A gene expression signature of resting NK cells from the peripheral blood of healthy donors and the changes in transcriptional profiles upon IL2 activation were obtained to overview the functional pathways underlying the biological properties of these cells. Others have addressed IL2 activation of NK cells for fixed time points of 4 hours  or 14 days with multiple activating stimuli with IL2, PHA and feeder cells  whereas our study is directed at early temporal regulation of pure NK cell activation. Our time course experiments demonstrated that IL2 exerts a broad range of effects on NK cells ranging from regulation of cell cycle, cell survival, cytotoxicity and secretion of immunologic and inflammatory effectors in a sequential manner. The use of two microarray platforms and independent NK cell populations validated the generated expression patterns and the biological properties that they suggested. Observed discrepancies between the two platforms indicate that technical variables and platform specific factors influence the large scale transcriptional profiles and sounds cautionary note for efforts to interpret differential gene expression. The technical variables include RNA amplifications protocols, which may also influence the gene expression profile . We have used very strict criteria in our comparative analysis of the two platforms to attend greater accuracy but this approach may lead to the lost of some information. However, the basic information generated from these platforms correlates well when gene signatures and biological pathways rather than single genes were compared. It is also important to keep in mind that high expression of components of a signaling pathway does not indicate activation of that pathway which may involve phosphorylation, specific intracellular localization or other posttranslational determinants. On the other hand, when group of genes subserving specific functional activities show altered expression patterns, it is indicative perturbation of the pathway in response to a given stimulus.
Resting NK cells are characterized by a set of genes that maintain the cells at quiescent state as exemplified by the expression of FOXO3A, SLA, KLF9 (BTEB1), PNRC1 and BTG1 (Figure 3Bright panel). An interesting finding is the high expression of many SMADs suggesting an active TGFβ pathway that may be part of the mechanism maintaining the resting profile and controlling the effector function of the NK cells. That these transcripts are involved in maintaining NK cells in the quiescent state is also supported by their rapid downregulation on IL-2 stimulation.
High expression of other effector-transcripts like cytotoxic effectors, cytokines and chemokines, NK receptors, unique surface markers and adhesion molecules illustrated the potential of circulating NK cells of the peripheral blood to catalyze and participate in the immediate immune responses. The presence of mRNAs encoding ligands like CCL5, CXCL7, TNFSF14, FASL and CCL4 might contribute to the killing of targets, activating other inflammatory cells and maintaining the circulating NK population in this reactive-prepared condition by autocrine stimulation loops. Thus, the CCR5 ligands CCL5 and CCL4 that are expressed in the resting NK cells may act directly on the growth and survival of neighboring NK cells expressing CCR5 at the initiation phase of an innate immune response. Such an autocrine loop is observed for CCL5-CCR5 in prostate cancer . The effector expression profiles shift when the cells are stimulated and more receptor transcripts are expressed preparing the circulating NK cells to take on other functional roles and adapt to increased paracrine stimulation from other infiltrating immune cells.
Another interesting observation is the high expression of GATA3 in resting NK cells, similar to observation in resting T cells . T-BET, on the other hand, had low expression in resting NK cells. There is evidence that both GATA3 and T-BET are important in the development of NK cells [36, 44] but they may also be important in the function of mature NK cells. GATA3 is downregulated in Th1 cells, but its expression is maintained in Th2 cells. This raised the intriguing possibility that downregulation of GATA3 and upregulation of T-BET (Th1-specific transcription factor) in IL2 stimulated NK cells is required for the elaboration of Th1 type of cytokines in activated NK cells. Together with the decreased expression of GATA3, activated NK cells appear to change to a more Th1-like expression profile. While IL2 is a well known Th1 activator, a similar role has not previously been reported or observed for NK cells. The regulation of the transcriptional profiles of pro and anti inflammatory cytokines and chemokines through these transcription factors is an interesting area of future investigation.
