Ex Vivo Expanded Adaptive NK Cells Effectively Kill Primary Acute Lymphoblastic Leukemia Cells Lisa L

Published OnlineFirst June 21, 2017; DOI: 10.1158/2326-6066.CIR-16-0296

Research Article Cancer Immunology Research Ex Vivo Expanded Adaptive NK Cells Effectively Kill Primary Acute Lymphoblastic Leukemia Cells Lisa L. Liu1, Vivien Beziat 2,3, Vincent Y.S. Oei4,5, Aline Pfefferle1, Marie Schaffer1, Soren€ Lehmann6,7, Eva Hellstrom-Lindberg€ 7, Stefan Soderh€ all€ 8, Mats Heyman8, Dan Grander 9, and Karl-Johan Malmberg1,4,5

Abstract

Manipulation of human natural killer (NK) cell repertoires eactivity against HLA-mismatched targets. The ex vivo expanded promises more effective strategies for NK cell–based cancer adaptive NK cells gradually obtained a more differentiated immunotherapy. A subset of highly differentiated NK cells, phenotype and were speciﬁcandhighlyefﬁcient killers of termed adaptive NK cells, expands naturally in vivo in response allogeneic pediatric T- and precursor B-cell acute lymphoblastic to human cytomegalovirus (HCMV) infection, carries unique leukemia (ALL) blasts, previously shown to be refractory to repertoires of inhibitory killer cell immunoglobulin-like recep- killing by autologous NK cells and the NK-cell line NK92 tors (KIR), and displays strong cytotoxicity against tumor cells. currently in clinical testing. Selective expansion of NK cells Here, we established a robust and scalable protocol for ex vivo that express one single inhibitory KIR for self-HLA class I would generation and expansion of adaptive NK cells for cell therapy allow exploitation of the full potential of NK-cell alloreactivity against pediatric acute lymphoblastic leukemia (ALL). Culture in cancer immunotherapy. In summary, our data suggest that of polyclonal NK cells together with feeder cells expressing adaptive NK cells may hold utility for therapy of refractory ALL, HLA-E, the ligand for the activating NKG2C receptor, led to either as a bridge to transplant or for patients that lack stem cell selective expansion of adaptive NK cells with enhanced allor- donors. Cancer Immunol Res; 5(8); 1–12. 2017 AACR.

Introduction CD94-NKG2A heterodimers, bind to groups of HLA class I alleles and the nonclassical HLA class I molecule HLA-E, Natural killer (NK) cells are innate lymphoid cells charac- respectively (8, 9). The variegated and random distribution of terized by a potent intrinsic cytotoxic potential and an ability to KIR in the NK-cell compartment endows the innate immune kill virus-infected and transformed cells (1–4). In humans, the system with a broad and highly diverse range of responsiveness functional potential of NK cells is regulated through expression without receptor rearrangement (10, 11). The functional poten- of germline-encoded activating and inhibitory receptors (5). tial of a given NK cell is quantitatively tuned by the net input Activating receptors bind to stress-induced ligands, which are obtained through interactions with ligands for activating recep- highly expressed on tumor cells (6, 7). Inhibitory receptors, tors and self-MHC molecules (12, 13). This functional calibra- including killer cell immunoglobulin-like receptors (KIR) and tion process is termed NK-cell education and allows the cell to sense a sudden decrease of normal HLA expression as a result of virus infection or tumor transformation (14). 1Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska In the context of allogeneic adoptive NK-cell therapy or stem Institutet, Stockholm, Sweden. 2Human Genetics of Infectious Diseases Labo- cell transplantation (SCT), such reactivity due to a lack of ratory, INSERM U1163, Imagine Institute, Paris, France. 3Paris Descartes Univer- recognized MHC-encoded inhibitory ligands is triggered when 4 sity, Imagine Institute, Paris, France. Institute for Cancer Research, Oslo NK cells are transferred across HLA barriers (15). There is com- University Hospital, Oslo, Norway. 5The KG Jebsen Center for Cancer Immuno- 6 pelling evidence that NK cells play an important role as anti- therapy, Institute of Clinical Medicine, University of Oslo, Oslo, Norway. Depart- – ment of Medical Sciences, Uppsala University, Uppsala, Sweden. 7Department of leukemic effector cells in T-cell depleted haploidentical, KIR – Medicine, Center for Hematology and Regenerative Medicine, Karolinska Insti- ligand mismatched, SCT for patients with acute myeloid leuke- tutet, Karolinska University Hospital Huddinge, Stockholm, Sweden. 8Depart- mia (AML; refs. 16–18). NK cell–mediated graft-versus-leukemia ment of Women's and Children's Health & the Pediatric Cancer Unit, Karolinska (GVL) effects have been less well documented in the context of 9 Institutet and Karolinska University Hospital, Stockholm, Sweden. Department acute lymphoblastic leukemia (ALL), in particular in adult ALL of Oncology and Pathology, Cancer Centre Karolinska, Karolinska Institutet, (19, 20). Being derived from lymphoid progenitors, ALL blasts Stockholm, Sweden. express more HLA class I than myeloid cells, potentially con- Note: Supplementary data for this article are available at Cancer Immunology tributing to its increased resistance to NK-cell killing (21). Other Research Online (http://cancerimmunolres.aacrjournals.org/). suggested mechanisms of resistance include insufﬁcient binding Corresponding Author: Karl-Johan Malmberg, Oslo University Hospital, to LFA-1 and lower expression of ligands for activating NK-cell Ullernchausseen 70, Oslo 0130, Norway. Phone: 474-539-0926; E-mail: receptors, including MICA/B on target cells (15, 19). [email protected] The KIR repertoire in any given donor is determined by KIR doi: 10.1158/2326-6066.CIR-16-0296 gene content, copy number variation, and a stochastic epigenetic 2017 American Association for Cancer Research. regulation at the promoter level (22, 23). Thus, the frequency of

