Apc anti human tim 3

The following cell surface fluorochrome-conjugated monoclonal antibodies were used to detect T cells phenotype: FITC anti-human CD4, PE/Cy7 anti-human CD8, PE anti-human CD3 (Biolegend, San Diego, CA, USA) and APC anti-human NKG2D (eBioscience). T cell activity was determined by staining the surface APC anti-human CD69, intracellular PE anti-human IFN-γ (BD Biosciences, San Diego, CA, USA), PE anti-human GzmB (eBioscience, San Diego, CA, USA) and PE anti-human Bcl-2 (Biolegend) antibodies. PE anti-human PD-1 and APC anti-human Tim-3 were purchased from Biolegend. FITC anti-human CD45RA, Percp-cy5.5 anti-human CCR7 and PE anti-human CD45RO were purchased from Biolegend to determine T cell subsets. APC Annexin-V and 7-Aminoactinomycin D (7-ADD) from BD Biosciences were used for apoptosis staining. APC mouse IgG1, κ isotype, PE mouse IgG1 isotype, PE/Cy7 mouse IgG2a, κ isotype, FITC mouse IgG1 isotype, PE/Cy7 mouse IgG1, κ isotype (Biolegend) were used as controls. Flow cytometry was performed on the BD LSRFortessa flow cytometer, and data were analyzed using FlowJo Version 10 software.

PE anti-human PD-1 (Cat.no: 329,906, clone EH12.2H7), APC anti-human Tim-3 (Cat.no: 364,804, clone A18087E), and isotype controls (Cat.no: 400,112 and 400,122, clone MOPC-21) were purchased from Biolegend. Alexa Fluor® 647 anti-human Adenosine A2aR (Cat.no: FAB94971R, clone #599,717), and isotype control (Cat.no: IC003R, clone #20,102) were purchased from R&D Systems. For T cell enrichment and stimulation, anti-human CD3 (Cat.no: 130-093-387, clone OKT3) and anti-human CD28 (Cat.no: 130-093-375, clone 15E8) antibodies and Recombinant human IL-2 IS, research grade (Cat.no: 130-097-743) were purchased from Miltenyi Biotec.