scholarly journals Cryopreservation impairs cytotoxicity and migration of NK cells in 3-D tissue: Implications for cancer immunotherapy

2019 ◽  
Author(s):  
Christoph Mark ◽  
Tina Czerwinski ◽  
Susanne Roessner ◽  
Astrid Mainka ◽  
Franziska Hörsch ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Cell Function ◽  
Nk Cell ◽  
Target Cells ◽  
Collagen Gels ◽  
Nk Cell Therapy

AbstractNatural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation for clinical use, the cells are cryopreserved to bridge the necessary evaluation time. While a degranulation assay confirms the ability of cryopreserved NK cells to kill target cells, we find a significant decrease of cytotoxicity after cryopreservation in a chromium release assay. We complement these standard assays with measurements of NK cell motility and cytotoxicity in 3-dimensional (3-D) collagen gels that serve as a substitute for connective tissue. We find a 5.6 fold decrease of cytotoxicity after cryopreservation and establish that this is mainly caused by a 6-fold decrease in the fraction of motile NK cells. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.SynopsisCryopreservation of natural killer (NK) cells dramatically impairs their motility and cytotoxicity in tissue. This finding may explain the persistent failure of clinical trials in which NK cell therapy is used for treating solid tumors.

scholarly journals 799 Neoantigen-cytokine-chemokine multifunctional natural killer cell engager for immunotherapy of solid tumors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A834-A834
Author(s):  
Xue Yao ◽  
Sandro Matosevic
Keyword(s):  
Tumor Microenvironment ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Nk Cell ◽  
Killer Cell ◽  
Nk Cell Therapy

BackgroundThe effectiveness of natural killer (NK) cell-based immunotherapy against solid tumors is limited by the lack of specific antigens and the immunosuppressive tumor microenvironment (TME). Glioblastoma multiforme (GBM) is one such heavily immunosuppressive tumor that has been particularly hard to target and remains without a viable treatment. The development of novel approaches to enhance the efficacy of NK cells against GBM is urgently needed. NK cell engagers (NKCE) have been developed to enhance the efficacy of NK cell therapy.MethodsTo improve the clinical efficacy of NK cell therapy, we are developing a new generation of multi-specific killer engagers, which consists of a neoantigen-targeting moiety, together with cytokine and chemokine-producing domains. Neoantigens are new antigens formed specifically in tumor cells due to genome mutations, making them highly specific tools to target tumor cells. Our engager has been designed to target Wilms' tumor-1 (WT-1), a highly specific antigen overexpressed in GBM among other solid tumors. This is done through the generation of an scFv specific targeting the complex of WT-1126-134/HLA-A*02:01 on the surface of GBM. On the NK cell side, the engager is designed to target the activating receptor NKp46. Incorporation of the cytokine IL-15 within the engager supports the maturation, persistence, and expansion of NK cells in vivo while favoring their proliferation and survival in the tumor microenvironment. Additionally, our data indicated that the chemokine CXCL10 plays an important role in the infiltration of NK cells into GBM, however, GBM tumors produce low levels of this chemokine. Incorporation of a CXCL10-producing function into our engager supports intratumoral NK cell trafficking by promoting, through their synthetic production, increased levels of CXCL10 locally in the tumor microenvironment.ResultsCollectively, this has resulted in a novel multifunctional NK cell engager, combining neoantigen-cytokine-chemokine elements fused to an activating domain-specific to NK cells, and we have investigated its ability to support and enhance NK cell-mediated cytotoxicity against solid tumors in vitro and in vivo against patient-derived GBM models. The multi-specific engager shows both high tumor specificity, as well as the ability to overcome NK cell dysfunction encountered in the GBM TME.ConclusionsWe hypothesize that taking advantage of our multi-functional engager, NK cells will exhibit superior ex vivo expansion, infiltration, and antitumor activity in the treatment of GBM and other solid tumors.