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alloreactive NK cells varies, ranging from a few percent to 60% to U-bottom plate (E:T ratio 10:1). Cells were labeled with CellTrace 70% of the NK-cell population in exceptional cases (24). There- violet (1 mmol/L, Life Technologies) prior to coculture. fore, expression of HLA class I on acute leukemia blasts remains an important limiting factor for the efficacy of NK-cell therapy Flow cytometry–based cytotoxic assay despite the use of allogeneic NK cells. ALL cells are resistant to Isolated NK cells, either rested overnight or expanded after the NK92 cell line (19). NK92 lacks KIRs, but expresses NKG2A, an 14 days, were labeled with CellTrace violet and used as effector inhibitory receptor that binds to HLA-E, which may contribute to cells at a final concentration of 1.25 106/mL. Blasts were the NK-cell resistance. However, pediatric ALL cells express little thawed and added at a final concentration of 2.5 105, and HLA-E (25). Thus, it remains unclear whether NK-cell recognition caspase-3 substrate was added at the beginning of the assay. After of ALL cells is functionally regulated through activating and 4 hours, blasts were stained with the live/dead cell marker aqua inhibitory input delivered through NKG2C and NKG2A receptors, (DCM) and relevant extracellular receptors. The percentage of respectively, binding to their shared ligand, HLA-E. cytotoxic activity was calculated using the following formula: þ þ Although several GMP-compliant NK-cell expansion protocols percent specific killing ¼ (C3 – spontaneous C3 ) þ þ þ þ þ þ yield large numbers of NK cells for therapy, it has proven difficult (C3 DCM spontaneous C3 DCM )/[live targets þ (C3 þ þ þ þ þ to selectively expand specific subsets of NK cells expressing a spontaneus C3 ) þ (C3 DCM spontaneous C3 DCM )]. single self-specific KIR, which would allow more effective targeting of allogeneic leukemia cells (26, 27). Insights into the natural KIR and KIR ligand typing immune response to human cytomegalovirus (HCMV), mani- KIR ligands were determined using the KIR HLA Ligand Kit fested as expansion and differentiation of highly cytotoxic NK (Olerup SSP; Qiagen) for the detection of, HLA-C1, HLA-C2, and fi cells with unique and predictable KIR expression pro les, may four HLA-Bw4 motifs. KIR genotyping was performed using a in vitro hold keys to the design of more selective expansion high-throughput technology called quantitative KIR automated protocols. Numerous reports have shown in vivo expansion of so þ typing (qKAT; ref. 34). called "adaptive" NKG2C NK cells expressing a single self-KIR in HCMV seropositive individuals (28–30). HCMV reactivation and HCMV serology the associated expansion of adaptive NK cells have been linked to CMV serology was determined using an ELISA-based assay on a lower relapse rate in AML patients after cord blood allogeneic plasma obtained during sample preparation. Purified nuclear SCT, a type of transplant in which one or more HLA mismatches CMV antigen (AD 169) was used, and the cut-off level for are common (31). This suggests that spontaneously occurring seropositivity was an absorbance of 0.2 at a dilution of 1/100. adaptive NK cells may have beneficial effects mediated through eradication of