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. 2016 Jul 8;65(9):1047–1059. doi: 10.1007/s00262-016-1864-z

Human NK cells maintain licensing status and are subject to killer immunoglobulin-like receptor (KIR) and KIR-ligand inhibition following ex vivo expansion

Wei Wang 1, Amy K Erbe 1, Kory A Alderson 1, Emily Phillips 1, Mikayla Gallenberger 1, Jacek Gan 1, Dario Campana 2, Jacquelyn A Hank 1, Paul M Sondel 1,3,✉
PMCID: PMC5477646  NIHMSID: NIHMS865312  PMID: 27392940

Abstract

Infusion of allogeneic NK cells is a potential immunotherapy for both hematopoietic malignancies and solid tumors. Interactions between killer immunoglobulin-like receptors (KIR) on human NK cells and KIR-ligands on tumor cells influence the magnitude of NK function. To obtain sufficient numbers of activated NK cells for infusion, one potent method uses cells from the K562 human erythroleukemia line that have been transfected to express activating 41BB ligand (41BBL) and membrane-bound interleukin 15 (mbIL15). The functional importance of KIRs on ex vivo expanded NK cells has not been studied in detail. We found that after a 12-day co-culture with K562-mbIL15-41BBL cells, expanded NK cells maintained inhibition specificity and prior in vivo licensing status determined by KIR/KIR-ligand interactions. Addition of an anti-CD20 antibody (rituximab) induced NK-mediated antibody-dependent cellular cytotoxicity and augmented killing of CD20+ target cells. However, partial inhibition induced by KIR/KIR-ligand interactions persisted. Finally, we found that extended co-cultures of NK cells with stimulatory cells transduced to express various KIR-ligands modified both the inhibitory and activating KIR repertoires of the expanded NK cell product. These studies demonstrate that the licensing interactions known to occur during NK ontogeny also influence NK cell function following NK expansion ex vivo with HLA-null stimulatory cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s00262-016-1864-z) contains supplementary material, which is available to authorized users.

Keywords: Ex vivo expansion, NK cells, Killer immunoglobulin-like receptors, HLA, ADCC

Introduction

Ex vivo expanded and activated NK cells for adoptive transfer is an immunotherapeutic option for cancer, mainly due to the augmented cytotoxic capabilities of these expanded cells [1–5]. Ongoing clinical efforts are investigating adoptive transfer of autologous and allogeneic NK cells following IL-2 stimulation or ex vivo expansion [6–9], with some early examples of potential clinical benefit [10, 11]. Due to the limited number of NK cells in peripheral blood, ex vivo expansion can serve to generate a better source of NK cells. In addition to the potential for generating a several hundred-fold increase in NK cell numbers, ex vivo expanded NK cells demonstrate augmented expression of activating receptors, such as NKG2D and natural cytotoxicity receptors (NCRs), as well as increased tumor cytolytic activity as compared to non-expanded NK cells [1, 2].

The killer immunoglobulin-like receptors (KIRs) are an important family of receptors on human NK cells. There are fifteen KIR genes and two pseudogenes on human chromosome 19 [12]. Individuals inherit widely different KIR genes, and the KIR haplotypes are highly diverse [13]. Moreover, within a single individual, the protein products of the KIR genes are expressed clonally and somewhat randomly on NK cell surfaces, with some NK cells only expressing the product of a single KIR gene and other NK cells expressing the products of two or many more KIR genes [14].

Interactions between KIRs and KIR-ligands, together with other receptors on NK cells, regulate NK cell education and activation. For some KIRs, especially the inhibitory KIRs (iKIRs), well-defined ligands are known; however, the ligands for some inhibitory and many activating KIRs (aKIRs) are still unidentified [12]. Some of the known KIR-ligands include those of the major histocompatibility complex (MHC) class I molecules, including HLA-C1 (C1), HLA-C2 (C2), and HLA-Bw4 (Bw4). Inhibitory KIR2DL1 recognizes HLA-C alleles with a lysine at amino acid position 80 (HLA-C2), and iKIRs 2DL2 and 2DL3 recognize HLA-C alleles with an asparagine at amino acid position 80 (HLA-C1) [15, 16]. Several groups have shown that iKIRs 2DL2 and 2DL3 may also cross-recognize HLA-C2 alleles, but not to the same degree as they recognize HLA-C1, or that iKIR2DL1 interacts with HLA-C2 [17, 18]. Inhibitory KIR3DL1 recognizes HLA-A and HLA-B alleles with a Bw4 motif within amino acid position 77–80 [19]. Finally, aKIR2DS1 shares the same ligand (C2) as the iKIR2DL1, but aKIR2DS1 has a lower binding affinity to this ligand [20].

