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. Author manuscript; available in PMC: 2011 May 1.
Published in final edited form as: Biol Blood Marrow Transplant. 2010 Feb 6;16(5):612–621. doi: 10.1016/j.bbmt.2010.01.019

NK cell killing of AML and ALL blasts by Killer-Immunoglobulin Receptor (KIR) negative NK cells after NKG2A and LIR-1 blockade

Robert Godal 1, Veronika Bachanova 1, Michelle Gleason 1, Valarie McCullar 1, Gong H Yun 1, Sarah Cooley 1, Michael R Verneris 1, Philip B McGlave 1, Jeffrey S Miller 1
PMCID: PMC2854246  NIHMSID: NIHMS177123  PMID: 20139023

Abstract

Although NK cell alloreactivity has been dominated by studies of KIR, we hypothesized that NKG2A and LIR-1, present on 53±13% and 36±18% of normal NK cells, plays a role in NK cell killing of primary leukemia targets. KIR− cells, which comprise nearly half of the circulating NK cell population, exhibited tolerance to primary leukemia targets, suggesting signaling through other inhibitory receptors. Both AML and ALL targets could be rendered susceptible to lysis by fresh resting KIR− NK cells when inhibitory receptor-MHC class I interactions were blocked by pan-HLA antibodies demonstrating that these cells were functionally competent. Blockade of a single inhibitory receptor resulted in slight increases in killing, while combined LIR-1 and NKG2A blockade consistently resulted in increased NK cell cytotoxicity. Dual blockade of NKG2A and LIR-1 led to significant killing of targets by resting KIR− NK cells showing that this population is not hyporesponsive. Together these results suggest that alloreactivity of a significant fraction of KIR− NK cells is determined by NKG2A and LIR-1. Thus strategies to interrupt NKG2A and LIR-1 in combination with anti-KIR blockade hold promise for exploiting NK cell therapy in acute leukemia.

Introduction

Human natural killer (NK) cells express several families of inhibitory NK cell receptors that recognize “self” human leukocyte antigen (HLA) class I ligands. These receptors are responsible for several mechanisms that determine whether or not a target will be susceptible to NK cells mediated lysis. Recognition of HLA class I by inhibitory receptors leads to self-tolerance by preventing cytolysis of normal cells(1–4). Although somewhat paradoxical, the same self-receptors that lead to tolerance also play a role in the acquisition of functional competence, a process termed NK cell education or licensing (5–7). Transiently interrupting NK cell inhibitory receptor signaling on educated NK cells may be a therapeutic strategy for augmenting anti-tumor activity.

There are three main inhibitory receptor families that recognize MHC class I molecules: killer immunoglobulin (Ig)-like receptors (KIRs), CD94/NKG2A and leukocyte Ig-like receptor-1 (LIR-1). KIRs display specificity for allele-specific variable regions of the alpha chain of classical HLA class I (HLA- A, -B, -C). CD94/NKG2A receptors recognize mainly non-classical HLA-E, whereas LIR-1 receptors recognize a broad spectrum of classical HLA –A, -B, -C and non-classical HLAE, -F and -G by binding to conserved regions of the alpha 3 domain(1, 2, 8–10). Although two studies investigating the inhibitory potential of LIR-1 on primary peripheral blood NK cells observed that NK cell inhibition is largely attributed to HLA-G recognition(11, 12), the functional role of LIR-1 interactions with primary leukemia cells is still poorly defined.

NK cells are the first immune cells to reconstitute after hematopoietic cell transplantation (HCT), representing the predominant lymphocyte population with potential to control leukemia relapse in the months preceding T-cell reconstitution(13–15). Despite the NK cell alloreactivity reported for acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) after KIR ligand mismatched HCT(16–20), not all reports agree(21–24) and the mechanism of apparent resistance in some studies is unclear. We hypothesize that NK cell receptors other than KIR may explain these clinical results.

