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. Author manuscript; available in PMC: 2015 Apr 15.
Published in final edited form as: J Immunol. 2014 Mar 14;192(8):3889–3897. doi: 10.4049/jimmunol.1302601

Functional NK cell repertoires are maintained through IL-2Rα and FasL

Martin Felices *, Todd R Lenvik *, Dave EM Ankarlo *, Bree Foley *, Julie Curtsinger *, Xianghua Luo §,, Bruce R Blazar *, Stephen K Anderson **, Jeffrey S Miller *
PMCID: PMC3979985  NIHMSID: NIHMS567077  PMID: 24634493

Abstract

Acquisition of a functional natural killer (NK) cell repertoire, known as education or licensing, is a complex process mediated through inhibitory receptors that recognize self. We found that NK cells containing self-killer immunoglobulin-like receptors (KIR) for cognate HLA-ligand in vivo were less susceptible to apoptosis. In vitro IL-15 withdrawal showed that uneducated NK cells upregulated Bim and Fas. Conversely, educated NK cells upregulated FasL under these conditions. Induction of cell death and Bim expression on uneducated cells correlated with increased IL-2Rα expression. Overexpression and knockdown studies showed that higher IL-2Rα limits NK cell survival in a novel manner that is independent from the role of IL-2 in activation induced cell death (AICD). To study the role of FasL in induction of IL-2Rαhi NK cell death, a co-culture assay with FasL blocking antibodies was used. IL-15 withdrawal led to FasL dependent killing of IL-2Rαhi NK cells by more educated IL-2Rαlo NK cells. Finally, CMV reactivation induces a potent long-lasting population of licensed NK cells with enhanced survival. These findings show education dependent NK cell survival advantages and killing of uneducated NK result in the maintenance of a functional repertoire, which may be manipulated to exploit NK cells for cancer immunotherapy.

Introduction

Natural Killer (NK) cell mediated immunotherapy is currently being tested in clinical trials (1, 2). Inhibitory KIR on NK cells induce function through a process termed education or licensing (3–5). The success of NK cell therapy depends on how functionally competent cells are homeostatically maintained after adoptive transfer. Although NK cell homeostasis has been studied on bulk NK cells, questions remain as to how the repertoire of functionally competent NK cells is maintained in order to exploit its differentiation state and previous exposure history (6–9).

Balance between more mature KIR+ NK cells and less differentiated KIR− NK cells could be regulated by three mechanisms in the periphery: proliferation, differentiation, and survival. Higher proliferation in the KIR− NK cell subset (10) and poor proliferation of more mature CD57+ NK cells have been described (11). This data suggests that the differentiation status of NK cells inversely correlates with proliferative potential. Differentiation could explain the persistence of KIR+ NK cells that develop from KIR− NK cells. Alternatively, it has been shown that KIR expression on T cells may inhibit AICD by inducing PI3K/Akt (12–15), suggesting that enhanced survival could control the balance between functional NK cells and their less differentiated counterparts. In support of this, H2Dd transgenic mice showed a dose dependent enrichment of educated Ly49A+ NK cells with increased sensitivity to IL-15 and reduced apoptosis (16). While these data suggest that education plays a role in NK cell survival, it is unknown whether KIR mediate such signals in humans.

IL-15 and IL-2 have prominent and overlapping roles in proliferation, survival, and NK cell activation (17). Similarities in function stem from shared usage of the common γ-chain (CD132) and IL-2Rβ (CD122) with nearly identical downstream signaling components (18). Specificity is determined by selective binding to IL-2Rα (CD25) or IL-15Rα (CD215). Only IL-15 is critical for NK cell development and homeostasis (19–24) indicating divergence in function. Negative effects on survival, such as the well-described role of IL-2 in FAS-mediated AICD (25, 26), have also been demonstrated. The role of NK cell education on cytokine-mediated survival remains unknown.

Materials and Methods

Cell Isolations

Peripheral blood from healthy human donors was obtained from the Memorial Blood Center (Minneapolis, MN). A Histopaque gradient (Sigma-Aldrich) was utilized to obtain peripheral blood mononuclear cells (PBMCs). For NK cell education studies PBMCs were HLA and KIR typed and cells were frozen down for later use. For experiments utilizing NK cells alone, NK cells were enriched using magnetic-activated cell sorting (MACS) NK Cell Isolation Kit as per the manufacturer’s protocol (Miltenyi Biotec). Where noted, cells were further sorted using a FACSAria II cell sorter (BD Biosciences) and processed for RNA or protein. For experiments (unless otherwise described) the following medium was used: complete DMEM (Cellgro) without exogenous cytokines (unless otherwise noted) supplemented with 10% human AB serum (Valley Biomedical), 30% Ham F-12 medium (Cellgro), 100 U/mL of penicillin (Invitrogen), 100 U/mL of streptomycin (Invitrogen), 24μM 2-β–mercaptoethanol, 50μM ethanolamine, 20 mg/L of ascorbic acid, and 50 μg/L of sodium selenate..