NK cells that express self-iKIRs (i.e., iKIRs that recognize the HLA expressed on normal tissues of the same individual) become “licensed” during their development [21]. NK cells are unlicensed when they do not express any iKIR, or only express non-self-iKIRs (i.e., iKIRs that do not recognize HLA expressed on normal tissues of the same individual). Once mature, licensed NK cells demonstrate augmented cytotoxic function (compared to unlicensed NK cells) against HLA-null tumor targets (or tumor cells that have downregulated their HLA expression) [22]. The differences between licensed and unlicensed NK cells have been intensively studied using freshly isolated NK cells; HLA-null tumor targets have been primarily used to differentiate the activation and cytotoxic capabilities of licensed versus unlicensed NK cells [21, 23, 24]. However, tumor cells in patients usually show some degree of HLA expression. Thus, interactions between KIR repertoire on NK cells and KIR-ligand repertoire on tumor cells, including licensing, play a considerable role in anti-tumor effects for immunotherapies that involve NK cells, in both the autologous and allogeneic settings [23, 25–29]. However, whether ex vivo expansion and activation, combined with tumor antigen-specific mAb, could mitigate inhibition from KIR/KIR-ligand interactions or alter NK cell licensing status needs further investigation.

Several different pathways were reported to contribute to NK-mediated tumor lysis, including degranulation of perforin- and granzyme-containing particles, cytokine release, and induction of tumor apoptosis by Fas-L and TRAIL interactions [30]. Among those mechanisms, degranulation and cytokine release have become important markers of NK cell activation and correlate with target cell lysis [29, 31]. Here, we use the NK cell degranulation marker, CD107a, to analyze the differences between the activation of licensed and unlicensed NK cells within the same donor, as well as between donors with various iKIR/KIR-ligand genotypes. Three different iKIR/KIR-ligand pairs (2DL1 with its ligand C2; 2DL2 and 2DL3 with their primary ligand C1; and 3DL1 with its ligand Bw4) have been the major focus for the licensing and inhibition interactions that involve iKIR [23, 28, 29]. In this study, we showed that ex vivo expanded NK cells maintained iKIR/KIR-ligand recognition specificities and prior licensing status. We also demonstrated that certain KIR/KIR-ligand interactions during the ex vivo expansion modify the inhibitory and activating KIR repertoire of the expanded NK cells.

Materials and methods

Cell lines and media

The K562-mbIL15-41BBL cell line was developed at St. Jude Children’s Research Hospital [32]. The 721.221 EBV transformed B cell line was a gift from Dr. Robert DeMars (University of Wisconsin–Madison) [33]. Both cell lines are maintained in RPMI with 10 % FBS, 2 mM L-glutamine, and penicillin/streptomycin.

Plasmid and transfection

HLA alleles were cloned into pcDNA3.0 (a gift from Dr. Deric Wheeler, University of Wisconsin–Madison) to generate HLA-C1 (C0801)-, HLA-C2 (C0401)-, and HLA-Bw4 (B4402)-carrying plasmids. All three plasmids, as well as an empty vector pcDNA3.0, were separately transfected into the 721.221 cell line by nucleofection (Mirus and AMAXA). Transfected 721.221 cells were then selected with 1700 mcg/mL geneticin (Teknova), and stable expressing cells were sorted for HLA expression using FACS. Sorted cells were maintained in RPMI with 1700 mcg/mL geneticin. CD20 expression and HLA expression levels of transfected variants are shown in Supplemental Figure 1.

KIR and HLA genotyping

DNA from healthy donors (Table 1) was isolated from buccal swab or whole blood. KIR genes were genotyped using a SYBR green real-time PCR method [34]. KIR-ligand genes were genotyped by a sequence-specific primer PCR method (Olerup).

Table 1.

Healthy donor KIR and KIR-ligand genotypes

Sample ID KIR 2DL1 KIR 2DL2 KIR 2DL3 KIR 3DL1 HLA-Ca HLA-Bw4 KIR haplotype
4 + + + + C1/C1 – B/x
8 + + + + C1/C2 + B/x
12 + + + – C1/C1 – B/x
14 + + + + C2/C2 + B/x
18 + – + + C2/C2 – A/A
23 + + – + C1/C2 + B/x
37 + – + + C1/C1 + B/x
38 + – + + C1/C1 – A/A
40 + – + + C1/C2 + A/A
44 + – + + C2/C2 + A/A
47 + – + + C2/C2 – A/A
48 + + + – C2/C2 + B/x
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aSince HLA-C1 and HLA-C2 are the two main supersets of alleles for HLA-C, all individuals should be either C1/C1, C1/C2, or C2/C2

Ex vivo expansion of NK cells

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor whole blood by Ficoll density gradient centrifugation. K562-mbIL15-41BBL cells were irradiated with 100 Gy and then mixed with 5 million PBMCs at a 1:1 ratio in 10 mL RPMI, with 100 IU/mL IL2. Mixtures of PBMCs and K562-mbIL15-41BBL cells were incubated at 37° on a rocker at a constant speed in an incubator. Fresh RPMI with IL2 was added every 2–3 days to the culture. All cells in the expansion cultures were harvested between expansion days 12–15 for CD3 and CD56 staining to determine NK cell percentage followed by functional analyses in the degranulation assay.

