Anti cd25 clone m a251

CD8⁺ T lymphocytes were activated for 12 hours in 96-well plates coated with anti-CD3 Ab (OKT3 clone, CRL-8001, ATCC) at different concentrations ranging from 50 mg/mL to 400 ng/mL. Immune checkpoints (ICs) expression was determined by labeling with anti-PD-1 (Clone EH12, BD Biosciences), anti-TIGIT (Clone A15153G, BioLegend), anti-LAG-3 (Clone 11C3C65, BD Biosciences), anti-Tim-3 (Clone F38-2E2, Biolegend) and anti-KLRG1 (Clone 14C2A07, Biolegend) antibodies. An anti-CD25 (clone M-A251, BD Biosciences) specific-antibody was also used as a T cell activation marker. The expression of the main costimulation molecules was assessed on resting T lymphocytes with anti-CD45RO (clone UCHL1, BD Biosciences), anti-CD28 (Clone CD28.2, BD Biosciences), anti-CD27 (Clone L128, BD Biosciences), anti-CD2 (Clone RPA-2.10, BD Biosciences) and anti-LFA-1 (Clone HI111, BD Biosciences) monoclonal antibodies. All the cytometric analyses were performed on a Facs Canto II (BD Biosciences).

PBMCs were isolated from healthy controls using density centrifugation as described previously. CD4⁺ T-cells were isolated from the PBMC fraction using the EasySep™ CD4⁺ T-cell isolation kit (Stemcell Technologies) as per the manufacturer’s instructions. The T-cells were stained with 2µM CellTrace Violet (Invitrogen). A 96-well plate (Eppendorf) was coated with anti-CD3 (1:1000, Clone OKT3, Invitrogen) for 90min. The coating solution was removed prior to use. Wells without coating served as negative controls.
Monocytes from patients or in vitro polarization, as described above, were counted (XN-350, Sysmex) and resuspended in RPMI-1640 supplemented with 10% foetal calf serum, 2mM L-glutamine and PenStrep. Next, monocytes and T-cells at a 1:10 ratio (monocytes:T-cells) were added to the coated plate in a total volume of 200µl. The cells were incubated for 72h, 37°C, at 5% CO₂. The cells were detached through gentle pipetting, centrifuged, and stained with anti-CD3 (clone UCHT1, alexa fluor 700, 1:200), anti-CD25 (clone M-A251, PerCP Cy5.51:200), anti-HLA-DR (clone G46-6, APC-H71:200) and anti-CTLA-4 (clone BNI3, PE, 1:50), all from BD, for 25min, RT. Finally, the cells were washed once with PBS and analysed using flow cytometry (CytoFLEX).