated or stimulated with R848 or poly I:C. After 4 h, intracellular staining for TNF and IL-12p40/p70 was performed. Expression of TNF was evaluated after gating on (A) pdcs and (B) CD11b-like DCs, and expression of IL-12P40/70 was evaluated after gating on (C) CD11b-like DCs from, TLR8 /, TLR8/9 /, or TLR9 / mice (n = 3 per genotype). Numbers in dot plots represent the percentage of cells that produce TNF or IL-12. Plots are representative of the data shown in Table S4. 4of6

A 10 nm R848 30 nm R848 500 ng/ml LPS B 0.20 RQ (tlr7/actb) 0.15 0.10 0.05 C 0.00 splenocytes B cells B cells 1 U/ml IFNa B cells 10 U/ml IFNa 10 nm R848 CD69 Fig. S4. expression by splenocytes and TLR7 expression and response of isolated B cells from, TLR8 /, TLR8/9 /, and TLR9 / mice. (A) Total splenocytes were left untreated or stimulated with R848 or LPS for 16 h. Representative flow cytometry plots of on (black line), TLR8 / (blue line), TLR8/9 / (red line), and TLR9 / (green line) CD119 + B220 + splenocytes. (B) Total RNA was extracted from, TLR8 /, TLR8/9 /, or TLR9 / splenic isolated B cells and the expression of TLR7 and β-actin were evaluated by quantitative PCR. P < 0.05. (C) Splenocytes or isolated B cells from (black line) or TLR9 / (green line) mice were left untreated or stimulated with the indicated amount of R848 in the presence or absence of IFN-α. The surface expression of CD69 was analyzed by flow cytometry on CD19 + cells. Data are representative of two independent experiments (n = 3 per group). Table S1. Spleen weight, total cell count, and major cell populations of, TLR8 /, TLR8/9 /, and TLR9 / mice TLR8 / TLR8/9 / TLR9 / Spleen weight (mg) 77.0 ± 19.5 116.9 ± 29.1, 145.7 ± 50.8,, 103.7 ± 17.8, Total cell count ( 10 6 ) 67.7 ± 26.4 108.6 ± 32.8, 143.0 ± 46.8, 113.4 ± 34.1 Cell type Cell surface markers Frequency (% of total) T cells CD3 + 25.7 ± 3.7 20.7 ± 5.4 21.3 ± 3.4 26.1 ± 1.8, CD4 T cells CD3 + /CD4 + 14.5 ± 2.7 12.3 ± 3.0 12.8 ± 1.7 15.9 ± 1.2, CD8 T cells CD3 + /CD8 + 9.7 ± 1.3 6.7 ± 2.3 6.5 ± 1.6 8.4 ± 1.1 NKT cells CD3 + /NK1.1 + 0.5 ± 0.1 0.6 ± 0.1 0.6 ± 0.1 ND NK cells CD3 /NK1.1 + 2.2 ± 0.1 1.8 ± 0.4 1.5 ± 0.2 ND B cells CD19 + 58.8 ± 3.7 65.9 ± 5.1 66.8 ± 5.5 63.4 ± 6.0 CD11c + cells CD11c + 3.9 ± 0.4 5.0 ± 0.5 5.3 ± 0.6 5.4 ± 0.9 DC CD11c hi /IA-IE + 1.6 ± 0.1 1.8 ± 0.1 2.1 ± 0.5 2.0 ± 0.1 pdc CD11c int /SiglecH + 0.7 ± 0.1 0.9 ± 0.3 0.8 ± 0.3 1.0 ± 0.2 Monocytes CD11b + /Ly6G /Ly6C + 0.4 ± 0.3 1.1 ± 0.4 0.8 ± 0.2 1.1 ± 0.2, Neutrophils CD11b + /Ly6G + /Ly6C 0.4 ± 0.3 0.9 ± 0.2, 0.5 ± 0.3 0.9 ± 0.2 Data are from 4- to 7-mo-old male or female mice. No differences were observed between males and females. Values shown are average of at least four mice ± SD. ND, not determined. P < 0.05 vs.. P < 0.05 vs. TLR8/9 /. P < 0.05 vs. TLR8 /. P < 0.05 vs. TLR9 /. 5of6