In separate experiments, 721.221 HLA variants were irradiated with 100 Gy and cultured together with fresh PBMCs at a 1:1 ratio for expansion, under the same conditions as described above for expansion with K562-mbIL15-41BBL cells with the addition of 1 ng/mL IL15. Expansion cultures were harvested between day 10 and 14 to determine inhibitory and activating KIR frequencies in the NK cells in the final product.

NK cell degranulation assay

Expanded NK cells were stimulated with 721.221 targets at a 1:1 ratio in 96-well round-bottom plates at 37°, in the presence of anti-CD107a antibody, with or without addition of rituximab. The lowest rituximab dose able to induce plateau-level CD107a expression of expanded NK cells stimulated with HLA-null 721.221 cells (E/T ratio = 1:1) was selected (0.5 μg/mL). After 1 h of incubation with 721.221 targets, protein transport inhibitor cocktail (500×, eBioscience) was added into the culture. After another 3 h of incubation, cells were washed, stained with live/dead Aqua dye (Biolegend) and various mAbs, then analyzed on a MACSQuant cytometer (Miltenyi) to determine the percentage of CD107+ NK cells in NK subpopulations defined by KIR staining [28, 29]. Flow cytometry data were analyzed using FlowJo software (Ashland OR).

In order to compare the relative amount of CD107 expression between different NK donors, as well as NK expansions and stimulations performed on different days, data for all donors were normalized as follows. For each individual NK expansion culture, the percent of CD107+ NK cells from each NK cell subset being evaluated (i.e., stimulation with HLA-C1-, HLA-C2-, or HLA-Bw4-expressing 721.221 variants, with or without addition of rituximab) was divided by the percent of CD107+ cells found in the iKIR-null NK cell subset stimulated by HLA-null 721.221 cells without rituximab from the same experiment (the same expansion from the same donor).

Staining mAbs for flow cytometry

The following reagents were used for flow cytometry: PE/Cy7 anti-CD3 (clone HIT3a, Biolegend), APC anti-CD56 (clone HCD56, Biolegend), PE anti-CD107a (clone H4A3, eBioscience), FITC anti-2DL1 (clone REA 284, Miltenyi), PerCP anti-2DL2/DL3 (clone DX27, Miltenyi), BV421 anti-3DL1 (clone DX9, Biolegend), Aqua fixable viability dye (Biolegend), BV605 anti-CD3 (clone UCHT1, Biolegend), PE anti-2DL1/DS1 (clone 11PB6, Miltenyi).

Cytotoxicity assay

Target cells were labeled with 51Cr for 1.5 h and mixed with expanded NK cells at various ratios (1:1, 2:1, 4:1) in 96-well plates for 4 h at 37°. Supernatants were harvested at the end of incubation and read by a gamma counter. Percent lysis of target cells was calculated as 100 × (treatment-spontaneous)/(maximum-spontaneous).

Statistical analyses

Statistical analyses were performed with GraphPad Prism 5.0, using the Student’s t test, one-way ANOVA, or two-way ANOVA as indicated within the figure legends. Standard errors are indicated as error bars for each comparison in figures.

Results

Selective NK cell outgrowth following ex vivo culturing of PBMCs with K562-mbIL15-41BBL cells

After 12 days of culturing PBMCs with irradiated K562-mbIL15-41BBL cells, NK cells were the major population identified to have expanded, with a purity between 70 and 95 % (Supplemental Figure 2a), and NK cell numbers increased by 150–250-fold (data not shown). This expansion and enrichment of NK cells in the final product enabled us to study rare NK cell subsets.

The majority of expanded NK cells showed a CD56high phenotype and similar CD16 expression level as compared to pre-expanded NK cells from the same donor (Supplemental Figure 2a). In addition, when we separated CD56highCD16neg and CD56lowCD16bright NK cells from PBMC (by flow sorting) and expanded them separately, the CD56lowCD16bright NK cells survived and expanded approximately ~ 100-fold, which is comparable to the level of expansion seen with bulk PBMCs. These CD56low cells became CD56high after expansion, while the CD56brightCD16neg population showed virtually no viable cell recovery after attempted ex vivo expansion under these conditions (data not shown). Therefore, it is primarily the CD56lowCD16bright NK cells that expanded ex vivo with engineered K562 cells.