Patients, materials and methods

Cell isolation and cell culture

All human samples were obtained after receiving informed consent under guidelines approved by the Committee on the Use of Human Subjects in Research at University of Minnesota and in accordance with the Declaration of Helsinki. Primary cells from 18 patients were collected by leukapheresis (AML [n=5], pre-B-ALL [n=3], T-ALL [n=1]) and bone marrow aspiration (AML [n=5], pre-B-ALL [n=4]). Blasts comprised greater than 80% of each sample. After thawing, necrotic blasts were removed by density gradient centrifugation using Ficoll-Histopaque and kept in a Ham's/F12 basal medium supplemented with 20% human AB serum for 12 hours. NK cells were isolated from peripheral blood mononuclear cells (PBMC) from 42 healthy donors using depletion of other cells by immunomagnetic beads (NK cell Isolation Kit, Miltenyi Biotech, Auburn, CA). KIR+ NK cells were positively separated by staining with phycoerythrin (PE)-conjugated antibodies against CD158a (HP-3E4), CD158b (CH-L), CD158e (DX9) (Becton Dickinson, San Jose, CA) and CD158i (FES172, Beckman Coulter, Fullerton, CA) and subsequent selection using anti-PE beads (Miltenyi Biotech, Auburn, CA). KIR− NK cells were isolated by positive depletion of KIR+ NK cells. We also tested NK cells from unseparated blood mononuclear cells 100 days after transplantation [autologous (n=1), umbilical cord blood (n=2) and unrelated adult donor(n=2)].

KIR-ligand typing, RT-PCR for HLA-G and Western Blotting

KIR-ligand typing was performed by pyrosequencing as described by Yun et al(25). HLA-G transcripts were amplified over 35 cycles of PCR using Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA) with published pan-HLA-G primer set G.257F and G.1004R as described(26). As an internal control, β-actin gene amplification was carried out for each sample. HLA-E and HLA-G expression of primary targets was determined by western blotting with the anti-HLA-E clone MEM–E/02 and anti-HLA-G clone MEM–G/01 (Abcam; Cambridge, MA) as described(27). The choriocarcinoma cell line JEG-3 was used as positive control. The mouse cell line EL08-1D2 was used as negative control(28).

Flow cytometry

Immunophenotypic analysis of cells was performed using 4-color analysis on a FACSCalibur (Becton Dickinson, San Jose, CA) with CELLQuest Pro software (Becton Dickinson). Cells were stained with the following monoclonal antibodies (mAbs) as indicated: fluorescein isothiocyanate (FITC)- or PE-conjugated DX9 (anti-CD158e), HP-3E4 (anti-CD158a/h), CH-L (158b); peridinin chlorophyll A protein (PerCP)-conjugated SK7 (anti-CD3); allophycocyanin (APC)-conjugated NCAM16.2 (anti-CD56) (Becton Dickinson, San Jose, CA); and PE-conjugated Z199 (anti-NKG2A) and HP-F1 (anti-LIR-1, Beckman Coulter, Fullerton, CA). HLA expression was analyzed by flow cytometry using PE-conjugated pan anti-HLA class 1 mAbs, W6/32 (Abcam, Cambridge, MA) and HP-1F7 (kindly provided by Lopez-Botet).

Determination of NK cell–mediated cytotoxicity

Flow-cytometric detection of NK cell-mediated cytotoxicity was performed as described(29–31). In brief, tumor target cells were labeled with 3 mMol (dissolved in DMSO) green lipophylic fluorescent dye DIOC18 (3,3'-dioctadecylox-acarbocyanine perchlorate; Sigma, St Louis, Mo). Effector cells and target cells were co-incubated at various ratios for 4 hours. Thereafter, propidium iodide was added at a final concentration of 5 μg/mL for 5 minutes to determine the proportion of dead cells. The proportion of propidium iodide-positive cells was determined by flow cytometry in the FL3 channel. The HLA class I-deficient cell line K562 was used as positive control. Primary targets that exhibited more than 10% specific cytolysis were considered sensitive to killing.

