Non-human primate models provide an invaluable tool for understanding and dissecting immune responses associated with lentivirus infection.15 The rhesus macaque in particular has been invaluable

in both SIV and SHIV vaccine and pathogenesis studies. The most effective use of the macaque model requires Akt inhibitor detailed knowledge of the cells that make up the immune system, including phenotypic identification and functional analysis of individual cell populations, and elucidation of the role they play during innate and adaptive immune responses. This knowledge enhances our understanding of both protective and non-protective immune mechanisms during viral exposure and on-going infection and contributes to the design of candidate prophylactic and therapeutic regimens.41,42 Natural killer cells are important for both the innate and adaptive lines of defence, and therefore represent a cell population of great interest. They have been shown to contribute to the control of both HIV and SIV infections,35,43–46

most likely because of their presence at mucosal effector sites.29,31,47 Despite their importance, only minimal efforts have been made to phenotypically identify and functionally characterize macaque NK cell subpopulations. In humans, NK cells can be categorized in multiple subsets by their surface expression patterns of CD56 and CD16 and by the expression AZD8055 mw of different types of NKRs.2,7,48 Recent reports have described rhesus macaque NK cells as CD3− CD8αα+ NKG2A+ lymphocytes present in the blood and tissues.29,30 However, study of NK cells in non-human primates has proven to be technically challenging for several reasons. First, CD56 in macaques is not only expressed by NK cells, but also by monocytes.49 Yet it has been recently shown that tissue NK cells are mostly CD16− CD56+,29 which indicates that CD56 is the most reliable marker for tissue NK cells. Therefore, use of anti-CD16 mAbs for depletion of NK cells in HIV/SIV in vivo studies may

not be providing correct information regarding the role played by these Cytidine deaminase cells in control of infection and the overall mucosal immune responses.50 Additionally, the presence of other CD3− cell subsets within the lymphocyte gate (B cells and monocytes), requires the use of specific lineage markers for the correct identification of NK cells.51 In the present study, our consistent gating strategy which eliminated dead cells, monocytes, T cells and B cells (Fig. 1a), left two distinct NK cell candidate populations based on their CD8α expression patterns. We subsequently found that a subset of the CD3− CD14− CD20−/dim CD8α− cells expressed NK-cell-associated lineage and activation markers, and responded to NK-cell-stimulating cytokines, making them a candidate macaque NK cell population. As mentioned, not all cells within the CD8α− gate were candidate NK cells because, as shown in Fig. 2, only a fraction of these cells expressed CD16, CD56, granzyme B and/or perforin.