Based on the expression of three iKIRs on these CD3−CD56+ NK cells, eight different NK cell subsets could be gated by flow cytometry analysis: 2DL1 single positive (2DL1sp), 2DL2/3 single positive (2DL2/3sp), 3DL1 single positive (3DL1sp), 2DL1 and 2DL2/3 double positive (2DL1+2DL2/3+), 2DL1 and 3DL1 double positive (2DL1+3DL1+), 2DL2/3 and 3DL1 double positive (2DL2/3+3DL1+), 2DL1 and 2DL2/3 and 3DL1 triple positive (2DL1+2DL2/3+3DL1+), iKIR negative (iKIR-null). For example, 2DL1sp NK cells were gated by first gating on the 2DL1+CD3−CD56+ NK cell subpopulation and then gating out both 2DL2/3- and 3DL1-expressing cells (Fig. 1a).

Fig. 1.

Fig. 1

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Inhibitory KIR frequencies are not affected by ex vivo expansion with K562-mbIL15-41BBL cells. PBMCs from healthy donors were expanded using K562-mbIL15-41BBL cells. a Cells were pre-gated by analyses of side scatter (SSC-A), forward scatter (FSC-A), live/dead, CD3 status and CD56 status in order to identify, sequentially (as shown by arrows in the panels on the left) the identification of: lymphocytes, live cells, singlets, and NK cells for KIR analyses. These CD3−CD56+ NK cells were then evaluated with mAbs specific for 2DL1, 2DL2/3, or 3DL1. KIR single-positive cells were then gated by gating out the CD3−CD56+ cells that express the two other iKIRs, as shown for identification of 2DL1sp NK cells in the right panels. A similar process was used to identify the eight separate NK cell subpopulations discussed in the text and shown in Fig. 2c. b The percentages of 2DL2/3+ cells in pre-expanded CD3−CD56+ cells were compared to the percentages of 2DL2/3+ cells in post-expanded NK cells for C1/C1 (n = 4), C1/C2 (n = 3), and C2/C2 (n = 5) donors, respectively. The percentages of 2DL2/3+ cells from C1/C1 (n = 4), C1/C2 (n = 3), and C2/C2 (n = 5) donors were also compared to each other. Each line represents an individual healthy donor. c The percentages of 3DL1+ cells in pre-expanded CD3−CD56+ cells were compared to the percentages of 3DL1+ cells in post-expanded CD3−CD56+ cells for Bw4+ (n = 6) and Bw4− (n = 4) donors, respectively. The percentages of 3DL1+ cells from Bw4+ donors (n = 6) were also compared to that from Bw4− donors (n = 4), pre- and post-expansion, respectively. d The percentages of 2DL1+ cells in pre-expanded CD3−CD56+ cells were compared to the percentages of 2DL1+ cells in post-expanded CD3−CD56+ cells for C1/C1 (n = 4), C1/C2 (n = 3), and C2/C2 (n = 5) donors, respectively. The percentages of 2DL1+ cells from C1/C1, C1/C2, and C2/C2 donors were also compared to each other. e The percentages of 2DL1+ cells in pre-expanded CD3−CD56+ cells were compared to the percentages of 2DL1+ cells in post-expanded CD3−CD56+ cells for 2DL2−2DL3+ (n = 6) and 2DL2+2DL3+ (n = 5), respectively. The percentages of 2DL1+ cells from 2DL2−2DL3+ (n = 6) donors were also compared to that from 2DL2+2DL3+ (n = 5) donors. Two-way ANOVA was used to compare KIR frequencies in NK cells expanded from donors with different genotypes or in NK cells pre- and post-expanded from donors with the same genotype. ns not significant, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

The distribution of iKIRs on NK cells is not affected by ex vivo expansion with K562-mbIL15-41BBL cells

We next evaluated whether distribution of specific KIRs on NK cells is affected by ex vivo expansion. There were no significant differences in the relative frequencies of KIR2DL1, KIR2DL2/3, and KIR3DL1 between pre-expanded and post-expanded NK cells, irrespective of donor HLA genotype (Fig. 1b–e). We did notice some trends in these data, in part, consistent with the prior observation that the KIR-ligand genotype of certain donors can influence the frequency of certain KIR-expressing subpopulations [35].

