Natural Killer Cells Generated from Cord Blood Hematopoietic Progenitor Cells Efficiently Target Bone Marrow-Residing Human Leukemia Cells in NOD/SCID/IL2Rgnull Mice

Natural killer (NK) cell-based adoptive immunotherapy is an attractive adjuvant treatment option for patients with acute myeloid leukemia. Recently, we reported a clinical-grade, cytokine-based culture method for the generation of NK cells from umbilical cord blood (UCB) CD34+ hematopoietic progenitor cells with high yield, purity and in vitro functionality. The present study was designed to evaluate the in vivo anti-leukemic potential of UCB-NK cells generated with our GMP-compliant culture system in terms of biodistribution, survival and cytolytic activity following adoptive transfer in immunodeficient NOD/SCID/IL2Rgnull mice. Using single photon emission computed tomography, we first demonstrated active migration of UCB-NK cells to bone marrow, spleen and liver within 24 h after infusion. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Thereafter, we showed that low dose IL-15 mediates efficient survival, expansion and maturation of UCB-NK cells in vivo. Most importantly, we demonstrate that a single UCB-NK cell infusion combined with supportive IL-15 administration efficiently inhibited growth of human leukemia cells implanted in the femur of mice, resulting in significant prolongation of mice survival. These preclinical studies strongly support the therapeutic potential of ex vivo-generated UCB-NK cells in the treatment of myeloid leukemia after immunosuppressive chemotherapy.


Introduction
Acute myeloid leukemia (AML) is a clonal disorder characterized by the accumulation of abnormal myeloid progenitor cells and suppression of normal hematopoiesis [1]. With a median age of ,70 years at diagnosis [2], AML is most common in the elderly and its yearly incidence is expected to increase as the population ages [3]. Current chemotherapeutic regimens lead to remission rates of 60-85%. However, relapse occurs in the vast majority of AML cases, resulting in a 5-year overall survival of 40% in patients ,60 years of age, which even drops to 10% in elderly patients due the higher prevalence of bad risk cytogenetics and poor chemotherapeutic tolerance [4]. Although allogeneic stem cell transplantation (alloSCT) is potentially curative, mostly younger patients can benefit from this therapeutic option due to high association with transplant-related morbidity and mortality [5]. Therefore, adjuvant and alternative treatment options are urgently needed.
Transfusion of allogeneic NK cells is a promising therapeutic approach for patients with AML. NK cells are major effector cells of the innate immune system and play a key role in control against virus infection and tumor immunosurveillance [6,7]. In the setting of haploidentical alloSCT, NK cell alloreactivity has proven to decrease relapse rate and improve survival among AML patients [8]. Therefore, there is an emerging interest in exploiting adoptive NK cell transfer in the treatment of AML. Clinical studies reported so far showed that infusion of haploidentical NK cells derived from leukapheresis products resulted in objective clinical responses in high-risk AML patients [9,10], as well as long term remissions in childhood AML [11].
However, further improvement of NK cell-based therapy is needed to increase the clinical effect. In this regard, NK cells generated ex vivo from hematopoietic progenitor cells (HPC) may have significant clinical benefits over enriched NK cells from adult donors, including the ability to choose an appropriate killer-cell immunoglobulin-like receptor (KIR)-ligand or KIR B haplotype alloreactive donor, as well as the capacity to reach high therapeutic dosages. Recently, we reported a GMP-compliant, cytokine/heparin-based culture protocol for the ex vivo generation of highly active NK cells from CD34 + HPC isolated from cryopreserved umbilical cord blood (UCB) units [12]. Expansion in closed, large-scale bioreactors yields a clinically relevant dose of NK cells with high purity and cytolytic activity against AML cells in vitro [13].
In the present study, we aimed at evaluating the anti-leukemic potential of UCB-NK cells in vivo in terms of biodistribution, survival and cytotoxicity following adoptive transfer in NOD/ SCID/IL2Rg null (NSG) mice. Therefore, we established an 111 Indium labelling protocol that enables specific and sensitive in vivo tracking of infused UCB-NK cells by single photon emission computed tomography (SPECT) imaging. Besides generating insight in UCB-NK cell trafficking, we demonstrated specific accumulation of UCB-NK cells in the bone marrow (BM) that matched their chemokine receptor profile. Moreover, we demonstrated that a single infusion of UCB-NK cells resulted in potent leukemia cell growth inhibition and significantly improved mice survival. These findings strongly support ex vivo-generated UCB-NK cells as promising immunotherapeutic products for the treatment of AML.