residual leukemic blasts. In vitro expansion of þ fl NKG2C adaptive NK cells can be achieved by coculturing na€ve Antibodies, tetramers, and ow cytometry analysis NK cells with CMV-infected fibroblasts or HLA-E transfected cell Stainings were performed using an appropriate mix of the lines (28, 32), but the robustness and efficacy of such expansion following antibodies (clone names are given in brackets): protocols remains to be determined. Herein, we have examined CD14-V500 (M5E2), CD19-V500 (HIB19), CD3-PE-Cy5.5 or the use of a feeder-based platform for selective expansion and PE.Cy5 (UCHT1), CD56-ECD or PE-Cy5.5 (N901), CD57 puri- fi differentiation of educated NK cells aiming to target primary ALL ed (TB01) or BV605 (NK-1), anti-mouse-IgM-EF650 (II/41), blasts. FCER1g-FITC (rabbit polyclonal), CD7-PE.Cy7 (8H8.1), ILT2-PE (HP-F1), CD161-BV605 (HP-3G10), NKG2A-APC or APC.AF750 or PE-Cy7 (Z199), NKG2C-PE (FAB138P, REA205), DNAM-1-PE. Materials and Methods vio770 (Dx11), NKG2D-BV711 (1D11), CD16-AF700 or BV785 Human participants and cells (3G8), 2B4-PE (C1.7), NKp46-BV786 (9E2), CD2-PB (TS1/8), This study was conducted in accordance with the Declaration KIR2DL3-FITC (180701), KIR2DL1-APC (143211), KIR3DL1- of Helsinki and was approved by the Regional Ethical Review AF700 (Dx9), KIR2DS4-QD585 (179315), KIR3DL2-biotin Board in Stockholm, Sweden. Healthy blood donors were used as (Dx31), KIR2DL2/L3/S2-PE.Cy5.5 (GL183), KIR2DL1/S1-PE.Cy7 a source for NK-cell expansion experiments. For all donors, (EB6), CD10 (HI10a), granzyme A (CB9), granzyme B (GB11), peripheral blood mononuclear cells (PBMC) were separated from perforin (B-D48), and TRAIL (RIK-1). Dead cells were labeled buffy coats by density gravity centrifugation (Ficoll–Hypaque; GE using live/dead aqua (Life Technologies). Biotin-conjugated anti- Healthcare) and were cryopreserved in 10% DMSO and 90% heat- bodies were visualized using streptavidin-Qdot 585 or 605 (Life fi inactivated FBS for later use. Bone marrow samples from patients Technologies). After staining, cells were xed and permeabilized with pediatric ALL and adult Myelodysplastic Syndromes (MDS) using a Fixation/Permeabilization Kit (eBioscience) prior to intra- were processed like the healthy PBMCs. When required, genomic cellular staining. Samples were acquired using an LSR-Fortessa fl DNA was isolated from 200 mL of whole blood using DNeasy 18-color ow cytometer (Becton Dickinson), and data were Blood and Tissue Kit (Qiagen). analyzed with FlowJo software version 9 (Tree Star, Inc.). The BD-LSR Fortessa instrument was equipped with a 100 mW Expansion of NK cells 405 nm laser, a 100 mW 488 nm laser, a 50 mW 561 nm laser, Thawed PBMCs from healthy blood donors were subjected to and a 40 mW 639 nm laser. NK-cell negative selection (NK Cell Isolation Kit, Miltenyi Biotec). NK cells were then cocultured with irradiated (40 Gy) 721.221. NK-cell functional assay AEH cells (33) in complete medium (RPMI, glutamine, 10% FCS) Isolated NK cells were rested overnight in complete medium in the presence of IL15 (final concentration 100 ng/mL, R&D and distributed at a final concentration of 2.5 105 cells/mL Systems) at a final concentration of 5 106/mL in a 96-well in a 96-well U-bottom plate. All target cells were added to NK