scholarly journals Natural Killer Cell Therapy: A New Treatment Paradigm for Solid Tumors

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1534 ◽  
Author(s):  
Sooyeon Oh ◽  
Joo-Ho Lee ◽  
KyuBum Kwack ◽  
Sang-Woon Choi
Keyword(s):  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Nk Cell ◽  
Ex Vivo ◽  
Killer Cell ◽  
Genetic Defects ◽  
New Paradigm ◽  
Nk Cell Therapy

In treatments of solid tumors, adoptive transfer of ex vivo expanded natural killer (NK) cells has dawned as a new paradigm. Compared with cytotoxic T lymphocytes, NK cells take a unique position targeting tumor cells that evade the host immune surveillance by down-regulating self-antigen presentation. Recent findings highlighted that NK cells can even target cancer stem cells. The efficacy of allogeneic NK cells has been widely investigated in the treatment of hematologic malignancies. In solid tumors, both autologous and allogeneic NK cells have demonstrated potential efficacy. In allogeneic NK cell therapy, the mismatch between the killer cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) can be harnessed to increase the antitumor activity. However, the allogeneic NK cells cause more adverse events and can be rejected by the host immune system after repeated injections. In this regard, the autologous NK cell therapy is safer. This article reviews the published results of clinical trials and discusses strategies to enhance the efficacy of the NK cell therapy. The difference in immunophenotype of the ex vivo expanded NK cells resulted from different culture methods may affect the final efficacy. Furthermore, currently available standard anticancer therapy, molecularly targeted agents, and checkpoint inhibitors may directly or indirectly enhance the efficacy of NK cell therapy. A recent study discovered that NK cell specific genetic defects are closely associated with the tumor immune microenvironment that determines clinical outcomes. This finding warrants future investigations to find the implication of NK cell specific genetic defects in cancer development and treatment, and NK cell deficiency syndrome should be revisited to enhance our understanding. Overall, it is clear that NK cell therapy is safe and promises a new paradigm for the treatment of solid tumors.

scholarly journals Cryopreservation impairs 3-D migration and cytotoxicity of natural killer cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Christoph Mark ◽  
Tina Czerwinski ◽  
Susanne Roessner ◽  
Astrid Mainka ◽  
Franziska Hörsch ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Cell Function ◽  
Nk Cell ◽  
Safety Evaluation ◽  
Collagen Gel ◽  
Target Cells ◽  
Effector Cells ◽  
Chromium Release

Abstract Natural killer (NK) cells are important effector cells in the immune response to cancer. Clinical trials on adoptively transferred NK cells in patients with solid tumors, however, have thus far been unsuccessful. As NK cells need to pass stringent safety evaluation tests before clinical use, the cells are cryopreserved to bridge the necessary evaluation time. Standard degranulation and chromium release cytotoxicity assays confirm the ability of cryopreserved NK cells to kill target cells. Here, we report that tumor cells embedded in a 3-dimensional collagen gel, however, are killed by cryopreserved NK cells at a 5.6-fold lower rate compared to fresh NK cells. This difference is mainly caused by a 6-fold decrease in the fraction of motile NK cells after cryopreservation. These findings may explain the persistent failure of NK cell therapy in patients with solid tumors and highlight the crucial role of a 3-D environment for testing NK cell function.

scholarly journals The Ly-49D Receptor Activates Murine Natural Killer Cells

1996 ◽  
Vol 184 (6) ◽  
pp. 2119-2128 ◽  
Author(s):  
L.H. Mason ◽  
S.K. Anderson ◽  
W.M. Yokoyama ◽  
H.R.C. Smith ◽  
R. Winkler-Pickett ◽  
...  
Keyword(s):  
Gene Family ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Cell Subset ◽  
Class I ◽  
Target Cells ◽  
Color Flow ◽  
Functional Capabilities

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immunoprecipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I–bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of FcγR+ target cells in a reverse antibody-dependent cellular cytotoxicity–type assay and induces apoptosis in Ly49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/IxYxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

