Ruxolitinib partially reverses functional NK cell deficiency in patients withSTAT1gain-of-function mutations

AbstractBackgroundNatural Killer (NK) cells are critical innate effector cells whose development is dependent on the JAK-STAT pathway. NK deficiency can result in severe or refractory viral infections. Patients with Signal Transducer and Activator of Transcription (STAT)1 gain of function (GOF) mutations have increased viral susceptibility.ObjectiveWe sought to investigate NK cell function in STAT1 GOF patients. Methods: NK cell phenotype and function were determined in 16 STAT1 GOF patients.MethodsNK cell phenotype and function were determined in 16 STAT1 GOF patients.NK cell lines expressing patient mutations were generated with CRISPR-Cas9 mediated gene editing. STAT1 GOF NK cells were treated in vitro with ruxolitinib.ResultsPeripheral blood NK cells from of STAT1 GOF patients had impaired terminal maturation. Specifically, patients withSTAT1 GOFmutations have immature CD56dimNK cells with decreased expression of CD16, perforin, CD57 and impaired cytolytic function. STAT1 phosphorylation was elevated but STAT5 was aberrantly phosphorylated in response to IL-2 stimulation. Upstream inhibition of STAT signaling with the small molecule JAK1/2 inhibitor ruxolitinibin vitroandin vivorestored perforin expression in CD56dimNK cells and partially restored NK cell cytotoxic function.ConclusionsProperly regulated STAT1 signaling is critical for NK cell maturation and function. Modulation of elevated STAT1 phosphorylation with ruxolitinib is an important option for therapeutic intervention in patients withSTAT1 GOFmutations.

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In Vivo Antagonistic Effects of Dexamethasone On Lenalidomide-Induced NK Cell Activation.

Blood ◽

10.1182/blood.v114.22.1639.1639 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1639-1639 ◽

Cited By ~ 1

Author(s):

Hang Quach ◽

Hsu Andy ◽

David Ritchie ◽

Paul Neeson ◽

Kevin Lynch ◽

...

Keyword(s):