Degranulation Assays

Degranulation was determined as previously described by Betts et al. and Rubio et al(32, 33). Degranulation assays have the advantage of being applicable to batch analysis of frozen samples and provides addition data, compared to cytotoxicity, on killing potential of NK cell subsets. Briefly, isolated resting or IL-2-activated CD56+/CD3− cells were co-incubated with target cells at 1:2 effector-target ratios under conditions identical to the cytotoxicity assays. Anti-CD107a–PE or –anti CD107 a, b FITC antibody clone H4A3 and clone H4B4 (BD Biosciences, San Jose, CA) was added during the entire incubation period. After 1 hour of incubation, monensin (Sigma-Aldrich, St Louis, MO) was added to a final concentration of 10 ug/ml. After an additional 5 hours, cells were washed and stained with anti-CD56 mAb. CD107 expression on NK cells was analyzed by flow cytometry.

Antibody blocking experiments

KIR blocking was performed with the mAb IgG4 clone 1–7F9 (blocking KIR2DL1/2DL2/2DL3). NKG2A blocking was performed with the mAb clone Z270 (both provided by Novo Nordisk, Copenhagen, Denmark). LIR-1 blocking was performed with the mouse mAb IgG2b clone 292319 (R&D Systems, Minneapolis, MN). Blocking experiments were performed in basal medium supplemented with 20% human AB serum. Mouse mAb IgG1 clone HP-1F7 (kindly provided by Lopez Botet) was used as positive control for pan-HLA blocking. The mAb clone HP-1F7 effectively binds to the alpha chain of HLA-A, -B, -C, -E and -G and blocks all NK cell inhibitory receptor engagement(10, 34, 35). NK cells were preincubated for 30 min with NK receptor blocking antibodies to a final concentration of 20 μg/ml and target cells with anti-HLA mAb to 20 μg/ml, which was at least 10-fold higher than the maximal saturating concentration. The mouse anti-hNCAM/CD56 IgG2b clone 301040 and IgG2b clone 20116.11 were used as isotype controls (R&D Systems, Minneapolis, MN).

Statistical Analysis

Student's t-test was applied for statistical evaluation of differences between groups.

Results

A high frequency of KIR− blood NK cells express NKG2A and LIR-1

NK cells receptors from three families recognize MHC class I molecules; KIR, NKG2A and LIR-1. The surface expression of these receptors on peripheral blood NK cells was measured from 42 healthy donors using flow cytometry (Table 1). Almost half of NK cells lacked KIR expression (45±11%), although there was variability amongst donors [range 28–79%]. NKG2A and LIR-1 were expressed on 53±13% (range 20–81%) and 36±18% (range 5–80%) of all NK cells, respectively. The likelihood of coexpression of NKG2A and LIR-1 varied amongst the KIR subsets. NKG2A was more commonly expressed on KIR− cells (ratio KIR+/NKG2A+: KIR−/NKG2A+= 1: 2.1, p<0.0001), whereas LIR-1 was more likely to be co-expressed on KIR+ NK cells (ratio KIR+/LIR-1+: KIR−/LIR-1+= 2.3: 1, p<0.0001). Some individuals displayed an inhibitory receptor pattern which was predominantly comprised of KIR while others expressed a decreased percentage of KIR+ cells and instead expressed an NKG2A dominant pattern (Figure 1A).

Table I.

Inhibitory NKR expression on KIR+ and KIR− NK cells

Receptor KIR+ population KIR− pulation n= p-value
NKG2A+* 35±13% 75±12% 42 p<0.0001
LIR-1+* 44±20% 27±14% 42 p<0.0001
NKG2A+LIR+* 11.2±7.4% 7.6±6.1% 7 p=NS
NKG2A+LIR−* 16.9±9.4% 44±15% 7 p<0.0001
NKG2A−LIR+* 30±19% 13±12% 7 p=0.01
NKG2A−LIR−* 41±18% 35±7.2 7 p=NS
KIR+** 87±7.6% 7.8±5.3% 15 p<0.0001
KIR−NKG2A−LIR− 16±2.7% of total NK cells (n=7)
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*

The mean % (± S.D.) of each receptor expressed gated on KIR+ and KIR− NK cells determined by flow cytometry before bead separation.