Ex Vivo Activated Natural Killer (NK) Cells from Myeloma Patients Kill Autologous Myeloma and Killing Is Enhanced by Elotuzumab

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3666-3666
Author(s):  
Tarun K. Garg ◽  
Susann Szmania ◽  
Jumei Shi ◽  
Katie Stone ◽  
Amberly Moreno-Bost ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Therapy ◽  
Nk Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
K562 Cells ◽  
Scid Mice ◽  
Target Cells ◽  
Recent Trial ◽  
Nk Cell Therapy

Abstract Immune-based therapies may improve outcome for multiple myeloma (MM) by eradicating chemo-resistant disease. Our recent trial utilizing IL2 activated, killer immunoglobulin-like receptor-ligand mismatched NK cell transfusions from haplo-identical donors yielded (n) CR in 50% of patients. Unfortunately, after NK cell therapy, 2/10 patients had progressive disease, and the median duration of response for the other 8/10 patients was only 105 days (range 58–593). This may have been due to an insufficient dose of alloreactive NK cells and early rejection. Furthermore, appropriate donors were identified for only 30% of otherwise eligible patients. We therefore investigated whether NK cells from MM patients could be expanded and activated to kill autologous MM. We then examined whether pre-treatment of MM cell targets with elotuzumab, a humanized antibody to the MM tumor antigen CS1, could further enhance NK cell-mediated lysis. PBMC from 5 MM patients were co-cultured for 14 days with irradiated K562 cells transfected with 4-1BBL and membrane bound IL15 in the presence of IL2 (300U/ml) as previously described (Imai et al, Blood2005;106:376–383). The degree of NK cell expansion, NK immunophenotype, and ability to kill MM (4 hour 51Cr release assays) were assessed. To determine the ability of ex vivo expanded NK cells to traffic to bone marrow, activated NK cells were injected into the tail vein of NK cell depleted NOD-SCID mice, which were then sacrificed after 48 hours. Flow cytometry for human CD45, CD3, and CD56 was performed on cells from blood, marrow and spleen. There was an average 64-fold expansion of NK cells (range: 8–200) after 2 weeks of co-culture with K562 transfectants. Expansion of T cells was not observed. The NK cell activating receptor NKG2D, and natural cytotoxicity receptors NKp30, NKp44, and NKp46 were up-regulated following the expansion. Expanded NK cells were able to kill autologous MM (E:T ratio 10:1, average 31%, range 22–41%), whereas resting NK cells did not. Pretreatment of autologous MM cells with elotuzumab increased the activated NK cell-mediated killing by 1.7-fold over target cells pretreated with an isotype control antibody. This level of killing was similar to that of the highly NK kill-sensitive cell line K562 (Figure). Autologous PHA blasts and CD34+ stem cells were not killed. Activated human NK cells were detectable in the bone marrow of NOD-SCID mice 48 hours after injection. Ex vivo activation of NK cells from MM patients with K562 transfectants can induce killing of autologous MM and produce large numbers of NK cells for potential therapy. The addition of elotuzumab to activated NK cell therapy enhances anti-MM effects by ADCC thus invoking an additional NK cell-mediated mechanism of MM killing. Importantly, ex vivo activated NK cells traffic to the bone marrow in mice. Autologous NK cell therapy eliminates the issues related to allo-donor availability and early NK cell rejection, and could provide an option for patients refractory to chemotherapy agents. Figure Figure

scholarly journals Glycosylation Affects Ligand Binding and Function of the Activating Natural Killer Cell Receptor 2B4 (CD244) Protein

2011 ◽  
Vol 286 (27) ◽  
pp. 24142-24149 ◽  
Author(s):  
Stefanie Margraf-Schönfeld ◽  
Carolin Böhm ◽  
Carsten Watzl
Keyword(s):  
Ligand Binding ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Function ◽  
Nk Cell ◽  
Negative Impact ◽  
Killer Cell ◽  
Target Cells ◽  
Functional Assay ◽  
Cell Responses