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cells at a final concentration of 2.5 105 cells/mL. For con- In vitro expansion differentiates cells toward an adaptive ventional functional assays, ALL blasts, PHA blasts, 721.221.wt, phenotype or 721.221.AEH cells were used as target cells. The cells were We monitored the phenotypic changes and granular load incubated for 6 hours (37 C, 5% CO2) after addition of in the NK cells pre- and post in vitro expansion and found monensin (GolgiStop, BD Biosciences, 1/1,500 final concen- that both conventional and adaptive NK cells displayed tration), brefeldin A (GolgiPlug, BD Biosciences, 1/1,000 final lower expression of CD7, Siglec-7, and PLZF and upregulation concentration), and CD107a-BV421 (H4A3, 1/100 final con- of CD2 and DNAM-1 (Fig. 2). Whereas the frequency of þ centration). After incubation, cells were washed and stained CD57 NK cells decreased somewhat, due to preferential – þ þ for extracellular receptors, permeabilized (fixation/perme- proliferation of CD57 NKG2C single-KIR NK cells abilization buffer, eBioscience) and stained for intracellular (Fig. 1B; ref. 28), the expression intensity of CD57 increased TNF-APC (MAb11) and IFNg-AF700 (B27) prior to analysis over the course of the 14-day culture. Loss of FceR1g and Syk with flow cytometry. are additional hallmarks of adaptive NK cells (35, 36). In contrast to the surface receptors, these adaptor molecules were Statistical analysis either stable or normalized during the 14-day culture. Expres- For comparisons of independent groups, the Student t test or sion of the inhibitory receptor LILRB1 did not show any the Mann–Whitney test was performed. For comparisons of consistent change. matched groups, the paired Student t test or the Wilcoxon Adaptive NK cells can express slightly more CD16 and matched test was performed depending on the sample size. perform better in antibody-dependent cytotoxicity assays For comparisons of qualitative variables, the Fisher exact t test assays (37). However, activation of NK cells with target cells was performed. In the relevant figures, n.s. indicates not signif- and/or cytokines leads to loss of CD16 through a metallo- icant; indicates P < 0.001; indicates P < 0.01; and indicates protease called ADAM17 (38). Indeed, the feeder-based P < 0.05. Analyses were performed using GraphPad software. expansion protocol led to a relative loss in CD16 expression down to approximately 50% positivity, in line with previously reported data (Fig. 2). Notably, CD16 was more stable in Results adaptive NK cells, leading to a relatively bigger difference þ Selective expansion of educated NKG2C NK cells expressing between adaptive and conventional NK cells after coculture a single KIR with HLA-E–expressing feeder cells. In addition to the Here, we set out to establish a platform for selective ex- observed changes in surface receptors and adaptor molecules, þ pansion of NKG2C NK cells expressing a single self-specific the levels of granzyme A and perforin increased or remained KIR (self-KIR). NK cells were cocultured together with 721.221 stable in all NK-cell subsets (Supplementary Fig. S3). Togeth- cells transfected with HLA-E (221.AEH) in the presence of er, these data suggest that the ligation of NKG2C under the IL15 for 14 days and stained for inhibitory KIRs, NKG2A, and influence of IL15 leads to a gradual shift toward an adaptive NKG2C. As previously reported (28), a profound skewing phenotype. þ toward NKG2C NK cells expressing self-KIR was seen after 14 days in culture (Fig. 1A). Stratification of NK-cell subsets Cytotoxicity and specificity of in vitro generated adaptive NK þ during proliferation revealed that CD57 NK cells dominate cells among nondividing NK cells (generation 0), whereas self- The dynamic change toward a more adaptive phenotype þ þ KIR NKG2C NK cells dominated in the fraction of NK cells prompted us to test the specificity and functional potential of reaching later generations (Fig. 1B). To explore the robust- in vitro generatedNKcells.Tothisend,expandedNKcells ness of this platform, we generated adaptive NK cells from expressing either KIR2DL3 or KIR2DL1, derived from HLA-C1/ 14 HCMV seropositive donors, with or without a preexisting C1 (C1/C1) or HLA-C2/C2 (C2/C2) donors, respectively, were þ þ þ þ NKG2C CD57 adaptive NK-cell populations. The 2-week tested against C1/C1 and C2/C2 PHA blasts in a flow þ culture protocol successfully expanded NKG2C NK cells from cytometry–based cytotoxicity assay. Target cell death was mea- all donors tested. The skewing of the KIR repertoire with a bias sured with caspase-3 (C3) activity and a live/dead cell marker toward expression of self-KIRs was noted both at the bulk NK- (DCM). The expanded NK cells displayed effective and specific þ cell level and in the NKG2C NKG2A NK-cell subset. In killing of mismatched PHA blasts, reaching nearly 100% donors with preexisting expansions of adaptive NK cells, the killing of C1/C1 blasts by KIR2DL1 single-positive NK cells skewing of the repertoire was further enhanced (Fig. 1C). This (Fig. 3B). Despite the 2-week culture in IL15, negligible killing was also observed in C1/C2 donors where the expanded phe- was seen in the KIR-HLA–matched setting (Fig. 3A and B). To notype was determined by the preexisting CMV-induced evaluate the impact of the selective expansion protocol, the imprint in the KIR repertoire (Supplementary Fig. S1). cytotoxicity of the expanded NK cells was compared with Although it cannot be excluded that the enrichment of the resting NK cells before expansion. All NK-cell expansions adaptive NK cells was partly a consequence of selective survival, displayed significantly more potent cytotoxicity against the protocol also led to a 2.4-fold expansion of the adaptive HLA-C–mismatched PHA blasts on a per cell basis compared NK-cell subset (Fig. 1D). Culture of adaptive NK cells in IL15 to the resting NK cells day 0 (Fig. 3B). In the presence of alone or in combination with 221.wt cells did not induce a HLA class I–blocking antibodies, expanded NK cells were similar skewing toward an end product enriched for NKG2A also able to kill PHA blasts in KIR-HLA–matched settings. In NK cells (Supplementary Fig. S2). These results demonstrate contrast, HLA class I blockade had no effect on the killing of þ the feasibility and robustness of the expansion of self-KIR HLA-mismatched PHA blasts, suggesting that the repro- þ NKG2C NK cells from HCMV-seropositive donors using HLA- grammed NK-cell cultures displayed a maximized alloreactive E–overexpressing feeder cells. response (Fig. 3C).