Mouse Studies

NOD scid IL2 receptor gamma chain knockout (NSG) mice were purchased from The Jackson Laboratory and maintained at the University of Minnesota under specific pathogen-free conditions utilizing protocols approved by the Institutional Animal Care and Use Committee. Mice were treated and assessed at different harvest times as described.

Patients and Samples

Adult donor HCT and umbilical cord blood allogeneic transplant samples were utilized from previously described studies (7, 8) and a detailed description of patients is included in supplementary table I. Briefly, after transplant patients were monitored weekly for CMV reactivation by quantitative PCR performed in the clinical virology laboratory, defined as CMV viremia (> 100 copies). CMV reactivation occurred prior to day 100 after transplantation and was treated with an 8-week course of ganciclovir. PBMCs were collected at noted times for analysis based on the time after transplantation. Samples were obtained after informed consent and approval from the University of Minnesota Institutional Review Board according to the declaration of Helsinki.

Flow Cytometry

All fluorochrome-conjugated antibodies were purchased from Biolegend, BD Biosciences, and Beckman Coulter. Staining, acquisition, and analysis were performed as previously described (7).

Proliferation and Differentiation Assays

PBMCs were labeled with CellTrace Violet Cell Proliferation Dye (Invitrogen) per manufacturer’s protocol, and placed in culture medium supplemented with IL-15 (10 ng/mL (R&D Systems)) for 7 days followed by surface staining. For the differentiation assay, sorted CD56dimCD3−KIR− NK cells were placed in the same conditions and assessed for KIR acquisition.

Cell Death Assay

To evaluate viability, we utilized the LIVE/DEAD Fixable Dead Cell stain (Invitrogen) per manufacturer’s protocol followed by labeling with desired surface stains under conditions described. Cell death was assessed on CD56dimCD3− cells. To study initiation of apoptosis in live cells, a similar approach was taken, but Annexin V antibody was added to the surface mix and gating was done as defined.

Quantitative RT-PCR And Western Blot Analysis

For evaluation of transcripts and protein, pure populations of CD56dimCD3−KIR− or CD56dimCD3−KIR+ NK cells were isolated as stated. RNA was processed and analyzed as previously described (7). For immunoblots, all procedures were done as previously described (27). Bcl-xL antibody was purchased from Cell Signaling.

Transfection Studies

Magnetically enriched NK cells were placed in medium with IL-15 (1 ng/mL) for 24 hours and transduced with the Amaxa Human Macrophage Nucleofector Kit (Lonza) in a Nucleofector II (Amaxa Biosystems). For overexpression studies, the GFP of the pmax vector was replaced with the PCR product of IL2RA. For knockdown studies, we utilized 300 picomoles (total) of FlexiTube siRNA’s specific for IL2RA, FAS, ALLStars Negative Control siRNA, and the Alexa Fluor-488 labeled ALLStars Negative Control siRNA (QIAGEN). Five hours post transduction, the cells were re-plated with media plus IL-15 (1 ng/mL). After 24 hours (48 hours for FAS studies), cells were spun and resuspended in RPMI-1640 with 10% serum to simulate IL-15 withdrawal. Forty-eight hours later, cells were stained (or incubated with Killers in the case of the FAS studies) and cell numbers and cell death were assessed by flow cytometry.

Statistical Analysis

Paired t tests were used for comparisons maintained internal pairing within each donor sample. For comparisons across different donors or same donor harvested at different time points an unpaired t test with Welch’s correction was utilized. Multiple comparisons were adjusted by using the method of Hommel (28). All tests were two-sided. Statistical significance is indicated as NS: P>0.05; *P<0.05; **P<0.01; and ***P<0.001. On all graphs bars represent the mean ± SEM. Statistical analyses were carried out with Prism software and SAS 9.3 (SAS Institute, Cary, NC).

Results

The homeostatic balance between KIR+ and KIR− NK cells is not fully explained by proliferation or continuous differentiation