2B4 (CD244) is an important activating receptor for the regulation of natural killer (NK) cell responses. Here we show that 2B4 is heavily and differentially glycosylated in primary human NK cells and NK cell lines. The differential glycosylation could be attributed to sialic acid residues on N- and O-linked carbohydrates. Using a recombinant fusion protein of the extracellular domain of 2B4, we demonstrate that N-linked glycosylation of 2B4 is essential for the binding to its ligand CD48. In contrast, sialylation of 2B4 has a negative impact on ligand binding, as the interaction between 2B4 and CD48 is increased after the removal of sialic acids. This was confirmed in a functional assay system, where the desialylation of NK cells or the inhibition of O-linked glycosylation resulted in increased 2B4-mediated lysis of CD48-expressing tumor target cells. These data demonstrate that glycosylation has an important impact on 2B4-mediated NK cell function and suggest that regulated changes in glycosylation during NK cell development and activation might be involved in the regulation of NK cell responses.

scholarly journals CD38 contributes to human natural killer cell responses through a role in immune synapse formation

2018 ◽  
Author(s):  
Mathieu Le Gars ◽  
Christof Seiler ◽  
Alexander W. Kay ◽  
Nicholas L. Bayless ◽  
Elsa Sola ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Tumor Cells ◽  
Synapse Formation ◽  
Cell Function ◽  
Nk Cell ◽  
Critical Role ◽  
Target Cells ◽  
Immune Synapse ◽  
Ifn Γ

AbstractNatural killer (NK) cells use a diverse array of activating and inhibitory surface receptors to detect threats and provide an early line of defense against viral infections and cancer. Here, we demonstrate that the cell surface protein CD38 is a key human NK cell functional receptor through a role in immune synapse formation. CD38 expression marks a mature subset of human NK cells with a high functional capacity. NK cells expressing high levels of CD38 display enhanced killing and IFN-γ secretion in response to influenza virus-infected and tumor cells. Inhibition of CD38 enzymatic activity does not influence NK cell function, but blockade of CD38 and its ligand CD31 abrogates killing and IFN-γ expression in response to influenza-infected cells. Blockade of CD38 on NK cells similarly inhibits killing of tumor cells. CD38 localizes and accumulates at the immune synapse between NK cells and their targets, and blocking CD38 severely abrogates the ability of NK cells to form conjugates and immune synapses with target cells. Thus, CD38 plays a critical role in NK cell immune synapse formation. These findings open new avenues in immunotherapeutic development for cancer and infection by revealing a critical role for CD38 in NK cell function.

scholarly journals CD38 knockout natural killer cells expressing an affinity optimized CD38 chimeric antigen receptor successfully target acute myeloid leukemia with reduced effector cell fratricide.

Haematologica ◽  
2020 ◽  
Author(s):  
Mark Gurney ◽  
Arwen Stikvoort ◽  
Emma Nolan ◽  
Lucy Kirkham-McCarthy ◽  
Stanislav Khoruzhenko ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Therapy ◽  
Nk Cells ◽  
Natural Killer ◽  
Chimeric Antigen Receptor ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Cd38 Expression ◽  
Nk Cell Therapy ◽  
Acute Myeloid

