BASIC STUDY

Enhanced Cytotoxic Activity of Ex Vivo-differentiated Human Natural Killer Cells in the Presence of HOXB4 Arash Nanbakhsh,* Ce´cile Pochon,* Sophie Amsellem,w Gianfranco Pittari,z Ania Tejchman,y Jean H. Bourhis,*w and Salem Chouaib*

Summary: We have previously shown that human umbilical cord blood CD34 + progenitor cells undergo in vitro differentiation into functional natural killer (NK) cells and that their coculture in the presence of HOXB4-transduced stromal MS-5 cells resulted in an increase in differentiated NK number. The present study was conducted to compare the stromal effect on NK lytic potential in the presence and absence of HOXB4. Our results provide evidence that HOXB4-transduced MS-5 cells as compared with transduced GFP (+) MS-5 cells induced highly differentiated cytotoxic NK cells. Importantly, this difference was not because of the expression of activating NK receptors but was associated with an increased induction of granzyme B degranulation in response to stimulation with NK cell susceptible targets. DNA microarray-based global transcriptional profiling confirmed the upregulation of granzyme B. These findings provide further evidence that HOXB4 is a crucial regulator of NK function and that its use in generating functional NK cells with increased lytic potential may be significant for cancer immunotherapy. Key Words: CD34 hematopoietic cells, HOXB4, NK cells, granzyme B, cancer immunotherapy

(J Immunother 2014;37:278–282)

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atural killer (NK) cells are a lymphoid population with potent cytotoxic activity against virus-infected or cancer cells, and which hold considerable potential for cellbased therapies targeting human malignancies. The natural cytotoxicity of NK cells is induced through the triggering of natural cytotoxicity receptors, including NKp46, NKp44, and NKp30. Mature NK cells induce target cell death upon binding of these receptors to their ligands, which induces Ca2 + -dependent granule exocytosis and release of cytotoxic proteins from these granules; Fas ligand (FasL)induced apoptosis; and production of membrane-bound or secreted cytokines including tumor necrosis factor-a and tumor necrosis factor–related apoptosis-inducing ligand.1 Granzyme B (GrB) is a unique serine protease that plays a

Received for publication July 5, 2013; accepted March 12, 2014. From the *Institut Gustave Roussy; wDepartment of Hematology & Bone Marrow Transplantation, Institut Gustave Roussy, Villejuif; yCentre de Biophysique Mole´culaire, Orle´ans, France; and zDepartment of Medical Oncology, National Cancer Center for Cancer Care and Research, Hamad Medical Corporation, Doha, Qatar. A.N. and C.P. contributed equally. A.N. and C.P. performed the experiments. S.C. designed the study. A.N., S.A, J.H.B, and S.C. wrote the manuscript. G.P. analyzed the data and all authors critically reviewed the manuscript and gave their final approval. Reprints: Salem Chouaib, Institut Gustave Roussy, INSERM U753, Villejuif 94800, France (e-mail: [email protected]). Copyright r 2014 by Lippincott Williams & Wilkins

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crucial role for target cell death. It directly activates Bid, a specific substrate for GrB, resulting in caspase activation.2 Although peripheral blood NK cell therapy following allogeneic stem cell transplantation for patients with acute myeloid leukemia has shown promise in clinical trials3; another potentially rich source of NK cells for adoptive immune therapy is cord blood.4 We previously provided evidence that NK cells could be differentiated from umbilical cord blood CD34 + cells in the presence of cytokines and stromal cells, yielding NK cells with phenotypic and functional features.5 Limitations in the use of NK cells for cell therapy relate to both the number of injected cells and to the killing potential of these cells. Generating high-quality NK cells in numbers sufficient to meet clinical requirements may be therefore problematic. To date, although many laboratories have reported expansion of NK cells,6,7 only a few have investigated the potentiation of the killing potential of cells differentiated from hematopoietic stem cells.8 Forced expression of the transcription factor HOXB4 homeoprotein has been reported to enhance the self-renewal capacity of mouse bone marrow hematopoietic stem cells.9 It is interesting to note that we have previously shown that it also improves ex vivo generation of functional human NK cell progenitors.10 Herein, we provide evidence that HOXB4 is a crucial regulator of NK lytic function and that its use in generating functional NK cells with increased lytic potential may be significant for hematologic malignancies.