NK cell function is determined by the repertoire of individual NK cells displaying a variety of receptors. We chose to focus on the more mature CD56dim cells throughout this study. Our first aim was to distinguish between proliferation, differentiation, and survival as possibilities to maintain the functional NK cell repertoire. Proliferation was studied first by labeling PBMCs from healthy donors with a proliferation dye followed by culture with 10 ng/ml IL-15 for 7 days. Similar to previous studies (10), KIR− NK cells proliferate better than KIR+ NK cells (Figure 1A). Approximately twice as many KIR− NK cells proliferated past cell division 3 when compared to KIR+ NK cell subsets (Figure 1B). Although KIR− NK cells were significantly increased after 7 days in culture with IL-15 when compared to day 0 (Figure 1C), the increase in KIR− frequencies is lower than expected based on the proliferation data. This discrepancy may be a result of differentiation from KIR− into KIR+ NK cells. To test for this possibility, KIR− NK cells were sorted (Supplemental Figure 1A) and placed into the same culture conditions for 7 days. Although a small amount of KIR acquisition was observed (Figure 1D and 1E), this amount does not account for the frequency of KIR+ NK cells after IL-15 culture given the marked proliferative advantage of KIR− NK cells. To determine if the in vitro proliferation discrepancy impacted KIR balance in a more physiological setting, a xenogeneic mouse model was utilized to track proportions of KIR− and KIR+ NK cells after transfer (Figure 1F). In this setting proportions of KIR− NK cells did not increase when compared to the starting population (Figure 1G) despite the enhanced proliferation seen with IL-15 in vitro. This inconsistency between proliferation in vitro and homeostasis of KIR proportions in vivo indicated that a mechanism other than proliferation is operant in maintaining the balance between the KIR+ and KIR− populations.

Figure 1. In vitro proliferation and xenogeneic transplantation studies show a discrepancy in the proportion of KIRs expressed by NK cells.

Figure 1

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(A) Representative histograms of KIR subset proliferation (measured by CellTraceTM dilution) on NK cells (PBMCs were gated on CD56+CD3− NK cells and the indicated KIR subset) after 7 days in culture with 10 ng/ml IL-15. (B) Aggregate data showing the proliferation (past division 3) of NK cells (CD56+CD3−) stratified by KIR and CD57 expression as indicated (n = 17). (C) Proportion of KIR+ and KIR− NK cells at day 0 (open circles) and day 7 (filled circles) after in vitro incubation of PBMCs with 10 ng/ml of IL-15 (n = 10). (D) KIR− NK cells were sorted using a FACSAria II sorter and placed in culture with 10 ng/ml IL-15. After 7 days of differentiation, KIR+ NK cells were assessed by flow cytometry as shown in the representative histogram. (E) Aggregate data of the differentiation assay results (n = 4). (F) Schematic of transplant methodology. NSG mice are irradiated (with 275 cGy) prior to I.V. injection of 1 × 106 Human NK cells. Mice are then injected I.P. Monday, Wednesday and Friday with 5 ug recombinant IL-15 per mouse (6 doses total) over 2 weeks. No treatment (NT) was given in the final two weeks. Blood was collected at day 7 and day 28 for flow cytometry analysis of KIR expression (n = 4). (G) KIR expression is shown on human CD56dimCD3− NK cells Day 0 prior to infusion (open bars, 1 human donor), and after infusion (n = 4 NSG mice) on day 7 (shaded bars) and day 28 (black bars).

NK cell education determines NK cell survival

NK cell survival was studied using an in vitro serum starvation assay. A marked difference in cell death was observed between KIR+ (Figure 2A and 2B; 8.9 ± 1.2%, 6.7 ± 0.5%, and 6.2 ± 0.9%) and KIR− NK cells (Figure 2A and 2B; 33 ± 2.4%). To exclude possible confounding issues with receptor down-regulation in dead cells, NK cells were evaluated for early indications of apoptosis (CD56dimCD3−LIVE/DEAD dye−Annexin V+ cells; Supplemental Figure 1B). We found that the majority of the live NK cells entering apoptosis belonged to the KIR− subset (Figure 2C; 73.9 ± 1.6%). These data indicate that KIR− NK cells are more prone to undergo cell death under the stress of serum starvation.

Figure 2. Preferential survival of educated NK cells.

Figure 2

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(A) Representative histograms of cell death (measure by LIVE/DEAD dye incorporation) in CD56dimCD3− KIR subsets after overnight serum starvation (RPMI-0%). (B) Aggregate cell death (from figure 2A) and (C) early apoptosis (Annexin V+Live/Dead−) data in CD56dim NK cells stratified by KIR expression after serum starvation (n = 9 for both). (D) CD56dimCD3− NK cells were broken down into subsets based on their expression of KIR and NKG2A, and cell death was measured after serum starvation (n = 9). (E) Donor cells were KIR and HLA-typed to determine the NK cell educational status. NKG2A− single KIR+ NK cells were considered educated when corresponding cognate HLA-ligand was present for self KIR, while those lacking cognate ligand are uneducated (non-self KIR). Educated and uneducated NK cells were analyzed for cell death after serum starvation (n = 30). (F) The proportion of CD57+ vs. CD57− NK cells undergoing cell death after serum starvation (n = 9). (G) The proportion and (H) intensity of CD57 on self KIR educated vs. non-self KIR uneducated NK cells (n = 20). For education experiments, in order to achieve internal comparison per donor, all samples selected had at least one uneducated KIR.