There is a strong biological rationale for the augmentation of allogeneic natural killer (NK) cell therapies with a chimeric antigen receptor (CAR) to enhance acute myeloid leukemia (AML) targeting. CD38 is an established immunotherapeutic target in multiple myeloma and under investigation as a target antigen in AML. CD38 expression on NK cells and its further induction during ex vivo NK cell expansion represents a barrier to the development of a CD38 CAR-NK cell therapy. We set out to develop a CD38 CAR-NK cell therapy for AML, first by using an NK cell line which has low baseline CD38 expression and subsequently healthy donor expanded NK cells. To overcome anticipated fratricide due to NK cell CD38 expression when using primary expanded NK cells, we applied CRISPR/Cas9 genome editing to disrupt the CD38 gene during expansion achieving a mean knockdown efficiency of 84%. The resulting CD38 KD expanded NK cells, after expression of an affinity optimized CD38 CAR, showed reduced NK cell fratricide and an enhanced ability to target primary AML blasts. Furthermore, the cytotoxic potential of CD38 CAR-NK cells was augmented by pre-treatment of the AML cells with all-trans retinoic acid which drove enhanced CD38 expression offering a rational combination therapy. These findings support the further investigation of CD38 KD - CD38 CAR-NK cells as a viable immunotherapeutic approach to the treatment of AML.

Artificial Antigen Presenting Cells (aAPC) Expanded NK-Mediated Enhanced Lysis of Allogeneic Tumor Cells Regardless of HLA Class I/KIR Mismatch and Its Implication of Use in Eradicating Acute Lymphoblastic Leukemia (ALL).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3023-3023
Author(s):  
Hua Zhang ◽  
Bruce Levine ◽  
Nga Voong ◽  
Alan S. Wayne ◽  
Carl H. June ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Nk Cells ◽  
Natural Killer ◽  
Solid Tumors ◽  
Tumor Cells ◽  
Fusion Proteins ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Target Cells ◽  
Activating Receptors

Abstract Abstract 3023 Poster Board II-999 NK Killer cell immunoglobulin-like receptors (KIRs) and their human leukocyte antigen (HLA) ligands play critical roles in maintaining natural killer (NK) cell tolerance, while providing surveillance against pathogens and malignant transformation. Natural killer (NK) cells have been explored as tools for adoptive anti-tumor or leukemia immunotherapy and current models hold that a mismatch or absence of KIR ligands on target cells is essential for efficient NK cell mediated cytolysis. However, new approaches are now available to activate NK cells and the role for KIR mediated signaling in regulating cytotoxicity of activated NK cells has not been well studied. In this study, aAPCs comprising IL15Ra+K562 cells engineered to express 4-1BBL activated and expanded peripheral NK cells in the presence of exogenous IL15 up to 1000-fold in 3 weeks. Compared to resting NK cells, 4-1BBL/IL15-activated NK cells upregulated TRAIL and NKp30, 44, 46 expression, and showed significantly enhanced cytotoxicity against a multitude of tumor targets including K562, Daudi, Ewing's tumors, osteosarcoma, as well as autologous tumors (50%-90% killing vs. 0%-8% with non-activated NK cells). Meanwhile we could detect little to no influence of KIR signaling in regulating cytotoxicity by aAPC activated NK cells, since sorted CD158a+ and CD158b+ activated NK cells showed similar killing of tumor cells expressing HLA group C1 (CD158b ligand) and/or C2 (CD158a ligand) antigens. In contrast, killer activating receptors (KARs) were indispensable for the cytolysis of solid pediatric tumors by aAPC-activated NK cells, since the killing was significantly inhibited by fusion proteins binding to the ligands of NKG2D, NK p30, p44, p46, p80 (KARs). About 20-40% inhibition of the killing was accomplished when all four activating receptors were blocked, though other activating receptors have not been well defined. Although acute lymphoblastic leukemia (ALL) blasts were refractory to fresh NK cytotoxicity, 4-1BBL/IL15 activated NK cells demonstrated higher lytic activities (20%-50%) against ALL blasts from either patients or cell lines. ALL blast lysis could be completely or partially inhibited by KAR-blocking fusion proteins, indicating that expression levels of KAR ligands vary among ALL cases and other solid tumors. We conclude that KIR ligand mismatch or absence is not essential for effective NK cytotoxicities on either solid tumors or ALL when fully activated NK cells are utilized. This suggests that adoptive therapy with autologous aAPC-activated NK cells may prove effective in some clinical settings, such as ALL, AML, or certain solid tumors. Further studies to assess the impact of KAR ligand expression on aAPC-activated NK killing of ALL blasts are in progress. Percentage of Activated NK Killings vs. Fresh NK's with/without KAR-Ig Fusion Proteins Activated NK (E:T=2.5:1) Fresh NK (E:T=25:1) -KAR-Ig Fc +KAR-Ig Fc SB tumor (Ewing's) 48% 30% 0.5% HOS (Osteo sarcoma) 63% 36% 0.7% Daudi (B. lymphoma) 78% 46% 0.2% REH (ALL) 54% 8% 3% Disclosures No relevant conflicts of interest to declare.

