Pd 1 apc clone eh12.2h7

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).

Analysis of the expression of cell surface and intracellular proteins in purified NK cells and in human carcinoma cell lines was performed by flow cytometry. Cells (1.0 × 10^{6 (link)}) were incubated with 1 μL per test of LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific, Waltham, MA) in 1 × phosphate buffered saline (PBS) for 30 min at 4°C to accomplish live versus dead cell discrimination. Cells were then centrifuged, washed twice with cold PBS, and then stained with primary antihuman mAbs in 1 × PBS +1% BSA (Teknova, Hollister, CA) for 30 min at 4°C. Binding of NEO-201 to human carcinoma cell lines was detected by Pacific Blue-conjugated NEO-201 antibody (BioLegend, San Diego, CA). To detect the NK markers modulated by ALT-803, purified NK cells were labeled with following antibodies: CD56-PE (clone 5.1H11), CD16-PerCP-Cy5.5 (clone 3G8), Tim-3-PE-Cy7 (clone F38–2E2), NKG2D-BV421 (clone 1D11), CD107a-APC-Cy7 (clone H4A3), Granzyme B-FITC (clone GB11), PD-1-APC (clone EH12.2H7), and CD158d-APC (clone mAb 33) (BioLegend). After staining, cells were washed twice with cold PBS and examined using a FACSVerse flow cytometer (BD Biosciences, San Jose, CA). Analysis of cellular fluorescence was performed using BD FACSuite software (BD Biosciences). Positivity was determined by using fluorescence-minus-one controls.