Cd11c apc

The phenotype of the cultured primary imDCs was confirmed at day 6 using flow cytometry analysis, essentially as described before [27] (link), [29] (link). The cells were analyzed using the primary labeled antibodies Lineage1-FITC, CD11c-APC, HLR-DR-V500, CD80-PE, CD83-PECy7 and CD86-V50 (Becton Dickinson). DC-SIGN levels of imDCs, Raji wild type, and Raji DC-SIGN cells were determined using an anti-DC-SIGN antibody and a secondary PE-labeled antibody (both R&D systems, MN, USA). Flow cytometry analysis was performed on a LSR-II (Becton Dickinson). Data was analyzed using Kaluza 1.2.

WT-1, BIRC5, MUC-1, and TERT were purchased from MyBioSource (San Diego, CA, USA). Recombinant mGM-CSF was purchased from PeproTech (Cranbury, NJ, USA). Cisplatin and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (Burlington, MA, USA). Fluorophore-labeled monoclonal antibodies (Annexin V-FITC, 7-AAD, CD3e-FITC, CD4-PE, CD8-Pacific blue, CD11c-APC, CD80-PE, CD86-FITC, Fixable Viability Stain 780, IFN-γ-BV605, MHC I-PE, MHC II-PerCP-Cy5.5) were purchased from BD Bioscience (Franklin Lakes, NJ, USA). Cell trace violet was purchased from Invitrogen (Waltham, MA, USA). LLC1 was purchased from ATCC (Manassas, VA, USA).

Single-cell suspension of 1×10⁶ cells from the draining pLNs were stimulated with 200 µg/ml phorbol myristate acetate (PMA) and 1mg/ml ionomycin for 2 hours followed by 4 hours incubation with 2mM monensin. The cells were stained with 50 µl FACs buffer containing 1% rat serum, 1% FC-γ blocker (clone 2.4G2) (homemade), and a cocktail of fluorophore-conjugated antibodies for 30 minutes at 4°C in the dark. Antibodies used included surface extracellular staining markers: Ly6C-PerCPCy5.5 (clone AL-21), CD11b-V450 (clone M1/70), MHCII-AF700 (clone, M5/114), CD11c-APC (clone, HL3), F4/80- PE-Cy7 (clone, BM8), CD44-FITC (clone IM7), CD4-V500 (clone RM4-5), CD3-A700 (clone 500A2), CD62L-V450 (clone MEL-14), CD19-PerCPCy5.5 (clone 1D3), CD8-APC (clone 53-6.7) (BD Biosciences). For intracellular cytokine staining, single-cell suspensions were fixed in 4%paraformaldehyde and permeabilized with 0.5% saponin buffer and stained with IFN-γ-AF700 (clone XMGL2), IL-4-APC (clone 11B11), and IL-13-PE (clone eBio13A) (BD Biosciences). 100,000 events were acquired on a BD Fortessa (BD flow Biosciences). Data were obtained as a percentage of the total cells acquired. The data were analyzed using FlowJo software version 10 (TreeStar). Absolute cell numbers were calculated as the product of the cell percentage and the total number of cells counted by trypan blue exclusion method in the isolated pLN.

PBMCs were isolated from whole blood collected from family members and healthy donors by ficoll-histopaque gradient centrifugation. For phenotypic staining, the following monoclonal antibodies were used: CD3-APC-H7, CD4-PerCP-Cy5.5, CD38-PerCP-Cy5.5, CD10-PECF594, CD21-APC, IgG PeCy7, CD14-PerCP, CD123-PE, CD56-PeCy7, CD11c-APC, CD16-APC-H7 (BD Biosciences, San Diego, CA, USA), CD8-APC-EF780, CD27-APC-EF780 (eBioscience, San Diego, CA, USA), CD19-BV650, CD24-BV605 (Biolegend, San Diego, CA, USA) and IgA-PE (Miltenyi Biotech, Bergisch Gladbach, Germany). Naïve B cells were enriched by negative selection using B-cell isolation kit (Stemcell, Vancouver, BC, Canada). Naïve B-cell purity was verified by flow cytometry to 98% purity.

Fresh isolated 2 × 10⁶ cells/mouse of splenocytes or MLNLs for each group were analyzed by flow cytometry. Separated cells were dyed with rabbit anti-mouse CD16/CD32, CD3e-PE, CD19-PE-Cy7, and CD11c-APC (BD Biosciences, Shanghai, China), as well as goat anti-mouse CCR10-Axlexa Flour 488 (Abcam, Cambridge, UK), at 4 °C for 30 min. The stained cells were washed and detected on a BD FACSAria III platform. The absolute number of CD3e⁺, CD19⁺, or CD11c⁺ cells (1 × 10⁵/mouse) from each immunization group and control group (pcDNA3.1) was analyzed by flow cytometry. CCR10 expression on CD3e⁺, CD19⁺, or CD11c⁺ cells was analyzed using FlowJo V10 software (BD Biosciences, Shanghai, China).