Bdca2

The phenotype of pDC and CD1c⁺ mDC populations was determined by flow cytometry. DC purity was assessed by double staining CD11c⁺/CD1c⁺ for CD1c⁺mDCs (above 95%) and BDCA2/CD123 for pDCs (above 95%; all Miltenyi Biotec) [56 (link)]. The following primary monoclonal antibodies (mAbs) were used to determine the maturation state of the DCs: anti–CD80-APC, anti–PD-L1-APC (all BD Bioscience Pharmingen, San Jose, CA); and anti–CD40-PE (Beckman Coulter, Marseille, France). Measurements were performed on FACSCalibur and FACSVerse flowcytometers (BD).

Paraffin-embedded human kidney sections and primary human renal proximal tubular cells (RPTECs) were stained or double stained for LMP7, MXA, p65, pNIK (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) and BDCA2 (Miltenyi Biotec, Calderara di Reno, Italy). For each experiment, 3 × 10⁵ cells were plated on a cover slip and fixed in 3.7% paraformaldehyde. The slides were incubated with the blocking solution, primary antibodies (anti-LMP7 1:500; anti-MXA 1:200; anti-p65 1:100, anti-pNIK 1:100 and anti -BDCA2 1:20) and the appropriate secondary antibodies (Alexa Flour 488 and 555 goat anti rabbit; Alexa Flour 555 and 488 anti mouse, Molecular Probes, Eugene, OR, USA). All sections were counterstained with TO-PRO-3 (Molecular Probes). Specific fluorescence was acquired using the confocal microscope Leica TCS SP2 (Leica, Wetzlar, Germany) using a 63 objective lens.
Quantification of p65 and pNIK nuclear fluorescence intensity was performed using Leica Software. We outlined cellular nuclei and then calculated the fluorescence intensity for p65 (green channel) and pNIK (red channel) within the nuclei. At least 10 cells in three different fields from each slide were measured to obtain the average quantification of nuclear signal intensity for p65 and pNIK.

Skin samples were fixed in 10% formalin and embedded in paraffin. 5-µm sections were de-waxed and rehydrated. After quenching endogenous peroxidase, achieving antigen retrieval, and blocking nonspecific binding sites, sections were incubated with monoclonal antibodies against CD15 (BD-Pharmingen, Franklin Lakes, NJ, 1∶100 dilution), CD3 (BD-Pharmingen, 1∶20 dilution), BDCA-2 (Miltenyi Biotec, Bergisch Gladbach, Germany), Ki67 (Dako, Carpinteria, CA, mib1 clone, 1∶50 dilution), IL-17 (R&D Systems, Minneapolis, MN, 1∶40) and perforine (Leica Biosystems, Milan, Italy, 5B10 clone, 1∶20). Secondary biotinylated mAbs and staining kits were obtained from Vector Laboratories. Stainings were developed using 3-amino-9-ethylcarbazole (Vector Laboratories, Burlinagame, CA, USA). Slides were counterstained with hematoxylin. As a negative control, primary antibodies were omitted or replaced with an irrelevant isotype-matched monoclonal antibody. Figures depict one experiment that is representative of all the patients investigated (n = 10). Positive cells were counted in two visual fields per condition by two independent investigators in a blinded manner.

All flow cytometry antibodies used to determine DC and T cell purities as well as Th cell surface marker expression were purchased from BD Biosciences, except for BDCA-2 (Miltenyi Biotech). Antibodies were used at the proper concentration either as fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein, allophyocyanin, allophyocyanin-H7, Horizon 450, Horizon 500, Alexa Fluor 488 or Pe-Cy7. Intracellular IFN-γ staining was performed as described previously [48] (link). Analyses were performed with BD FACS Canto II and analyzed by BD FACSDiva Software v6.1.2 (BD Biosciences).