Stimulation of resting NK cells with IL2 triggered an expression pattern consistent with the NK cells as important mediators of pro-inflammatory and innate immune response. Hence, the pro-inflammatory cytokines like IFNγ, CCL5, CCL4, LTA and CCL3 were upregulated whereas anti-inflammatory cytokines and receptors like IL18BP and TNFRSF1B [45, 46] were downregulated. Combined with the activated innate immune response mediated by increased TLR signaling (TLR2, Myd88, TIRAP, TICAM1, TICAM2, IRAK1, -2 and -3, Tollip, TRAF6, TAK1(MAP3K7) and TAB2(.MAP3K7IP2) and the enhanced direct (GZMA, PRF1, GZMB, GNLY and TNFSF6 (FASL)) and indirect ERK enhanced (PI3Ks, AKTs, RAC1 and MEK2 (MAP2K2)) NK cell cytotoxicity  the stimulation of the circulating NK cells resulted in a significant shift in transcript profile reflecting the cells adapting to new functional roles. Strikingly, the cytolytic profile exhibited by activated NK cells resemble closely that of IL2 activated CD8+ T-cells . In CD8+ T-cells, the cytotoxic effectors in granules (GZMB, GNLY and PRF1) and TNF family members (FAS, LTA, TRAIL and TNF-α) were induced whereas GZMK and CD27 (TNRSF7) were downregulated after 4 hours of stimulation (300 IU/ml IL2) (see figure 5B in ). In our study, the above mentioned genes were also upregulated but CD27 showed upregulation at 24 hours.
IL2 stimulation mediated early activation of the JAK/STAT signaling pathway (upregulated JAK3, STAT3, STAT4, STAT5A, and STAT1) hence affecting down stream transcription of many target genes (Figure 4A). When we examined STAT target gene expression, many targets of STAT1, -4 and -5 are upregulated providing confirmatory evidence of STAT1, -4 and -5 activation. Upregulation of JAK3, STAT-1, 3, and 5A was also observed by Jin and colleagues with purified CD8+ and CD4+ T cells stimulated with IL2 , indicating a common usuage of the JAK/STAT pathway in the activation in these cells, at least initially (see figure 1 in ). In our study the inhibitors of JAK/STAT signaling (PIAS1, -2, SOCS3, -5 and -6) were downregulated but PIAS3, -4 and SOCS7 were upregulated illustrating a balance that may limit the degree of JAK/STAT activation. IL2 can also activate the Ras-->RAF-->MEK-->ERK signaling pathway via JAK phosphorylation of SHC leading to stimulation of proliferation in T cells . This may happened in NK cells and this is suggested by our study by the observed upregulated MEK2 (MAP2K2) and ERK1 (MAPK1).
Powerful pro-survival signals were induced by IL2
Four different genes of the PI3K family were upregulated as were the three isoforms of AKT kinases and simultaneously there was decreased expression of the AKT target genes (FOXO1A, -3A and p27). Together with the facts that activated AKT promotes cell survival through 1) phosphorylation dependent dissociation of the BAD/BCLXL complex  (Bad upregulated) and 2) activation of NF-κB through phosphorylation of IKK-β  (IKKβ upregulated) IL2 induced PI3K resulted in the expression patterns of transcripts that appear to promote survival and proliferation. Interestingly, when CTLs were simulated in the presence of other cells in PBMC, the upregulation of both PI3K and AKT were not detected even though IL2 induced a general T cell activation and anti-apoptotic effect illustrating the importance of interactions between effector and bystander cells. Our study was focused on purified NK cells and the effects of bystander cells will not be observed.