MATERIALS AND METHODS NK Cell Differentiation CD34 + cells were separated from mononuclear cells by using the MACS system (Miltenyi Biotec, Auburn, CA). Isolated cells with >90% purity (104 cells) were transferred into 24-well plates in a coculture system containing MS-5 cells engineered to actively secrete the HOXB4 protein or the GFP protein, as previously described.11 CD34 + cells were cultured for 3 weeks in RPMI 1640 media containing 10% pooled human serum and 5% SVF, human recombinant SCF (50 ng/mL), interleukin-2 (IL-2, 100 U/mL), IL15 (20 ng/mL), and FLT3 (50 ng/mL). Cells were replated with new media once a week. After 3 weeks, NK cells were isolated for further analysis.

Slide Preparation and Confocal Microscopy K562 leukemia cells and NK cells were spread on polycoverslips at a 2:1 effector-to-target ratio and incubated for 1 and 2 hours at 371C for conjugates formation. Cells were stained with anti-GrB mAb (green fluorescence). Nuclei were stained with TO-PRO-3 iodide (blue fluorescence), and K562 cells were stained with cell L-lysine–coated

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FIGURE 1. Cytotoxic activity of natural killer (NK) cells derived from cocultures with MS-5/HOXB4 stromal cells (NK-HOXB4) compared with NK cells derived from cocultures with MS-5/-GFP stromal cells (NK-GFP). A, NK cell cytotoxicity of NK-GFP and NK-HOXB4 cells was determined by a conventional 4-hour 51Cr release assay using AML cell targets. B, NK cell activity counted in lytic units. Data are expressed in lytic units. One lytic unit was defined as the number of effector cells required for 20% lysis of 103 target cells.

tracker red. Data were analyzed by a fluorescence microscope (Leica TCS Confocal System, Wetzler, Germany).

performed to identify genes differentially expressed between NK cells (GFP and HOXB4) with a fold-change >2.

Gene Expression Microarray

We first investigated the effect of HOXB4-transduced and GFP-transduced MS-5 cells on the cytotoxic activity of NK cells by conventional 51Cr release assay. The results shown in Figure 1 indicate that NK cells derived from cocultures with MS-5/HOXB4 cells were more

RESULTS AND DISCUSSION DNA microarray was performed (Agilent Human Whole Genome Microarray: 44,000 spots) to assess and compare overall gene expression profiles of GFP-differentiated and HOXB4-differentiated NK cells. Analysis was r

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NKG2D

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FIGURE 2. Granzyme B degranulation and gene expression in HOXB4-NK cells. A, Expression of NK receptors by differentiated NK cells (CD3  CD56 + cells). B, Granule accumulation and granzyme B polarization at the immune synapse. C, Granzyme B released by NKHOXB4 and NK-GFP cells measured using the ELISPOT assay. Data are presented as average number of spots per well ± SD and are representative of 3 experiments with similar results (*P < 0.05). D, Granule exocytosis assay in CD3  CD56 + -gated cells for CD107a expression. NK cells indicates natural killer cells.

cytotoxic than NK cells derived from cocultures with MS-5/ GFP control cells. Increased cytolytic activity as illustrated in Figures 1A and B was observed against all acute myeloid

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leukemia cell targets tested, including Molm-13, K562, HL60, KG-1, and U937. Modulation of the relative frequency and intensity of expression of the NK cell receptors may r