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Adaptive NK Cells Recognize Primary ALL

Bulk Conventional Adaptive Bulk Conventional Adaptive Bulk Conventional Adaptive

100 100 *** *** 100 *** *** *

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Figure 2. In vitro expansion is associated with cellular differentiation toward an adaptive phenotype. Compiled graph from three experiments (n between 5 and 10) showing the expression of the indicated surface receptors and adaptor molecules on bulk, conventional, and adaptive NK cells at days 0, 7, and 14.

Adaptive NK cells efﬁciently recognize primary pediatric somes (Heh) or (t12;21) (Table 1). All samples were taken at ALL blasts. the time of diagnosis and were >80% blasts. We established a Previous studies have reported that ALL blasts are relatively FACS-based killing assay using an individualized gating strat- resistant to lysis by allogeneic NK cells and the NK cell line egy for each ALL sample, based on the immune phenotyping NK92 (19, 39). Therefore, KIR2DL1- and KIR2DL3 single- developed by the Nordic society of pediatric hematology and positive, in vitro expanded adaptive NK cells were tested against oncology (Fig. 4A). Expanded NK cells displayed high killing a panel of blasts from 23 pediatric, primary ALL bone marrow activity against HLA-mismatched primary ALL blasts (Fig. 4B samples of three different subgroups; T-cell ALL and B-cell and C). The average killing was approximately 70% at an precursor cells with either high hyperdiploidy 47-50 chromo- effector to target ratio of 5:1 for ALL blasts in the mismatched

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A PHA Blasts (C1/C1) PHA Blasts (C2/C2) B 2DL3+ NK Cells 2DL3+ NK Cells 100 100 100 **100 Caspase-3 80 80 80 80 Caspase-3 60 DCM 60 60 60 Live cells 40 40 40 40 20 20 %Specific killing %Specific killing

%Specific killing 20 %Specific killing 20 0 0 + + + + 2DL3 2DL1 2DL3 2DL1 0 0 (C1/C1) (C2/C2) (C1/C1) (C2/C2) Day 0 Day 14 Day 0 Day 14 NK Cells NK Cells PHA Blasts PHA Blasts C2/C2 C1/C1 C 2DL1+ NK Cells 2DL1+ NK Cells 2DL1+ NK Cells ns Isotype control 100 *** 100 100 * HLA Class I blocking 80 80 * 60 60

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0 0 0 Day 0 Day 14 Day 0 Day 14 PHA Blasts PHA Blasts PHA Blasts PHA Blasts C1/C1 C2/C2 C1/C1 C2/C2

Figure 3. In vitro expanded adaptive NK cells are highly cytotoxic and specifically recognize HLA-mismatched targets. A, Representative graph showing specific lysis of PHA blasts derived from C1/C1 or C2/C2 donors using KIR2DL1þ or KIR2DL3þ expanded NK cells. B, Compiled graph of specific lysis using KIR2DL3þ and KIR2DL1þ NK cells expanded from different donors (n ¼ 7, n ¼ 3, respectively) against PHA blasts derived from C1/C1 or C2/C2 donors. C, Specific lysis of C1/C1 or C2/C2 PHA blasts in a FACS-based killing assay with KIR2DL1þ NK cells in the presence or absence of HLA class I–blocking antibodies. The figure shows a representative experiment out of three. Error bars represent the SD between three donors.

setting, independent of disease subgroup (Fig. 4B and C). lation was seen with or without anti-CD94 mAb in either – þ þ – Asimilarefficiency was noted when selectively expanded NK NKG2A NKG2C or NKG2A NKG2C NK cells when stimu- cells were tested against primary MDS and AML blasts (Sup- lated with ALL blasts (Fig. 4F). Taken together, these results plementary Fig. S4), suggesting the possibility of a potentially suggest that although NKG2C is functional on the in vitro broader clinical implementation of in vitro expanded adaptive expanded adaptive NK cells, its ligation is not required for the NK cells. potent killing of ALL blasts in an HLA-mismatched setting. ALL blasts from pediatric patients express little HLA-E (25). Thus, it remained an open question whether NKG2C contrib- Adaptive NK cells display an unleashed allogeneic response uted to the recognition of the mismatched targets. In line The finding that NKG2C appeared redundant for the ability with previous reports, we observed lower expression of HLA-E of the expanded adaptive NK cells to kill ALL blasts led us to on B-ALL blasts compared with healthy controls (Fig. 4D). To examine the impact of the size of the alloreactive subset in more address the relative contribution of the NKG2C–HLA-E inter- detail. To this end, we generated parallel cultures and moni- þ – action, degranulation was monitored in NKG2C NKG2A tored the ability of resting, short-term IL15 stimulated and – þ and NKG2C NKG2A NK-cell subsets following stimulation expanded NK cells to kill mismatched ALL blasts (Fig. 5A). The with ALL blasts and 221.AEH cells in the presence or absence killing of primary ALL blasts correlated with the generation of a of anti-CD94 blockade (Fig. 4F). Although predictable recog- large alloreactive subset (Fig. 5B). To further explore the impact – þ nition patterns were observed for NKG2A NKG2C NK cells of allorecognition and the intrinsic cytotoxic potential of adap- þ – þ – þ and NKG2A NKG2C NK cells against 221.AEH cells overex- tive NK cells, we sorted NKG2C NKG2A KIR2DL1 NK cells pressing HLA-E (Fig. 4E), no significant difference in degranu- from C2/C2 donors to 100% purity (Supplementary Fig. S5)

