Pe cy 5 mouse anti human cd4

The cells were stained with FITC-, phycoerythrin (PE)-, Phycoerythrin-TexasRed (ECD)-, or Phycoerythrin-Cyanin (PC5)-conjugated mAb specific for CD3, CD4, CD8, CD27, CD45RA, CD56, HLA-DR (Beckman Coulter, Marseille, France), CD28, NKG2D (eBioscience, CA, USA) and CCR7 (R&D systems, MN, USA). To determine the regulatory T cell (Treg), we used the following antibodies: PE-Cy™5 mouse anti-human CD4, PE mouse anti-human CD25, and Alexa Fluor® 488 mouse anti-human Foxp3 (BD Biosciences Pharmingen, CA, USA). Cells were incubated at 5°C for 30 min, then washed with phosphate-buffered saline and analyzed by Cytomics FC500 (Beckman Coulter). For Foxp3 detection, cells were permeabilized overnight in Fix/Perm buffer (eBioscience) and then stained with anti-Foxp3. Data acquisition and analysis were conducted with the CXP Software, version 2.2 (Beckman Coulter). The flow data was analyzed by the following method: lymphocyte gates were determined visually in FSC/SSC dot plot after which gates with a positive region of CD3, HLA-DR were determined by staining cells with isotype control antibodies. The others (CD4, CD8, CD28, CD45RA, CD56, CD28, NKG2D and CCR7) were determined visually at a valley point of histogram or dot plot. Tregs phenotype was defined as CD4+ CD25+Foxp3+ cell.

To analyze their capacity to decrease T cell proliferation, we cocultured macrophages (previously cocultured with MSCs in the experimental conditions for the generation of T-regs, Section 2.10) with CD4⁺ T lymphocytes isolated by CD4 MicroBeads (Miltenyi Biotec) in a 1:1 ratio (macrophages:T lymphocytes) without cellular contact using a 0.4-μm Transwell system (Corning), for 6 days. The cocultures were grown in the presence of inducing medium M0, M1 or M2. T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE, Thermo Fisher Scientific, Waltham, MA, USA) 2.5 μM and cultured in RPMI medium (HyClone) containing 10% FBS (Biowest) activated with anti-CD2/CD3/CD28 beads (Miltenyi Biotec) (1 bead for each T cell). After 4 days of coculture, T lymphocytes were collected, washed and blocked with FBS (Biowest) at 4 °C for 15 min. After blocking, PE-Cy™5 mouse anti-human CD4 (BD Biosciences) was added for 20 min, then were washed. Acquisitions were made on a spectral flow cytometer Aurora (Cytek Biosciences) and analyzed with FlowJo (Ashland). Macrophages cultured in the absence of MSCs and different polarization conditions were used as controls. Activated T cells cultured in the absence of macrophages were used as a control to normalize percentages.