Cell Proliferation ◽

Nk Cells ◽

K562 Cell ◽

Cell Function ◽

Nk Cell ◽

Cell Lysis ◽

Cell Numbers ◽

And Function

Abstract Abstract 1639 Poster Board I-665 Dexamethasone (dex) and lenalidomide (len) is a potent treatment for multiple myeloma (MM). In vitro, len directly inhibits MM tumor cell proliferation via cell cyle arrest, and can also costimulate T cells and augment natural killer (NK) cell activity, leading to enhanced anti-tumour immunity. Conversely, dex also directly inhibits MM cell proliferation but is profoundly immuno-suppressive and may therefore subvert the full capacity of len to act via immune mechanisms against MM. We previously reported that MM patients responding to len-dex combination show an increase in Treg numbers, and little evidence in recovery of their B and T cell numbers (Quach et al. Blood 2008; 112: abstract 1696). We have since undertaken a prospective and systematic analysis of NK cell number and function in MM patients treated with len-dex, and evaluated the mechanisms by which dex downregulates len-induced NK activation in in vitro assays using patients' and normal donors' blood samples. 25 relapsed MM patients (aged 58-77 years) were treated with low dose len (15mg Days 1-21 of each 28-day cycle) and dex (20mg/day, Days 1-4,9-12,17-20). After a median of 9 (2-19) cycles, 19 patients responded (24% CR/VGPR, 52% PR). At baseline, NK cell numbers and function [assessed by % lysis of 51Cr labelled K562 target cells at 50 (effector):1 (target) ratio] in MM patients were similar to age matched controls (0.2 vs. 0.3× 105/ml in controls, p=0.09 and 49% K562 cell lysis vs. 58% in controls, p=0.44 respectively) (fig.1A). Whilst NK cell numbers slightly increased in vivo after len-dex treatment [2.0 (baseline) vs. 3.9×105/l (cycle 6), p=0.04, paired t test] (fig.1A), mean NK cell function progressively decreased compared to baseline after 6 and 9 len-dex cycles [mean 49% K562 cell lysis at baseline vs. 28% after 6 cycles (p=0.007) and 31% after 9 cycles (p=0.02)] (fig.1B). Following 72 hours of in vitro treatment with len (10mM), there was increased NK function in healthy donor peripheral blood mononuclear cells (PBMC) [mean 54% K562 cell lysis from len-treated PBMC vs. 38% lysis in untreated PBMC, p=0.04] (fig. 2). In PBMCs from MM patients at baseline, ex vivo treatment with len (10mM) did not significantly increase NK cell function [mean 47% K562 cell lysis (untreated) vs. 52% (len treated), p=0.17], nor did it increase NK cell function after 6 len-dex treatment cycles [mean 32% K562 cell lysis (untreated) vs. 30% (treated), p=0.4].Conversely, dex (0.1mM) decreased NK cell function in healthy donors' PBMC [mean 7.6% K562 cell lysis (dex treated) vs. 38% (untreated) p=0.01], even in the presence of len [mean 7% K562 cell lysis (len+dex) vs. 38% (untreated), p=0.002] (fig. 2). Dex-induced in vitro NK inhibition was dose dependent and could be rescued by the addition of IL-2 to normal donors [mean 7.6 % K562 cell lysis (dex) vs. 28% lysis (Dex +IL2),p=0.03] as well as PBMC from MM patients at baseline [mean lysis 16% (dex) vs. 59% (Dex+IL2) p=0.0002]. However, IL-2 was less able to rescue dex-induced NK dysfunction in PBMC from patients post 6 treatment cycles compared to patients at baseline [mean 59% K562 cell lysis (baseline) vs. 28% (C6), p=0.03]. Dex induced NK dysfunction was reversible as NK cell function recovered after a 3 days dex washout. In summary, NK function in MM patients, whilst similar to healthy controls at baseline, progressively decreases after prolonged len-dex treatment despite a clinical response. The observed decrease in NK function in vivo and in vitro is directly due to the effects of dex, which could not be reversed by the NK activating effects of len. Our results suggest that the efficacy of len and dex co-therapy is not due to augmentation of NK cytolytic activity, due to the immunosuppressive effects of dex against NK cells. This suggests that alternative dosing schedules of dex, after initial induction with len and dex co-therapy, may optimise len-induced immunostimulation of NK cells and subsequent sustained disease control via anti-MM immunity. Disclosures Lynch: Celgene Corporation: Employment. Prince:Celgene Corporation: Research Funding.

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Contribution of Natural Killer Cells to Inhibition of Angiogenesis by Interleukin-12

Blood ◽

10.1182/blood.v93.5.1612 ◽

1999 ◽

Vol 93 (5) ◽

pp. 1612-1621 ◽

Cited By ~ 127

Author(s):

Lei Yao ◽

Cecilia Sgadari ◽

Keizo Furuke ◽

Eda T. Bloom ◽

Julie Teruya-Feldstein ◽

...

Keyword(s):

Endothelial Cells ◽

Nk Cells ◽

Natural Killer ◽

Cell Function ◽

Nk Cell ◽

Primary Cultures ◽

Interleukin 12 ◽

Ifn Γ

Abstract Interleukin-12 (IL-12) inhibits angiogenesis in vivo by inducing interferon-γ (IFN-γ) and other downstream mediators. Here, we report that neutralization of natural killer (NK) cell function with antibodies to either asialo GM1 or NK 1.1 reversed IL-12 inhibition of basic fibroblast growth factor (bFGF)-induced angiogenesis in athymic mice. By immunohistochemistry, those sites where bFGF-induced neovascularization was inhibited by IL-12 displayed accumulation of NK cells and the presence of IP-10–positive cells. Based on expression of the cytolytic mediators perforin and granzyme B, the NK cells were locally activated. Experimental Burkitt lymphomas treated locally with IL-12 displayed tumor tissue necrosis, vascular damage, and NK-cell infiltration surrounding small vessels. After activation in vitro with IL-12, NK cells from nude mice became strongly cytotoxic for primary cultures of syngeneic aortic endothelial cells. Cytotoxicity was neutralized by antibodies to IFN-γ. These results document that NK cells are required mediators of angiogenesis inhibition by IL-12, and provide evidence that NK-cell cytotoxicity of endothelial cells is a potential mechanism by which IL-12 can suppress neovascularization.

