Lag 3 fitc

PBMC samples at the indicated visits pre- and post-pembrolizumab treatment were thawed and stained with a fixable Aqua viability dye (Invitrogen) and a cocktail of antibodies to the following surface markers: CD8–Qdot605 (Invitrogen, 3B5), CD4–Qdot655 (Invitrogen, S3.5), PD-1–PE (BD, MIH4), LAG-3–FITC (Enzo, 17B4), ICOS–PE-Cy7 (eBioscience, ISA-3), TIM-3–APC (R&D Systems, 344823). Cells were next fixed and permeabilized with the FOXP3/Ki67 Fixation/Permeabilization Concentrate and Diluent (eBioscience), and subsequently stained intracellularly with CD3–BV570 (Biolegend, UCHT1), Ki67–AlexaFluor700 (BD), FOXP3-eFluor450 (eBioscience), and CTLA-4–PerCP–eFluor710 (eBioscience). Stained cells were acquired on a BD Biosciences LSRFortessa and analysed using FlowJo software (FlowJo, LLC).

Human peripheral blood mononuclear cell (PBMC) samples were thawed and stained with a fixable Aqua viability dye (Invitrogen) and a cocktail of cell surface antibodies. For measurement of T cell activation/exhaustion status, fluorescent activated cell sorting (FACS) analysis was performed using the following cell surface markers: CD8-Qdot605 (Invitrogen, 3B5), CD4-Qdot 655 (Invitrogen, S3.5), PD-1-PE (BD, MIH4), LAG-3-FITC (Enzo, 17B4), ICOS-PE-Cy7 (eBioscience, ISA-3), and TIM-3-APC (R&D Systems, 344823). For multiplex cytokine measurements, validated V-PLEX Proinflammatory Panel 10-plex (human) kits (for IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFNγ, TNFα, IL-1β, and IL-13) were used (MSD, Cat #K15049D-1). See Supplementary Materials and Methods for all FACS and cytokine assay details.