UCB-NK Cell Generation
UCB units were obtained at birth after normal full-term delivery after written informed consent with regard of scientific use from the cord blood bank of the Radboud University Nijmegen Medical Centre (RUNMC, Nijmegen, The Netherlands). The use of UCB units was approved by the RUNMC Institutional Review board. NK cells were generated from cryopreserved UCB-derived HPC as previously reported [12,13]. Briefly, expanded CD34 + UCB cells were differentiated and further expanded using NK cell differentiation medium which consists of Glycostem Basal Growth Medium (GBGMH) for cord blood (Clear Cell Technologies) supplemented with 2% human serum (HS; Sanquin Blood Supply Foundation, Nijmegen, The Netherlands), low-dose GM-CSF (Neupogen), G-CSF, IL-6 (both CellGenix) and a high-dose cytokine cocktail consisting of IL-7, SCF, IL-15 (all CellGenix) and IL-2 (ProleukinH). The cell density was checked two times a week and adjusted to .1610 6 cells/ml by the addition of GBGMH NK cell differentiation medium. For experiments, CD56 + CD3 2 UCB-NK cells were used at the end of the culture process with .90% purity, what was typically achieved within 3-4 weeks in GBGMH NK cell differentiation medium.

In vitro Cell Migration Assay
UCB-NK cells were resuspended in GBGM/2% HS and loaded into transwell inserts (10 5 cells/well, 5 mm pore filter transwell, 24-well plate, Corning). The human chemokines CCL4, CCL20, CXCL10, CXCL11 and CXCL12 (all Immunotools) were diluted at 10-250 ng/ml and added to the lower compartment (600 ml/well) in triplicates. After 2 h at 37uC, inserts were removed; cells in lower compartments were collected, stained for CD56 and quantified by flow cytometry. Percentage of migrated cells was calculated as the number of CD56 + cells in the lower compartment divided by the total number of CD56 + loaded cells.

Mice
NOD/SCID/IL2Rg null (NSG) mice were originally purchased from Jackson Laboratories, and housed and bred in the RUNMC Central Animal Laboratory. Male NSG mice were used from 6 to 12 weeks of age (weight was 20-30 g). All animal experiments were approved by the Animal Experimental Committee of the RUNMC and were conducted in accordance with institutional and national guidelines under the university permit number 10300.
NK Cell Labeling with 111 Indium, SPECT-CT Imaging and Biodistribution Analysis UCB-NK cells were labeled with 111 Indium-oxinate ( 111 In; GE Healthcare) in PBS Tris 0.1 M HCl, pH 7.4 for 15 min at RT at doses mentioned in the text. After incubation, cells were washed twice with PBS/2% HS and resuspended in PBS before use. Viability was assessed by trypan blue exclusion and cell-associated activity was quantified using a dose calibrator VDC-404 (Veenstra Instruments, The Netherlands). Lysates were obtained after three freezing/thawing cycles of 111 In-NK cells previously resuspended in distilled water. Whole body scans of isoflurane gas anesthetized (2% in air) mice were acquired with a SPECT-CT dual-modality scanner (U-SPECT II, MiLabs) for 30-45 min using a 1.0 mm diameter pinhole mouse collimator cylinder. Scans were reconstructed with MiLabs reconstruction software and analyzed using Inveon Research Workplace software. For biodistribution analysis, mice were euthanized by cervical dislocation, tissues of interest were dissected, weighed, and analyzed for their 111 In content using a shielded 3-inch-well-type gamma counter (Wizard; Pharmacia LKB). The 111 In activity in each tissue was expressed as percentage of the injected dose (%ID) per gram of tissue and was normalized to the blood level. Values for the total blood and BM fraction were extrapolated according to physiological values, with blood being 6% of the total body weight, and one femur being 6.7% of the total BM fraction [14].