NF-κB activation could be mediated by pathways other than IL2 induced PI3K activation, namely the TLR/IL1R pathway, the TNF pathway and possibly a NK specific surface receptor pathway involving BCL10. We have observed good evidence of NF-κB activation through the first two pathways. It is well established that in both T and B cells, BCL10 specifically mediate antigen receptor-induced NF-κB activation  In NK cells, BCL10 has been observed in the cytoplasm of normal NK cells, and in the nuclei of tumor cells of nasal NK/T-cell lymphomas . Since we observed upregulation of BCL10, NF-κB1 and NF-κB2 upon stimulation of NK cells with IL2, it is possible that cytokine receptor mediated signaling also involve BCL10. The expression pattern of CARD11, a participant in BCL10 induced NF-κB activation in T and B cells  also supports this idea. In support of NF-κB activation was the upregulation of NF-κB1, NF-κB2 and the upregulation NF-κB target genes (Figure 4E II). In CD8+T cells, Jin et.al observed increased expression of several mediators of NF-κB pathway, possibly through modulation of TCR signaling (see figure 3 in )
Activation of NFAT signaling pathway
PI3K can promote NFAT nuclear accumulation in two ways: by enhancing Ca2+ mobilization and calcineurin-dependent NFAT dephosphorylation leading to nuclear import, or by AKT-mediated inactivation of GSK-3 that can phosphorylate NFAT and drive nuclear export . The increased expression of positive regulators of intracellular Ca2+ release, activators of calcineurin, catalytic calcineurin A subunits and kinases for NFAT nuclear shuttling in activated NK cells suggested both mechanisms to be involved in the NFAT signaling induced by IL2. NFATC1 showed highest expression in resting NK cells, similar to a prior observation in resting T cells, where it is the predominant NFAT protein . NFATC1 interacts with GATA3 to maintain the differentiated Th2 phenotype . This supports the notion of suppressed expression of Th1 type cytokines in resting NK cells. The profile shifted with the downregulation of GATA3 and upregulation of T-BET on activation.
Temporal pattern of transcript expression
The early increase in transcription (within the first 2 hours) is likely regulated directly by signaling events - "first wave" induced transcription. Genes that were upregulated only after 8–24 hours are likely to represent activation of "second wave" genes and these were numerous in our study and especially represented genes involved in adhesion, secretory pathway, cytotoxicity and cell cycle control. These "second wave" transcripts are probably under the control of upstream genes or induced by factors such as cytokines or chemokines that are elaborated by the cells at a later time point as illustrated by general upregulation of target genes of STAT1 and NF-κB. Many genes had expression patterns where transcript levels were upregulated at 2 hours, low at 8 hours and then high again at 24 hours (genes important for cytotoxicity (TIA1 and PAF1), cytokine signaling (IL2RA and IL18R1), secretory signaling (SLC3A2, HM13 and DEGS1), cell cycle regulation (MCM6 and CCND2)). The control of transcription of these genes is more complex and may involve feedback inhibition or the induction of inhibitors. Time course experiments are therefore important to gain a more complete picture of the biology and function of the cell of interest.
The effect of IL2 on cytotoxic cells have been reported by several groups [16, 33, 39, 54, 55] showing many similarities as well as differences. For example, CD8+ T cells stimulated with 300 IU/ml of IL2 for 4 hours either alone or cocultured with PBMC upregulate IL7, IL13, TNFα and IFNγ whereas the NK cells in our study did not express IL7 or IL13, and the upregulation of TNFα and IFNγ was registered at 8 hours. The cytokine profile of activated NK cells showed marked differences with that observed in activated CD8+T cells by Jin et.al, with the exception of a few genes (e.g. downregulation of IL8 and CXCR4, and upregulation of IL15 and IFN-γ). A few chemokines e.g.CCL3 and CCL4 were only observed to be upregulated at 24 hours in activated NK cells, whereas in CD8+ T cells, these chemokines are upregulated early (4 hours). On the other hand CCL5, IL16 and CXCR3 were downregulated in isolated CD8+ T cells, but upregulated in NK cells. In general, a detailed comparison is very difficult because of the different systems used and the different time point at which the assays were performed. The expression of many of the genes are highly variable over a 24 hr period so there is a significant degree of uncertainty when compared with results from a particular time point.
In most of the studies, the ethnic composition of the subjects studied was not specified but a systematic comparison of two ethnic groups was performed by Jin and co-workers . The latter study is important in highlighting racial differences in immune response and may provide insight into the differences in disease susceptibility and response to immune modulation.