Inhibitory KIRs on ex vivo expanded NK cells retain inhibition specificity of their ligands

Following ex vivo activation and expansion, NK effector cells were stimulated with various HLA-expressing target cells. The iKIRs expressed on expanded NK cells inhibited NK cell activation, via interaction with the cognate KIR-ligand expressed on the 721.221 targets for each iKIR analyzed (Fig. 2a and Supplemental Figure 3). Consistent with the degranulation data, the cytotoxic function of sorted KIR single-positive NK cell subsets was also specifically inhibited by KIR-ligand on target cells (Fig. 2b). Our results indicate that NK cell ex vivo expansion and activation (by K562-mbIL15-41BBL cells and IL2 for 2 weeks) does not prevent the iKIR on these NK cells from causing NK cell inhibition when they encounter their cognate KIR-ligand. The amount of NK cell inhibition due to specific KIR-ligand recognition by iKIRs on ex vivo expanded NK cells was relatively similar to the amount of inhibition on freshly harvested NK cells from PBMCs cultured overnight in IL2 (Supplemental Figure 4).

Fig. 2.

Fig. 2

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Inhibitory KIRs on expanded NK cells retain inhibition specificity. a PBMCs from donor #18 were expanded with K562-mbIL15-41BBL cells. Expanded NK cells were stimulated with either HLA-null 721.221 cells or C1-, C2-, or Bw4-expressing 721.221 variants. CD107a+ cells were gated for 2DL1 single-positive (sp), 3DL1sp, 2DL2/3sp, and iKIR-null NK cell subsets by flow cytometry, and the percentage of CD107a+ cells was shown within the gate. b Expanded NK cells from donor #38 were sorted into four NK cell subsets: 2DL1sp, 2DL2/3sp, 3DL1sp, and iKIR-null. These four different NK cell subsets were incubated with 51Cr labeled HLA-null 721.221 cells or C1-, C2-, or Bw4-expressing 721.221 variants, at various E/T ratios. The cytotoxic functions of these NK cells were shown as percent lysis (see method section “Cytotoxicity assay”). The same result has been repeated. c Similar relationships as in Fig. 2a were found for expanded NK cells from 12 healthy donors using K562-mbIL15-41BBL cells, when stimulated with the 721.221-C1, 721.221-C2, and 721.221-Bw4 cell lines, respectively, in the absence or presence of rituximab (RT: 0.5 μg/mL). Single-positive (sp), double-positive and triple-positive as well as iKIR-null NK cell subsets were gated based on KIR2DL1, KIR2DL2/3, and KIR3DL1 expressions by flow cytometry analysis. The table legend within Fig. 2c shows the ligand for each inhibitory KIR receptor that was analyzed. Two-way ANOVA analyses were used to compare normalized CD107a level between NK cells that only express cognate iKIR and NK cells that express different combinations of iKIRs. Statistics are only shown for the RT group (black bars). ns not significant, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Ex vivo expanded NK cell-mediated ADCC is subject to iKIR/KIR-ligand inhibition

After NK cell expansion and activation, the expression of CD16 was similar to that measured on NK cells from freshly isolated PBMCs from the same donor (Supplemental Figure 2a). As expected, exposure of target cells to rituximab (an anti-CD20 monoclonal antibody) resulted in increased percentage of degranulating NK cells (Supplemental Figure 2b). Despite the increase in degranulation, rituximab-induced ADCC is still subject to iKIR/KIR-ligand inhibitions (Fig. 2c). The three inhibitory KIRs and their KIR-ligand pairs that we analyzed are listed in the legend box of Fig. 2c. This suggests that after a 12-day ex vivo expansion, where NK cells are in an activated state due to the continuous stimulation with both IL-2 and K562-mbIL15-41BBL, iKIR expressed on the expanded NK cells maintain their characteristics as on unstimulated NK cells in both natural cytotoxicity and ADCC settings.

Ex vivo expanded NK cells retain their licensing status determined by iKIR/KIR-ligand repertoire of the NK cell donor

To compare licensed NK cells from unlicensed NK cells, we grouped 12 healthy donors (Table 1) according to their KIR-ligand (HLA-C1, HLA-C2, and HLA-Bw4) genotypes. The 4 C1/C1 donors (thus not carrying C2) have licensed NK cells (NK cell subsets that express iKIR2DL2/3) that were more activated than their unlicensed NK cells (NK cell subsets that do not express iKIR2DL2/3), when stimulated with HLA-null 721.221 cells (Fig. 3a, HLA-null 721, no rituximab). Although this was not seen for the C2/C2 donors (Fig. 3b, discussed below), a similar licensing effect was also seen for the five donors that are Bw4+, with respect to its corresponding iKIR (3DL1) (Fig. 3c, HLA-null 721, no rituximab). Rituximab increased NK cell activation, but the unlicensed NK cells still demonstrated less activity than the licensed NK cells from the same donors on HLA-null 721.221 cells (Fig. 3a, c, HLA-null 721, +rituximab). These results are consistent with previous studies that utilized unstimulated NK cells [21] and demonstrate that the effect of NK cell licensing that occurred in vivo is retained after 12 days of ex vivo expansion with HLA-null cells (K562-mbIL15-41BBL).

