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. Eos, eosinophils; GC, germinal center B cells; MFI, mean

fluorescence intensity; MZ, marginal zone B cells; Tfh, T follicular helper cells; PMN, polymorphonuclear leukocytes. Indented entries are gated on the populations highlighted in bold immediately above.

Celhar et al. www.pnas.org/cgi/content/short/1507052112 5of10 Table S3. Complete list of probes used for Nanostring gene- expression analysis Cell Type Tg7

Gene Adjusted P value

DC and macrophage ontogeny Cd64 (FcGR1) 4.75E-11 3.30E-05 Rbpj 1.64E-13 3.30E-05 Irf4 2.70E-12 0.0004899 Kmo 1.64E-13 0.001432 C/ebp-b 5.67E-12 0.003363 Esam 1.93E-06 0.003809 Zbtb46 2.15E-18 0.004737 Batf3 2.12E-16 P > 0.05 Btla 4.76E-13 P > 0.05 C/ebp-a 4.13E-10 P > 0.05 Ccr7 1.97E-11 P > 0.05 Cd14 4.65E-12 P > 0.05 Cd24a 1.23E-04 P > 0.05 Csf1 4.70E-02 P > 0.05 Csf1r(Mcsfr/Cd115) 9.63E-12 P > 0.05 Cx3cr1 2.72E-09 P > 0.05 Flt3 1.43E-16 P > 0.05 Gmcsfr (Csf2ra) 4.05E-07 P > 0.05 Id2 3.54E-12 P > 0.05 Ikzf1 1.20E-10 P > 0.05 Irf2 5.28E-04 P > 0.05 Irf8 3.48E-07 P > 0.05 Kit 8.59E-09 P > 0.05 Klf4 1.39E-12 P > 0.05 Lyz2 2.11E-07 P > 0.05 Mafb 2.34E-08 P > 0.05 Maf-v 1.90E-06 P > 0.05 Mertk 7.62E-08 P > 0.05 Pu.1(Sfpi1) 1.97E-11 P > 0.05 RelB 1.74E-13 P > 0.05 Runx1 2.11E-07 P > 0.05 Runx2 1.93E-06 P > 0.05 Runx3 1.07E-07 P > 0.05 Spib 1.60E-11 P > 0.05 Tcf4(E2.2) 3.28E-08 P > 0.05 Tcf7 1.78E-07 P > 0.05 Tlr5 7.95E-05 P > 0.05 Traf1 3.25E-13 P > 0.05 Il34 Gmcsf (Csf2) Inflammation Ebi3 4.64E-13 3.30E-05 Fcer1g 2.48E-08 0.002286 Il1b 1.23E-03 0.00401 Ccl2(Mcp1) 1.07E-07 0.004239 Ccl5(Rantes) 1.01E-07 P > 0.05 Il10 3.97E-08 P > 0.05 Il12p40 (Il2b) 3.80E-05 P > 0.05 Il15 1.15E-03 P > 0.05 Il15ra 8.26E-05 P > 0.05 Il18 1.03E-06 P > 0.05 Ly6C 2.27E-03 P > 0.05 Tgfb1 2.73E-06 P > 0.05 Tnf 1.94E-05 P > 0.05 Il12p35(IL12a) Il6 Il17a Il23p19(Il23a) Il9

Celhar et al. www.pnas.org/cgi/content/short/1507052112 6of10 Table S3. Cont. Cell Type Tg7

Gene Adjusted P value

Kc Ifna1 Ifna4 Ifna5 Ifnb Lab/SLE interest Tlr7 4.01E-07 0.000273 Ccr9 1.28E-05 P > 0.05 Cd229 (Slamf3) 1.26E-02 P > 0.05 Cd244 1.07E-07 P > 0.05 Cd48 8.91E-07 P > 0.05 CD84_v1 3.52E-11 P > 0.05 Cd84_v2 6.01E-09 P > 0.05 Cd9 3.79E-14 P > 0.05 Dap12(Tyrobp) 9.63E-06 P > 0.05 Dr6 3.82E-05 P > 0.05 SiglecE 4.42E-07 P > 0.05 SiglecG 1.43E-16 P > 0.05 Slamf7 (Cracc) 2.97E-15 P > 0.05 Slamf9 2.72E-09 P > 0.05 Tlr9 5.21E-13 P > 0.05 SiglecH Housekeeping genes B2m, Gapdh, Cltc, Tbp, Tubb4, Eefg1

Genes in boldface were not expressed above background levels in the cell populations analyzed. Two-way ANOVA analysis of the Nanostring expres- sion data across all four populations and adjusted P values based on com- parison between cell types or between normal or increased TLR7 copy number (Tg7).

