Unless

otherwise specified, all data reported were averag

Unless

otherwise specified, all data reported were averaged from the number of macaques indicated in the figure legends. Results are shown as means ± SEM. Data were analysed using Prism (v5.03; GraphPad Software, La Jolla, CA). A P-value of ≤ 0·05 was considered statistically significant. Previous studies have identified macaque NK cells as CD3− lymphocytes that are positive for CD8α and CD159a, while lacking CD14 and CD8β expression.29 However, expression of the NK cell-associated lineage markers R788 datasheet CD16 and CD56, as well as perforin, have also been detected in CD8α− NK cells of humans.32,33 Given this, and in view of the increasing interest in elucidating NK effector mechanisms in SIV and SHIV macaque models, we investigated whether rhesus macaque CD3− CD8α− cells also included NK cells. Two candidate NK subpopulations,

based on their CD8α expression patterns, were identified in rhesus macaque PBMCs as CD3− CD14− CD20−/dim cells within a large side-scatter versus forward-scatter lymphocyte singlet gate (Fig. 1a). Cells in these two subsets were negative for the common lineage markers CD4, CD8β, CD123, γδTCR and CD19 (data not shown). Proportionally, CD3− lymphocytes accounted for 28·62 ± 6·92% of CD14− circulating lymphocytes (Fig. 1b).Within the CD3− compartment, CD8α− and CD8α+ cells represented 19·8 ± 7·1% and 34·3 ± 17·4% of CD3− CD14− CD20−/dim cells, respectively (Fig. 1c). Natural killer cells can be identified by surface expression of the classical cell lineage markers CD16 and CD56, as well as a number of inhibitory/activating receptors and intracellular cytotoxic proteins.8 To determine if CD8α− NK cells comprise Temsirolimus molecular weight PIK3C2G a subpopulation of macaque NK cells, we used polychromatic flow cytometry to detect co-expression of NK cell-associated markers. As shown in the representative histograms (Fig. 2a), CD8α− NK cells expressed

CD16, CD56, granzyme B and perforin, but no expression of NKG2A, CD161, NKp46 and NKp30 was detected. On the other hand, CD8α+ NK cells stained positively for all of the above-mentioned molecules (Fig. 2a, bottom row). Further analysis revealed that CD8α− and CD8α+ NK cells expressed comparable levels of the Integrin α-X (CD11c) on their surface; while NKG2D expression was more abundant on CD8α+ NK cells (approximately 85%) compared with CD8α− NK cells (approximately 18%, Fig. 2b). Only CD8α− NK cells expressed HLA-DR on their surface (Fig. 2b). Given the fact that granzyme B and perforin are crucial for NK cell cytolytic function,38 we evaluated the co-expression of these two proteins in the NK cell subpopulations. Approximately 10% of CD8α− NK cells co-expressed granzyme B and perforin (Fig. 2c), indicating cytolytic potential for this NK cell subpopulation. On the other hand, in agreement with their known cytolytic capability,30 approximately 46% of macaque CD8α+ NK cells co-expressed these two proteins.

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