Mice were housed in a Canadian Council on Animal Care approved facility at the Toronto Western Research Institute, part of the University Health Network. All mice used and experiments performed in this study were approved by the Animal Care Committee of the University Health Network (Animal Use Protocol #123).
B6 and NZB mice were purchased from Taconic Farms (Germantown, NY) and Harlan-Sprague-Dawley (Blackthorne, England), respectively, and subsequently bred in our facility. Congenic mice were produced by separately backcrossing NZB chromosome 1 and 13 intervals onto the B6 background, using the speed congenic technique 
. Mice were genotyped at each successive generation using polymorphic microsatellite markers that discriminate between NZB and B6 DNA, spaced at ~20 cM intervals throughout the genome, except for regions containing lupus susceptibility genes where more densely spaced markers were used. Fully backcrossed mice were produced within 6 or 7 generations, for chromosome 1 and 13 intervals respectively, and then intercrossed to produce congenic mice that were homozygous for the NZB intervals. For NZB chromosome 1 congenic mice (previously called B6.NZBc1(35-106) but here denoted as B6.NZBc1 for simplicity) the NZB interval extends from between rs13475886 (61.2 Mb) and D1Mit303 (63.0 Mb) to between D1Mit223 (190.5 Mb) and D1Mit210 (192.1 Mb). NZB chromosome 13 congenic mice (B6.NZBc13) have an NZB interval extending from between D13Mit117 (37.7 Mb) and D13Mit92 (46.9 Mb) to D13Mit78 (119.6 Mb). B6.NZBc1c13 bicongenic mice were produced by intercrossing B6.NZBc1 and B6.NZBc13 mice and selecting for homozygous NZB chromosome 1 and 13 intervals in successive crosses. Typing of all polymorphic markers (spaced on average ~5–6 Mb apart) in the NZB chromosome 1 and 13 intervals of bicongenic mice was identical to that of the parental monocongenic mice. The mice were housed in microisolators in the animal facility at the Toronto Western Hospital (Toronto, Canada) and were specific-pathogen free. All of the mice that were examined in this study were female.
Flow cytometry analysis
RBC-depleted splenocytes, bone marrow cells, or BMDC (5×105) were incubated with 10 µg/ml mouse IgG (Sigma-Aldrich, St Louis, MO) for 15 min to block Fc receptors and stained with various combinations of directly-conjugated mAbs. Following washing, allophycocyanin-conjugated streptavidin (BD Biosciences, San Diego, CA) was used to reveal biotin-conjugated Ab staining. Dead cells were excluded by staining with 0.6 µg/ml propidium iodide (PI; Sigma-Aldrich). For intracellular staining, cells were fixed and permeabilized using Cytofix/Cytoperm (BD Biosciences), and washed and stained in Perm/Wash buffer (BD Biosciences), according to the manufacturer's protocol. Flow cytometry of the stained cells was performed using a FACSCalibur or LSRII (BD Biosciences, Mountain View, CA) and analyzed using Cell Quest Pro (BD Biosciences) or FlowJo (Treestar) software. Live cells were gated on the basis of PI exclusion and scattering characteristics, with 10,000 or 25,000 events being acquired for each sample. The following directly conjugated mAbs were purchased from BD Biosciences: biotin conjugated anti-CD11c (N418), -CD11b (Mac1), -CD4 (L3T4), -CD8 (53-6.7), and -CD62L (MEL-14); PerCP conjugated anti-B220 (30-F11); PE conjugated anti-B7.1 (16-10A1), -B7.2 (GL1), -IA/IE (M5/114.15.2), -CD3 (145-2C11), -CD23 (B3B4), -IgMb (AF6-78), -CD69 (H1.2F3), -CD44 (IM7), -ICAM-1 (3E2), -NK1.1 (PK136), and -CD4 (H129.19); FITC conjugated anti-CD3 (145-2C11), -CD4 (L3T4), -CD8 (53-6.7), -CD21/CD35 (7G6), -CD25 (7D4), -B220 (RA3-6B2), and -CD11c (N418); PE-Cy7 conjugated anti-CD19 (1D3); Pacific Blue conjugated anti-B220 (RA3-6B2); and APC-Cy7 conjugated anti-CD11b (M1/70). Allophycocyanin conjugated anti-CD9 (eBioKMC8) was purchased from eBioscience, and Pacific Blue anti-SiglecH (551) and APC-Cy7 anti-IA/IE (M5/114.15.2) were purchased from BioLegend. Biotinylated antibiodies were revealed with streptavidin conjugated with allophycocyanin or PerCP from BD Biosciences. FITC and biotin conjugated PDCA-1 (Miltenyi Biotec) was a generous gift from Dr. Eleanor Fish. FITC anti-CD62L (MEL-14) and anti-CD11b (M1/70.15) were purchased from Cedarlane (Hornby, Ontario, Canada). All isotype controls, with the exception of hamster IgG controls (BD Biosciences), were purchased from Cedarlane.