The most functionally differentiated NK cells are those that express an inhibitory receptor with specificity for self-HLA molecules (29–31). Therefore, cell death was measured after overnight serum starvation in NK cell subsets based on the presence or absence of educating signals. In addition to education through KIR, NK cells can acquire function through CD94/NKG2A heterodimers (3). The KIR−NKG2A− NK cells, which lack all education signals, consistently exhibited more cell death compared to the KIR−NKG2A+, KIR+NKG2A+, and KIR+NKG2A− subsets (Figure 2D). It has been previously published that additive educating signals yield higher functionality (4). Consistent with that finding, cells educated through both NKG2A and KIR have better survival than those that receive signals through NKG2A or KIR alone. Although differences in survival were seen based on NKG2A and KIR staining in the absence of ligand determination, education needs to be determined based on the presence or absence of self-ligand in the environment. Therefore, donors were HLA- and KIR-typed to define educated [NKG2A− NK cells containing a single KIR corresponding to self ligand (self KIR)] and uneducated cells [NKG2A− NK cells containing a single KIR without ligand (non-self KIR)], and survival was assessed after overnight serum starvation. Educated NK cells survived better than uneducated NK cells (Figure 2E), indicating that signaling through inhibitory KIR is necessary for the enhanced survival. More differentiated CD57+ NK cells also survived better than CD57− NK cell after serum starvation (Figure 2F). As previously reported, there was a higher frequency of CD57 expression on KIR+ NK cells (Supplemental figure 2C). Educated NK cells displayed a higher proportion of CD57+ cells (Figure 2G) and more CD57 on a per cell basis (Figure 2H). Taken together, these data show that NK cell education enhances survival and CD57 expression.

KIR+ NK cells express more anti-apoptotic molecules

To understand differences in survival, an apoptosis PCR array was used to look at survival-related molecules in sorted CD56dimCD3−KIR+ vs. CD56dimCD3−KIR− NK subsets (supplemental Figure 1D). While no expression differences were seen in pro-apoptotic genes, the anti-apoptotic genes BCL2 and BCL2L1 [BCLXL] differed (data not shown). The increases in BCL2 and BCL2L1 transcripts in the KIR+ population when compared to the KIR− population were independently confirmed with standard Q-PCR (Figure 3A). Increased Bcl-xL protein expression was observed in KIR+ compared KIR− NK cells (Figure 3B). Bcl-2 protein expression was next analyzed by MFI (Median Fluorescence Intensity) on NK subsets under different settings. While no significant difference was observed under basal conditions, serum starvation or treatment with 10 ng/ml IL-15 overnight (Figure 3C and data not shown) induced a small increase in Bcl-2 expression on KIR+ NK cells. Differential elevation in Bcl-2 could also be seen on educated NK cells under basal, serum starving or IL-15 treatment conditions overnight (Supplemental Figure 2B–D). When evaluating proteins involved in cell death under basal conditions, Fas (CD95) was expressed at higher levels on KIR− than KIR+ NK cells (Figure 3D) and there was no differences for FasL (CD178) or Bim expression. These results indicate that KIR expression and education equips NK cells, under basal conditions, for survival with increased anti-apoptotic mechanisms and decreased expression of Fas.

Figure 3. KIR positive NK cells express more anti-apoptotic molecules and less Fas.

Figure 3

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NK cells were enriched using the Miltenyi human NK cell isolation kit and sorted into CD56dimKIR− and CD56dimKIR+ populations. (A) mRNA was isolated and expression of different anti-apoptotic genes (BCL2 and BCL2L1 (BCLXL)) was normalized to 18s ribosomal RNA. The ratio of KIR+/KIR− expression is shown (n = 5). (B) Representative western blot of Bcl-xL protein expression in KIR− vs. KIR+ NK cells (n = 6). (C) Bcl-2 protein expression (shown as Bcl-2 MFI) on NK cells was analyzed after PBMCs were incubated overnight with 10 ng/ml IL-15 (n = 9). (D) Fas expression (MFI) was analyzed on different subsets of NK cells under basal conditions (n = 9).

Cytokine receptor levels correlate with KIR expression and education

In order to investigate differences in cytokine receptor signaling between the KIR+ and KIR− subsets, we examined the expression of IL-15Rα [IL15RA (CD215)] and IL-2Rα [IL2RA (CD25)] and of common signaling components utilized in both pathways: the common γ-chain [IL2RG (CD132)] and IL-2Rβ [IL2RB (CD122)]. Small and variable differences were found in all components (Supplemental Figure 2A). No difference in IL-15Rα or IL-2Rβ protein expression was found between KIR+ and KIR− subsets at basal levels (Figure 4A and 4B). KIR+ NK cells expressed more common γ-chain (Figure 4C). In contrast, decreased IL-2Rα protein was observed in the KIR+ NK cells when compared to the KIR− NK cells (Figure 4D). Educated NK cells expressed more common γ-chain, but no differences were seen for IL-2Rα under basal conditions (Supplemental Figure 2F and data not shown).

