Cd83 pe

HLA-ABC FITC (MHC I), HLA-DP, DQ, DR-FITC (MHC II), CD80 FITC, CD86 FITC, CD83 PE, CD1a PE, CD14 PE, Langerin PE, E-cadherin PE, CD8-FITC, CD3-PE-Cy5, and CCR7 PE were purchased from BD Biosciences (San Jose, CA). CD40 PE, purified rat IgG2a, goat anti-rat IgG PE, mouse IgG1 FITC, and mouse IgG1 PE were purchased from Biolegend (San Diego, CA). Recombinant human (rhu)-CCL21 was purchased from R&D Systems (Minneapolis, MN); rhu-GM-CSF was purchased from Berlex (Seattle, WA); and rhu-transforming growth factor-β1 (TGFβ1) and rhu-IL-4 were purchased from Biosource (Carlsbad, CA).

Thioacetamide (TAA), Oligomycin, DMSO, and CCCP were purchased from Sigma‐Aldrich. Vinculin #V9264, STAT1 #SAB4300326, and CYP2E1 HPA009128 were obtained from Sigma‐Aldrich, while p100/p52 #06‐413 from Millipore (Billerica, MA, USA) and p105/p50 #D4P4D from Cell Signaling (Danvers, MA, USA). PRC antibody was described previously.⁵ Anti‐mouse: CD45 (30‐F11), CD11c (HL3), Ly6c (AL‐21), Ly6g (1A8), MHCII I‐A/I‐E (M5/114.15.2), CD19 (ID3), CD8 (53‐6.7), CD4 (RM4‐5), TCRβ (H57.597), CD11b (M1/70) were purchased from BD Biosciences and F4/80 (BM8) from eBioscience. Anit‐human: CD14‐v500, CD1a‐FITC, HLA‐DR‐APC‐H7, CD86‐BV421, CD83‐PE were acquired from BD Biosciences and CD40‐APC from eBiosciences.

The dendritic cell (DC) maturation experiments were contracted to and run by AllCells (Alameda, CA) using standard company protocols. Briefly, human PBMCs from 3 separate donors were cultured in RPMI medium supplemented with 10% FBS, 100 ng/ml human IL-4, and 140 ng/ml human granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days, with medium supplementation on day 3. After the 7-day differentiation period, DCs were stimulated with BECC-derived TLR4Ls for 24 h at 37°C, 5% CO₂. Cells were then lifted off the culture well using Accutase reagent, and EDTA added to halt enzymatic digestion. Cells were washed twice with PBS, nonspecific binding was inhibited using FcBlock (Miltenyi), and finally, cells were stained with CD80-fluorescein isothiocyanate (FITC), CD40-phycoerythrin (PE), and CD83-PE (BD Biosciences). Flow cytometry analysis was performed using an LSRII (BD Biosciences). Dead cells/debris were excluded using a forward scatter/side scatter (FSC/SSC) gate, 10,000 events were collected for each sample, and isotype controls for each antibody were run and determined to contain <1% of events recorded.

Migration of MUTZ‐LCs from the epidermis in RHS and maturation of MUTZ‐LCs were assessed by flow cytometry. Cell staining was performed using mouse antihuman CD1a‐PE (IgG1; BD Pharmingen), intracellular CD68‐PE (kit 556 079, IgG2b, κ; BD Pharmingen), langerin APC (IgG1; Miltenyi Biotec), CD86‐FITC, and CD83‐PE (IgG1; BD Biosciences). Isotype controls to assess nonspecific binding were mouse IgG1‐PE, IgG2b, κ (BD Pharmingen), and mouse IgG2a‐FITC (Miltenyi Biotec). Cells were washed and resuspended in FACS buffer (PBS supplemented with 0.1% bovine serum albumin and 0.1% sodium azide) and incubated for 30 minutes at 4°C in the presence of the antibodies. Subsequently the cells were resuspended in the same FACS buffer with an abundance of 123count eBeads (Thermo Fisher) before analysis using a FACSCalibur flow cytometer (Beckton Dickinson, San Jose, California). In addition, a propidium iodide (GIBCO) staining was performed to confirm the viability of MUTZ‐LC monocultures. All data were analyzed using CellQuest Pro FACS analysis software (BD Pharmingen).

For analysis of the phenotype of BDCA-1⁺/BDCA-3⁺ myDC, cells were stained with CD11c-Alexa Fluor 700 (BD Biosciences, clone B-ly6), CD1c-Brilliant Violet 510 (BD Biosciences, clone F10/21A3), CD141-PE/Cy7 (Invitrogen, clone JAA17), CD274-PE-CF594 (BD Biosciences clone MIH1), CD86-Brilliant Violet 421 (BD Biosciences, clone 2331 (FUN-1)), CD83-PE (BD Biosciences, clone HB15e), CD40-APC (BD Biosciences, clone 5C3), CD80-PE/Cy5 (BD Biosciences, clone L307.4), HLA-ABC-FITC (BD Biosciences, clone G46-2.6), Zombie Yellow (Biolegend) for 20 minutes at 4°C. After washing, cells were resuspended in PBS/0.5%BSA and acquired on a BD LSR Fortessa instrument. Data analysis was performed using FlowJo software. The gating strategy was as follows: cells were first gated on FSC/SSC characteristics, followed by gating on single cells. Next, dead cells were excluded and subsequently we gated on the CD11c⁺ population. On this gate, CD1c⁺ CD141^- cells were identified as BDCA-1⁺ myDC and CD1c^- CD141⁺ cells as BDCA-3⁺ myDC. Subsequently, we evaluated for each myDC subtype the expression of HLA-ABC, CD83, CD274/PD-L1, CD80, CD40 and CD86.