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Table 1. Patient characteristics lated (41). In particular, it represents an exciting possibility Sex Age of diagnosis Diagnosis HLA-C typing to selectively expand and/or guide differentiation of adaptive M 7 B cell (t12;21) C2/C2 NK cells showing high cytolytic potential and unique KIR M 1 B cell (t12;21) C1/C2 specificities (42–44). In this study, we have shown the robust- M 5 B cell (t12;21) C1/C2 ness of a preclinical platform for selective expansion of adap- M 4 B cell (t12;21) C1/C1 þ F 11 B cell (t12;21) C1/C2 tive NKG2C NK cells and its potential in targeting primary F 7 B cell (t12;21) C1/C2 ALL blasts. F 3 B cell (t12;21) C1/C2 The protocol developed in this study yielded NK cells with a M 6 B cell (t12;21) C1/C2 differentiated phenotype displaying profound skewing toward F 2 B cell (t12;21) C1/C2 expression of a single self-KIR. Consequently, the expanded M 4 B cell (t12;21) C2/C2 fi F 8 B cell (t12;21) C1/C1 adaptive NK cells displayed an elevated speci city for allogeneic F 4 B cell (Heh) C1/C2 targets lacking the cognate HLA ligand, demonstrating the main- F 5 B cell (Heh) C1/C1 tained signaling potential of inhibitory KIRs, despite the 2-week M 3 B cell (Heh) C1/C1 culture in IL15. This is an important and unique feature of this M 7 B cell (Heh) C2/C2 particular strategy for expanding adaptive NK cells, because M 5 B cell (Heh) C2/C2 other ex vivo expansion protocols have been associated with M 16 B cell (Heh) C1/C1 loss of self-tolerance by overcoming KIR inhibition (45–48). It is F 1 T cell C1/C2 þ F 3 T cell C1/C2 well documented that the proliferative capacity of the CD57 M 7 T cell C1/C1 adaptive NK-cell subset is limited (49). This raises the question M 12 T cell C1/C1 whether the observed enrichment of adaptive NK cells is merely M 2 T cell C1/C1 a consequence of selective survival. We have previously shown F 5 T cell C1/C2 that a proportion of CMV-seropositive patients with TAP defi- þ Abbreviations: F, female; Heh, high hyperdiploidy; M, male. ciency also displays expansion of NKG2C NK cells with phenotypic hallmarks of adaptive NK cells (50). However, these expansions had polyclonal KIR repertoires, suggesting that the and compared their ability to kill HLA-mismatched PHA blasts selective skewing in MHC-sufficient individuals depends on a with unsorted, expanded adaptive NK cells from the same selective advantage of inhibition through self-specificKIRs. donor. Notably, the sorted adaptive NK cells were highly potent This aligns with earlier studies in the mouse showing preferen- þ against mismatched C1/C1 PHA blasts, although expanded tial proliferation and expression of Bcl-2 in NK cells expressing þ NK cells showed slightly higher killing against the same targets self-MHC Ly49 in the mouse (51, 52). Notably, the current (Fig. 5C–D). These results show that adaptive NK cells, both protocol yielded a small, but significant increase in absolute resting and expanded, represent a unique subset with enhanced numbers of adaptive NK cells, reflecting their expansion in alloreactive capacity due to the near complete lack of inhibitory combination with differentiation from less differentiated þ MHC-binding receptors when transferred across HLA-C bar- NKG2C precursors, as previously suggested (28). NKG2C is riers. The selective expansion and repertoire skewing using unique among activating NK-cell receptors, and is the only NK- HLA-E–expressing feeder cells leads to further potentiation of cell receptor in addition to CD16, that can activate resting NK the adaptive NK cells and holds promise for the next-generation cells (53, 54). Thus, we propose that the enrichment of adaptive NK cell–based cancer immunotherapy. NK cells under the current protocol is a combined effect of survival, NKG2C-driven expansion, and differentiation from earlier precursors. Discussion The most appealing setting for clinical implementation of The survival rate for pediatric ALL currently exceeds 90%, but the current protocol is to transfer ex vivo expanded adaptive NK the remaining patients who relapse remain a major clinical cellsacrossHLA-Cbarrierstopatientswhoarerefractoryto challenge requiring effective treatment modalities. Allogeneic conventional treatment modalities. In such protocols, cells SCT has become the established procedure for treating children generatedfromaC1/C1donorcouldbetransferredtopatients with high-risk and relapsed leukemia. Over the past decade, with C2/C2 genotypes and vice versa. Notably, C1/C2 donors accumulating evidence indicates that NK cells may contribute could also be used pending on their CMV imprinted repertoire. þ to the GVL effect associated with allogeneic SCT. However, the In C1/C2 donors with a preexisting expansion of KIR2DL1 contribution of NK cells in the GVL effect against ALL appears adaptive NK cells, the current protocol expanded the same limited, possibly reflecting the poor ability of heterogeneous phenotype, possibly extending the number of possible donors NK-cell populations to kill ALL blasts in vitro (39, 40). A large for C1/C1 patients, which may be of clinical relevance given number of clinical trials are currently exploring the potential of that the C2/C2 genotype is less common. Although not inves- resting and cytokine-activated NK cells as a therapeutic modal- tigated here, it is possible to expand adaptive NK cells from ity against various cancer types. Most of the existing protocols CMV-na€ve repertoires, albeit the robustness and efficiency is involve transfer of highly diverse populations of NK cells. much lower (28). Although currently available clinical NK-cell products contain Although no off-target effects have been noted in more than cells with the desired alloreactive specificity, it is obvious that 200 patients that have so far received polyclonal conventional further refinement of the procedures hold promise to improve allogeneic NK cells, it is possible that the highly potent adaptive the efficacy of NK cell–based immunotherapy (26). Indeed, the NK cells, lacking all inhibitory receptors to recipient HLA class I, size of the alloreactive NK-cell subset and induction of molec- may cause tissue damage from targeting mismatched normal cells. ular remission and prolonged disease-free survival are corre- It will be essential to design phase I/II studies with transfer of low

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Liu et al.

