Be0083

Rag2−/− mice (008449), Tcrδ−/− mice (002120), muMT−/− mice (002288), Tcrα−/− mice (002116), Il17a−/− mice (016879) were from Jackson Lab. Il17f−/− mice were from Dr. Sarah L. Gaffen and Dr.Yoichiro Iwakura’s lab, Il17rc−/− and Il17ra−/− were from Amgen, Il17RC^flox/flox mice were from Dr. Jay K. Kolls’ lab³¹ . Vγ6Vδ1 Tg mice were from Dr. Diane Mathis’ lab. Adiponectin Cre mice (028020) and UCP1 Cre mice (024670) were from Jackson lab³² . VGLUT3-ires-Cre mice were from Dr. Bradford Lowell’s lab³³ . TGFβ antibody (Bioxcell # BE0057 Clone 1D11.16.8) and Isotype control antibody (Bioxcell # BE0083) were i.p. injected to WT B6 mice for 3 weeks at 10mg/kg 3 times a week. sb431542 was i.p. injected to WT B6 mice at 4.2mg/kg/day for 3 weeks. All animal studies were approved by the Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center.

Paraffin-embedded cutaneous lupus skin samples were obtained as above. Paraffin slides were heated at 60°Cfor 30 minutes, then rehydrated and rinsed. Heat-induced antigen retrieval was performed using a pH 6.0 citrate buffer x30 min. Slides were rinsed, washed, and blocked with 10%FBS in PBS+0.025% Tween x1 h. Primary antibody (mouse monoclonal IL-6, Abcam 9324) or isotype (mouse IgG, BioxCell #BE0083) was added overnight at 4°C. Slides were then washed and HRP-conjugated rabbit anti-mouse secondary antibody (Jackson ImmunoResearch #315-035-003) was added x1 h at room temperature. Slides were washed and stained with DAB+chromagen, hematoxylin counterstained, washed, air-dried, and mounted with Permount.

Bone marrow cells were isolated from wildtype mice by snipping the end of the femur and centrifuging at 5000 rpm for 1min. Monocytes were differentiated into macrophages by culturing in non-TC treated plates (Corning, 430597) for 6 days in RPMI medium containing M-CSF (10ng/ml). Media containing M-CSF were replaced after 3 days. Where indicated, BMDM’s were treated with recombinant mouse IFN-beta1 (8234-MB, R&D systems) for 4hrs (10ng/ml). KMC tumour cells were used to generate conditioned media. Fresh media (DMEM+20%FBS) were added 24hrs after transfection with siMYC or non-targeting control siRNA. 24hrs later, conditioned media from MYC or non-targeting control depleted cells were collected, centrifuged (1200 rpm, 5mins), supplemented with M-CSF and, where indicated, anti-IFNAR1 blocking antibody (20ng/ml, BE0241, BioxCell) or mIgG (BE0083) prior to BMDM treatment. BMDMs were additionally pre-treated with anti-IFNAR1 or mIgG overnight. BMDM’s were treated with conditioned media for 24hrs. To detect Cxcl13 expression in BMDM’s, cDNA was synthesized with Oligo-dT primer (M510, PromegA), pre-amplified (40nM of each primers, SYBR Green buffer, 1mg/ml BSA, 2.5% glycerol) for GusB and Cxcl13 for 20cycles. Diluted cDNA (1:20 in 10mMTris and 1mM EDTA) was quantified by real time PCR using SYBR Green method (VWR QUNT95072).