Supporting Information Celhar et al. 10.1073/pnas.1507052112 SI Materials and Methods using a Nanodrop spectrophotometer (Thermo Fisher Scien- Proteinuria. Proteinuria was assessed using Albustix (Bayer). Al- tific). A TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems) bumin levels in urine were assayed using an Albumin Mouse was used to perform the reverse transcription and quantitative ELISA Kit (Abcam) according to the manufacturer’s instructions; PCR reactions according to the manufacturer’s instructions samples were assayed at a dilution of 1:400. Samples were nor- using TaqMan gene expression assays (Applied Biosystems) to malized for creatinine using a Creatinine (urinary) Colorimetric either Tlr7 (Mm00446590) or the B2m housekeeping gene Assay Kit (Cayman Chemical) according to the manufacturer’s (Mm00437762). Real-time PCR was performed on the 7900H instructions; initial sample dilution of 1:10. fast real-time PCR system and analyzed using SDS 2.4 (Applied Biosystems). Relative mRNA expression was calculated using the Cell Sorting, RNA Isolation, and RT-PCR. Splenic B cells were comparative C method. + − + + t sorted as live CD45 Gr1 B220 CD19 , splenic T cells as live + − + + CD45 Gr1 CD3 CD5 and peritoneal macrophages as live Imaging. Kidney sections from OCT embedded tissue were fixed + − CD45 Gr1 CD11bhiF4/80hi. Sorted cells were centrifuged, re- with 4% paraformaldehyde before permeabilization with acetone suspended in TRIzol (Life Technologies) and stored at −80°. RNA and stained with Phalloidin (AF647) and anti-CD3d (unlabeled was extracted by TRIzol/chloroform and purified with the Qiagen Ab followed by secondary staining with donkey anti-goat Dylight RNeasy Mini purification kit according to the manufacturer’s 550). Images were captured with a Zeiss LSM800 microscope at instructions (Qiagen). Concentration and purity were measured 200× magnification and processed with Zen imaging software. Celhar et al. www.pnas.org/cgi/content/short/1507052112 1of10 Fig. S1. TLR7 mRNA expression levels in lymphoid and non-DC myeloid lineages and autoimmune pathology. (A) TLR7 mRNA levels were assessed by quantitative RT-PCR in B- and T-lymphoid cells and macrophages from CD11cSle1Tg7 and control mouse spleens at 1–4 mo of age (n = 4–6 per strain for B cells and n = 4–5 mice per strain for T cells and macrophages). (B) B-cell and macrophage TLR7 mRNA levels were assessed by quantitative RT-PCR at 11 mo of age in Sle1 (n =2) and CD11cSle1 mice (n = 4). (C) Splenic weight and analysis of T-cell subsets by flow cytometry in Sle1 (n =12) and CD11cSle1 mice (n = 8) at a median of 11 mo of age (all mice aged between 8 and 13 mo). (D) Proteinuria in Sle1, Sle1Tg7 and CD11cSle1Tg7 mice (Albustix, n = 11–14 mice per strain, albumin/ creatinine ratio n = 6–10 per strain). (E) Frozen kidney sections were assessed for complement activation by staining with anti-C3d antibody. C3d deposition is evident in the enlarged glomeruli of Sle1Tg7 mice and to a lesser extent in the glomeruli of Sle1 and CD11cSle1Tg7 mice. Microscopy magnification: 200×, images are representative of two to three mice per strain. Bars represent mean + SEM. *P < 0.05 (Kruskal–Wallis test with post-hoc Dunn’s multiple correction test); #P < 0.05 (multiple t test corrected for multiple comparisons using the Holm–Sidak method); ns, not significant. Celhar et al. www.pnas.org/cgi/content/short/1507052112 2of10 Fig. S2. M-lysozyme-Cre-normalization of TLR7 does not ameliorate severe autoimmune disease pathogenesis. (A) Splenic weight, GN score, and BUN in Sle1, Sle1Tg7, and LyzMSle1Tg7 mice (6-mo-old mice, n ≥ 12 per strain). (B) LyzM-Cre-normalization does not lead to significant changes in ANAs and anti-snRNP levels as determined by ELISA (n ≥ 7 mice per strain). Bars represent mean + SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA); ns, not significant. Celhar et al. www.pnas.org/cgi/content/short/1507052112 3of10 + Fig. S3. Quantification of kidney pDCs and CD64 expression in sorted renal cell subsets. (A) To