Paired samples that were directly cotransferred were analyzed using paired Students tests. Acknowledgments L.L. and naive Ly49H+ NK cells in tissues at day 5 after infection with influenza. All of the NK cells detected in the BAL expressed CD69 (Fig. 2 A), indicating that the MCMV-memory and naive NK cells present in the airway were similarly activated. However, in the lung, a significantly higher percentage of the naive NK cell subset was CD69+, compared with MCMV-memory NK cells (Fig. 2 B), after influenza infection. Surprisingly, this was also observed in Abiraterone (CB-7598) the spleen, suggesting that activation was not dependent on interaction with influenza-infected cells (which are restricted to the lung) but is likely driven by systemic cytokines. Endogenous Ly49H? NK cells showed a similar level of activation as the adoptively transferred naive Ly49H+ NK cells (Fig. 2 B). We examined the expression of cell Abiraterone (CB-7598) surface markers associated with MCMV-induced activation and MCMV-memory, including KLRG1 and Ly6C, before and after influenza infection (Fig. 2 D), as well as activating receptors implicated in response to influenza-infected cells (Draghi et al., 2007; Mendelson et al., 2010), for example, NKG2D and NKp46 (Fig. 2 C). MCMV-memory NK cells (CD45.1+) clearly showed a distinct phenotype, compared with naive cells, before Abiraterone (CB-7598) influenza infection, in all tissues analyzed. As reported previously (Bezman et al., 2012), they expressed high levels of KLRG1 and Ly6C compared with naive NK cells (Fig. 2 D). After influenza infection, the cell surface density of KLRG1 and Ly6C was not remarkably changed on either the MCMV-memory or adoptively transferred naive Ly49H+ NK cells (Fig. 2 D), or endogenous Ly49H? cells (our unpublished data). Expression of the activating NKG2D and NKp46 receptors was slightly increased in the lungs, but to a similar extent in naive and MCMV-memory NK cells (Fig. 2 C). These results indicate that Rabbit Polyclonal to NOTCH2 (Cleaved-Val1697) the expression of markers associated with MCMV-memory was not impacted significantly by influenza infection. Open in a separate window Figure 2. Activation of MCMV-memory NK cells is reduced compared with naive Ly49H+ NK cells during influenza infection. MCMV-memory NK cells were generated as described in Fig. 1. At day 29 after infection, 105 naive Ly49H+ NK cells were transferred into these hosts and mice were infected with 50 PFU of PR8 influenza virus. NK cell populations were analyzed at day 5 after infection with influenza. (A) A representative histogram showing CD69 expression on memory and naive Ly49H+ NK cells in the BAL; uninfected splenic NK cells are shown as a control. (B) The percentages of CD69+ NK cells in the MCMV-memory and naive Ly49H+ cells and endogenous Ly49H? NK cells are presented for lung and spleen. (C) The expression of NKG2D and NKp46 was assessed in MCMV-memory and naive Ly49H+ and endogenous Ly49H? NK cells in the lung and graphed as median fluorescence intensity (MFI). (D) The expression of Ly6C and KLRG1 on memory (CD45.1+) and naive (CD45.1?) Ly49H+ NK cells in flu-infected and uninfected mice are shown in representative flow cytometry plots. For all panels, = 3C4 mice and data shown are the mean the SEM. MCMV plus influenza infection data are representative of three independent experiments. *, P < 0.05. Response of MCMV-memory NK cells to Listeria infection As influenza viral replication is strictly limited to the lungs and airway, we examined a second model, systemic infection by = 3C4 mice for all panels, uninfected mice were pooled from independent experiments, and data shown are the mean the SEM. **, P < 0.005; *, P < 0.05. MCMV-memory NK cells mount a diminished functional response after Listeria infection To assess differential NK cell activation and function in naive versus MCMV-memory NK cells in response to Listeria infection, we analyzed the expression of CD69 at day 4 after infection and the in vivo production of IFN- at 24 h after infection. We readily detected MCMV-memory and adoptively transferred naive Ly49H+ NK cells in the spleen in both uninfected and infected mice (Fig. 4 A) at this early time point, before any significant proliferation or loss through apoptosis. The percentage of NK cells expressing IFN-, directly ex vivo, was significantly higher in the naive subset of Ly49H+ cells than in the MCMV-memory subset, comparable to endogenous naive Ly49H? NK cells in the spleen (Fig. 4 B). We.