Novel Approach for NK Cell Therapy for Cancer

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3836-3836 ◽  
Author(s):  
Reshmi Parameswaran ◽  
David N. Wald ◽  
Marcos De Lima ◽  
Dean A. Lee ◽  
Stephen Moreton
Keyword(s):  
Cell Therapy ◽  
Nk Cells ◽  
Nk Cell ◽  
Model Systems ◽  
Target Cells ◽  
Adhesion Assay ◽  
Mouse Bone Marrow ◽  
Clinical Potential ◽  
Gsk3 Inhibitor ◽  
Nk Cell Therapy

Abstract Novel therapeutic approaches are urgently needed for many malignancies such as Acute Myeloid Leukemia (AML). We have developed a new therapeutic strategy based upon NK cell immunotherapy that exhibits high clinical potential based upon cell and animal studies. While the harnessing of NK cells for cellular therapy against malignancies has been a topic of interest for several decades, our approach overcomes a major hurdle of insufficient NK cell cytotoxic activity. We have identified that targeting the kinase GSK3 through pharmacologic and genetic approaches leads to the hyperactivation of human blood derived NK cells and a significant improvement in efficacy as compared to traditionally used activated NK cells or chemotherapy in our mouse AML model systems. Importantly this GSK3 inhibition can be achieved through a short ex-vivo incubation of NK cells with a GSK3 inhibitor paving the way for a rapid implementation into a clinical trial. Utilizing both in vitro studies with AML cell lines (ex. OCI-AML3 and HL-60)) and primary human AML cells we observe approximately a 50% increase in efficacy with GSK3 inhibited NK cells as compared to untreated NK cells. Further, we demonstrate significant efficacy of GSK3 inhibited NK cells in a mouse model of circulating human AML. After 4 weekly injections of human NK cells, there is a 50% greater reduction in human AML cells present in the mouse bone marrow with GSK3 inhibited NK cells as compared to vehicle treated NK cells. Besides efficacy studies, our work has led a model of how GSK3-inhibition enhances NK cell activity as depicted in figure 1. GSK3 inhibition leads to a dramatic increase in adhesion of NK cells to target cells as demonstrated by a flow cytometric adhesion assay (49% vs 83% after 20 min incubation) as well as live cell imaging. Consistent with the increased adhesion, GSK3 inhibited NK cells as well as target cells (after co-incubation) exhibit increased expression of essential NK cell-target adhesion molecules including L-selectin (on NK cells) and ICAM (on target cells). The induction of ICAM on target cells is due to a marked induction in TNFa production from the NK cells upon incubation with target cells (>7 fold increase in TNFa production). TNFa neutralization impairs the NK activity of the GSK3 inhibited NK cells (~30%) but not vehicle treated cells. Finally, GSK3 inhibition also leads to changes in the NK cells that enhance activity such as increased expression of granzyme and perforin and secretion of IFNg. Overall, our work has a revealed a novel strategy for NK cell therapy that holds high clinical potential. Figure 1. Model of how GSK3 inhibition leads to hyperactive NK cells. GSK3I - GSK3 inhibitor Figure 1. Model of how GSK3 inhibition leads to hyperactive NK cells. GSK3I - GSK3 inhibitor Disclosures No relevant conflicts of interest to declare.

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