Cd25 pecy7 clone bc96

Twenty thousand immDCs, chemotherapy-treated HL-60, KG-1 or primary AML cells pulsed DCs (as described in Section 4.6) were co-cultured for 5 days in complete RPMI with 200,000 autologous CD3⁺ T cells at the ratio of 1:10. The CD3⁺ T cells alone were used as negative control. After 5 days of co-culture, T cells were stained for 15 min in the dark using the following anti-human mAbs: CD4 APCH7 (clone SK3; BD Biosciences), CD25 PeCy7 (clone BC96; Biolegend), CD127 PerCP 5.5 (clone A019D5; Biolegend), CD45RA V500 (clone HI100; Biolegend), PD-1 APC (clone EH12.2H7; Biolegend). Intracellular staining of FOXP3 using Foxp3/Transcription Factor Staining Buffer Set (eBioscience/ThermoFisher) was performed as follows. For each sample, unstained CD3 cells were used as negative fluorescence control. At least 5000 events of Total Tregs in each sample were collected and analyzed using a FACS Canto II Flow Cytometer (BD Biosciences).

CD4+CD25− T cells (Teff cells) were sorted from PBMCs using human CD4+CD25^hi T cell isolation kit (Stem cell technologies). The percent purity of cellular subsets was assessed using anti-human CD3 Alexa Fluor 700 (Clone OKT3), CD4 APC-Cy7 (clone RPA-T4) and CD25 PE-Cy7 (clone BC96) (Biolegend Inc., San Diego, CA) antibodies by flow cytometry (Suppl. Fig. 6). Purity of isolated cells ranged between 90–98%.

Total CD4⁺ T cells were enriched using magnetic beads and column purification (Miltenyi Biotec) from the peripheral blood, lymph node, and spleen cells. Enriched CD4⁺ T cells were then stained with previously determined volumes of the following fluorescently conjugated antibodies: CD3-AF700 (clone SP34-2), CD8-APC-Cy7 (clone SK1), CD69-PE-CF594 (clone FN50), HLA-DR peridinin chlorophyll protein (PerCP)-Cy5.5 (clone G46-6) (all from BD Biosciences), and CD4-BV650 (clone OKT4) and CD25-PE-Cy7 (clone BC96) (both from BioLegend). Resting CD4⁺ T cells were defined as CD3⁺ CD4⁺ CD8⁻ CD69⁻ CD25⁻ HLA-DR⁻. Sorting was performed on a FACSAria LSR II system (BD Biosciences) equipped with FACSDiva software.