Fig. 3.

Fig. 3

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Inhibition from iKIR/KIR-ligand interactions can override the licensing effect, and exposure to rituximab does not overcome differences due to licensing or iKIR inhibition. a Normalized CD107a levels of licensed NK cells (2DL2/3sp and 2DL1+2DL2/3+ NK cells, blue bars) expanded from C1/C1 donors (n = 4) were compared to normalized CD107a levels of unlicensed NK cells (gray bars) expanded from the same donors, following stimulation with HLA-null, HLA-C1-, or HLA-C2-expressing 721.221 cells, either with or without rituximab (RT) (0.5 μg/mL). b Normalized CD107a levels of licensed NK cells (blue bars) expanded from C2/C2 donors (n = 5) were compared to normalized CD107a levels of unlicensed NK cells (gray bars) expanded from the same donors, following stimulation with HLA-null, HLA-C1, or HLA-C2 721.221 cells, either with or without RT (0.5 μg/mL). c Normalized CD107a levels of licensed NK cells (3DL1sp, blue bars) expanded from Bw4+ donors (n = 5) were compared to normalized CD107a levels of unlicensed NK cells (iKIR-null, gray bars) expanded from the same donors, following stimulations with HLA-null, HLA-C1, HLA-C2, or HLA-Bw4 721.221 cells, either with or without RT (0.5 μg/mL). In a–c, one-way ANOVA analyses (matched by donor) were used to compare CD107a levels between licensed and unlicensed NK cell subsets expanded from the same donor. Since every donor is normalized to their iKIR-null NK cells stimulated with HLA-null 721.221 cells without RT, the normalized value of each donor’s iKIR-null NK cells stimulated with HLA-null 721.221 cells without RT is defined as “1.0”. Therefore, no error bar is shown for iKIR-null NK cells stimulated with HLA-null 721.221 cells without RT. ns not significant, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

In contrast, in C2/C2 donors, there was no difference in the amount of activation of the subset that should be licensed (2DL1sp NK cells) from the subset that should be unlicensed (2DL2/3sp NK cells) (Fig. 3b, HLA-null 721). Previously published data demonstrate that there is some cross-reactive recognition of HLA-C2 by iKIR2DL2/3 [17, 18]. Thus, the 2DL2/3sp NK cells in Fig. 3b might be considered “partially licensed,” thereby potentially accounting for their similar level of activation as the licensed 2DL1sp NK cells.

Inhibition due to iKIR/KIR-ligand interaction negates the augmented function of licensed versus unlicensed NK cells following expansion

Inhibition resulting from iKIR and KIR-ligand interactions was reported to dominate over the licensing effect seen with unexpanded NK cells [23, 29]. In this study, the same phenomenon is observed with expanded NK cells. When expanded NK cells from C1/C1 and C2/C2 donors were stimulated with C1- or C2-expressing 721.221 cells, respectively, NK cells lacking self-iKIR (unlicensed) were more activated than NK cells expressing self-iKIR (licensed) due to KIR-ligand inhibition via self-iKIRs on licensed NK cells (Fig. 3a HLA-C1 721, and Fig. 3b HLA-C2 721). Similarly, the interaction between Bw4 and self-iKIR 3DL1 inhibited activation of licensed 3DL1sp cells down to the same level as their unlicensed iKIR-null NK cells (Fig. 3c, HLA-Bw4 721). These results suggest that expanded NK cells that are unlicensed may play a more important role when the KIR-ligand that matches the NK donor (matched KIR-ligand) is expressed on target cells.

Altogether, the data in Figs. 2, 3 suggest that the difference between licensed and unlicensed NK cells detected in unstimulated NK cells [21, 23, 24] still applies for expanded and activated NK cells, even though they are cultured ex vivo with HLA-null cells (K562-mbIL15-41BBL) for 12–15 days. It could be, in part, due to maintenance of licensing from the autologous KIR-ligands expressed on fellow expanded NK cells. Whether a given target cell causes differential activation of licensed or unlicensed NK cells depends on what KIR-ligand the target cells express. In most combinations, the inhibitory effect caused by iKIRs recognizing their cognate ligand can override the augmented capabilities associated with prior licensing.