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Loss of CD20 Expression and Exhaustion of Effector Cells Limit ADCC in CLL Patients Treated with Rituximab.

Blood ◽

10.1182/blood.v114.22.1610.1610 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1610-1610 ◽

Cited By ~ 1

Author(s):

Berengere Vire ◽

Justin SA Perry ◽

Elinor Lee ◽

Lawrence S Stennett ◽

Leigh Samsel ◽

...

Keyword(s):

Nk Cells ◽

Effector Cell ◽

Cell Function ◽

Nk Cell ◽

Cell Surface Expression ◽

Target Cells ◽

Effector Cells ◽

Cell Functions ◽

Anti Cd20

Abstract Abstract 1610 Poster Board I-636 A major mechanism how the chimeric anti-CD20 monoclonal antibody rituximab (RTX) depletes B-cells is antibody-dependent cellular cytotoxicity (ADCC). ADCC has been modeled in-vitro and in mouse models. However, investigations on ADCC directly in patients treated with RTX are scarce. Recent efforts have focused on improving ADCC through modifications in the Fc binding portion of novel antibodies or through stimulation of effector cell functions with GM-CSF. A more detailed understanding of ADCC as a therapeutic process is needed to optimize such strategies and to identify biomarkers of improved efficacy. Here we report a comprehensive analysis of ADCC in previously untreated CLL patients during the first two RTX infusions (375mg/m2) given in combination with fludarabine every 4 weeks. Following the initial infusion of RTX the absolute lymphocyte count (ALC) decreased by a median of 74% at 2h, followed by a partial recrudescence of cells so that by 24h the median decrease in ALC reached 39% (n=11). ADCC is mediated by effector cells that include NK cells, monocytes/macrophages, and granulocytes. First, we investigated changes in NK cell function: consistent with NK cell activation we found an increase in CD69 at 2, 6 and up to 24h (median 4.2-fold, p=0.005, n=10) after RTX administration and increased expression of the degranulation marker CD107a/b (median 1.9-fold, p<0.001, n=5) and down-regulation of perforin expression (median decrease 63%, p<0.001, n=5) at 4h from treatment start. Activation of NK cells is triggered by the engagement of CD16/FcγRIIIa by RTX coated CLL cells. Interestingly, CD16 expression on NK cells was rapidly lost, already apparent at 2h and maximal at 6h from the start of the RTX infusion (median decrease 82%, p=0.02, n=10) and was not completely recovered by 24h. We also found a significant decrease in expression of CD16 on granulocytes (78%, p<0.001, n=5) but an increase in monocytes (3.9-fold, p<0.001, n=5). In addition to loss of CD16, we found that the cytotoxic capacity of the effector cells was rapidly exhausted: in an oxidative-burst assay, monocytes showed a significant decrease in the production of reactive oxygen species 4h after initiation of RTX infusion (median 60% decrease, p=0.043) and at 6h from the start of the RTX infusion NK cell-mediated killing of K562 target cells was reduced by half (p<0.001, n=3). Interestingly, both the acute reaction to RTX infusions that manifest as a cytokine release syndrome and changes in effector cell function peaked during the first hours of the RTX infusion. We hypothesized that this might be due to the process of CD20 shaving, a rapid and pronounced decrease of CD20 cell surface expression modeled in-vitro and in mice as the result of a mechanism called trogocytosis that relies on the direct and rapid exchange of cell membrane fragments and associated molecules between effectors and target cells (Beum, J Immunol, 2008). First, we used western blot analysis of total CD20 protein in CLL cells and found a rapid loss of CD20 that was apparent already at 2h resulting in virtually complete loss of expression at 24h. Next, we used ImageStream technology to directly visualize ADCC interactions in-vivo. We indeed detected transfer of CD20 from CLL cells to NK cells and monocytes, resulting in complete CD20 loss in circulating CLL cells. While we detected transfer of CD20 into both cell types, monocytes were much more engaged in trogocytosis than NK cells. Consistently, 4h post RTX infusion we found a significant increase in intracellular RTX in granulocytes and monocytes using intracellular staining for human IgG. CD20 shaving appears to be of particular importance given that immunohistochemical analyses revealed that persistent disease in the bone marrow aspirates after 4 cycles of RTX treatment was mostly CD20 negative. Collectively, our results identify loss of CD20 from CLL cells by trogocytosis and exhaustion of immune effector mechanisms as limitations for anti-CD20 immunotherapy. These data identify possible avenues for improving CD20 mediated immunotherapy and characterize endpoints on which different anti-CD20 antibodies can be compared. Given that trogocytosis appears to be a common occurrence our findings likely have general importance to immunotherapy of hematologic malignancies. Disclosures No relevant conflicts of interest to declare.

