Anti ifn γ biotinylated detection antibody clone 7 b6 1

PBMCs were pulsed either with 1 µg/ml or with 5 µg/ml of HLA class I or HLA class II peptide, respectively. Irrelevant peptides with the respective HLA restrictions were used as negative control (YLLPAIVHI for HLA-A*02 (source protein: DDX5_HUMAN), and ETVITVDTKAAGKGK for HLA class II (source protein: FLNA_HUMAN)). Cells were cultured for 12 days adding 20 U/ml IL-2 (Novartis, Basel, Switzerland) on days 2, 5, and 7. Peptide-stimulated PBMCs were analyzed by IFN-γ enzyme-linked immunospot (ELISPOT) assay on day 12^{67 (link)}, with anti-IFN-γ antibody (clone 1-D1K, 2 µg/mL, MabTech), anti-IFN-γ biotinylated detection antibody (clone 7 B6 1, 0.3 µg/mL, MabTech), ExtrAvidin-Alkaline Phosphatase (1:1000 dilution, Sigma-Aldrich) and BCIP/NBT (5 bromo 4-chloro 3 indolyl-phosphate/nitro-blue tetrazolium chloride, Sigma-Aldrich). Spots were counted using an ImmunoSpot S6 analyzer (CTL, Cleveland, OH, USA) and T cell responses were considered positive if >10 spots/500,000 cells were counted, and the mean spot count was at least three-fold higher than the mean spot count of the negative control.

PBMCs from AML patients after DNMTi therapy were pulsed with 5 µg/ml per HLA class II peptide and the HLA class II negative control peptide ETVITVDTKAAGKGK (source protein: FLNA_HUMAN) was also used for stimulation. Cells were cultured for 12 days adding 20 U/ml IL-2 (Novartis, Basel, Switzerland) on days 2, 5, and 7. Peptide-stimulated PBMCs were analyzed by IFN-γ ELISpot assay on day 12 with anti-IFN-γ antibody (clone 1-D1K, 2 µg/mL, MabTech), anti-IFN-γ biotinylated detection antibody (clone 7 B6 1, 0.3 µg/mL, MabTech), ExtrAvidin-Alkaline Phosphatase (1:1000 dilution, Sigma-Aldrich) and BCIP/NBT (5 bromo 4-chloro 3 indolyl-phosphate/nitro-blue tetrazolium chloride, Sigma-Aldrich)^{78 (link)}. Spots were counted using an ImmunoSpot S6 analyzer (CTL, Cleveland, OH, USA) and T cell responses were considered positive when >10 spots/500 000 cells were counted, and the mean spot count was at least three-fold higher than the mean spot count of the negative control.