Specific KIR-ligands modify the distribution of both inhibitory and activating KIRs on expanded NK cells

Besides K562-mbIL15-41BBL cells, many other cell lines can also serve as stimulatory cells for NK expansion, such as 721.221 cells [36]. We expanded NK cells from a donor who expressed iKIR2DL1 and aKIR2DS1 using either HLA-null 721.221, or HLA-C2-expressing 721.221 stimulatory cells. Unlike the expansion results obtained when PBMCs are cultured with K562-mbIL15-41BBL cells, 721.221 cell lines as stimulants resulted in a greater number of contaminating T cells in the expansion product (data not shown). To analyze NK cell subset frequencies, we gated out CD3+ cells. We found that the frequency of 2DL1+2DS1− NK cells following ex vivo expansion with 721.221 cells was significantly reduced when HLA-C2 was expressed (Fig. 4b). Conversely, 2DL1−2DS1+ NK cell frequency was increased when NK cells were expanded with C2+ cells. The frequency of 2DL1+2DS1+ NK cells was reduced when expanded with C2+ cells (Fig. 4b), which suggests that the inhibitory effect from 2DL1/C2 is stronger than the corresponding activating 2DS1/C2 interaction. The frequencies of 2DL2/3+ and 3DL1+ NK cells were decreased by expansion with C1+ and Bw4+ 721.221 cells, respectively (Fig. 4b), but the decrease in 3DL1+ cells by Bw4+ stimulatory cells was not significant.

Fig. 4.

Fig. 4

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Inhibitory and activating KIR frequencies are affected by KIR-ligands expressed on stimulatory cells. PBMCs from healthy donors were expanded using 721.221 HLA-null or 721.221 HLA-C1/HLA-C2/HLA-Bw4 variants as stimulatory cells, at PBMC: stimulatory ratios of 1:1. a KIR2DS1 and KIR2DL1 stainings were distinguished by a mAb specific for 2DL1 and a mAb recognizing 2DL1 and 2DS1. b The frequency of 2DL1+2DS1+, 2DL1+2DS1−, and 2DL1−2DS1+ cells in NK cells expanded with HLA-null 721.221 cells was compared to the frequency of the same subset in NK cells expanded with HLA-C2 721.221 cells, regardless of expression of other iKIRs on NK cells (n = 4). The frequency of 2DL3+ cells and the frequency of 3DL1+ cells in NK cells expanded with HLA-null 721.221 cells were compared to the frequency of the same subset in NK cells expanded with HLA-C1 721.221 or HLA-Bw4 721.221 cells, respectively, regardless of expression of other iKIRs on NK cells (n = 4). Paired t test or two-way ANOVA (matched by donor) was used to compare KIR frequencies in NK cells expanded with various stimulatory cell lines. ns not significant, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)

Discussion

Current clinical trials are investigating the therapeutic potential of ex vivo expanded NK cells. Here, we focused on ex vivo expanded and stimulated NK cells, and examined their licensing status as well as recognition specificity of each iKIR/KIR-ligand interaction pair.

Ex vivo expanded NK cells have upregulated activating receptors, such as NKG2D and NKp44, and have superior tumor killing compared to freshly isolated NK cells [2]. In our NK cell expansion model using K562-mbIL15-41BBL cells, we showed that expanded NK cells still maintain FcR expression, and their target cell lysis can be improved by including a target-cell antigen-specific mAb, in this case, rituximab. These data further support that adoptive transfer of expanded NK cells has the potential to be combined with tumor antigen-specific mAb to treat cancers via augmented ADCC [37].

Our data suggest that activation of ex vivo expanded NK cells is dependent upon the KIR-ligand expressed on the target cells, and on whether the iKIR receptors on the NK cells recognize the KIR-ligand on the target cells. However, the three 721.221 variants we used only represent one allele from each KIR-ligand group (C0801, C0401, and B4402 [threonine80]). Whether other alleles, especially Bw4-isoleucine80 alleles that show differential interaction with 3DL1 than threonine80 alleles [19, 38, 39], would have different impacts on NK cell degranulation needs further investigation.

Even though K562 and 721.221 cells have both been used to stimulate NK cells and as targets for NK cells in many studies [21, 23, 24, 28, 29], they may have different profiles that may interact with NK cell receptors. A comparison between the abilities of K562 and 721.221 cells to stimulate NK cells has previously been reported [40]. Since our current study includes analyses of interactions between KIR on ex vivo expanded NK cells and KIR-ligands transfected into 721.221 cells, we have used 721.221-null cells as control target cells, allowing a consistent comparison focused on the roles of the KIR-ligands transfected into the 721.221 cells.

In this study, exposure of target cells to antigen-specific mAb to augment killing via ADCC did not overcome the influence of the iKIR/KIR-ligand interactions. It suggests that iKIR/KIR-ligand mismatch between donor and recipient may still be beneficial when adoptive transfer of ex vivo expanded NK cells and mAb therapy are combined.