**

The % of each receptor on KIR+ and KIR− NK cells after bead separation.

Figure 1. NK cells express KIR, NKG2A, and LIR-1 and leukemia targets minimally express HLA-G transcripts.

Figure 1

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A) NK cells were evaluated by flow cytometry for the expression of class I recognizing inhibitory receptors (n=42). Each CD56+/CD3− NK cell population was gated to evaluate the expression of NKG2A and LIR-1 on KIR (using a cocktail recognizing KIR2DL1/S1, KIR2DL2/L3/S2 and KIR3DL1) expressing cells. Shown is a representative example from a donor with a i) KIR and ii) NKG2A dominant phenotype. B) NK cells were then enriched into a KIR+ and KIR− population using anti-PE conjugated immunomagnetic beads recognizing a cocktail of anti-KIR mab. Representative examples of a KIR+ and KIR− population are shown. KIR− NK cells co-express a high proportion of NKG2A and less LIR-1. C) RT-PCR and Southern blot analysis was used to test for alternatively spliced HLA-G on nine leukemia samples, the erythroleukemia cell line K562, two normal peripheral blood mononuclear cell populations (PBMC) and a positive control choriocarcinoma cells line (JEG-3). β-actin was used as an internal control.

Primary acute leukemia blasts express NKG2A and LIR-1 ligands

The cognate ligands for NKG2A and LIR-1 were measured on primary AML (n=5) and primary ALL blasts (n=4). All targets expressed HLA class I molecules on their surface as detected by a pan-HLA antibody recognizing HLA-A, B, C, E, and G. Class I expression was consistently higher on ALL (MFI 244±52, n=4) compared to AML blasts (MFI 70±30, n=5; p=0.0004) suggesting that more inhibition by class I molecules may explain the relative resistance of ALL to NK cell mediated killing(36). All leukemia blasts also expressed HLA-E, the ligand for NKG2A, as measured by Western blotting ((21) and data not shown). LIR-1 ligands are less definitively characterized, but are thought to include HLA-G or other classical MHC ligands. Although MHC is variably expressed by leukemia, the expression of HLA-G on primary leukemia is still unclear(37, 38). We did not detect HLA-G protein by Western blot in any of the primary leukemia samples used in this study. Since the sensitivity of the anti-HLA-G antibody is not well documented and the specificity of anti-HLA-G antibodies is uncertain(39), we also used PCR to measure HLA-G transcripts in the samples. No HLA-G transcripts were found in 5 of 9 samples tested supporting the conclusion that they were truly HLA-G negative (Figure 1C). The low levels of HLA-G1 (770 bp) and HLA-G2/-G4 (490 bp) transcripts detected in 4 out of 9 leukemia samples did not result in expression of detectable HLA-G protein and probably represent low level HLA-G transcripts that have been found in normal peripheral blood(40).

KIR, NKG2A and LIR-1 all contribute to alloreactivity against primary AML and ALL blasts

To characterize the contributions of KIR, NKG2A and LIR-1 blockade to NK cell mediated alloreactivity against leukemia we tested the ability of polyclonal allogeneic NK cells to kill primary AML and ALL blasts in the presence and absence of blocking mAbs. Cytotoxicity and degranulation were tested using fresh resting NK cells without confounding effects of exogenous cytokines. For pan-HLA blockade, we selected the antibody clone HP-1F7 because it binds to the alpha chain of HLA-A, B, C, E and G to functionally block multiple HLA interactions(10, 34, 35). For anti-KIR blockade, no single reagent can block all KIR so we focused on an antibody being developed clinically (anti-2DL1/2DL2/2DL3) that interacts with all HLA-C1 and HLA-C2 ligands. Other blocking antibodies were used individually or in combinations as indicated.

