Bdca 1

To determine the numbers of cDCs and pDCs in the skin, we applied an immunostaining technique to serial cryosections (6 μm). Slides were warmed up at room temperature (RT) for 30 min, fixed in ice cold (4°C) acetone for 5 min, air dried for 45 min, and washed in PBS. Sections were then blocked with normal goat serum (S-1000, Vector Laboratories) and incubated overnight at 4°C with unconjugated mouse anti-human CD1c (BDCA-1) for the determination of cDCs or mouse anti-human CD303 (BDCA-2) for pDCs (130-090-695 and 130-090-690, resp., Miltenyi Biotec). A secondary goat anti-mouse antibody conjugated with AlexaFluor568 or goat anti-mouse conjugated with AlexaFluor488 (A21134 and A21121, resp., Molecular Probes) was applied for 30 min at RT. Negative controls were obtained by substitution of the primary antibody with the same concentration of the corresponding IgG isotype control (MAB003 and MAB002, resp., R&D Systems). Slides were rinsed and then counterstained with Hoechst (H3569, Molecular Probes).

Peripheral blood lymphocyte, monocyte, peripheral blood dendritic cell (PBDC) and monocytoid myeloid-derived suppressor cell (mMDSC) frequencies and activation status were assessed before and during treatment by four-color flow cytometry staining. Cell surface antibody staining of PBMC was performed in PBS/0.1% BSA/0.02% Sodium-Azide for 30 min at 4 °C. The following antibodies were used: FITC, PE, PerCP-Cy5.5 or APC-labeled Abs directed against human CD3, CD4, CD8, CD11c, CD14, CD15, CD16, CD19, CD25, CD27, CD33, CD45, CD45RO, CD45RA, CD56, CTLA4, CD123, HLA-DR, PD-1 (all BD Biosciences), CD11b, FoxP3 (eBioscience, San Diego, CA), CD40 (Beckman Coulter, Marseille, France), Fab-M-FITC (Southern Biotec, Birmingham, AL), and blood DC antigens BDCA1, BDCA2, BDCA3 (all from Milteny Biotec, Bergisch Gladbach, Germany) and MDC8 (a kind gift from Dr. E.P. Rieber, Dresden, Germany) and matching isotype control antibodies. Intracellular FoxP3 and CTLA-4 staining was conducted with the anti-human FoxP3 staining kit (eBioscience, San Diego, CA) according to the manufacturers’ protocol. Stained cells were analyzed on a FACScalibur (BD Biosciences) using Cell Quest software.

At 24, 48 and 96 hours post-infection, PBMC were stained with LIVE/DEAD cell blue viability dye (Invitrogen, Carlsbad, CA) and monoclonal antibodies directed against CD11c (Biolegend), CD14 (BD), CD40, CD83, CD86, HLA-DR (Biolegend), and BDCA-1 (Miltenyi Biotec) and subsequently analyzed on a Fortessa cytometer (BD Biosciences, San Jose, CA). For intracellular cytokine staining, cells were treated with a commercial fixation/permeabilization kit (BioLegend) according to the manufacturer’s protocol. Data were analyzed with FlowJo software (Tree Star). cDCs were identified from bulk PBMCs as a population of viable CD14^- lymphocytes expressing high levels of CD11c and HLA-DR and the cDC specific marker BDCA-1.