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Exhausted CD8+ T cells exhibit low and strongly inhibited TCR signaling during chronic LCMV infection

Nature Communications ◽

10.1038/s41467-020-18256-4 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Ioana Sandu ◽

Dario Cerletti ◽

Manfred Claassen ◽

Annette Oxenius

Keyword(s):

T Cells ◽

T Cell ◽

Cell Function ◽

Viral Infections ◽

Cell Receptor ◽

Target Cells ◽

Tcr Signaling ◽

And Function

Abstract Chronic viral infections are often associated with impaired CD8+ T cell function, referred to as exhaustion. Although the molecular and cellular circuits involved in CD8+ T cell exhaustion are well defined, with sustained presence of antigen being one important parameter, how much T cell receptor (TCR) signaling is actually ongoing in vivo during established chronic infection is unclear. Here, we characterize the in vivo TCR signaling of virus-specific exhausted CD8+ T cells in a mouse model, leveraging TCR signaling reporter mice in combination with transcriptomics. In vivo signaling in exhausted cells is low, in contrast to their in vitro signaling potential, and despite antigen being abundantly present. Both checkpoint blockade and adoptive transfer of naïve target cells increase TCR signaling, demonstrating that engagement of co-inhibitory receptors curtails CD8+ T cell signaling and function in vivo.

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Azacytidine Compromises NK-Cell Activity in AML and MDS Patients Undergoing MRD-Based Pre-Emptive Treatment After Allogeneic Stem Cell Transplantation

Blood ◽

10.1182/blood.v120.21.4122.4122 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4122-4122

Author(s):

Katja Sockel ◽

Claudia Schönefeldt ◽

Sieghart Sopper ◽

Martin Wermke ◽

Marc Schmitz ◽

...

Keyword(s):

Stem Cell ◽

Stem Cell Transplantation ◽

Nk Cells ◽

Cell Function ◽

Nk Cell ◽

Cell Activity ◽

Nk Cell Activity ◽

Activating Receptors

Abstract Abstract 4122 The hypomethylating agent azacytidine (AZA) represents the standard treatment for many high-risk MDS and AML patients. While the clinical efficacy has been confirmed in several studies, the precise molecular mechanism of action has not been fully understood yet. Human NK-cells play an important role in the regulation of immune responses against malignant cells. Their function is controlled by a complex interplay of activating and inhibitory receptors - some of them being regulated by methylation of the respective genes. We, therefore explored, whether AZA modulates in vitro NK-cell function as well as in vivo during minimal-residual disease (MRD)-guided treatment of imminent relapse in MDS and AML patients treated within the prospective RELAZA trial (NCT00422890). Methods: After purifying NK-cells of healthy donors by MACS (magnetic cell sorting), NK-cells were exposed in vitro to different concentrations of AZA (100nM, 1μM, 3μM) with or without IL-2. In parallel, the NK-cell phenotype of patients (n=12) with AML or MDS, undergoing MRD-guided treatment with AZA after stem cell transplantation was monitored by FACS from peripheral blood samples on day 1, 5 and 7 of the first and second AZA cycle. All patients were still in complete haematological remission at the time of therapy. Results: In vitro, we observed a significant reduction (3,1% to 1,8% p=0.028) of the immature and cytokine-regulating CD56bright NK-cell subpopulation with increasing concentrations of AZA. There was a trend towards a reduced expression of the death-ligand TRAIL, the activating receptors NKG2D and NKp46 and for an increased expression of the inhibitory KIR CD158b1/b2, whereas we could not detect any changes in the expression of FAS-L, Perforin, Granzyme B, NKp30, NKp44, CD69, CD57, DNAM-1, CD16, and NKG2A-CD94. Confirmatory, we observed a significant decrease in the expression of TRAIL (p=0.003), NKG2D (p=0.03) and NKp46 (p=0.006) during AZA treatment in-vivo. Interestingly, these changes appeared to be reversible. The observed reduction of NK-cell activating receptors and TRAIL during AZA treatment correlated with a reduction or stable course of MRD in all analyzed patients. Conclusion: In summary these data suggest that the clinical effects of AZA are not mediated by enhancing NK-cell activity. In fact, the drug may have inhibitory effects on NK-cell function which should be considered when applying AZA in the post-transplant setting. Disclosures: Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Optimizing NK Cell-Based Immunotherapy in Myeloid Leukemia: Abrogating an Immunosuppressive Microenvironment

