1. Volume 26, Issue 6, Pages 798-811 (June 2007)
Acquisition of Murine NK Cell Cytotoxicity Requires the Translation of a Pre-existing Pool of Granzyme B and Perforin mRNAs 
Todd A. Fehniger, Sheng F. Cai, Xuefang Cao, Andrew J. Bredemeyer, Rachel M. Presti, Anthony R. French, Timothy J. Ley 
Immunity 
Volume 26, Issue 6, Pages (June 2007)
DOI: /j.immuni
Copyright © 2007 Elsevier Inc. Terms and Conditions

2. Figure 1
Resting Murine NK Cells Express Abundant Gzma but Little Gzmb or Prf1 Protein
Resting splenocytes from B6 mice (A), freshly isolated mononuclear cells from various B6 mouse tissues (B), or resting splenocytes from the indicated strains of mice (C) were gated on NK1.1+CD3− or DX5+CD3− NK cells and analyzed for expression of intracellular Gzma, Gzmb, or Prf1. Individual flow-cytometry plots are shown and are representative of at least 30 (A), 5 (B), or 3 (C) independent experiments (see Results for summary statistics). Numbers indicate the percentage of cells in each quadrant.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

3. Figure 2
Cytokine-Activated Murine NK Cells Express Abundant Gzmb and Prf1 Protein
(A) Cytokine-activated NK cells express Gzmb and Prf1 protein by flow cytometry. Splenocytes were cultured with no cytokines (media) or the following cytokines for 48 hr: rhIL-2 (1000 IU/mL), rmIL-12 (100 ng/mL), rmIL-15 (100 ng/mL), rmIL-18 (100 ng/mL), or rmIFN-α (100 U/mL), and flow-cytometry plots of NK1.1+CD3− NK cell expression of Gzma, Gzmb, and Prf1 are shown. Results from an individual experiment are shown and are representative of at least three independent experiments. Numbers indicate the percentage of cells in each quadrant.
(B and C) IL-15-treated NK cells express Gzmb and Prf1 protein in a time-dependent fashion. Splenocytes were stimulated with 100 ng/mL IL-15 for 0 (resting), 6, 12, 18, 24, or 48 hr, and NK1.1+CD3− NK cells were analyzed for expression of intracellular Gzma, Gzmb, and Prf1. Representative flow-cytometry plots from individual splenocyte cultures (B), and summary scatter plots of the percent positive NK1.1+CD3− NK cells for Gzma, Gzmb, and Prf1 from at least six independent experiments (C) are shown, with each data point representing an individual mouse. Horizontal bars represent mean percent positive NK1.1+CD3− cells. ∗p < 0.01; ∗∗p < 0.001; ∗∗∗p <
(D) IL-15-activated NK cells express Gzmb and Prf1 in a dose-dependent fashion. Splenocytes were stimulated with the indicated concentrations of rmIL-15 for 48 hr, and NK1.1+CD3− NK cells were analyzed by flow cytometry for intracellular Gzma, Gzmb, and Prf1. Results shown are representative of at least three independent experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

4. Figure 3
IL-15 Induces Gzmb and Prf1 Protein Expression and Granule Formation in Murine NK Cells
(A) Immunoblots of resting and activated NK cells or bulk splenocytes for Prf1 (∼66 kDa) and Gzmb (∼30 kDa). Protein lysates were made from bulk splenocytes or flow-sorted NK cells at rest (fresh) or after stimulation with IL-15 for 24 hr or 48 hr. The murine NK cell line 36.2 (which constitutively expresses Prf1 and Gzmb) is shown as a positive control (+ control), and actin is used as a loading control. NK, NK cells; SP, spleen; NS, nonspecific band. Results shown are representative of two independent experiments.
(B) IL-15 induces the formation of azurophilic granules in purified murine NK cells. Light-microscopic evaluation (1000×) of flow-sorted murine NK cells at rest or after 24 hr stimulation with IL-15 (100 ng/mL). Note the large size, open nuclear chromatin, prominent Golgi, and abundant azurophilic granules in the IL-15-stimulated NK cells, compared with resting NK cells. Results shown are representative of three independent experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

5. Figure 4
Resting Murine NK Cells Express Abundant Gzma, Gzmb, and Prf1 mRNA, but Only Gzmb mRNA Increases with IL-15 Stimulation
(A and B) Gzma, Gzmb, and Prf1 mRNA expression detected on microarrays from resting and IL-15-activated NK cells.
(A) Flow-sorted NK cells were stimulated with 100 ng/mL of IL-15 for 0 hr (rest) or 24 hr, and total mRNA was isolated, labeled, and hybridized with Affymetrix 430v2.0 expression microarrays. Signal intensities of probe sets for Gzma, Gzmb, Prf1, and β-actin (control for [D]) from three independent NK cell experiments are shown with the mean indicated by horizontal bar. ∗p < 0.05.
(B) Representative scatter plot of probe set values from three resting (x axis) and IL-15-activated (y axis) NK cell pairs, with Gzma, Gzmb, and Prf1 probe set values designated with arrows.
(C) Microarray signal intensity (mean ± SD) from three independent resting and IL-15-activated (24 hr) NK cell pairs for Prf1 and murine Gzm probe sets on the Affymetrix 430v2.0 microarray. Also shown is the signal intensity of resting, unmanipulated WT spleen cells (n = 2 spleens, with arrays for both performed in duplicate) for comparison. Values in italics indicate that these signals were Affymetrix “absent” calls (no specific detectable signal present) on the microarrays.
(D) Quantitative real-time RT-PCR comparing resting and 24 hr IL-15-activated NK cell expression of Gzma, Gzmb, and Prf1 mRNAs. The mean ± SD expression from at least four independent experiments is shown as fold-change of IL-15-activated (24 hr) compared to resting NK cells, utilizing the comparative Ct method, with β-actin used as the control. ∗p < 0.05.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

