Quantifying T cell populations in rhesus macaques

Whole blood was stained at specific pre- and during treatment time points to monitor the impact of treatment on major immune cell populations. The two-step TruCount (BD Bioscience) technique was used to enumerate the absolute number of CD4⁺ and CD8⁺ T cells in blood^{18 (link),60 (link)}. Blood was stained with fluorescently-labeled antibodies (antibodies from BD Bioscience, San Jose, CA, USA, except where noted): CD4 (APC, cat# 551980, 2.5 μl), CD8 (PE-CF594, cat#: 562282, 3 μl), CD3 (V450, cat#: 560351, 2 μl), CD45 (PerCP, cat#: 558411, 3 μl), NKG2A (PE, Beckman Coulter Life Sciences, Indianapolis, IN, USA, cat#: IM3291U, 5 μl). In addition, Ki-67 (PE, cat#: 556027, 20 μl) was stained post fixing and permeabilizing cells after the surface staining. Flow cytometry acquisitions were performed on an LSR II flow cytometer (BD Biosciences, with FACSDiva v8.01, and FlowJo v10.4, v10.7.1). All antibodies were used following the manufacturers’ recommendations, and validation for use of these antibodies in rhesus macaques was ascertained by the NIH (as indicated in www.nhpreagents.org/ReactivityDatabase).