Figure 4. Differential IL-2 cytokine receptor component expression on educated NK cells.

Figure 4

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Healthy donor PBMCs were gated on CD56dimCD3− NK cells and KIR subsets were analyzed for protein expression (MFI) of (A) IL-15 receptor alpha (n = 9), (B) IL-2 receptor beta (n =7), (C) the common-gamma chain (n =15), and (D) IL-2 receptor alpha (n = 7). (E) IL-2 receptor alpha MFI on NK cells stratified by KIR expression (n=9) or (F) NK cell education (n=18) after 24 (left panel) or 72 (right panel) hour treatment with IL-15 (1 ng/ml). For education experiments, in order to achieve internal comparison per donor, all samples selected had at least one uneducated KIR.

Since IL-15 signaling on NK cells can alter IL-2Rα levels (32), we set out to investigate if it could differentially regulate IL-2Rα expression on NK cells based KIR and education. PMBCs were treated for 24 or 72 hours with IL-15 and then assessed for IL-2Rα levels on different NK cell subsets. KIR− NK cells had 1.26 fold more IL-2Rα than KIR+ NK cells after 24 hours of treatment and the difference increased to 1.45 fold after 72 hours (Figure 4E). At 24 hours, no significant differences in IL-2Rα levels could be noted based on education, but a 3 fold increase was seen on uneducated NK cells after 72 hours of treatment, while no increase occurred on educated NK cells (Figure 4F). These results indicate that NK cell education yields differential regulation of cytokine receptor genes that might be involved in survival.

IL-15 withdrawal promotes cell death on IL-2Rαhi uneducated NK cells through differential Bim and FasL expression

Our results suggest that NK cell education influences IL-2Rα expression, but the role of IL-2Rα on survival remains unclear. To address this point in a more physiologic setting, we used a cytokine withdrawal assay to model the contraction phase after virus infection in which antigen depletion results in decreased inflammation. NK cells were treated for 48 hours with 1 ng/ml IL-15 and then IL-15 was withdrawn for 48 hours to look at cell death. IL-15 treatment followed by withdrawal yielded a 1.45 fold differential in IL-2Rα on uneducated NK cells when compared to their educated counterparts (Figure 5A). The amount of cell death paralleled the fold difference seen in IL-2Rα expression (Figure 5B: 1.44 fold increase in cell death on uneducated NK cells). To test if IL-2Rα expression itself directly correlated with cell death, we gated on IL-2Rαlo and IL-2Rαhi NK cell populations and evaluated cell death post IL-15 withdrawal. IL-2Rαhi NK cell death was 1.7 fold higher than IL-2Rαlo NK cell death (Figure 5C), establishing a definitive link between IL-2Rα expression and cell death.

Figure 5. IL-15 withdrawal leads to a disparity in survival proteins on educated versus uneducated NK cells that correlates with IL-2R alpha expression.

Figure 5

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Healthy donor PBMCs were cultured with 1 ng/ml (A–G) or 10 ng/ml (H–K) IL-15 for 48 hours, cytokine was washed away and then they were cultured in RPMI-10% (serum) without cytokine for another 48 hours before analysis. (A) IL-2 receptor alpha expression and (B) cell death on self KIR educated versus non-self KIR uneducated NK cell subsets after 1 ng/ml IL-15 withdrawal (n = 18). (C) Percentage of NK cell death post 1 ng/ml IL-15 withdrawal based on IL-2 receptor alpha (IL-2RA) expression (n = 10). (D & H) Bim expression based on NK cell education after 1 and 10 ng/ml IL-15 withdrawal (n = 18). (E & I) Bim expression based on NK cell IL-2 receptor alpha expression after 1 and 10 ng/ml IL-15 withdrawal (n = 10). (F & J) Fas expression based on NK cell education after 1 and 10 ng/ml IL-15 withdrawal (n = 18). (G & K) FasL expression based on NK cell education after 1 and 10 ng/ml IL-15 withdrawal (n = 18). For education experiments, in order to achieve internal comparison per donor all samples selected had at least one uneducated KIR.