Intra-femoral K562 Model, Bioluminescence Imaging and UCB-NK Cell Adoptive Transfer
The NK-sensitive leukemia cell line K562 (ATCC) was cultured in Iscove's modified Dulbecco's medium (IMDM; Invitrogen) containing 50 U/ml penicillin, 50 mg/ml streptomycin and 10% fetal calf serum. Green fluorescent protein (GFP) and Luciferase expressing K562 cells (K562.LucGFP) were generated by stable transduction of parental cells with lentiviral particles LVP20 encoding the reporter genes under control of the CMV promoter (GenTech). To establish a preclinical AML xenograft model, adult NSG mice were injected in their right femur with 10 5 K562.GFPLuc cells (injection volume = 5 ml), by insertion of a 25G Hamilton needle through the knee joint of isoflurane gas anesthetized mice. Using this procedure, leukemia cell growth remained localized to BM up to 5 weeks. Thereafter, tumor cells eventually overgrow outside the bone forming a palpable tumor.
Mice were sacrificed (cervical dislocation) when the palpable tumor reached 1 cm in diameter or when one of the following criteria was observed: severe weight loss, poor coat and skin condition, static activity or paraplegia. Leukemia load was monitored by bioluminescence imaging (BLI) following injection of Luciferine (3.5 mg per mouse, Caliper Life Science) using the IVIS system (Xenogen). Images were analyzed using Living Image Software 2.5 (Xenogen). Leukemia load was quantified in the region of interest with subtraction of background signal, and expressed as photons per second. For adoptive transfer, UCB-NK cells were resuspended in PBS and injected i.v. via the tail vein of

Statistical Analysis
Statistical analyses were performed using Graphpad Prism 5 software. Biodistribution of 111 In-associated activity following 111 In-NK cell infusion was compared to that observed after lysate or free 111 In injection using one way-analysis of variance (ANOVA) and Dunnett's multiple comparison tests. Two way-ANOVA followed by Bonferroni post-hoc test, and log rank Mantel Cox tests were used in anti-leukemic studies as indicated in the figure legends. Differences were considered to be significant for p values ,0.05.

Characterization of the Homing Receptor Expression Profile of UCB-NK Cells
We reported previously that the cytokine-based culture system that we established allows generation of CD3 2 CD56 + NK cells with high purity, that express typical inhibitory and activating NK receptors and display similar cytotoxic gene expression profile to peripheral blood NK cells [15]. To further characterize UCB-NK cell products and to investigate their biodistribution potential upon adoptive transfer, we examined the expression and functionality of a panel of receptors that were previously described to participate in the regulation of human NK cell trafficking in vivo [16,17]. As shown in Figure 1A-B, the chemokine receptors CCR2, CCR5, CCR7, CXCR6 and CX3CR1 were virtually absent. However, high proportion of UCB-NK cells expressed CCR6 (52612%) and CXCR3 (65621%). Expression of CXCR4 as well as the adhesion molecule CD62L (L-selectin) were also detected typically on 10-20% of UCB-NK cells at the end of the culture process. Results obtained from in vitro migration assays were consistent with this chemokine receptor profile ( Figure 1C). Notably, the proportion of CD56 + UCB-NK cells migrating in response to the chemokine CXCL12 was similar to level of CXCR4-expressing cells, and specific migration towards the chemokines CCL20 (CCR6 ligand), CXCL10 and CXCL11 (both CXCR3 ligands) were confirmed. These data indicate that UCB-NK cells functionally express CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, data suggest that UCB-NK cells have the capacity to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis.

Development of in vivo UCB-NK Cell Tracking
Next, we aimed to establish a method using 111 In-oxinate as radiolabel and SPECT-CT imaging that could be exploited both at the pre-clinical level in mice and for clinical studies in humans, to monitor early distribution of UCB-NK cell following infusion in relation to anti-leukemic potency in BM. To address the feasibility of this methodology, increasing doses of UCB-NK cells previously labeled with 111 In-oxinate ( 111 In-NK cells; 2MBq of 111 In-oxinate was added per 10 6 cells and labeling efficiency was 55%) were injected i.v. into NSG mice. Whole body scans acquired 1 and 24 h after infusion showed that the 111 In-activity first localized in the lungs, and thereafter redistributed to the spleen, liver and BM (Figure 2A). At 24 h, liver and spleen were visible at all doses of infused 111 In-NK cells, while the activity present in BM was detected only in mice injected with $5610 6 111 In-NK cells. To address the specificity of 111 In-NK cell in vivo imaging, we also analyzed the distribution of 111 In-activity following injection of either a lysate obtained from 111 In-NK cells or 111 In-oxinate ( Figure 2B). In both cases, after 24 h, the activity was mainly visualized in kidneys. Weak uptake of 111 In was noticed in liver and lungs following injection of 111 In-lysate and 111 In-oxinate, respectively.
After imaging, mice were euthanized, organs were collected, weighted, and used to analyse quantitatively the distribution of the 111 In-activity as described in materials and methods. All tissues examined following injection of 111 In-oxinate exhibited similar level of 111 In compared to that measured in blood, indicating that the 111 In activity remained in the circulation, resulting in visualization of well perfused organs. In mice injected with 111 In-lysate, the activity mainly accumulated in the kidneys, representing ,20% of the injected dose (data not shown). Slightly enhanced 111 In activity levels were also measured in liver and lymphoid organs. In contrast, following infusion of 111 In-NK cells, the proportions of activity quantified in BM, spleen, liver and lungs were strongly increased compared to blood level. Values were also significantly higher compared to those determined following injection of 111 In-lysate and 111 In-oxinate ( Figure 2C), indicating that the accumulation of 111 In activity in these organs could be attributed to the accumulation of 111 In-NK cells. Notably, the distribution of 111 In activity was similar at all doses of infused 111 In-NK cells ( Figure S1). Moreover, human CD45 + CD56 + NK cells were clearly identified in the same organs, except kidneys, by ex vivo flow cytometric analysis performed 24 h after infusion of UCB-NK cells (Figure 3). All together, these data show that SPECT-CT imaging allows tracking of 111 In-NK cells in vivo with good sensitivity and specificity, and that UCB-NK cells, after a brief period of retention in the lungs, rapidly traffic to the liver, spleen and BM.