ALL Blasts only

NK Cells + ALL blasts SSC SSC

CD10 (matched) Caspase-3

NK Cells + ALL blasts (mismatched)

FSC Cell Trace CD19 Dead cell marker

B C + ALL 3140 (C1/C1) ALL 3856 (C1/C2) ALL 3084 (C2/C2) 100 2DL1 NK cells 100 100 100 *** *** T-ALL 80 80 80 B-ALL (t12;21) Caspase-3 80 B-ALL (Heh) 60 60 60 Caspase-3 DCM 60 40 40 40 Live cells 20 20 20

%Specific killing 40 %Specific killing %Specific killing

0 0 0 %Specific killing 2DL3+ 2DL1+ + + + + 2DL3 2DL1 2DL3 2DL1 20 (C1/C1)(C2/C2) (C1/C1)(C2/C2) (C1/C1)(C2/C2) NK Cells NK Cells NK Cells 0 * ns C1/C1 C1/C2 C2/C2 D 300 3,000 E E FMO 200 LA- 2,000 ALL 221.AEH 100

MFI HLA-I 1,000 MFI H MFI % of Max

0 0 HLA-E

B cells L blasts B cells AL ALL blasts

F *** - + 80 NKG2A NKG2C **** + - ** NKG2A NKG2C

60 *

NK cells 40 +

20 %CD107a

+aCD94 +aCD94 ALL blasts 221.AEH

Figure 4. Primary pediatric ALL blasts are susceptible to selectively expanded NK cells. A, Example of gating strategy for a pre-B-ALL sample using FACS-based killing assay. B, Representative graphs showing frequency of specific cytolysis using expanded KIR2DL1þ or KIR2DL3þ NK cells against three different ALL patients being C1/C1, C1/C2, or C2/C2. C, Compiled graph showing frequency of specific lysis using KIR2DL1þ NK cells against three different HLA subgroups of primary pediatric ALL blasts being C1/C1, C1/C2, or C2/C2. D, MFI of HLA-E and HLA class I on healthy B cells and B-ALL blasts. Data generated in five experiments on 23 patients (E) Representative graph showing HLA-E level on ALL blasts compared with 721.221.AEH cells. F, Frequency of CD107aþ NKG2CNKG2Aþ and NKG2CþNKG2A NK cells stimulated with primary ALL blasts or 221.AEH with or without blocking antibody to CD94. Shown is a representative experiment out of three.

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Adaptive NK Cells Recognize Primary ALL

+ A 2DL3 NK Expansions B ** 100 100 * Resting NK cells ns ** NK cells + 221.wt 80 80 NK cells + 221.AEH

60 60

40 40 % Specific k illing % Specific k illing 20 20

0 0 Resting IL15 221.wt 221.AEH 0 20406080100 ALL blasts Alloreactive subset in CD56dim NK cells (%) (C2/C2) Figure 5. C Unleashed alloreactive response by Expanded (C2/C2) Sorted (C2/C2) resting and expanded adaptive NK cells. A, Killing of mismatched ALL 70.8 4.7 blasts by resting NK cells, overnight- activated IL15-stimulated NK cells, or cells expanded by the indicated feeder cells in IL15 for 14 days. Data are NKG2A KIR2DL1 compiled from three experiments and NKG2A include n ¼ 4 to 8 donors for the 69.8 99.7 different conditions. B, Graph shows correlation between the size of the 20.4 4.1 alloreactive subset and the killing of NKG2C KIR2DL3 NKG2C mismatched ALL blasts. Data are 92.1 3.3 94.6 5.4 compiled from three experiments and 4 to 8 donors. C, Expression of NKG2C and KIR on expanded NK cells at day 14 (left) and sorted adaptive NK cells from the same donor. D, Speciﬁc lysis KIR2DL1 of C1/C1 or C2/C2 PHA blasts by KIR2DL1 expanded and sorted NK cells in a FACS-based killing assay. C and D, 3.7 0.9 0.0 0.0 Data are representative of two KIR2DL3 independent sorts and cytotoxicity KIR2DL3 assays.

D PHA Blasts (C1/C1) PHA Blasts (C2/C2) 100 100

80 80 Caspase-3 60 60 Caspase-3 DCM 40 40 Live cells

20 20 % Specific killing % Specific killing

0 0 Expanded Sorted Expanded Sorted KIR2DL1+ NK cells KIR2DL1+ NK cells (C2/C2) (C2/C2) cell doses of expanded NK cells using gradually increasing doses of Another attractive and potentially safer clinical indication for preconditioning with chemotherapy as a means to indirectly expanded adaptive NK cells is for patients with tumors that titrate the length of persistence of this highly cytotoxic subset in display downregulation or loss of HLA class I (55, 56). Resistance vivo. Paralleling the clinical testing of new chimeric antigen to checkpoint inhibition therapy with anti-PD-1 for patients with receptors by means of mRNA electroporation, any off-target melanoma is associated with point mutations causing decreased effects will be transient in the nonconditioned host, as the antigen presentation and in one case the complete loss of HLA allogeneic NK cells will be rapidly rejected by recipient T cells. class I (57). If this observation were common across more tumors,

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Liu et al.

