Pe anti human cd25

Phenotyping of resting and activated moDCs was performed by flow cytometry using anti-human CD1d-phycoerythrin (PE), CD103-FITC, HLA-DQ-FITC, PD-L1-PE (BD Biosciences, Franklin Lakes, NJ, USA), CD1a-allophycocyanin (APC), CD40-FITC (BioLegend, San Diego, CA, USA), CX₃CR1-PE, CD80-FITC, CD83-FITC, CD86-PE, DC-SIGN-FITC, CCR7-PE, CD14-PE (R&D Systems, Minneapolis, MN, USA), B7RP1 (ICOSL)-PE (EBiosciences, Santa Clara, CA, USA), and isotype-matched control antibodies. The ratio of regulatory T-lymphocytes was measured by flow cytometry using anti-human CD25-PE (BD Pharmingen), CD4-FITC (BioLegend), FoxP3-APC (R&D Systems), and anti-IL-10-AlexaFluor488 (BioLegend). The viability of moDCs was determined with 2 μg/ml 7-amino-actinomycin D (LKT Laboratories Inc., St. Paul, MN, USA) dye followed by a 24-h activation period with live bacteria or LPS. Fluorescence intensities were measured by FACSCalibur (BD Biosciences), and data were analyzed by the FlowJo software (Tree Star, Ashland, OR, USA).

Peripheral blood was diluted with PBS to the same volume, and PBMCs in diluted peripheral blood were isolated with lymphocyte separation fluid (lymphocyte separation fluid volume:diluted peripheral blood volume = 1:1). The finally about 0.5-1×10⁶ cells per tube were used for flow cytometry analysis. PBMCs were labeled with APC-anti-human CD4, Perp-anti-human CD8, PE-anti-human CD25 and FITC-anti-human CD127 antibodies (all flow cytometry reagents came from BD Biosciences by following the manufacturer’s protocols). The percentages of CD4⁺, CD8⁺, CD4⁺ CD25⁺ and CD4⁺ CD25⁺CD127^-/low cells were assessed by flow cytometry. The specificity of immunostaining was ascertained by the background fluorescence of cells incubated with CD25⁺ and CD127⁺ Ig isotype controls. FACSCaliber (BD Biosciences, Mississauga, Canada) was used to quantify the percentage of cells in all groups. CD8⁺Tregs had a low abundance (0.2- 2% of CD8⁺ T cells) in both the circulation and periphery in healthy controls. In comparison, the well-studied CD4⁺ Tregs comprise approximately 5-12% of the CD4⁺ T cell population. In our study, Tregs indicate CD4⁺ Treg cells.

After 15 h of co-culture, cells were stained with the following antibodies: PE anti-human CD25/ APC anti-human CD3/ V450 anti-human CD45 (BD Biosciences). FACS Aria Fusion Cell Sorter was used to sorter the different populations. The sorting strategy was: First, the viable region FSC/SSC, doublet zone FSC-A/FSC-H and positive region for CD45 was selected. Then, double positive cells (CD3+PKH+) and non-doublet cells (CD3+PKH-) were gated. Finally, within the non-doublet cells (CD3+PKH-), two different populations were sorted using the antibody CD25. The nozzle size used to sorter the doublet cells was 85 μm.

Aliquots (2 × 10⁵) of PBMCs were placed in wells of 96-well-plates, and T cells in PBMCs were activated by human CD3/CD28 T cell activator (#10971, Stemcell Technologies) for 6 h at 37°C and then coincubated with MKN74-derived NC exosomes or PD-L1-KD exosomes for 48 h in the presence or absence of nivolumab (Bristol-Myers Squibb Company, Princeton, NJ, USA). PBMCs were stained with the following antibodies: FITC antihuman CD3 (561806, BD), APC antihuman CD69 (560967, BD), and PE antihuman CD25 (560989, BD). Isotype control Ab staining was used as a negative control.

Cells were harvested 24 hours post reactivation and washed in cold PBS and 0.5 x 10⁶ cells were allotted to each tube. Cells were stained with PE mouse anti-human CD69 (555531, BD Biosciences), PE anti-human CD25 (555432, BD Biosciences), FITC rat anti-mouse HSA (553261, BD Biosciences), or fixed immediately to analyze GFP expression. Cells were fixed in 2% paraformaldehyde and analyzed using the BD Biosciences FACScaliber and CellQuest Pro software at the UCSF Parnassus Flow Cytometry Core. Cells were gated on the live lymphocyte gate using the forward and side scatter plot, and the percentage of live lymphocytes in 10,000 collected total cells was used as an estimate for cell viability.