A Consistent Proportion of UCB-NK Cells Accumulates in Mouse BM Following Adoptive Transfer
After demonstrating the feasibility of 111 In-NK cell tracking in vivo, we aimed to determine quantitatively the biodistribution of UCB-NK cells in 2 independent experiments. To this end, we first optimized the labelling procedure by incubating increasing numbers of NK cells with 2 MBq 111 In. Good 111 In labelling efficiency and cell recovery were achieved using .4610 6 cells ( Figure 4A). Based on these findings, 0.4 MBq of 111 In-oxinate was added per 10 6 cells for subsequent experiments where labeling efficiency always exceeded 80%, cell viability .90% and cell recovery .95% (data not shown). In addition, this procedure did not affect the migration capacity of UCB-NK cells towards the prototypic BM-chemokine CXCL12 in vitro ( Figure 4B).
As observed in the pilot studies, we found the highest activity concentration in the spleen, followed by liver, BM and lungs at 24 h after infusion. Taking into account total organ weights, nearly 70% (64.962.1) of the ID was present in liver, while spleen and lungs contained 3.161.2%ID and 3.961.9%ID, respectively ( Figure 4C). For BM, 0.3260.05%ID was quantified per femur (data not shown). Assuming that one femur accounts for 6.7% of the total BM in adult mouse [14], we estimated at ,5% the fraction of 111 In-NK cells accumulating within 24 h in the total BM compartment. Accordingly, a homogenous 111 In-signal was visualized by SPECT-CT imaging in all bones ( Figure 4D). These data indicate that a significant percentage of UCB-NK cells are able to migrate to the mouse BM following adoptive transfer.

Low Dose Human IL-15 Drives UCB-NK Cell Expansion in vivo
Next to BM homing, we aimed to evaluate the survival potential of UCB-NK cells following adoptive transfer. Indeed, clinical responses reported so far always occurred with concomitant donor NK cell persistence and even expansion within the first two weeks after infusion. Interestingly, conditioning regimens that allowed successful alloNK cell engraftment also resulted in transient elevation of endogenous IL-15 [9]. Therefore, we examined UCB-NK cell survival potential upon adoptive transfer in NSG mice in the presence of low dose IL-15 support. In a first experiment, we observed that daily administration of IL-15 mediated efficient expansion of infused UCB-NK cells in vivo ( Figure S2). In contrast to mice injected with UCB-NK cells alone, a clear population of human CD45 + CD56 + cells could be visualized in mice co-injected with IL-15, representing 3.5% of total leukocytes at day 7, which Ex vivo flow cytometric analysis confirmed trafficking of UCB-NK cells through lymphoid tissues, liver and lungs following adoptive transfer in NSG mice. Two adult NSG mice were infused i.v. with 10610 6 UCB-NK cells. The day after, mice were sacrificed, organs collected and used to prepare cell suspension for ex vivo flow cytometric analysis following erythrocyte lysis. One additional non-injected mouse was used as control. Presence of human CD45 + CD56 + NK cells was confirmed in all examined tissues except kidneys. Dot plots gated on total living cells are shown. doi:10.1371/journal.pone.0064384.g003 further increased till 4.5% at day 14. Nevertheless, NK cell numbers rapidly declined after removal of IL-15 ( Figure S2). We next intended to confirm these findings with a different UCB donor and to examine UCB-NK cell engraftment level in lymphoid tissues. To this end, mice received a single infusion of 5610 6 UCB-NK cells, and percentages of circulating human NK cells were monitored in peripheral blood collected at 1, 7 and 14 days later. Here, NSG mice were given intermittent injections of IL-15, thereby reducing the IL-15 support compared to the first experiment. Consistent with previous observations, human NK cells were almost absent at day 7 in mice co-treated with PBS. In contrast, mice co-injected with IL-15 displayed stable levels of NK cells from day 1 to day 7, which even increased in 4 out 5 mice at day 14 ( Figure 5A). In addition, significant percentages of UCB-NK cells were identified in spleen and BM by day 14 ( Figure 5B). Moreover, examination of the NK cell phenotype in these tissues revealed high expression level of CD16 and KIR receptors, which were strongly increased compared to the infused UCB-NK cell product ( Figure 5C). All together, these data demonstrate that low dose IL-15 mediates efficient UCB-NK cell survival and expansion in vivo, and that further differentiation of UCB-NK cell products occurs rapidly following adoptive transfer.

