Igg1 k

PBMC were obtained one day after the hemolysis of red blood cells in ACK lysing buffer (Thermo Fisher Scientific K.K., Tokyo, Japan) according to the manufacturer’s instructions. PBMC were incubated with antibodies that included 7-AAD (BioLegend, San Diego, USA), APC-anti-CD3e (BioLegend), FITC-anti-CD8a (BioLegend), and PE-anti-HLA-C (BD Biosciences) or PE-HLA-C-isotype control by IgG1 k (BioLegend) for 30 min on ice, washed twice with staining buffer, and then analyzed using a BD FACSCanto™ II flow cytometer (BD Biosciences, New Jersey, USA). HLA-C expression was determined by median fluorescence intensity on CD3e⁺CD8a⁺ lymphocytes, CD3e⁺CD8a⁻ lymphocytes, neutrophils, and macrophages in relation to the isotype control. The gating strategy is shown in Fig. 1a. Obtained data were analyzed by FlowJo software (BD Biosciences). All samples were simultaneously stained and analyzed in a one-day experiment.

Cells were plated in a Labtek Chamber slide at a density of 2 × 10⁴ cells/mL After three days, when the cells had reached near confluency, medium was removed from the cells and they were washed with PBS. After fixation with methanol 100% for 5 min, hPDLSC were incubated over night at 4 °C with primary antibodies. The antibodies used in this study include the following: anti-CD9 (monoclonal mouse, IgG1k, 1:1000, BioLegend, San Diego, CA, USA), anti-CD63 (monoclonal mouse, IgG1k, 1:500, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-ALIX (monoclonal mouse, IgG1k,1:500, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). For negative controls, the primary antibody was replaced by non-immune serum (mouse IgG, 1:500, Sigma-Aldrich, Saint-Louis, MO, USA). Then a secondary goat anti mouse AlexaFluor488 antibody (LSBio, Seattle, WA, USA) was applied for 1 h at RT. Counterstaining was performed using Hoechst-dye (1:2000, Sigma-Aldrich, Saint-Louis, MO, USA). Observations were performed using an Upright Widefield Microscope Leica DMRXA2 (Leica Microsystemes, Wetzlar, Germany).

Mouse anti‐IA/IE mAb (HB‐225™ hybridoma: Mus musculus (myeloma), hamster, Armenian B cell, reacts with a monomorphic determinant on the I‐A and I‐E region, IgG isotype, generous gift from Dr R Steinman, Rockefeller University, NY) was purified from culture supernatants, used at the concentration 0.1 μg/mL in ELISA experiments and was covalently linked to magnetic beads coupled with Sheep anti‐Mouse IgG for protein purification procedures (see below).
For immunofluorescence experiments, FITC‐labeled rat anti‐mouse CD4 (IgG2b, Eurobioscience, Germany) and FITC‐labeled rat anti‐mouse CD8 (IgG2α, k, Biolegend, San Diego, CA) were used at the concentration of 1 µg/mL. Furthermore, rat anti‐mouse IL2 (IgG2a, k, Biolegend), rat anti‐mouse IL4, IL‐6, IL‐10, and IFN‐γ (IgG1, k, Biolegend), were used at the concentration of 0.1μg/mL in ELISA experiments.