In addition to evaluating degranulation frequencies of expanded NK cells during stimulation by target cells and rituximab exposure, we also evaluated IFNγ as a measure of NK activation. High levels of IFNγ were detected by ELISA in the supernatant of the expanded NK cells even without any target cell stimulation, and the amounts of IFNγ detected were not affected by rituximab or by KIR-ligand inhibition (data not shown). This indicated that IFNγ secretion by these NK cells was potently activated by the ex vivo expansion and could not be used as a measure of interaction with target cells during the 4-h assay.

Besides iKIRs, there are other inhibitory receptors on NK cells that could be inhibited by HLA alleles, such as LIR1 and NKG2A [41]. Even though the HLA alleles expressed on the 721.221 variants we made gave relatively specific inhibition to their cognate iKIR receptors, there remains some low level of HLA-induced inhibition mediated by inhibitory receptors other than the iKIRs we evaluated on these cells (for example, the decreased CD107a of iKIR-null NK cells by HLA-C1 and HLA-C2). LIR1/HLA and NKG2A/HLA-E interactions might account for some of this inhibition, but further validation by excluding the roles of LIR1 and NKG2A receptors on NK cells would be helpful.

Besides inhibitory function, iKIRs have also been shown to play a role in NK cell licensing [21, 22, 24]. We hypothesized that during 12 days of ex vivo expansion with HLA-null cells, expanded NK cells might lose their original licensing status, because long-term stimulation might overcome the tolerance of unlicensed cells. However, we found that NK cells preserve their original licensing status after ex vivo expansion with HLA-null cells. It is possible that the autologous KIR-ligands expressed on the surface of fellow NK cells might play a role in maintaining their licensing status. Consistent with data published on unexpanded NK cells [23, 29, 42], we found that inhibition induced by iKIR recognition of its cognate ligand normally dominated over the anticipated increase in effector function due to licensing effects. Interestingly, the impact from Bw4 inhibition on licensed iKIR3DL1sp cells seemed less potent at overriding the licensing difference compared to HLA-C inhibition for iKIR2DL1 or iKIR2DL2/3.

Finally, our expansion results using KIR-ligand-expressing 721.221 cells suggest that expression of KIR-ligand, HLA-C1 or HLA-Bw4, on the stimulatory cells selectively suppresses the expansion of NK cells with iKIR that recognize them (iKIR2DL2/3 and iKIR3DL1, respectively). Interestingly, the presence of the KIR-ligand, HLA-C2, on the stimulatory cells selectively inhibits expansion of NK cells with iKIR2DL1 but augments expansion of NK cells with aKIR2DS1. A larger sample size will be important to confirm our observations, as there may also be an effect from allelic variation within KIRs and HLA that is overlooked with the small number of healthy donors we have evaluated.

Ex vivo expanded NK cells may differ from fresh PBMC derived NK cells in many ways, but they both express KIRs. Our data demonstrate that ex vivo expanded NK cells are still influenced by KIR/KIR-ligand interactions and by rules similar to the way KIR/KIR-ligand interactions regulate unstimulated NK cells.

Overall, we have analyzed the effect of KIR/KIR-ligand interactions on ex vivo expanded NK cells in multiple ways. These in vitro studies demonstrate that: (1) NK cells expanded with HLA-null cells retain their licensing status; (2) iKIR recognition of KIR-ligands on target cells partially inhibit effector function of ex vivo expanded NK cells, even when licensed and involved in ADCC; and (3) KIR-ligands expressed on the stimulatory cells used for expansion can modify both inhibitory and activating KIR repertoires. Understanding the influence of the ex vivo NK cell expansion on the functional interactions of their KIR and KIR-ligands may be of use in future clinical applications.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 428 kb) (428.3KB, pdf)

Acknowledgments

The authors thank all healthy donors participating in this study. Thanks also to Dr. Robert DeMars, who provided 721.221 cells, and Dr. Deric Wheeler who shared the empty vector. This research was supported by a Hyundai Hope on Wheels Grant; Midwest Athletes Against Childhood Cancer; NIH Grants R01 CA166105 and R35 CA197078; and a grant from The Stand Up To Cancer Foundation and St. Baldrick’s Foundation.

Abbreviations

ADCC

Antibody-dependent cellular cytotoxicity

aKIR

Activating killer immunoglobulin-like receptor

Bw4

HLA-Bw4

C1

HLA-C1

C2

HLA-C2

iKIR

Inhibitory killer immunoglobulin-like receptor

KIR

Killer immunoglobulin-like receptor

mbIL15

Membrane-bound interleukin 15

MHC

Major histocompatibility complex

NCR

Natural cytotoxicity receptor

RT

Rituximab

41BBL

41BB ligand

Compliance with ethical standards

Conflict of interest

The authors have no financial conflicts of interest.

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Supplementary material 1 (PDF 428 kb) (428.3KB, pdf)

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