Adoptive Transfer of UCB-NK Cells Inhibits Growth of BMresiding Human Leukemia Cells in Mice
Finally, to evaluate the cytolytic activity of UCB-NK cells in vivo, we developed a leukemia xenograft model by injecting adult NSG mice with leukemia cells intra-femorally (i.f.), therefore requiring BM homing by infused UCB-NK cells to achieve anti-leukemic response. For this, we used K562 leukemia cells expressing  Figure 6A). A dose of 20610 6 NK cells per mouse was defined based on biodistribution studies to approach an effector to target ratio of 1:1 in vivo. The antileukemic potential of UCB-NK cells to target K562.LucGFP cells was evaluated in two independent experiments with similar outcomes ( Figure 6B). Comparable K562 cell engraftment was observed in both experiments and all control mice treated with PBS and IL-15 except one displayed detectable i.f. tumors by day 15 (Figure 6B-C). In striking contrast, 8 out of 12 mice treated with UCB-NK cells had undetectable tumor load by day 15. Significant inhibition of K562.LucGFP cell progression following UCB-NK cells infusion was demonstrated by BLI in time ( Figure 6D). Most importantly, a single infusion of ex vivogenerated UCB-NK cells in combination with low-dose IL-15 prolonged survival of K562 i.f. injected mice of which 25% showed long-term protection ( Figure 6E-F). These results demonstrate that UCB-NK cells are functional following adoptive transfer, and that they are able to target and eliminate BM-residing leukemia cells in vivo.