identify pDCs, CD45 singlets which did not express CD11b were gated for B220 expression. These cells were subsequently gated as CD19−CD11c+ and finally pDCs identified as the population of F480−SiglecH+ cells. (B) Flow cytometry was used to assess the frequency of pDCs in the kidneys of Sle1, Sle1Tg7, and CD11cSle1Tg7 mice (n = 6 mice per strain). (C) mRNA expression levels assessed by Nanostring as described. (D) Surface CD64 expression levels assessed by flow cytometry. Representative histograms with median fluorescence intensities are shown. *P < 0.05 (computed as described under Nanostring Gene-Expression Analysis); ns, not significant (one-way ANOVA). Celhar et al. www.pnas.org/cgi/content/short/1507052112 4of10 Table S1. Kidney supernatant analysis by Luminex assay Sle1 (n = 13) Sle1Tg7 (n = 12) CD11cSle1Tg7 (n = 11) Chemokines and cytokines Mean ± SEM (pg/kidney) Mean ± SEM (pg/kidney) Mean ± SEM (pg/kidney) Eotaxin 12.27 ± 0.9962 22.03 ± 3.154 13.39 ± 1.216 G-CSF 10.23 ± 2.221 24.32 ± 7.513 9.7 ± 1.264 GM-CSF 4.446 ± 2.083 4.358 ± 1.735 6.582 ± 2.101 IFNg 5.662 ± 1.073 4.65 ± 1.565 3.891 ± 1.08 IL-10 8.258 ± 1.186 5.882 ± 1.361 8.145 ± 1.775 IL-12p40 26.99 ± 4.156 21.65 ± 5.169 22.35 ± 3.257 IL-12p70 25.88 ± 3.014 19.17 ± 2.546 18.44 ± 3.634 MCP-1 8.354 ± 0.8829 24.78 ± 5.536 8.873 ± 1.247 IP-10 19.15 ± 1.507 74.44 ± 13.59 27.59 ± 5.012 LIF 2.015 ± 0.281 63.18 ± 17.94 4.991 ± 0.9669 MIG 264.6 ± 22.76 784.4 ± 158.5 370.3 ± 61.87 Rantes 5.792 ± 0.7022 19.77 ± 3.366 9.3 ± 2.416 IL-1a 40.32 ± 6.746 33.07 ± 7.838 41.7 ± 8.666 IL-1b 19.9 ± 2.407 26.59 ± 6.167 28.89 ± 4.421 MIP1a 3.346 ± 0.7272 9.317 ± 2.376 3.6 ± 0.897 IL-15 20.8 ± 1.6 17.6 ± 2.4 20.6 ± 2.4 IL-2 9.6 ± 1.2 7.6 ± 1.6 9.8 ± 1.5 IL-7 4.2 ± 0.4 4.0 ± 0.5 3.7 ± 0.5 IL-9 272.6 ± 19.8 264.8 ± 23.5 257.2 ± 17.0 KC 28.2 ± 1.9 30.7 ± 4.2 26.6 ± 2.3 M-CSF 3.0 ± 0.4 6.0 ± 1.0 3.6 ± 0.3 Kidney supernatants from 5- to 7.5-mo-old mice were assayed for the presence of chemokines and cytokines. IL-3, IL-4, IL-5, IL-6, MIP-1b, MIP-2, TNFa, VEGF, and IL-13 were all undetectable. Table S2. Splenic cell counts and expression of activation markers in 5- to 7-mo-old female mice assessed by flow cytometry Sle1 (n = 10) Sle1Tg7 (n = 9) CD11cSle1Tg7 (n = 9) Cells Mean ± SEM Mean ± SEM Mean ± SEM − + Plasma (B220 CD138 )(106) 1.8 ± 0.1 15.7 ± 4.6** 3.9 ± 1.0ns Plasmablast (B220+CD138+)(106) 2.3 ± 0.5 18.2 ± 11.2* 4.4 ± 1.1ns + + B220 CD19 B cells (107) 7.5 ± 0.6 18.1 ± 2.8*** 9.7 ± 1.2ns + + GC (Fas GL7 )(106) 3.0 ± 0.3 11.6 ± 2.7** 3.4 ± 0.6ns MZ (106) 6.8 ± 0.7 7.5 ± 2.0ns 7.8 ± 1.1ns CD4+ T cells (107) 2.8 ± 0.5 8.8 ± 1.8*** 3.2 ± 0.8ns PD-1 (MFI) 88.1 ± 9.8 276 ± 34.4**** 142.4 ± 24.6* ICOS1 (MFI) 891 ± 85.9 2771.1 ± 380.1**** 1513.2 ± 217.5* CD69+ (% CD4+) 3.1 ± 0.3 8.3 ± 2.0** 4.7 ± 0.9ns Effector memory CD4+ T (CD62LloCD44hi) (106) 12.3 ± 2.1 77.2 ± 15.7**** 22.2 ± 5.6ns + Naïve CD4 T (CD62LhiCD44lo) (106) 12.6 ± 2.7 4.0 ± 0.7** 6.3 ± 1.3ns 6 ns Tfh (10 ) 1.1 ± 0.2 11.7 ± 3.2*** 2.5 ± 0.5 CD8+ T cells (107) 1.7 ± 0.3 3.2 ± 0.9ns 1.5 ± 0.3ns PD-1 (MFI) 66.6 ± 9.4 135.8 ± 16.3**** 71.0 ± 14.8ns ICOS1 (MFI) 578.1 ± 35.6 644.4 ± 131.4ns 593.0 ± 67.5ns CD69+ (% CD8+) 2.1 ± 0.2 4.0 ± 0.7** 2.9 ± 0.4ns Effector memory CD8+ T (CD62LloCD44hi) (106) 2.2 ± 0.3 22.3 ± 7.2**** 4.3 ± 0.8ns + Naïve CD8 T (CD62LhiCD44lo) (106) 9.7 ± 2.5 2.4 ± 0.5* 5.5 ± 1.3ns + CD11b total myeloid (107) 2.3 ± 0.4 24.5 ± 4.9**** 3.7 ± 0.7ns Eos (106) 1.7 ± 0.4 22.3 ± 7.2**** 3.2 ± 0.5ns PMN (106) 5.8 ± 1.3 36.8 ± 8.9*** 9.1 ± 2.4ns + Gr1 monocytes (106) 2.4 ± 0.3 39.6 ± 7.9**** 4.7 ± 0.7ns + + + CD11b CD11c MHCII DC (106) 2.2 ± 0.31 29.3 ± 6.9*** 4.8 ± 1.1ns CD8+ DC (106) 0.7 ± 0.16 3.4 ± 0.7*** 0.8 ± 0.2ns pDC (106) 0.7 ± 1.5 0.7 ± 1.1ns 0.8 ± 1.5ns One-way ANOVA was used to analyze data as described in Materials and Methods. Significant differences between Sle1 and Sle1Tg7 are shown in the “Sle1Tg7” column and significant differences between Sle1 and CD11cSle1Tg7 are shown in the “CD11cSle1Tg7” column: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.