Anti human pd l1

For programmed death ligand-1 (PD-L1) blocking, 10 μg/mL of anti-human PD-L1 (Biolegend, clone 29E.2A3, San Diego, CA, USA) blocking antibody was added to the surface of PTEC and incubated for 2 h. Following incubation, the PTEC wells were extensively washed with 5 changes of warm CM to remove all traces of unbound antibody and the co-cultures were established. To neutralise soluble human leukocyte antigen-G (sHLA-G) and IDO, 10 μg/mL of anti-human sHLA-G (Biolegend, clone 87G) blocking antibody and 1500 μM/mL IDO inhibitor 1-methyl-tryptophan (1-MT) (Life Technologies, CA, USA) respectively was added into the PTEC or control wells immediately following co-culture establishment. The effective blocking concentration levels of 1-MT were determined via titration using cell viability and tryptophan-kynurenine ratios monitored by HPLC (data not shown).

Engraftment of human Kasumi-1 and FA-AML1 cells was verified by flow cytometric analysis of peripheral blood (PB). Briefly, 100 μL of blood was collected from the submandibular vein every week from 8 weeks after cell injection. Following lysis of red blood cells by RBC lysis buffer according to manufacturer’s protocol (Biolegend, 420301), the blood was washed twice by Cell Staining Buffer (Biolegend, 420201), then stained with anti-human HLA-DR (BD, 560744). Human HLA-DR positivity (>1% in PB) was used to confirm Kasumi-1 and FA-AML1 engraftment [19 (link)]. Engraftment of hPBMCs was determined by flow cytometric analysis. Following peripheral blood collection and RBC lysis, the cells were stained with anti-human CD3 (Biolegend, 317306). Cell surface level PD-L1 was measured after stimulating Kasumi-1 and FA-AML1 with 10 ng/mL IFNγ (R&D Systems, 285-IF) for 24 hours and staining with anti-human PD-L1 (Biolegend, 374514). Flow cytometry was carried out in the Immune Monitoring and Flow Cytometry Shared Resource at the Dartmouth Cancer Center, with Cancer Center Support Grant 5P30 CA023108-41.