Anti hla dr antibody

After resuspending PBMC to 0.9 - 1.8 million per ml in RPMI/10% Human AB serum, two 2 ml fractions were transferred to fresh tubes and incubated with anti-HLA-DQ antibody at 10 μg/ml (clone SPVL3; Beckman Coulter) or anti-HLA-DR antibody at 10 μg/ml (clone L243; BioLegend) for one hour at 37°C. Test conditions were assessed in duplicate wells.

UCMSCs and BMDMs were isolated with 0.25% trypsin and resuspended in PBS containing 3% FBS for flow cytometry analysis of cell surface markers. UCMSCs were then incubated in the dark for 30 min with phycoerythrin (PE)-conjugated human anti-CD34 antibody (Biolegend, 343506, USA), anti-CD45 antibody (Biolegend, 304058, USA), anti-CD105 antibody (Biolegend, 800504, USA), anti-CD90 antibody (eBioscience, 12-0909-41, USA); allophycocyanin (APC)-conjugated human anti-CD14 antibody (eBioscience, 17-0141-81, USA), anti-CD19 antibody (Biolegend, 302212, USA); and fluorescein isothiocyanate (FITC)-conjugated human anti-CD73 antibody (Biolegend, 344015, USA) and anti-HLA-DR antibody (Biolegend, 307603, USA). BMDMs were incubated with APC-conjugated mouse anti-F4/80 antibody (Biolegend, 123116, USA) and anti-CD11b antibody (Biolegend, 101206, USA) in the dark for 30 min. Associated conjugated immunoglobulins, provided by eBioscience and Biolegend, USA, were used as negative controls. Finally, cells were washed twice in PBS, and flow cytometry was used to detect positive cells (Beckman Coulter, USA).

On D0, PBMCs from 6 healthy women donors (from 25 to 35 years old) were incubated at the density of 200,000 cells/well in 96-well plates in Roswell Park Memorial Institute (RPMI) 1640 medium (Panbiotech, P04-17500), added with 2% human decomplemented serum, 1 mM non-essential amino acids, 1 mM pyruvate, 2 mM L-glutamine, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, in the presence of HPV16_(L1) peptide mix at the final dilution of 1/450 v/v. On D2, either the Veh., MIM-1; -2; -3; -4 or -5 were added to the medium and incubated for the next 72 h at the final sucrose–lactose concentration of 11 mM. Controls with either HPV16_(L1) or IL-2 (20 ng/mL) were also run in parallel. On D5, the cells were harvested, saturated with FcBlock solution (BD, 564220), immune-stained with fluorescent anti-CD3, anti-CD4, anti-CD8, anti-CD71, anti-CD95, anti-CD28 and anti-HLA-DR antibodies (all purchased from BioLegend), fixed, and analyzed by flow cytometry. The discrimination of the cell sub-populations is as follows: CD4⁺: CD3^high; CD4^high; CD8^low; SSC^low, CD8⁺: CD3^high, CD4^low, CD8^high, SSC^low, CD4⁻/CD8⁻: CD3^high; CD4^low; CD8^low; SSC^low. The supernatants (SNs) were retrieved and the cytokine levels of IFN-γ, IL-6, and interferon- γ inducible protein (IP-10) were assessed by enzyme-linked immunosorbent assay (ELISA).