Table S2. Frequency of splenic marginal zone (MZ) B cells and peritoneal B1a and B1b B cells of, TLR8 /, TLR8/9 /, and TLR9 / mice Genotype MZ B cells B1a B cells B1b B cells 4.7 ± 0.2 (n = 8) 18.8 ± 1.2 (n = 9) 10.9 ± 0.4 (n = 5) TLR8 / 2.1 ± 0.2, (n = 8) 9.1 ± 1.2,, (n = 8) 5.1 ± 1.0 (n = 4) TLR8/9 / 1.5 ± 0.3, (n = 8) 4.7 ± 0.6,, (n = 7) 7.2 ± 1.7, (n = 3) TLR9 / 4.8 ± 0.5, (n = 3) 14.9 ± 1.5, (n = 7) 11.7 ± 0.8, (n = 3) Data are from 4- to 7-mo-old female mice. Values shown are average ± SD. In parenthesis is the number of mice per group. P < 0.05 vs.. P < 0.05 vs. TLR9 /. P < 0.05 vs. TLR8/9 /. P < 0.05 vs. TLR8 /. Table S3. Renal scores for, TLR8 /, TLR8/9 /, and TLR9 / female 6-mo-old mice Glomerular lesions Tubulo-interstitial lesions Genotype Glomerular cellularity Extracapillary proliferation Glomerular depositis Tubular atrophy Fibrosis Interstitial infiltration Total score 0 0 0 0 0 0 0 TLR8 / 1.33 ± 0.57 0 1.00 ± 0.00 0 0 0.66 ± 0.57 2.9 TLR8/9 / 1.66 ± 0.57 0.33 ± 0.57 1.66 ± 0.57 0 0 1.00 ± 0.00 4.5 TLR9 / 1.00 ± 0.00 0 1.00 ± 0.00 0 0 0 2 Pathologist scored H&E and periodic acid-schiff stained paraffin embedded kidney sections displayed as mean ± SD, n = 3 mice per group shown. Pathologist scoring method and definitions can be found in SI Materials and Methods. Table S4. Percentages of TNF- and IL-12p40/p75-expressing splenic pdcs, CD8α + -like DCs, and CD11b + -like DCs from, TLR8 /, TLR8/9 /, and TLR9 / mice upon R848 or poly I:C stimulation Genotype Untreated R848 Poly I:C Untreated R848 Poly I:C TNF by pdc IL-12 by pdc 2.5 ± 0.7 10.9 ± 2.3 7.4 ± 1.3 0.3 ± 0.2 5.0 ± 2.8 0.4 ± 0.2 TLR8 / 1.4 ± 0.1 44.1 ± 21.5, 10.2 ± 1.3 0.5 ± 0.2 8.2 ± 1.6 0.9 ± 0.1 TLR8/9 / 2.9 ± 1.4 30.1 ± 4.0, 12.9 ± 4.5 0.2 ± 0.1 12.4 ± 2.1 1.2 ± 0.4 TLR9 / 0.8 ± 0.1,, 11.5 ± 0.7, 5.8 ± 0.6 0.2 ± 0.1 4.9 ± 1.0 0.6 ± 0.2 TNF by CD8α + -like DC IL-12 by CD8α + -like DC 3.5 ± 2.3 2.7 ± 1.5 6.7 ± 0.8 2.1 ± 1.0 3.4 ± 1.4 10.9 ± 2.4 TLR8 / 1.0 ± 0.3 1.5 ± 0.3 9.7 ± 1.5 4.2 ± 0.8 5.4 ± 0.6 13.8 ± 0.4 TLR8/9 / 1.3 ± 0.1 2.1 ± 0.3 7.2 ± 0.4 4.4 ± 0.1, 5.4 ± 0.8 13.2 ± 1.4 TLR9 / 0.9 ± 0.1, 1.2 ± 0.2 8.2 ± 1.9 3.8 ± 0.2, 4.5 ± 0.8 9.9 ± 1.5 TNF by CD11b + -like DC IL-12 by CD11b + -like DC 2.1 ± 1.1 7.1 ± 1.9 2.1 ± 0.2 0.1 ± 0.0 3.9 ± 1.5 0.7 ± 0.1 TLR8 / 1.2 ± 0.2 14.6 ± 3.7 2.2 ± 0.1 0.6 ± 0.1, 10.7 ± 0.6, 1.0 ± 0.2 TLR8/9 / 1.6 ± 0.4 16.5 ± 1.4, 3.0 ± 0.2, 0.6 ± 0.1, 12.9 ± 1.9, 0.8 ± 0.3 TLR9 / 0.7 ± 0.1,, 8.1 ± 1.2 2.4 ± 0.4 0.2 ± 0.1, 5.3 ± 1.0, 0.1 ± 0.1 Data represent the average percentage ± SD of TNF or IL-12p40/p70- expressing pdcs, CD8α + -like DCs, or CD11b + -like DCs after stimulation of splenocytes from three mice per group with 30 nm R848 for pdcs and CD8α + -like DCs, 100 nm R848 for CD11b + -like DCs, or 100 μg/ml poly I:C. P < 0.05 vs. TLR9 /. P < 0.05 vs.. P < 0.05 vs. TLR8/9 /. P < 0.05 vs. TLR8 /. 6of6

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