Fresh polyclonal NK cells potently lysed class I negative K562 cells targets as a control for their broad cytotoxicity. Lysis of primary leukemia targets was always significantly less. Pan-HLA blockade resulted in increased target lysis by resting NK cells for both AML (~3-fold; p<0.0005; n=2) and ALL targets (~6 fold:p<0.0005; n=2) compared to no blockade (Figures 2A, Supplemental 1A). Clinically achievable KIR blockade using an anti-KIR reagent slightly increased the level of cytotoxicity against AML and ALL blasts, but not to the level of pan-HLA blockade, suggesting that other class I recognizing receptors were functionally operant. AML blasts (AML3) lacking two KIR ligands (HLA-C2 and HLA-Bw4) were slightly more sensitive to killing without blockade by allogeneic NK cell donors mismatched in both ligands (n=3) compared to completely matched donors (n=3) 15.6± 2.1 vs 9.4±1.1% (p=0.06). The effect of NKG2A blockade was similar to that of KIR blockade. The combination of LIR-1 blockade with either KIR or NKG2A blockade resulted in a potent increase in killing of both AML and ALL targets. These results indicate that all three receptors have the potential to inhibit NK cell cytotoxicity.

Figure 2. KIR, NKG2A and LIR-1 blockade all contribute to cytotoxicity against primary AML.

Figure 2

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A) Primary AML blasts from 2 patients, AML4 (i, ii) (tested with 5 allogeneic NK cell donors) and AML3 (iii, iv) (tested with 6 allogeneic donors), were investigated for their susceptibility to cytolysis mediated by resting (i, iii) NK cells at effector to target (E:T) ratio of 5:1. B) Degranulation of resting (ii, iv) NK cells was determined by CD107a expression following co-incubation with AML4 (ii) (n=3 NK cell donors) and AML3 blasts (iv) (n=6 NK cell donors). Mean ± SEM from different NK cell donors are shown. Pan-HLA blockade was compared to blockade of KIR, NKG2A and LIR-1 individually or in combinations. Statistically significant differences between groups are marked (* p< 0.05; ** p<. 0.01; *** p<0.005; **** p<0.0005). HLA-B and -C ligand status of leukemia blasts are shown. There was KIR ligand match in three donors and mismatch in 2 KIR ligands in three donors used for AML3.

In samples negative for HLA-G transcripts and protein, indicating a lack of HLA-G expression, LIR-1 blockade enhanced killing of blasts from these patients. This suggests that the engagement of classical and/or non-classical HLA class I molecules other than HLA-G on primary blasts can inhibit NK cell killing through LIR-1. Measurement of the degranulation response by CD107a yielded similar results (Figures 2B, Supplemental 1B). Dual blockade of KIR and LIR-1 or NKG2A and LIR-1 resulted in an increased proportion of degranulating NK cells comparable to that achieved with pan HLA blockade.

KIR− NK cells exhibit significant cytotoxicity against primary leukemia blasts

To further study the differential function of non-KIR receptors against primary leukemia targets, KIR+ and KIR− NK cells were separated from 15 healthy donors using a cocktail of anti-KIR mAbs (Figure 1B and Table 1). Both populations lysed class I negative K562 targets, however killing by KIR− NK cells was lower than KIR+ cells (56±8% [n=5] vs 39±16% at E:T 5:1 [n=11], P=0.02). Cytotoxicity assays were then performed against primary AML and ALL blasts (Figures 3A, 4A). Pan-HLA blockade of both the AML and ALL blasts resulted in potent cytotoxicity by resting KIR+ and KIR− NK cells against both targets. Similar to the results obtained using unsorted NK cells, blockade of both NKG2A and LIR-1 in the KIR− NK cell population resulted in a 3.1 fold (p<0.01) and 2.3 fold (p<0.01) increase in cytotoxicity against both AML and ALL blasts compared to no blockade. Similar results were obtained using CD107a degranulation (Figures 3B, 4B).

