Cd27 pe cy7 clone m t271

Human HER2 and PD-L1 expression on tumor cells was detected using human HER2-PE-CY7 (clone 24D2, Biolegend) and PD-L1-APC (clone MIH1, eBioscience). HER2 4D5 CAR was detected using the anti-trastuzumab idiotype Alexa Fluor 647-conjugated antibody (clone 2661E, R&D system). HER2 4D5 CAR was also detected using human recombinant HER2 protein conjugated with Alex Fluor 647. Human T cells surface phenotype and transduction efficiency were assessed using the following antibodies: NGFR-FITC (clone ME20.4, Biolegend), Q8 (clone QBEND/10, ThermoFisher), CD45-AF700 or CD45-BV605 (clone HI30, Biolegend), CD3-APC (clone SK7, Biolegend), CD4-PerCP (clone SK3, Biolegend), CD4-BB700 (clone SK3, BD Bioscience), CD8-BV510 (clone SK1, Biolegend), CD27-PE-CY7 (clone M-T271, Biolegend), CD28-BV605 (clone CD28.2, Biolegend. Expression of T cell inhibitory receptors was analyzed using PD-1-BV421 or PD-1-BV605 (clone EH12.2H7, Biolegend), TIM-3-BV605 or TIM-3-PE-CY-7 (clone F38-2E2, Biolegend), CD39 (clone A1, Biolegend), LAG-3-BV711 (clone 11C3C65, Biolegend). Live/dead discrimination was determined using LIVE/DEAD fixable Near-IR dead cell stain kit (ThermoFisher). Flow cytometry results were analyzed using Kaluza software (Beckman Coulter).

Peripheral blood samples were collected from each participant (6x10 ml in lithium-heparin tubes, 3x6 ml in serum tubes). Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Paque PLUS (GE Healthcare) gradient centrifugation. The cell suspension was washed twice in PBS and resuspended in RPMI 1640 containing 5% fetal bovine serum (FBS) (Sigma-Aldrich).
All measurements were performed on freshly prepared PBMCs. The times from sample acquisition to measurement were standardized throughout the entire protocol. PBMCs (8 x 10⁶ cells) were stained with the appropriate combination of fluorochrome-conjugated anti-human antibodies in 100 µl of media in order to differentiate B lymphocyte subsets: CD5 PerCP (clone UCHT2), CD19 PE (clone SJ25C1), IgD APC (clone IA6-2), CD25 BV421 (clone BC96) (all from Sony Biotechnology), CD27 PE-Cy7(clone M-T271, BioLegend). The cells were incubated with the antibodies for 30 minutes in the dark at room temperature (RT), according to the manufacturer’s instructions. Following labeling, samples were washed twice in RPMI.