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FIGURE 3. Genome-wide analysis of target genes regulated by HOXB4. Gene expression of 2 independent experiments using the Agilent Human Whole Genome Microarray. Samples from natural killer (NK) cells derived from cocultures with MS-5/ HOXB4 stromal cells (NK-HOXB4) compared with NK cells derived from cocultures with MS-5/-GFP stromal cells (NK-GFP). Top-scoring genes were defined by a minimal fold-change of 2.

increase NK-mediated cytotoxicity in cancer patients. Several mechanisms are involved in the alterations of NK cytotoxicity including a decreased expression of activating receptors,12 increased expression of inhibitory receptors,13 or defective expression of NK ligands on target cells.14 Attempts to increase the expression of activating receptors, to counteract inhibitory receptors expression, or to increase NK cell cytotoxic capacities could overcome tumor escape from innate immunity. To gain insight into the mechanisms associated with potentiation of cytotoxicity in NK cells derived from cocultures with MS-5/HOXB4 cells, we asked whether the increased lytic capability was associated with r

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HOXB4 Increases NK Cell Cytotoxic Activity

an upregulation in NK receptors. As depicted in Figure 2A, CD3  CD56 + -gated HOXB4-derived and GFP coculturederived NK cells express a similar pattern of receptors important for NK activity and maturation, including NKG2D, NKp30, NKp44, CD16, KIR2DL1/S1, KIR2DL2-3/S2, KIR3DL1/S1, CD159a, CD161, CXCR1, CCR7, and DNAM1. It is well established that NK cells require contact with target cells to form an immunologic synapse. This interface formed between NK and target cells by receptor recognition and adhesion molecule binding is essential for NK cell cytotoxicity, which is mediated through degranulation and release of cytolytic enzymes. We, therefore, investigated immune synapse formation and GrB polarization under our experimental conditions. For this purpose, NK cells derived by coculturing with GFP or HOXB4 were cocultured with K562 target cells, and confocal microscopy analysis was performed. The results shown in Figure 2B indicate increased polarization and accumulation of GrB in HOXB4-derived NK cells as compared with GFP-derived NK cells. In addition, Elispot analysis further indicates that HOXB4-derived NK cells produce more GrB when cocultured with K562 cells. Cellsurface expression of CD107a on NK cells after 4-hour coculture with K562 cells was assessed to examine the degranulation capacity of NK cells. The results shown in Figure 2D revealed that HOXB4 coculture-derived NK cells degranulate more effectively than GFP coculturedderived NK cells in the presence of target cells. We next analyzed the gene expression changes induced by HOXB4 using gene expression microarrays. The microarray analysis identified 75 downregulated and 51 upregulated genes with >2-fold changes in NK-HOXB4 compared with the GFP control. Data shown in Figure 3 demonstrate that GrB is one of the target genes regulated by HOXB4. More importantly, these data also illustrate an increase in CXCL10 gene expression in NK-HOXB4 cells. This chemokine, which is structurally and functionally related to CXCL9 and CXCL11 and acting through the CXCR3, has been reported to enhance NK cell cytotoxicity against leukemic cells and dormant tumor cells.15 Experiments conducted to further characterize the NK-HOXB4 cells indicate that cells bind to endothelial cells, induce autophagy in target cells, and induce DC maturation in common comparable to NK-GFP control cells (data not shown). Our studies clearly indicate that in vitro differentiation of NK cells in the presence of the homeoprotein HOXB4 could provide a powerful strategy for NK cell-mediated stimulation of the antileukemic immunologic response. This study shows, for the first time, the functional duality of HOXB4 in regulating the differentiation of NK cells in number and in cytolytic activity. This differentiation system may be a promising approach for generating increased numbers of allogenic NK cells with enhanced lytic potential. The NK-differentiated cells in the presence of HOXB4 displaying an enhanced lytic activity may be suitable candidates for immune intervention in graft-versus-leukemia and in maintaining remission of minimal residual disease.