Frontiers in Immunology ◽

10.3389/fimmu.2021.683381 ◽

2021 ◽

Vol 12 ◽

Author(s):

Natasha Mupeta Kaweme ◽

Fuling Zhou

Keyword(s):

Tumor Microenvironment ◽

Nk Cells ◽

Myeloid Leukemia ◽

Cell Function ◽

Nk Cell ◽

Ex Vivo ◽

Substantial Improvement ◽

Effector Cells ◽

Cell Sources

Natural killer (NK) cells are prominent cytotoxic and cytokine-producing components of the innate immune system representing crucial effector cells in cancer immunotherapy. Presently, various NK cell-based immunotherapies have contributed to the substantial improvement in the reconstitution of NK cells against advanced-staged and high-risk AML. Various NK cell sources, including haploidentical NK cells, adaptive NK cells, umbilical cord blood NK cells, stem cell-derived NK cells, chimeric antigen receptor NK cells, cytokine-induced memory-like NK cells, and NK cell lines have been identified. Devising innovative approaches to improve the generation of therapeutic NK cells from the aforementioned sources is likely to enhance NK cell expansion and activation, stimulate ex vivo and in vivo persistence of NK cells and improve conventional treatment response of myeloid leukemia. The tumor-promoting properties of the tumor microenvironment and downmodulation of NK cellular metabolic activity in solid tumors and hematological malignancies constitute a significant impediment in enhancing the anti-tumor effects of NK cells. In this review, we discuss the current NK cell sources, highlight ongoing interventions in enhancing NK cell function, and outline novel strategies to circumvent immunosuppressive factors in the tumor microenvironment to improve the efficacy of NK cell-based immunotherapy and expand their future success in treating myeloid leukemia.

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Enhancing Human NK Cell Function and Specificity for Cancer Immunotherapy

Blood ◽

10.1182/blood-2018-99-120173 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2044-2044

Author(s):

Pomeroy Emily ◽

Hunzeker John ◽

Kluesner Mitchell ◽

Crosby Margaret ◽

Laura Bendzick ◽

...

Keyword(s):