Next we asked what molecules could be involved in this process. Although Bcl-2 is found at slightly higher levels on educated NK cells early on (Supplemental figure 2B–D), under IL-15 withdrawal conditions no change was seen (Supplemental figure 2E). Previous publications indicate that NK cell withdrawal from cytokines results in pro-apoptotic Bim upregulation (16, 33). Withdrawal from a 1 ng/ml IL-15 treatment yields a 1.2 fold increase of Bim on uneducated (Figure 5D) and IL-2Rαhi (Figure 5E) NK cells. Withdrawal from a 10 ng/ml IL-15 treatment further augments the Bim differential to 1.4 fold and 1.3 fold increases on uneducated (Figure 5H) and IL-2Rαhi (Figure 5I) NK cells, respectively. Differences can be further augmented by longer initial IL-15 incubation periods followed by withdrawal from both IL-15 and serum (Supplemental figure 3A–C). While withdrawal from 1 ng/ml IL-15 resulted on higher Fas on uneducated NK cells (Figure 5F), initial incubation with 10 ng/ml IL-15 negated this difference (Figure 5J). Surprisingly, educated NK cells expressed more FasL when IL-15 was withdrawn (Figure 5G and 5K; 1.4 fold and 1.5 fold respectively). This data indicates that when cytokine is limiting, NK cell education and absence of IL-2Rα expression is advantageous for cell survival through decreased Bim and Fas expression. Furthermore, educated NK cells might actively kill uneducated NK cells through FasL when IL-15 is scarce, perhaps to compete for cytokine.

IL-2Rα directly impacts survival through FasL mediated by NK-NK interactions

To test if IL-2Rα had a direct role in NK cell survival, NK cells were purified and IL-2Rα was knocked down or overexpressed in the IL-15 withdrawal setting (Figure 6A). Although IL-2 signaling can enhance AICD there was no IL-2 or crosslinking signals present in these cultures. Therefore, cell death in this setting is independent of IL-2 signaling and must be attributed to another mechanism. The majority of the cells expressed siRNAs on the day harvested (Supplemental figure 3D). Knockdown of IL-2Rα (siIL2RA) lead to a 1.5 fold increase in NK cell numbers (Figure 6B) when compared to the control (siC). Similarly, the control had 1.5 fold more cells than the samples in which IL-2Rα was overexpressed (oIL2RA). To ensure that the changes were due to differential cell death (and not proliferation), the proportion of dying cells was evaluated. A 1.4 fold decrease in cell death was observed in the IL-2Rα knockdown when compared to the control (Figure 6C). The data indicate that the mechanism, at least in part, for differential survival of NK cells is controlled by the amount of IL-2Rα expressed, independent of IL-2 signaling and AICD.

Figure 6. IL-2RA expression determines NK cell survival and renders IL-2RAHi NK cells sensitive to FasL mediated “attack” from KIR+ NK cells.

Figure 6

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NK cells were enriched by bead separation and placed in 1 ng/ml IL-15 overnight. The next day they were transduced with a negative control siRNA containing an Alexa Fluor 488 tag (siC), a set of 4 siRNA’s specific for human IL-2 receptor alpha (siIL2RA), or a plasmid to overexpress IL-2 receptor alpha (oIL2RA). After transduction, the cells were kept in 1 ng/ml IL-15 for another 24 hours. IL-15 was then withdrawn for the next 48hrs and cells were analyzed using flow cytometry. (A) Representative histograms of transduction efficiency showing IL-2 receptor alpha levels in the control (siC; shaded line), the knockdown (siIL2RA; solid line), and the overexpression vector (oIL2RA; dashed line). (B) Events were collected for 60 seconds at the same rate for each sample and the total number of NK cells collected during this period was calculated by gating on live CD56+CD3− NK cells. Aggregate data is presented for each of the three treatment groups (n = 4). (C) The proportion of dead NK cells, 3 days post transduction, was measured by looking at LIVE/DEAD dye incorporation in CD56+CD3− NK cells. Aggregate data for each of the three treatment groups is shown (n = 4). (D) Representative dot plots showing IL-2 receptor alpha expression in mock-transduced (left) or IL-2 receptor alpha transduced (center) KIR+ vs. KIR− NK cells. The dot plot on right shows eGFP levels in a representative control eGFP transduction in KIR+ vs. KIR− NK cells. Large bolded numbers show proportion of expression (IL-2 receptor alpha (left and center) or eGFP (right)) in the positive population of KIR+ or KIR− NK cells. (E) Quantification of differential expression of overexpressed proteins in KIR+ vs. KIR− NK cells. The graph shows the ratio (KIR+/KIR−) of protein expression after transduction where a value of 1 indicates equal protein expression in both populations (n = 4). (F) Schematic for FasL NK-NK killing experiment. NK cells were enriched from healthy donor PBMCs, and for each donor cells were either frozen for use as “targets” or bead separated into KIR+ and KIR− NK cells for use as “killers”. Killers were treated with 10 ng/ml IL-15 for 72 hours (in RPMI-10% serum) and then IL-15 was withdrawn for 48 hours by changing media to RPMI-10 to drive FasL expression on killers. Killers were then labeled with CellTrace and co-cultured with NK targets from the same donor in the presence or absence of FasL blocking antibody (NOK-1, BioLegend). After 24 hours of co-culture, cells were harvested and stained. For analysis, killers (CellTrace+ NK cells) were excluded and the proportion of live cells entering apoptosis (Live/Dead dye−/Annexin V+) was evaluated on IL-2RAlo and IL-2RAhi targets by flow cytometry. (G) Analysis of Annexin V expression on targets after 24 hour incubation with killers, with or without FasL-blocking antibody (n = 4).