6. Figure 5
Resting Murine Splenocytes Are Minimally Cytotoxic, and IL-15-Activated Splenocytes Require Gzmb and Prf1 to Efficiently Kill Tumor Targets In Vitro
(A) Resting murine splenocytes exhibit minimal cytotoxicity against the NK-sensitive RMAS and YAC1 tumor cell lines that is increased by IL-15 activation. FloKAs (4 hr) were performed with resting or activated (48 hr IL-15) B6 splenocytes as effectors and the indicated tumor cell line as targets, at E:T ratios of 5:1, 10:1, and 20:1. The NK-insensitive EL4 cell line is included as a negative control. For (A), (B), (C), (E), and (F), FloKA results shown are the percent target cells that are 7-AAD positive (mean ± SD of replicate wells) from one experiment. The data (mean ± SD) shown are representative of at least three independent experiments.
(B) Multiple strains of mice exhibit minimal resting NK cell cytotoxicity, which is increased by IL-15 activation. Splenocytes from the indicated mouse strains isolated fresh (resting) or after 48 hr of IL-15 activation were used as effector cells in a 4 hr FloKA against RMAS targets at the indicated E:T ratios. Results (mean ± SD) shown are representative of three independent experiments.
(C and D) FloKA and Cr51-release assays yield similar results, showing a time-dependent increase in NK cell cytotoxicity after IL-15 stimulation. WT B6 splenocytes were isolated and used fresh (0 hr) or at 12 hr, 24 hr, or 48 hr of IL-15 stimulation as effector cells in both FloKA (C) and Cr51-release (D) assays against identical RMAS targets. Results (mean ± SD) shown are representative of two independent experiments.
(E) Splenocytes from WT, Gzmb−/−, and Prf1−/− (B6) were used as effector cells in a 4 hr FloKA at rest or after 48 hr of IL-15 stimulation against RMAS tumor targets at 5:1, 10:1, and 20:1 E:T ratios. Results (mean ± SD) shown are from one experiment representative of at least three independent experiments.
(F) Splenocytes from WT, Gzma−/−, Gzmb−/−, Gzma−/−Gzmb−/−, and Prf1−/− (129/SvJ) were used as effector cells in a 4 hr FloKA after 48 hr of IL-15 stimulation against RMAS tumor targets. Results (mean ± SD) are representative of at least three independent experiments. For all experiments, spontaneous target cell death (FloKAs, percent targets positive for 7-AAD in target-only wells; Cr51-release, CPM in target-only wells) was routinely <10% and subtracted to provide specific cytotoxicity data. Summary and comparative statistics are detailed in the Results section.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

7. Figure 6
In Vivo Treatment with Poly I:C Induces NK Cell Gzmb and Prf1 Protein and Cytotoxicity
(A and B) Mice were injected i.p. with 200 μg poly I:C or PBS (vehicle control), and after 24 hr, splenic NK1.1+CD3− NK cell expression of intracellular Gzma, Gzmb, and Prf1 was analyzed by flow cytometry. Flow-cytometry data representative of at least three independent experiments are shown (A), with a significant increase in the percent NK cells positive (mean ± SD) for Gzmb and Prf1 after poly I:C injection (B). ∗p <
(C) Splenocytes from poly I:C- or PBS-injected mice were used as effectors in a FloKA against RMAS target cells at a 100:1 E:T ratio. Results (mean ± SD) are representative of two independent experiments.
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions

8. Figure 7
MCMV Infection Induces NK Cell Gzmb and Prf1 Protein, which Are Required for Host Defense and Survival
(A and B) NK cell expression of Gzma, Gzmb, and Prf1 protein by flow cytometry after MCMV infection. Mice were infected with 5 × 104 pfu MCMV/mouse, and splenocytes were harvested from naive mice or at days 1, 2, 4, 6, and 8 after infection (n = 3 mice/time point) and analyzed for NK cell expression of intracellular Gzma, Gzmb, and Prf1. Representative plots from individual mice are shown (A), and the percent NK cells positive for Gzma, Gzmb, and Prf1 are summarized for one experiment (B), with each dot indicative of one mouse and the mean shown as a horizontal bar with SD. These data are representative of 2–5 independent experiments.
(C) MCMV titers in wild-type (WT), Gzmb−/−, and Prf1−/− mice (B6 background). Mice were infected with 1 × 105 pfu MCMV/mouse. Spleens were harvested on day 3 after infection and analyzed by plaque assay for MCMV titers. Combined results from two independent experiments demonstrate that Gzmb−/− (n = 9, p < 0.001) and Prf1−/− (n = 5, p < 0.01) had significantly higher MCMV titers in the spleen compared to WT mice (n = 9).
(D) Survival after MCMV infection in WT, Gzmb−/−, and Prf1−/− mice. Groups of WT, Gzmb−/−, and Prf1−/− (B6 background) mice were infected with MCMV (day 0) and followed for survival. Kaplan-Meier analysis is shown, with significantly inferior survival compared to WT in both Gzmb−/− and Prf1−/− mice (p < 0.001) at two different MCMV doses: left, 1 × 105 pfu/mouse (females); right, 2.5 × 105 pfu/mouse (males). All p values shown on the survival curves indicate significance compared to WT mice. There is also a significant difference between the Gzmb−/− and Prf1−/− curves (p = 0.003) at the 1 × 105 pfu dose in females (left).
Immunity  , DOI: ( /j.immuni )
Copyright © 2007 Elsevier Inc. Terms and Conditions