Figure 1|.

Gating strategy for the identification of human and rhesus T_SCM cells. (a,b) Human and NHP PBMCs were stained as indicated in Table 1. In this example, both panels included anti-CD4 conjugated to Qdot 585 in addition to anti-CD8 conjugated to Pacific Blue in order to allow the simultaneous identification of CD4⁺ and CD8⁺ T cells. These T cells were identified by first gating on singlets (FSC-H versus FSC-A), live CD3⁺ T cells (CD3 versus Dump/AQUA ) and lymphocytes (SSC versus FSC). Naive-like T cells were defined as CD45RO–CCR7⁺ CD62L⁺ in humans and as CD45RA⁺ CCR7⁺ CD28⁺ in rhesus macaques. In these gated populations, T_SCM cells express CD95, whereas T_N cells are CD95–. In NHP CD8⁺ T cells, CXCR3 is coexpressed with CD95 and thus helps identify CD8⁺ T_SCM cells but not CD4 + T_SCM cells, as not all CD95⁺ T_SCM in naive-like CD4⁺ cells express CXCR3⁺ (black arrow). Red straight arrows indicate the sequential gating strategy. The curved red arrows indicate the gate to be copied on gated ‘naive-like’ cells. (c) CD95 FMO control in human T cells. Dashed bars indicate the threshold for positivity for CD95 expression, whereas the diagonal red bar indicates the T_SCM gate. (d) Identification of human antigen–specific T_SCM cells. Human PBMCs were stained as indicated in Table 1. CD8⁺ T cells were identified as in a. T cells specific for the influenza matrix protein_58–66 (Flu GL9) were identified using an MHC class I tetramer (blue dots overlaid on total CD8⁺ T cells in gray). In this case, frozen PBMCs were used, and CD27 replaced CD62L for the identification of T_SCM cells. Blue straight arrows indicate the sequential gating strategy. FSC, forward scatter; SSC, side scatter.