Bdca4

Human PBMCs were separated from whole blood by a density gradient centrifugation method using Leucosep tubes (Greiner Bio-One). pDCs were purified from freshly isolated PBMCs by negative selection using the Diamond Plasmacytoid Dendritic Cell Isolation Kit II (Miltenyi Biotec). Naive CD4⁺ T cells were purified by negative selection using the Naive CD4⁺ T Cell Isolation Kit II (Miltenyi Biotec). Pre-enriched pDCs were sorted using an antibody to BDCA-4 (Miltenyi Biotec). Cell sorting was carried out at the SCIF Flow Cytometry and Imaging Facility of the Wellcome Trust Brenner Building, University of Leeds, with a BD Influx 6 Way Cell Sorter (BD Biosciences).

Frozen samples were thawed and stained in PBS 2% foetal calf serum (FCS) with several fluorochrome‐labelled anti‐human antibodies depending on the DC subset and analyses. We developed a novel multi‐parametric flow cytometry approach (11 colours) allowing depicting the 3 DC subsets simultaneously. The combination of the following surface markers allowed to define cDC1s, cDC2s and pDCs: CD11c, HLA‐DR (BD Biosciences, Le Pont de Claix), Lin (Biolegend, Paris), CD45, cDC1/BDCA1 (Beckman, Roissy), BDCA2, BDCA3 and BDCA4 (Miltenyi, Paris). We used the same fluorochrome for BDCA2 and BDCA3 antibodies because the corresponding DC subsets were distinguishable by different intensities of labelling. To assess the basal activation status, CD86 (BD), CD40 and CD80 (Beckman) fluorochrome‐labelled anti‐human antibodies were used. Stained cells were then analysed using LSRII Flow Cytometer and FACSDiva software v.8 (BD). Isotype controls were used to differentiate positive cells from nonspecific background staining (CD45⁺ cells also served to determine the threshold of positivity). Dead cells were excluded with live and dead staining. To ensure quality control during the study, we performed a standardisation of the fluorescence intensities using cytometer setup and tracking beads (CST) (BD).

PBMCs were separated on Ficoll-Hypaque (Amersham Biosciences) from buffy coats (New York Blood Center). pDC were purified by BDCA-4 magnetic bead separation (Miltenyi Biotec) as described previously [12 (link)], with a purity ranging from 80 to 95%. Cells were cultured in RPMI 1640 Glutamax (Invitrogen) with 5% PHS (Innovative Research, MI), gentamycin, and HEPES.

Monoclonal antibodies anti-Lineage cocktail (Lin), PD-L1, CD80, CD86, HLA-DR, CD123, CXCR-3, CXCR-4, CD62-L and CCR7 as well as IgG1 or IgG2a isotype controls were purchased from BD Biosciences, while BDCA4 from Miltenyi Biotech. To establish cell viability and exclude dead cells from flow cytometry analyses, Fixable Viability Dye (FvDye, eBioscience) was always included in antibody cocktails. In the mixed cell population of PBMC, pDC were considered as those cells in live/single FvDye^-Lineage^-CD123⁺BDCA4⁺HLADR⁺ gate (S3A Fig). Cells (1.5x10⁶ for PBMC or 5x10⁴ for isolated pDC) were incubated with monoclonal antibodies at 4°C for 30 min and then fixed with 4% paraformaldehyde before analysis on a Cytoflex cytometer (Beckman Coulter). Data were analyzed by Flow Jo software v.10.7 (BD Biosciences). Expression of analyzed cell surface molecules was evaluated using the median fluorescence intensity (MFI). Only viable and single cells were considered for further analysis.

Monoclonal Ab specific for CD14, CD19, CD38, CD27, PD-L1, CD80, CD86, IL-T7, HLA-DR, CD123 as well as IgG1 or IgG2a isotype controls were purchased from BD Biosciences (San Diego, CA, USA), BDCA2 from Biolegend (Fell, DE), BDCA4 from Miltenyi Biotech and IgM by Jackson Laboratories (Bar Harbour, Maine, USA). To establish viability of cells and to exclude dead cells from flow cytometry analysis Fixable Viability Dye eFluor780 (FvDye) (eBioscience, San Diego, CA, USA) was used as previously described [68 (link)]. Briefly, cells (10⁵ for PBMC and 5x10⁴ for isolated pDC) were incubated with monoclonal Abs at 4°C for 30 min and then fixed with 2% paraformaldehyde before analysis on a Gallios cytometer (Beckman Coulter). Data were analyzed by Kaluza software (Beckman Coulter). The expression of cell surface molecules was evaluated using the median fluorescence intensity (MFI) after subtraction of the values of the isotype Ab controls. Only cells present in the viable cell gate were considered for the analysis.