Figure 3. KIR− NK cells exhibit significant alloreactivity against primary AML blasts.

Figure 3

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Primary AML blasts (AML3) were investigated for their susceptibility to KIR enriched (KIR+) and KIR− resting (n=4 NK cell donors) NK cells in A) cytotoxicity and B) degranulation assays. There was KIR ligand match in one KIR+ donor and mismatch in 1 KIR-ligand in one and in 2 KIR-ligands in two KIR+ donors.

Figure 4. KIR− NK cells exhibit significant alloreactivity against primary ALL blasts.

Figure 4

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Primary ALL blasts (ALL9) were investigated for their susceptibility to KIR enriched (KIR+) and KIR− resting (n=4 NK cell donors) NK cells in A) cytotoxicity and B) degranulation assays.

Maximal killing of primary AML and ALL blasts requires NKG2A and LIR-1 blockade in addition to KIR blockade

The above studies show that KIR− NK cells are not hyporesponsive and are capable of lysing targets. To better characterize the contribution of anti-leukemic killing by these 3 families of inhibitory receptors we measured the sensitivity of additional primary AML and ALL blasts to killing by polyclonal resting NK cells (Supplemental Figure 2). One of 9 AML and 1 of 7 ALL samples were completely resistant to NK cell mediated lysis. Consistent with the cytotoxicity findings, neither resistant sample induced degranulation of NK cells (data not shown). We then characterized sensitivity to alloreactive NK cell mediated killing by setting a threshold of at least a 10% increment in specific lysis with pan-HLA blockade. Two AML samples that were killed by resting NK cells at baseline were not sensitive to NK alloreactivity after pan-HLA blockade or combined blockade of inhibitory receptors based on this definition. In sum, 6 of 9 AML and 6 of 7 ALL (including one T-ALL) samples demonstrated sensitivity to alloreactive NK cells, as pan-HLA blockade resulted in increased killing. Individual blockade of KIR, NKG2A or LIR-1 resulted in a modest increases in leukemia lysis, but the effects were significantly less that those seen with pan-HLA blockade in AML 23.9±3.7% vs 7.8±2.3% (p<0.005, n=6) and in ALL 24.7±3.9% vs 8.21±2.3% (p<0.005, n=6). Blockade of 2 or more receptors resulted in higher leukemia killing. Furthermore, blockade of KIR, NKG2A and LIR-1 achieved the same level of killing obtained with the pan-HLA antibody, suggesting that 3 receptor families account for the MHC class I interactions between bulk NK cells and leukemia blasts.

Augmented killing by day 100 post-transplant NK cells with inhibitory receptor blockade

We have previously shown that despite a 4-fold increase in the percentage of NK cells reconstituting in the peripheral blood 100 days after transplantation, the NK cell receptor repertoire in recipients of T-cell replete transplants is perturbed. KIRs are diminished and NKG2A is the dominant inhibitory receptor (expressed on > 80% NK cells)(15). We find that LIR-1 expression is diminished and is on only ~20% of NK cells at this time point post-transplant (not shown). Based on these results, NKG2A and LIR-1 may be ideal for exploiting post-transplant NK cell reactivity hematopoietic cell transplantation. Five patients were studied at ~100 days after transplantation to understand if NK cells are functional and to determine their tolerance mechanism against primary allogeneic leukemia blasts. AML or ALL targets already shown to be sensitive to enhanced killing after interrupting class I interactions between normal donor NK cells and targets were used. Given the unscheduled nature of receiving patient samples, blood mononuclear cells were incubated in 5 U/ml IL-2 for 48 hours prior to testing to allow thawing and preparation of targets. This concentration and duration of IL-2 exposure did not change the expression of KIR, NKG2A and LIR-1 (n=5, data not shown). KIR blockade in this setting had no effect on killing compared to control conditions. However, the addition of NKG2A, LIR-1 or a combination of all three blocking antibodies significantly increased killing showing that NK cells are functional in a setting where non-KIR class I recognizing receptors dominate (Figure 5).