Discussion
To date, it is well established that NK cells mediate efficient graft-versus-leukemia reactivity with improved control of relapse in AML patients following alloSCT. This raises the interest in exploiting NK cells for adoptive immunotherapy, particularly as an adjuvant treatment approach to chemotherapy for elderly, high-risk and refractory AML patients. Most trials reported so far employed peripheral blood derived-NK cells enriched by CD3 depletion with or without CD56 selection from donor apheresis products, and showed that enriched NK cell infusions are well tolerated, without induction of GVHD or severe toxicity [9][10][11]18]. Nevertheless, the clinical impact of NK cell-based therapy remains inconsistent and several issues need to be optimized to achieve clinical efficacy. In addition to conditioning regimens that prevent rapid graft rejection and supply of exogenous cytokines  like IL-2 supporting NK cell survival upon adoptive transfer, the purity, the activation and the number of cells that can be infused in patients are critical factors determining clinical response [9,11]. Since, new methodologies are currently emerging to generate higher numbers of activated NK cells, including in vitro expansion of donor-derived NK cells before infusion [19], as well as NK cell generation from CD34 + hematopoietic stem/progenitor cells [12,20,21]. In particular, the availability of cryo-preserved UCB units and the development of GMP-compliant culture systems constitute a very attractive approach to exploit the potential of NK cell-based adoptive immunotherapy, with generation of a clinically relevant dosages of UCB-NK cells with high purity and cytolytic activity in vitro [13].
The present study was designed to pre-clinically evaluate the in vivo anti-leukemic potential of UCB-NK cells generated with our GMP-compliant culture system. To this end, we developed a mouse model in which human K562.LucGFP leukemia cells are directly implanted into the femur of NSG mice, thus requiring BM-specific homing of infused UCB-NK cells to achieve antitumor response. In this model, we showed that treatment with UCB-NK cells in combination with supportive IL-15 potently inhibited progression of K562 cells, thereby demonstrating that UCB-NK cells are functional in vivo and have the capacity to target leukemia cells within BM upon adoptive transfer. Importantly, prolongation of mice survival including complete and persistent response in 25% of the mice was achieved following a single infusion of UCB-NK cells and in the presence of low dose IL-15 support. Considering that complete protection against K562 cells in vivo was mostly reported following multiple NK cell infusions and/or prolonged high-dose IL-2 administration [21][22][23][24], we believe that our results nicely illustrates the therapeutic potential of UCB-NK cells in the clinic. In addition, biodistribution analysis showed that a relatively small proportion of our current UCB-NK cell product accumulates in one femur within 24 h (,0.3% of 20610 6 cells, i.e. ,1610 5 UCB-NK cells per femur), suggesting that inhibition of leukemia cell growth primarily happened at a low effector to target ratio what also illustrates the high cytolytic potential of ex vivo generated UCB-NK cells.
Our phenotypical analysis and in vitro migration studies support that UCB-NK cells can actively home to BM following adoptive transfer. Consistent expression of the chemokine receptor CXCR4 was detected on UCB-NK cells at the end of the culture process, and robust migration in response to its ligand CXCL12 was shown in vitro. Implication of the CXCR4/CXCL12 axis in regulating the migration of human T and NK cells to BM has been reported in several studies [25,26], including in human metastatic prostate cancer [27]. Therefore, it is likely that UCB-NK cell homing to BM is CXCR4/CXCL12-dependent. Nevertheless, we also showed that UCB-NK cells display high expression of CXCR3 and CCR6, two receptors that could also regulate UCB-NK cell trafficking to inflamed tissues in vivo. Future studies are now warranted to demonstrate the role of the CXCR4/CXCL12 axis in UCB-NK cell homing to BM and to examine its implication in patients among other inflammatory pathways like CXCR3/ CXCL10-11. Also, trafficking of adoptively transferred NK cells has not been addressed in leukemia patients yet. Use of 111 In for in vivo tracking of adoptively transferred NK cells has already been reported in patients with renal cell carcinoma and liver metastases [28,29], and at the pre-clinical level in xenograft mouse models of leukemia [23]. Similar to our findings, early distribution of NK cells in the lungs, and later in the liver, spleen and eventually BM was visualized following systemic infusion. However, detailed organ distribution of NK cells as well as demonstration of the specificity of the visualized signal, particularly in BM, were not available. Here, we showed that 111 In-based NK cell tracking provides good specificity and sensitivity, and will constitute a useful method to study and correlate effective BM targeting with clinical response in patients.
We reported previously that UCB-NK cells generated with our GMP-compliant culture system display a relatively low expression level of CD16 and KIRs at the end of the culture process [12]. However, we observed that the proportions of UCB-NK cells expressing these markers were strongly increased two weeks after adoptive transfer, indicating that UCB-NK cells can further differentiate in vivo into a more mature cell population. These data are in agreement with Huntington et al. who showed that rapid IL-15 driven-transition of human CD56 hi CD16 2 KIR 2 to CD56 dim CD16 + KIR + NK cells occurs in vivo [30]. Since KIRligand mismatch triggers NK cell alloreactivity towards AML, such phenotypic modifications might have important implications in patients. In that view, UCB-NK cell reactivity towards AML in vivo should be further addressed using HLA-expressing AML cells in our i.f. NSG model.
In conclusion, our results strongly support that UCB-NK cells constitute promising immunotherapeutic products to improve the treatment of AML, as demonstrated by their capability to migrate to BM and to inhibit progression of human leukemia cells following adoptive transfer. In addition, we demonstrated efficient UCB-NK survival in vivo in the presence of low dose human IL-15. Transient elevation of IL-15 plasma levels was reported in AML patients following immunosuppressive Cy/Flu conditioning [9,10], what could favor in vivo expansion and maturation of UCB-NK cells as well as clinical responses following UCB-NK cell adoptive transfer. Since BM is the primary site of AML development and encloses niches essential for leukemic stem cells causing relapse [31], we believe that BM targeting is essential for elimination of minimal residual disease and induction of optimal and persistent clinical responses against AML. Strategies that aim at increasing BM-specific chemokine receptors, like CXCR4, on UCB-NK cells are now considered to enhance BM targeting. In addition, the methodologies that we report here for UCB-NK cell tracking and anti-leukemic effect monitoring will be instrumental to validate future findings and to fully exploit the potential of UCB-NK cells against AML and other hematological malignancies. Figure S1 The biodistribution of 111 In-NK cells upon adoptive transfer is reproducible between animals and independent of the dose of injected cells.