Overexpression of IL-2Rα was not as efficient in the KIR+ NK cells as it was in the KIR− NK cells (Figure 6D: center panel). To ensure that this was not due to a differential transduction of KIR+ vs. KIR− NK cells, we transduced cells with the same construct expressing eGFP alone. There were no significant differences in transduction efficiency between KIR+ and KIR− cells excluding this possibility (Figure 6D: right panel), and leading to the conclusion that there is an active mechanism for modulation of IL-2Rα expression in KIR+ NK cells despite forced expression (Figure 6E).

To connect the role of IL-2Rα in sensitizing NK cells to death with the finding that educated NK cells upregulate FasL when IL-15 is limiting, KIR+ containing educated NK cells and KIR− containing uneducated NK cells were purified to test their ability to kill NK cells (Figure 6F). Donor NK cells were saved as “targets” before separation into KIR+ or KIR− “killers”. Killers were subjected to IL-15 withdrawal, to induce FasL expression, and labeled, to allow for exclusion during analysis, and incubated overnight with NK targets from the same donor with or without FasL antibody blockade. As expected, IL-2Rαhi targets were more sensitive to apoptosis than IL-2Rαlo targets (Figure 6G). Death in IL-2Rαlo targets remained low regardless of treatment. In contrast, IL-2Rαhi targets were more sensitive (by about 1.65 fold) to apoptosis when incubated with KIR+ containing educated NK cell killers than with KIR− NK cells. This fold difference is similar to the difference in apoptosis in IL-2Rαhi targets when comparing NK targets alone versus NK targets incubated with KIR+ killers overnight (Supplemental Figure 3E). The IL-2Rαhi target sensitivity to apoptosis was decreased when FasL was blocked on KIR+ killers, but no changes were seen when FasL was blocked on KIR− killers. Knockdown of Fas on NK cell targets (Supplemental Figure 3F) similarly decreased the ability of KIR+ killers to induce apoptosis on targets (Supplemental Figure 3G), confirming involvement of this pathway in NK cell fratricide. These results indicate that populations enriched with educated NK cells induce cell death autologously on IL-2Rαhi NK cells when IL-15 is limiting, partially through FasL signaling.

NK cell survival is enhanced after CMV reactivation

We wanted to investigate if survival of educated NK cells is enhanced under pathologic settings known to activate NK cells. We have shown that CMV reactivation after transplant results in a clonal expansion of educated NK cells that are maintained long-term after transplant, suggesting NK cell memory (7, 8). In an adult donor allogeneic hematopoietic transplantation setting, expression of KIR (Figure 7A) or CD57 (Figure 7B) on NK cells, both markers of maturity, resulted in diminished cell death at all time points after transplant. We hypothesized that CMV reactivation would result in selection of educated of NK cells through better survival. In an adult allogeneic transplantation setting where 50% of normal adult donors may have had prior exposure capable of passing on memory NK cells, CMV reactivation resulted in better NK cell survival, which was statistically significant at 6 months after transplantation (Figure 7C). To differentiate between primary and secondary CMV activation, we used umbilical cord blood transplantation where donor units are CMV naïve and activation represents primary infection in the recipient of the newly developing donor NK cells. CMV activation before day 100 after umbilical cord transplantation protected day 100 post transplantation NK cells from cell death (Figure 7D). These results indicate that enhanced survival of more differentiated NK cells after primary and secondary CMV activation promotes the long-lived repertoire of functional NK cells seen in this setting (7, 8).

Figure 7. Increased survival properties of NK cells can be seen post transplantation and NK cell survival is accentuated by CMV reactivation.

Figure 7

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NK cell survival was measured based on (A) KIR or (B) CD57 expression on NK cells from adult donor allogeneic transplant patients at day 28 (n = 28), day 100 (n = 25), and 6 months (n = 27) post transplant. (C) NK cell survival was segregated by CMV reactivation (CMVR) in adult donor allogeneic transplantation patients (no reactivation=CMVR- (filled circles): day 28 (n = 19), day 100 (n = 18), and 6 months (n = 19); CMV reactivation=CMVR+ (empty circles): day 28 (n = 9), day 100 or 8 weeks post viral diagnosis (n = 7), and 6 months (n = 8). (D) NK cell survival at a 100 days (CMV- (n = 12)) or 8 weeks post viral diagnosis (CMV+ (n = 11)) after umbilical cord allogeneic transplant.