Figure 5. Enhanced killing by NK cells 100 days after allogeneic transplantation by NKG2A and LIR-1 blockade.

Figure 5

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PBMC from patients 100 days after transplantation were tested against NK cells susceptible AML (n=3) and ALL (n=2) primary blasts alone or with anti-KIR, LIR-1, NKG2A blockade or the combination (Combo). A) Representative and B) aggregate data are shown. P-values less than 0.05 are shown (*).

Discussion

Tolerance to allogeneic NK cell killing is generally attributed to the inhibitory effects of killer immunoglobulin receptors (KIR) in most of the current literature on this topic. Our studies suggest that overcoming KIR inhibition may be insufficient to maximize killing of primary leukemia targets. In fact, in some settings were KIR expression is low, this tolerance mechanism may be minor. Although it have recently been published that a humanized anti-KIR reagent (binding to KIR2DL1, L2, L3) can effectively enhance killing of leukemia targets, it does not block all inhibitory KIR, as KIR3DL1 is not bound(41). However, nearly half of our AML targets were Bw4 negative, supporting the notion that KIR3DL1 interactions could not explain the lack of response to KIR blockade shown here. This finding prompted us to assess the importance of other inhibitory NK cell receptors in recognition and killing of acute leukemia cells. Here we demonstrate that two other families of inhibitory receptors—NKG2A and LIR-1—are present on both KIR+ and KIR− NK cells and are implicated in NK cell tolerance against primary AML and ALL blasts.

Our findings show that LIR-1 interactions can occur in the absence of HLA-G (high affinity ligand) supporting the observation that LIR-1 recognizes other HLA class I molecules(9, 10, 35, 42). This is in contrast to studies by Riteau et al(11, 12) reporting that a “dim” level of LIR-1 expression on primary peripheral blood NK cells is unable to mediate significant inhibitory signals by HLA class I molecules. The KIR−/NKG2A+ NK cell population are dominant in NK cells residing in lymphoid tissue, in peripheral blood of some healthy individuals and in most patients after transplantation(15, 43, 44). All AML and ALL samples tested expressed the NKG2A ligand HLA-E. Our findings show that resting polyclonal KIR−/NKG2A+ NK cells populations from donors exhibit potent cytotoxicity against primary blasts in agreement with the NK cell clone work by Yawata et. al. using HLA class I deficient targets (45). Our KIR− NK cell results may be particularly important in patients reconstituting early after T-cell replete transplantation where KIR− NK cells predominate. In a T-cell depleted setting, results may be different. Yu et al have recently shown that tolerance to self may be broken at early time points after transplantation but is re-established later (46). Further kinetic studies will be needed with various graft sources (adult donor or umbilical cord blood) to fully explore NK cell function in vivo but our data supports the notion that NKG2A and LIR-1 blockade may be more important post-transplant given the perturbed repertoire seen in these settings.

Our results show that primary AML and ALL blasts effectively protect themselves against cytolysis from KIR− NK cells through MHC interactions with NKG2A and LIR-1. This resistance can be diminished by combined blockade of these inhibitory receptors. The tight correlation between cytotoxicity and degranulation will allow us to measure NK cell subsets responsible for this activity to better understand this process. We acknowledge that other factors are important in determining tumor kill. NK cell function is determined by the next sum of inhibitory signals but activating receptor signals are also required for NK cell killing. Studies of activating receptors and how they reconstitute after hematopoietic transplantation will be needed to fully understand the role of NK cells in a graft-versus leukemia effect. Although the functional competence of KIR−/NKG2A+ NK cell reconstitution after HCT could be adversely affected by post-transplant immunosuppression, our data provides proof of concept that inhibitory receptor blockade enhances leukemia killing. A comprehensive analysis of NK cell function after transplantation has been initiated and will determine whether combining inhibitory receptor blockade with other strategies, such as IL-2 or IL-15 administration, may be needed to improve transplantation outcomes and whether in vitro function correlates with clinical efficacy.

