Tim 3 bv650

PBMCs were incubated with directly conjugated antibodies for 30 min at 4°C. The cells were washed before flow cytometry analysis. Antibodies used included anti-human CD3-BV786, CD4-APC-Fire750, CD8-BV510, CD45RA-AF700, CD70-PE, PD-1-BV711, 2B4-FITC, CD160-AF488, TIM-3-BV650, CD95-PE-CY7 (BD Biosciences, San Diego, CA, USA), CCR7-BV421, HLA-DR-AF700, CD38-BV421, CD28-BV711, CD27-BV650 (BioLegend, San Diego, CA, USA), TIGIT-PE-Cy7, LAG-3-APC (Ebioscience, San Diego, CA, USA) and the corresponding isotype controls. Data acquisition was performed on an LSR Fortessa flow cytometer (BD Biosciences), and data was analyzed with FlowJo software (Tree Star, Ashland, OR, USA).

Anti-CD123 CAR T cells were harvested at serial time points during the expansion period and analyzed for surface expression of TIM-3, CTLA-4, LAG-3, and PD-1. Briefly, CAR T cells were harvested, CD3/CD28 dynabeads removed, and stained for CTLA-4-BV786, CD8-BV711, TIM-3-BV650, CD3-PerCPCy5.5, PD-1-PE, LAG-3-AlexaFluor 647 (BD Biosciences), and live dead aqua fixable viability stain (Life Technologies) prior to flow cytometric analysis. Unstained and FMO controls were used to identify gating boundaries.

All experiments and assays were performed on freshly isolated samples. Isolated PBMCs were incubated with directly conjugated fluorescent antibodies for 30 min at 4 °C. The cells were washed before flow cytometry analysis. Monocytes were separated from other cells by gating on CD3/15/19⁻ cells combined with forward scatter (FSC)/ side scatter (SSC) characteristics and CD45 expression. The gating strategy used is shown in Fig. S1. Antibodies used included anti-human CD160-Alexa Fluor 488, CD4-APC-Fire750, CD8-BV510, HLA-DR-Alexa Fluor 700, CD14-APC, PD-1-PE, 2B4-PE-CF594, CD16-BV711, TIM-3-BV650, CD200R-PE, BTLA-BV650, CD45-BV786 (BD Biosciences, San Diego, CA, USA), CX3CR1-BV421, CD3-PerCP-Cy5.5, CD15-PerCP-Cy5.5, CD19-PerCP-Cy5.5, CD29-Alexa Fluor 488, CD62L-BV650, CD11b-BV605, CCR2-PE (BioLegend, San Diego, CA, USA), TIGIT-PE-Cy7, and LAG-3-APC (eBioscience, San Diego, CA, USA), along with the corresponding isotype controls. BD Trucount Tubes (BD Biosciences), combined with specific antibodies (CD45/3/4/8 cocktail; BD Biosciences), were used to determine the absolute counts of leukocytes in the blood with flow cytometry according to the manufacturer’s instructions. The absolute numbers (cells per microliter) of leukocytes and T cells were determined by comparing cellular and bead events.