Measurement of Ab production
Serum levels of IgM, IgG, and IgA anti-chromatin, -dsDNA and -ssDNA Ab were measured by ELISA. ssDNA was prepared by boiling dsDNA (isolated from calf thymus DNA) for 10 min and quick cooling on ice for 2 minutes. H1-stripped chromatin was prepared from chicken RBC, as previously described 
. ELISA plates were coated overnight with chromatin (8 µg/ml), dsDNA (40 µg/ml) or ssDNA (20 µg/ml) diluted in PBS at 4°C. The plates were then washed with 0.05% Tween 20/PBS, and blocked with 2% BSA/PBS for 1 h at room temperature. After further washing, serum samples, diluted 1/100 in 2% BSA/Tween 20/PBS, were added. Bound Ab were detected using alkaline phosphatase-conjugated anti-IgG, -IgM or -IgA Ab (Caltag, Burlingame, CA) as secondary reagents. For measurement of total IgM, IgG, and IgA, plates were coated with goat anti-mouse IgM or IgG (Jackson ImmunoResearch, West Grove, PA, USA) or rat anti-mouse IgA (BD Biosciences) respectively, and the serum was diluted 1/1000. The amount of bound IgM, IgG or IgA was calculated from a standard curve using purified class-specific controls, and the Ab concentration was calculated from a plot of concentration versus absorbance.
Bone marrow-derived DC (BMDC) cultures and CD11c+ splenic DC isolation
Bone marrow cells were isolated by flushing femurs of 8–12 week-old mice. After RBC lysis, ex vivo cells were resuspended to 106 cells/mL and cultured for 7 days with recombinant human Flt3L (20 ng/mL; R&D Systems) in complete RPMI 1640 media (10% fetal bovine serum (FBS), non-essential amino acids, L-glutamine, β-mercaptoethanol, penicillin, and streptomycin). Splenic DC were positively selected using biotin-conjugated anti-CD11c antibody (N418) and Dynabeads Biotin Binder kit (Invitrogen), following the manufacturer's instructions.
TLR stimulation and cytokine blockade
TLR ligands used were imiquimod R837 (2 µM; TLR7), poly I:C (50 µg/mL; TLR3), CpG ODN 2216 or control (250 nM; TLR9), CpG ODN 1826 or control (250 nM; TLR9) all from InvivoGen (San Diego, CA), and LPS (25 µg/mL; Sigma-Alderich) as a positive control. For splenocytes cultures, 2×106 cells were cultured in 96-well flat-bottom plates for 48 hours with various TLR ligands or in media alone (0.5% normal mouse serum in complete RPMI1640 without FBS). For BMDC cultures, 4×105 cells were cultured in 96-well flat-bottom plates for 24 or 48 hours with various TLR ligands or in media alone (10% FBS in complete RPMI 1640). For CD11c+ splenic DC cultures, 2×105 cells were cultured in 96-well flat-bottom plates for 24 hours with CpG 2216 or in media alone (10% FBS in complete RPMI 1640). To assess IFN-α, IL-10 and TNF-α production following TLR stimulation, cytokine levels in tissue culture supernatants were measured by ELISA using commercially available kits as follows: IFN-α, PBL Biomedical Laboratories (Piscataway, NJ); IL-10, SABiosciences Corporation (Frederick, MD) or eBioscience (San Diego, CA); and TNF-α, BD Biosciences or eBioscience. Assays were performed as per the manufacturer's recommendations with the concentration of cytokine calculated from a standard curve of absorbance versus concentration of recombinant cytokine.