Celhar et al. www.pnas.org/cgi/content/short/1507052112 7of10 Table S4. Nanostring expression data of all of the genes analyzed in Sle1 vs. Sle1Tg7 kidney DCs and Sle1 vs, Sle1Tg7 kidney macrophages Sle1 Sle1Tg7

Symbol Average ± SD Average ± SD Adjusted P value

+ + − Kidney DCs (CD11c MHCII F4/80 ) Ebi3 2.22 ± 0.07 1.91 ± 0.05 0.023 Slamf9 2.70 ± 0.07 2.39 ± 0.06 0.023 Irf4 2.38 ± 0.08 1.86 ± 0.15 0.035 Rbpj 2.86 ± 0.04 2.55 ± 0.08 0.035 Batf3 2.15 ± 0.09 1.86 ± 0.09 0.042 Kmo 2.63 ± 0.11 2.29 ± 0.10 0.042 Btla 2.36 ± 0.14 1.93 ± 0.17 0.071 Cd84_v2 1.25 ± 0.12 1.60 ± 0.03 0.071 Zbtb46 2.41 ± 0.10 2.12 ± 0.12 0.071 SiglecG 2.18 ± 0.13 1.86 ± 0.12 0.071 Flt3 2.52 ± 0.08 2.14 ± 0.17 0.073 Tcf4(E2.2) 2.06 ± 0.05 1.87 ± 0.09 0.073 Il18 2.13 ± 0.06 1.91 ± 0.10 0.080 Cd64(Fcgr1) 2.43 ± 0.13 2.69 ± 0.05 0.092 Cd84_v1 1.89 ± 0.15 2.19 ± 0.05 0.092 Runx3 1.54 ± 0.11 1.79 ± 0.12 0.097 C/ebp-b 2.79 ± 0.24 3.23 ± 0.06 0.113 Slamf7(Cracc) 2.67 ± 0.07 2.51 ± 0.10 0.151 Cd229 2.10 ± 0.16 2.35 ± 0.11 0.158 Cd48 2.72 ± 0.05 2.63 ± 0.05 0.158 Cd9 2.63 ± 0.08 2.76 ± 0.07 0.158 SiglecE 2.01 ± 0.11 1.84 ± 0.04 0.158 Tlr5 1.12 ± 0.10 1.29 ± 0.11 0.158 Esam 0.85 ± 0.25 0.49 ± 0.17 0.162 Klf4 2.68 ± 0.12 2.44 ± 0.16 0.162 Mafb 2.42 ± 0.16 2.63 ± 0.06 0.162 RelB 2.48 ± 0.14 2.67 ± 0.08 0.162 Tlr7 1.95 ± 0.31 2.38 ± 0.12 0.162 Traf1 2.44 ± 0.13 2.62 ± 0.07 0.162 Lyz2 3.22 ± 0.13 3.39 ± 0.05 0.165 Ly6c 1.97 ± 0.16 2.27 ± 0.23 0.171 Fcer1g 3.37 ± 0.07 3.45 ± 0.04 0.197 Cd14 2.27 ± 0.16 2.54 ± 0.23 0.206 Il15 1.57 ± 0.15 1.27 ± 0.26 0.206 Gmcsfr(Csf2ra) 3.01 ± 0.01 2.95 ± 0.05 0.228 Irf2 2.44 ± 0.08 2.35 ± 0.07 0.254 Ccl2(Mcp1) 1.56 ± 0.30 1.92 ± 0.32 0.274 Csf1r(Mcsfr/Cd115) 3.10 ± 0.13 3.22 ± 0.02 0.274 Id2 2.95 ± 0.15 3.09 ± 0.03 0.274 C/ebp-a 2.59 ± 0.03 2.54 ± 0.04 0.279 Pu.1(Sfpi1) 3.11 ± 0.08 3.18 ± 0.02 0.279 Tcf7 −0.21 ± 0.92 0.54 ± 0.10 0.323 Il10 0.47 ± 0.42 0.81 ± 0.24 0.342 Runx1 2.28 ± 0.07 2.21 ± 0.08 0.343 Ccr7 1.85 ± 0.30 2.06 ± 0.13 0.373 Tlr9 2.47 ± 0.07 2.39 ± 0.10 0.373 Cx3cr1 2.70 ± 0.11 2.62 ± 0.05 0.390 Il1b 2.91 ± 0.25 3.08 ± 0.12 0.390 Runx2 1.68 ± 0.08 1.77 ± 0.12 0.390 Spib 2.03 ± 0.13 1.92 ± 0.13 0.401 Mertk 1.94 ± 0.35 2.15 ± 0.07 0.401 Ikzf1 2.10 ± 0.05 2.13 ± 0.05 0.431 Kit 1.62 ± 0.15 1.71 ± 0.15 0.510 Ccr9 0.87 ± 0.17 0.75 ± 0.23 0.565 Il12p40(Il12b) 1.05 ± 0.20 1.16 ± 0.23 0.597 Il15ra 1.32 ± 0.17 1.39 ± 0.11 0.597 Ccl5(Rantes) 2.26 ± 0.23 2.16 ± 0.16 0.600 Tgfb1 3.00 ± 0.08 3.02 ± 0.05 0.688 Cd24a 1.39 ± 0.14 1.33 ± 0.17 0.707