Our work also provides insight into ALL resistance to NK alloreactivity. ALL blasts exhibit substantially higher HLA class I expression compared to myeloid blasts as shown here. Importantly, the resistance of ALL blasts to NK cell mediated killing can be overcome by pan-HLA blockade. The blockade of multiple class I MHC recognizing receptors can mediate robust NK cytotoxicity against ALL blasts, similar to that achieved in AML, supporting the therapeutic potential of inhibitory NK cell receptor blockade in patients with ALL.

In summary, our results show that inhibitory receptors other than KIR endow polyclonal, bulk NK cells with self tolerance mechanisms through NKG2A and LIR-1. The pattern of expression of KIR, NKG2A, and LIR-1 on NK cells and the respective expression of their cognate ligands on leukemia blasts determine the magnitude of potential inhibition by self MCH class I. Screening of donors and patient blast ligand interactions for their impact on functional killing may guide future trials and could possible spare patients who exhibit a primary NK cell resistance pattern. Simultaneous blockade of more than one inhibitory receptor family significantly increases the frequency of alloreactive NK cells. Our study warrants clinical testing of KIR, NKG2A and LIR-1 blockade (alone and in combination) to increase the efficacy of NK cell-based therapies in the treatment of leukemia and other class I expressing malignancies, especially in patients undergoing hematopoietic cell transplantation where non-KIR mechanisms may dominate.

Supplementary Material

01. Supplemental Figure 1. KIR, NKG2A and LIR-1 blockade all contribute to cytotoxicity against primary ALL blasts.

A) Primary ALL blasts, ALL9 (i, ii) (n=6 NK cell donors) and ALL8 (iii, iv) (n=4 NK cell donors) were investigated for their susceptibility to cytolysis mediated by resting NK cells at an E:T ratio of 5:1. B) Degranulation of resting NK cells was investigated following co-incubation with ALL9 (n=4 NK cell donors) and ALL8 (n=4 NK cell donors). There was a KIR ligand match in two donors and mismatch with 1 KIR-ligand in two donors used for ALL8.

02. Supplemental Figure 2. Maximal killing of primary leukema blasts requires blockade of multiple receptors.

Primary A) AML (n=7) and B) ALL blasts (n=5) were investigated for their susceptibility to resting NK cell cytolysis at an effector to target ratio of 10:1.

Grant acknowledgements

This work was supported in part by National Institutes of Health Grant P01-CA-65493 (JSM, SC, PBM), and P01-CA-111412 (JSM, SC, MRV).

Footnotes

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Conflict of interest statement: There is no conflict of interest to declare on behalf of the authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Supplemental Figure 1. KIR, NKG2A and LIR-1 blockade all contribute to cytotoxicity against primary ALL blasts.

A) Primary ALL blasts, ALL9 (i, ii) (n=6 NK cell donors) and ALL8 (iii, iv) (n=4 NK cell donors) were investigated for their susceptibility to cytolysis mediated by resting NK cells at an E:T ratio of 5:1. B) Degranulation of resting NK cells was investigated following co-incubation with ALL9 (n=4 NK cell donors) and ALL8 (n=4 NK cell donors). There was a KIR ligand match in two donors and mismatch with 1 KIR-ligand in two donors used for ALL8.

NIHMS177123-supplement-01.tif (1.5MB, tif)
02. Supplemental Figure 2. Maximal killing of primary leukema blasts requires blockade of multiple receptors.

Primary A) AML (n=7) and B) ALL blasts (n=5) were investigated for their susceptibility to resting NK cell cytolysis at an effector to target ratio of 10:1.

NIHMS177123-supplement-02.tif (1.5MB, tif)

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