Antibodies and flow cytometry.

The following dyes, antibodies, and secondaries were used to stain cells: Zombie Violet (1:500 in 100 μL/10⁶ cells, BioLegend), Zombie Yellow (1:500 in 100 μL/10⁶ cells, BioLegend), APC anti-human CD4 (1:100, 300514, BioLegend), PerCP/Cy5.5 anti-human CD8a (1:100, 301031, BioLegend), APC anti-human CD8a (rhesus cross-reactivity, 1:100, 301014, BioLegend), CD8-biotin (1:100, 130–098-556, Miltenyi), anti-mouse CD16/CD32 Fc block (1:100, 14–0161-86, eBioscience), FITC anti-mouse CD3e (1:50, 100305, BioLegend), BV421 anti-mouse CD8a (1:50, 100737, BioLegend), FITC mouse anti-human CD3e (rhesus cross-reactivity, 1:20, 55611, BD Biosciences), Purified anti-human CD3 (Clone UCHT1, 300402, BioLegend), Purified anti-human CD8a (Clone RPA-T8, 301002, BioLegend), Super Bright 600 anti-human CD19 (1:20, 63–0198-42, eBioscience), Super Bright 702 anti-human CD56 (1:100, 67–0566-42, eBioscience), PE anti-human CD3 (1:100, 300308, BioLegend), APC/Cy7 anti-human CD14 (1:40, 325619, BioLegend), FITC anti-human CD16 (1:50, 302006, BioLegend), Alexa Fluor 700 anti-human CD3 (1:50, 300424, BioLegend), Brilliant Violet 785 anti-human CD4 (1:50, 317442, BioLegend), PE/Cy7 anti-human CD8a (1:200, 300914, BioLegend), BUV737 mouse anti-human CD45RA (1:25, 564442, BD Biosciences), BUV395 mouse anti-human CD45RO (1:25, 564291, BD Biosciences), PE anti-human CD62L (1:400, 304806, BioLegend), Brilliant Violet 421 anti-human CCR7 (1:25, 353208, BioLegend), Erbitux-biotin (1:500, Jensen Lab), PE-Cy7 mouse anti-Ki-67 (1:20, 561283, BD Biosciences), BUV737 mouse anti-human PD-1 (1:20, 565299, BD Biosciences), Brilliant Violet 785 anti-human TIM-3 (1:20, 345032, BioLegend), PE mouse anti-human LAG-3 (1:20, 565616, BD Biosciences), Brilliant Violet 785 anti-human CD45RA (1:160, 304140, BioLegend), NeutrAvidin Protein DyLight 633 (1:500, 22844, Invitrogen), Alexa Fluor 647 Streptavidin (1:500, 405237, BioLegend), and PE Streptavidin (1:500, 405204, BioLegend). OneComp eBeads (Invitrogen) were used to prepare single-color controls for compensation, if needed. Stained samples were analyzed with either a MACSQuant Analyzer 10 (Miltenyi), Attune NxT (Invitrogen), or BD LSRFortessa (BD Biosciences) flow cytometer.