ACKNOWLEDGMENTS The authors thank Dr Guillaume Meurice and Cedric Orear for DNA microarray analysis, Sylvie Rusakiewicz and Nathalie Chaput for DC maturation assay, and Claudine Kieda for the adhesion assay. www.immunotherapy-journal.com |

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CONFLICTS OF INTEREST/ FINANCIAL DISCLOSURES This work was supported by grants from “Institut National du Cancer” (INCA), Association Laurette Fugain, and Qatar foundation. All authors have declared that there are no financial conflicts of interest with regard to this work. REFERENCES 1. Zamai L, Ahmad M, Bennett IM, et al. Natural killer (NK) cell-mediated cytotoxicity: differential use of TRAIL and Fas ligand by immature and mature primary human NK cells. J Exp Med. 1998;188:2375–2380. 2. Sutton VR, Davis JE, Cancilla M, et al. Initiation of apoptosis by granzyme B requires direct cleavage of bid, but not direct granzyme B-mediated caspase activation. J Exp Med. 2000; 192:1403–1414. 3. Koehl U, Esser R, Zimmermann S, et al. Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children. Klin Padiatr. 2005;217:345–350. 4. Pinho MJ, Punzel M, Sousa M, et al. Ex vivo differentiation of natural killer cells from human umbilical cord blood CD34 + progenitor cells. Cell Commun Adhes. 2011;18:45–55. 5. Carayol G, Robin C, Bourhis JH, et al. NK cells differentiated from bone marrow, cord blood and peripheral blood stem cells exhibit similar phenotype and functions. Eur J Immunol. 1998; 28:1991–2002. 6. Spanholtz J, Preijers F, Tordoir M, et al. Clinical-grade generation of active NK cells from cord blood hematopoietic progenitor cells for immunotherapy using a closed-system culture process. PLoS One. 2011;6:e20740.

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7. Miller JS, McCullar V. Human natural killer cells with polyclonal lectin and immunoglobulin like receptors develop from single hematopoietic stem cells with preferential expression of NKG2A and KIR2DL2/L3/S2. Blood. 2001;98: 705–713. 8. Perez SA, Mahaira LG, Sotiropoulou PA, et al. Effect of IL-21 on NK cells derived from different umbilical cord blood populations. Int Immunol. 2006;18:49–58. 9. Oshima M, Endoh M, Endo TA, et al. Genome-wide analysis of target genes regulated by HoxB4 in hematopoietic stem and progenitor cells developing from embryonic stem cells. Blood. 2011;117:e142–e150. 10. Haddad R, Caignard A, Visentin G, et al. The HOXB4 homeoprotein improves ex vivo generation of functional human NK-cell progenitors. Leukemia. 2007;21:1836–1839. 11. Amsellem S, Pflumio F, Bardinet D, et al. Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein. Nat Med. 2003;9:1423–1427. 12. Carlsten M, Baumann BC, Simonsson M, et al. Reduced DNAM-1 expression on bone marrow NK cells associated with impaired killing of CD34 + blasts in myelodysplastic syndrome. Leukemia. 2010;24:1607–1616. 13. Junevik K, Werlenius O, Hasselblom S, et al. The expression of NK cell inhibitory receptors on cytotoxic T cells in B-cell chronic lymphocytic leukaemia (B-CLL). Ann Hematol. 2007; 86:89–94. 14. Costello RT, Sivori S, Marcenaro E, et al. Defective expression and function of natural killer cell-triggering receptors in patients with acute myeloid leukemia. Blood. 2002;99: 3661–3667. 15. Saudemont A, Jouy N, Hetuin D, et al. NK cells that are activated by CXCL10 can kill dormant tumor cells that resist CTL-mediated lysis and can express B7-H1 that stimulates T cells. Blood. 2005;105:2428–2435.

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2014 Lippincott Williams & Wilkins

Enhanced cytotoxic activity of ex vivo-differentiated human natural killer cells in the presence of HOXB4.

We have previously shown that human umbilical cord blood CD34 progenitor cells undergo in vitro differentiation into functional natural killer (NK) ce...
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