NK cells may find their therapeutic niche as a combination with apy. These results may also guide future attempts to develop checkpoint inhibition or a rescue therapy following relapse. In induced pluripotent stem cell–derived off-the-shelf adaptive fact, total or partial loss of HLA class I has been reported at NK cells (26). diagnosis in numerous cancer types (55, 58, 59). Previous obser- vations show a correlation between HLA-I expression and NK-cell Disclosure of Potential Conflicts of Interest susceptibility in pediatric B-ALL (20). Extending those results, we fi V. Beziat has ownership interest (including patents) in Patent WO2014037422 found that adaptive NK cells ef ciently lyse HLA-matched targets A1. K.-J. Malmberg is a visiting professor at Karolinska Institute, reports receiving a in the presence of HLA class I blockade, mimicking the selective commercial research grant from, has ownership interest (including patents) in, loss of HLA class I in HLA-matched tumor cells. The fact that in and is a consultant/advisory board member for Fate Therapeutics. No potential vitro expanded NK cells retained their sensitivity to cognate KIR– conflicts of interest were disclosed by the other authors. HLA interactions despite culture in IL15 suggests that they will not be toxic to normal tissues when transferred to HLA-matched Authors' Contributions patients. Furthermore, targeting patients whose tumors displayed Conception and design: L.L. Liu, V. Beziat, S. Soderh€ €all, K.-J. Malmberg low or absent HLA class I will facilitate treatment of patients who Development of methodology: L.L. Liu, V. Beziat, K.-J. Malmberg are C1/C2, the most common KIR ligand genotype. Acquisition of data (provided animals, acquired and managed patients, Reusing and colleagues reported low expression of HLA-E on provided facilities, etc.): L.L. Liu, V.Y.S. Oei, A. Pfefferle, M. Schaffer, primary ALL blasts (25). This raised the question whether S. Lehmann, E. Hellstrom-Lindberg,€ S. Soderh€ €all, M. Heyman, D. Grander NKG2C was involved in the recognition of the ALL blasts. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L.L. Liu, V.Y.S. Oei, A. Pfefferle, M. Schaffer, Antibody-blocking experiments suggested a redundant role for in vitro D. Grander, K.-J. Malmberg NKG2C in the activation of the expanded adaptive NK Writing, review, and/or revision of the manuscript: L.L. Liu, V.Y.S. Oei, cells. NK-cell recognition of ALL blasts depends on DNAM-1 A. Pfefferle, S. Lehmann, S. Soderh€ €all, M. Heyman, D. Grander, K.-J. Malmberg (40). Adaptive NK cells highly express DNAM-1 and CD2 Administrative, technical, or material support (i.e., reporting or organizing (28, 53, 60), of which CD2 acts in synergy with both CD16 data, constructing databases): V.Y.S. Oei and NKG2C (53). Both these receptors are stable or induced Study supervision: K.-J. Malmberg fi during the in vitro expansion and may contribute to the efficient Other (Patient clinical subgroup identi cation during project planning): S. Soderh€ €all killing of ALL cells. Although our expansion protocol resulted in highly specific cytotoxicity against all targets tested, the frequency of cells that Grant Support mobilized CD107a to the cell surface was relatively low, ranging This work was supported by grants from the Swedish Research Council, the between 5% and 30%. A previous report showed a subpopulation Swedish Children's Cancer Society, the Swedish Cancer Society, the Tobias of NK cells, termed "serial killers" that can deliver multiple lytic Foundation, the Karolinska Institutet, the Wenner-Gren Foundation, the Nor- wegian Cancer Society, the Norwegian Research Council, the South-Eastern hits in a row before exhaustion (61). It is tempting to speculate Norway Regional Health Authority, the European Commission Horizon 2020 that the some of the expanded adaptive NK cells are "serial killers" Programme 692180-STREAMH2020-TWINN-2015, grant from National Sci- and display different killing kinetics that give rise to the discrep- ence Center, Poland 2014/13/N/NZ6/02081 (to M. Schaffer), and Stiftelsen KG ancy between high specific cytolysis and measured CD107a Jebsen. V. Beziat was supported by the French National Research Agency (ANR; mobilization. grant no. NKIR-ANR-13-PDOC-0025-01). In conclusion, we have demonstrated the robustness of a The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked culture platform yielding expansion and differentiation of adap- advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate tive NK cells with high cytotoxic potential and with predictable this fact. specificity. The feeder cell–based expansion platform should be scalable into a GMP-compliant process, having immediate impli- Received October 27, 2016; revised April 13, 2017; accepted June 14, 2017; cations for harnessing adaptive NK cells in cancer immunother- published OnlineFirst June 21, 2017.

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Ex Vivo Expanded Adaptive NK Cells Effectively Kill Primary Acute Lymphoblastic Leukemia Cells

Lisa L. Liu, Vivien Béziat, Vincent Y.S. Oei, et al.

Cancer Immunol Res Published OnlineFirst June 21, 2017.

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