Discussion

We propose a model of long-term NK cell homeostasis determined largely by NK cell survival mechanisms. In this model, proliferation and differentiation form the functional NK cell repertoire, while enhanced survival by interaction with self MHC maintains these educated NK cells. This process is tunable and additive signals through NKG2A and KIR enhance survival, much like what is seen in terms of function (4, 29–31). Sensitivity to cell death is linked to reduced Bim expression, in agreement with another report (16), and modest increases in anti-apoptotic proteins Bcl-2 and Bcl-xL. Survival is negatively modulated by IL-2Rα expression, and educated NK cells are less susceptible to Fas-driven AICD. Educated NK cells have the capacity to dominate over their hyporesponsive counterparts by inducing death of IL-2Rαhi NK cells, which are enriched in uneducated NK cell populations, through FasL when IL-15 is limiting. These data show that educated NK cells persist longer and control the survival of uneducated cells, a mechanism we refer to as NK cell probation. The rapidly proliferating uneducated NK cells are in a “probationary” phase marked by high expression of IL-2Rα and Fas, while educated NK cells expressing FasL are the “probation officers” that remove hyporesponsive NK cells. In this context, NK cell education is required for acquisition of function while NK cell probation shapes the repertoire by preferentially maintaining more functional cells and allowing clonal dominance by direct NK-NK interactions.

NK cell probation could be explained by activation of the PI3K/Akt pathway through cross-linking of KIR on NK3.3 cells (12). However, this data is at odds with murine SHIP−/− data, which is activated downstream of Ly49 in mice and analogous to SHP-1 in humans, showing that SHIP opposes PI3K-mediated Akt activation in mice (34, 35). A more recent study showed that dose dependent escalation of cognate MHC signals yielded increased selection of educated NK cells (16), consistent with the probation hypothesis.

Cytokine receptor balance plays a key role in probation, with IL-2Rα expression leading to increased cell death, while the common-gamma chain is associated with enhanced survival. The lack of change observed in IL-15Rα on NK cells might be explained by the fact that this protein is thought to be trans-presented (36, 37). Given these results, we hypothesize that IL-2Rα might directly influence survival by competing for common signaling components with trans-presented IL-15Rα, whose signals yield greater survival (18, 19, 38). This hypothesis is supported by the fact that cell death and Bim expression correlate well with IL-2Rα levels after withdrawal and the finding that manipulation of IL-2Rα directly influences survival in an IL-2 free system, but more experiments are needed to fully prove the mechanism. The concept is that IL-2Rαhi NK cells are sensitized to pro-apoptotic signals, particularly FasL, presented by more educated NK cells during times of stress.

These findings have important clinical implications in NK cell mediated immunotherapies (1, 2). In both T and NK cell mediated cell therapy, efficacy seems to correlate best with the persistence of lymphocytes after adoptive transfer (39, 40). For NK cells, survival could be modulated by targeting expression of IL-2Rα through lentiviral knockdown in the NK cell infusion product or manipulating IL-2 signaling negative feedback loops (32) by treating with IL-2, perhaps following pharmacologic “inflammation” using IL-15, to reduce the effect of IL-2Rα on NK cells in both infusion and transplant patients. In addition, blocking Fas/FasL interactions during the NK cell expansion stage during transplants could lead to increased NK cell reconstitution. Conversely, maximal NK cell functionality in infusion products could also be achieved by treating NK cells with multiple rounds of IL-15 followed by IL-15 withdrawal, resulting in an enriched population of educated NK cells. Finally, the concept of NK cell memory has relevance for the longevity of cell-based immunotherapy, as well as the observation that memory cells exhibit enhanced function (7–9). We show here that CMV reactivation results in better survival of NK cells. In some settings, this correlates with protection from leukemia relapse (41, 42). Gaining a better understanding of how survival is mediated will help enhance future use of NK memory products for immunotherapy. In conclusion, this study presents novel findings showing that NK cell differentiation and education have an important role in NK cell survival through the process of education/probation, which is mediated by IL-2Rα expression, Bim, minor changes in anti-apoptotic molecules, and “predatory” FasL expression when cytokine is limiting.

Supplementary Material

1

Acknowledgments

This project has been funded in part with federal funds from the National Cancer Institute (NCI), NIH, under contract HHSN261200800001E, 2T32HL007062-36, CA111412 and CA65493. This research was supported by the Intramural Research Program of the NIH, NCI, Center for Cancer Research.

We thank the Tolar lab for assistance with the mouse experiments.

Footnotes

The authors declare no competing financial interests.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

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