Celhar et al. www.pnas.org/cgi/content/short/1507052112 8of10 Table S4. Cont. Sle1 Sle1Tg7

Symbol Average ± SD Average ± SD Adjusted P value

Csf1 1.09 ± 0.10 1.17 ± 0.36 0.761 Dr6 2.11 ± 0.08 2.09 ± 0.09 0.825 Dap12(Tyrobp) 3.58 ± 0.06 3.57 ± 0.04 0.827 Irf8 2.15 ± 0.14 2.16 ± 0.06 0.947 Maf-v 1.67 ± 0.11 1.67 ± 0.14 0.952 Tnf 2.01 ± 0.21 2.01 ± 0.08 0.952 Cd244 1.14 ± 0.17 1.15 ± 0.31 0.963 Kidney macrophages (CD11c+MHCII+F4/80+) Tlr7 2.40 ± 0.06 2.70 ± 0.02 0.0331 Ccl2(Mcp1) 1.83 ± 0.12 2.32 ± 0.10 0.0331 Lyz2 3.25 ± 0.07 3.50 ± 0.07 0.0452 Gmcsfr(Csf2ra) 3.01 ± 0.05 2.84 ± 0.04 0.0461 Rbpj 2.80 ± 0.06 2.57 ± 0.10 0.0499 Cd64(Fcgr1) 2.86 ± 0.08 3.08 ± 0.03 0.0499 Cd84_v1 2.18 ± 0.05 2.31 ± 0.03 0.0499 Slamf7(Cracc) 1.71 ± 0.14 2.14 ± 0.02 0.0499 Cd229 1.96 ± 0.12 2.31 ± 0.11 0.0499 Cd48 2.49 ± 0.03 2.58 ± 0.03 0.0499 Fcer1g 3.32 ± 0.04 3.44 ± 0.05 0.0499 Cd14 3.13 ± 0.02 2.86 ± 0.08 0.0499 Dr6 1.69 ± 0.11 1.95 ± 0.06 0.0499 Cd244 0.38 ± 0.15 0.81 ± 0.15 0.0499 IL15ra 1.11 ± 0.10 1.34 ± 0.09 0.0581 Relb 1.98 ± 0.05 2.13 ± 0.08 0.0895 Ebi3 2.31 ± 0.05 2.18 ± 0.07 0.1030 Id2 2.35 ± 0.13 2.58 ± 0.06 0.1133 C/ebp-b 2.37 ± 0.24 2.77 ± 0.16 0.1235 MerTK 2.19 ± 0.12 2.42 ± 0.11 0.1235 Csf1r(Mcsfr/Cd115) 3.48 ± 0.05 3.40 ± 0.04 0.1327 Irf8 2.43 ± 0.08 2.28 ± 0.09 0.1401 Il1b 2.82 ± 0.12 3.00 ± 0.07 0.1520 Maf-v 1.92 ± 0.07 2.04 ± 0.06 0.1653 Cx3cr1 3.09 ± 0.05 3.01 ± 0.05 0.1731 Il12p40(Il12b) 0.85 ± 0.11 0.49 ± 0.27 0.1731 Tlr5 1.08 ± 0.07 0.83 ± 0.21 0.2191 Slamf9 3.01 ± 0.04 2.91 ± 0.09 0.2390 Irf4 1.45 ± 0.14 1.26 ± 0.14 0.2390 Csf1 0.85 ± 0.22 1.12 ± 0.18 0.2391 Batf3 2.11 ± 0.08 1.99 ± 0.10 0.2457 Tcf7 0.30 ± 0.13 0.14 ± 0.15 0.3230 Mafb 2.95 ± 0.09 2.87 ± 0.02 0.3434 Traf1 1.99 ± 0.03 1.94 ± 0.06 0.3434 Pu.1(Sfpi1) 2.87 ± 0.03 2.91 ± 0.03 0.3434 CCL5(Rantes) 1.35 ± 0.40 1.68 ± 0.09 0.3434 Tgfb1 2.79 ± 0.04 2.83 ± 0.04 0.3434 Ly6c 0.70 ± 1.14 1.58 ± 0.17 0.3730 Runx2 0.48 ± 0.99 1.26 ± 0.28 0.3730 Runx1 2.47 ± 0.08 2.38 ± 0.11 0.3758 Kmo 1.70 ± 0.14 1.81 ± 0.12 0.4385 Spib −0.32 ± 0.79 0.18 ± 0.30 0.4731 Btla −0.14 ± 0.59 0.23 ± 0.25 0.4733 Il10 0.52 ± 0.40 0.77 ± 0.21 0.4733 Runx3 0.88 ± 0.16 0.36 ± 0.92 0.5027 Tlr9 2.66 ± 0.03 2.68 ± 0.03 0.5096 C/ebp-a 2.54 ± 0.11 2.61 ± 0.09 0.5105 Tnf 1.91 ± 0.21 1.74 ± 0.29 0.5241 Cd9 2.43 ± 0.09 2.35 ± 0.14 0.5427 Il18 2.03 ± 0.08 1.99 ± 0.03 0.5849 CD24a 0.01 ± 0.71 0.51 ± 1.00 0.5849 Dap12(Tyrobp) 3.44 ± 0.06 3.46 ± 0.03 0.6564 Tcf4(E2.2) 2.15 ± 0.06 2.12 ± 0.08 0.6614

Celhar et al. www.pnas.org/cgi/content/short/1507052112 9of10 Table S4. Cont. Sle1 Sle1Tg7

Symbol Average ± SD Average ± SD Adjusted P value

SiglecE 2.52 ± 0.06 2.49 ± 0.03 0.6614 Zbtb46 0.65 ± 0.15 0.72 ± 0.15 0.6692 SiglecG 0.23 ± 0.19 0.32 ± 0.28 0.7057 Esam 0.77 ± 0.30 0.68 ± 0.16 0.7057 Ccr7 0.32 ± 0.31 0.42 ± 0.38 0.8093 Flt3 0.76 ± 0.22 0.72 ± 0.22 0.8642 Cd84_v2 1.63 ± 0.08 1.62 ± 0.11 0.9159 Ccr9 0.34 ± 0.26 0.32 ± 0.14 0.9588 Irf2 2.24 ± 0.03 2.23 ± 0.09 0.9740 Kit −0.02 ± 0.66 0.02 ± 0.69 0.9740 Klf4 1.71 ± 0.15 1.71 ± 0.16 0.9931 IL15 1.70 ± 0.08 1.70 ± 0.08 0.9931 Ikzf1 1.82 ± 0.08 1.82 ± 0.07 0.9974

Average ± SD values are shown (n = 4 per strain). Genes that are significantly differentially expressed are in boldface (based on adjusted P values).

Celhar et al. www.pnas.org/cgi/content/short/1507052112 10 of 10