Immunofluorescence staining of tissue sections
Spleens were snap frozen in OCT compound (Sakura Finetek, Torrance, CA) at the time of sacrifice. Cryostat sections (5 µm) were fixed in acetone, washed with PBS, and blocked with 5% normal goat serum/PBS or 5% fetal bovine serum/PBS. Sections were stained with biotinylated anti-CD11b and FITC anti-CD11c to detect myeloid DC. BAFF production was assessed by staining in tandem with rabbit IgG anti-BAFF (Sigma) followed by AMCA-conjugated goat anti-rabbit IgG Ab (Jackson ImmunoResearch). Biotin staining was revealed using rhodamine-conjugated streptavidin as a secondary reagent (Molecular Probes, Eugene, OR). Stained sections were mounted with Mowiol (Calbiochem, La Jolla, CA) and tissue fluorescence visualized using a Zeiss Axioplan 2 imaging microscope (Oberkochen, Germany). Digital images were obtained using the manufacturer's imaging system.
Grading of kidney sections
For immunofluorescence score, kidneys were snap frozen in OCT compound (Sakura Finetek) and sectioned (3 µm). For glomerular score, kidneys were fixed in formalin, paraffin embedded, sectioned (3 µm), and stained with periodic acid-Schiff. Grading was performed by a renal pathologist (G. Lajoie) who was blinded as to the strain of origin of the tissue section. Glomerular staining of kidney sections stained with FITC anti-IgG was graded by immunofluorescence microscopy. Sections with no or only trace deposits were graded as 0; those with mesangial deposits, grade 1 (B more extensive than A); those with mesangial and segmental capillary wall deposits, grade 2; those with diffuse mesangial and capillary wall deposits, grade 3; and those with crescents, grade 4. The grading scale used for glomerular score using light microscopy was as follows: grade 0, normal glomeruli; grade 1, mesangial expansion and/or proliferation; grade 2, focal segmental (endocapillary) proliferative glomerulonephritis; grade 3, diffuse (endocapillary) proliferative glomerulonephritis; and grade 4, diffuse proliferative glomerulonephritis with crescents.
Measurement of mRNA expression
RNA was purified from splenocytes and bone marrow cells of 8-month-old mice using the RNeasy Mini Kit (Qiagen, Basel, Switzerland), treated with DNaseI (Invitrogen, Canada), and converted to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Forster City, CA), according to the manufacturer's instructions. Quantitative real-time PCR was performed with SYBR Green Master Mix on a 7900HT Fast Real-Time PCR System (Applied Biosystems) using default cycling conditions. Primer sequences were designed to span exon-to-exon and were as follows: β-actin forward, TTGCTGACAGGATGCAGAAG, β-actin reverse, GTACTTGCGCTCAGGAGGAG; baff forward, CAGGAACAGACGCGCTTTC, baff reverse, GTTGAGAATGGCGGCATCC; tnf-α forward, GCCACCACGCTCTTCTGTCT, tnf-α reverse, TCTGGGCCATAGAACTGATGAGA; pkr forward, TGAGCGCCCCCCATCT, pkr reverse, TATGCCAAAAGCCAGAGTCCTT; 2′-5′ oas forward, TGAGCGCCCCCCATCT, 2′-5′ oas reverse, CATGACCCAGGACATCAAAGG; ifn-α4 forward, CTTGTCTGCTACTTGGAATGCAA, ifn-α4 reverse, AGGAGGTTCCTGCATCACACA; and ifn-β1 forward, TGACGGAGAAGATGCAGAAGAG, ifn-β1 reverse CACCCAGTGCTGGAGAAATTG. Gene expression was analyzed using the relative standard curve method and was normalized to β-actin expression.
Statistical significance of comparisons between groups of mice was determined using the Mann-Whitney non-parametric two-tailed test with the exception of comparisons between kidney tissue section grades where Fisher's exact test was used.