Nk Cells ◽

Cancer Immunotherapy ◽

Peripheral Blood ◽

High Efficiency ◽

Cell Function ◽

Nk Cell ◽

Negative Regulator ◽

Car T

Abstract Natural Killer (NK) cells are cytotoxic lymphocytes capable of immune surveillance and represent an excellent source of cells for cancer immunotherapy for numerous reasons: 1) they mediate direct killing of transformed cells with reduced or absent MHC expression, 2) they can carryout antibody-dependent cell-mediated cytotoxicity (ADCC) on cells bound by appropriate antibodies via CD16, 3) they are readily available and easy to isolate from peripheral blood, 4) they can be expanded to clinically relevant numbers in vitro. Moreover, as NK cells do not cause graft versus host disease, they are inherently an off-the-shelf cellular product, precluding the need to use a patient's own NK cells to treat their cancer. In light of these attributes, NK cells have been used in many clinical trials to treat a number of cancer types; however, the results have not been as successful as other cellular based immunotherapies, such as CAR-T. In light of this, many groups have taken approaches to augment NK cell function, such as high dose IL15, CARs and Bi- or Tri-specific killer engagers. A synergistic or even alternative approach to these technologies is the use of CRISPR/Cas9-based genome editing to disrupt or manipulate the function of NK genes to improve their utility as an immunotherapeutic agent. In order to enhance the immunotherapeutic efficacy of NK cells we have implemented the CRISPR/Cas9 system to edit genes and deliver CARs. To this end, we have developed methods for high efficiency nucleic acid delivery to NK cells using electroporation. First, primary human NK cells are immunomagnetically isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors. Purified NK cells are then activated and expanded using artificial antigen presenting cells (aAPCs) expressing membrane bound IL21 and 41BB for 7 days and subsequently electroporated (Figure 1A). Using this approach with EGFP encoding mRNA, we achieve high rates of transfection (>90%) and high viability (>90%) (Figure 1B). We next developed gRNAs targeting PD1, CISH, and ADAM17. PD1 is a negative regulator of NK cell function and its cognate receptor, PD-L1, is upregulated in a number of cancers. ADAM17 mediates CD16 cleavage on NK cells to negatively regulate their ability to perform ADCC. CISH is a recently described negative regulator of NK cell activation and integrates cytokine signals, including IL-15. We consistently achieved high rates (up to 90%) of gene inactivation in primary human NK cells across multiple donors (Figure 1C). Importantly, these gene edits do not affect expansion potential and are stable over several rounds of expansion (Figure 1D, E). Moreover, ADAM17 KO NK cells are highly resistant to CD16 cleavage upon activation (Figure 2A-E) and PD1 KO NK cells demonstrate significantly enhanced function against PD-L1 expressing cancer cell lines in vitro and in vivo (Figure 2F-J). These data demonstrate that high efficiency gene editing of NK cells can significantly enhance their function while maintaining in vitro expansion. In an effort to engineer NK cell specificity for cancer immunotherapy, we recently developed CAR molecules designed for use in NK cells (Li et al., 2018, Cell Stem Cell 23, 1-12). To this end, we engineered and tested 10 mesothelin CAR molecules with NK specific transmembrane domains (CD16, NKp44, NKp46, or NKG2D) and intracellular signaling domains (2B4, DAP10, DAP12, CD3ζ, and/or CD137). Utilizing several cancer models, we identified an architecture that significantly enhanced NK activation compared to T-CAR architectures (CAR4: scFv-NKG2D-2B4-CD3ζ). Moreover, NK-CAR4 cells demonstrated increased in vivo expansion, improved activity, and reduced toxicity compared to CAR-T cell therapy. In our studies to develop novel NK CARs, CARs were delivered to iPSC derived test NK cells (iNKs) using the PiggyBac transposon system. In order to deliver NK-CAR4 to peripheral blood NK cells we developed methods for high frequency, site specific integration. To this end, we utilized CRISPR/Cas9 combined with non-integrating recombinant Adeno-Associated Virus (rAAV) DNA donor for homologous recombination. Using an EGFP reporter we were able to optimize this process and deliver EGFP reporter to the AAVS1 safe harbor site with efficiencies >80% in NK cells. We are now utilizing our optimized gene editing approaches to generate multiplex edited CAR-NK cells and results from these studies will be presented. Disclosures Webber: BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership. Felices:GT Biopharma: Research Funding. Moriarity:BEAM Therapeutics: Consultancy; B-MoGen Biotechnologies: Employment, Equity Ownership.

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Eomes and T-Bet Expression Are Required By Mature Primary Human NK Cells for Anti-Leukemia Responses In Vivo

Blood ◽

10.1182/blood-2021-149693 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 194-194

Author(s):

Pamela Wong ◽

Carly C. Neal ◽

Lily Chang ◽

Julia A Wagner ◽

Melissa M. Berrien-Elliott ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

T Box ◽

Nsg Mice ◽

Healthy Donors ◽

And Function

Abstract Natural Killer (NK) cells are innate lymphoid cells that respond to hematologic cancers via cytotoxicity (perforin/granzyme and death receptors) and cytokine/chemokine production, yet the molecular determinants underlying their proliferation, function, and persistence are poorly understood. There are promising reports of pre-clinical and clinical NK cell responses to leukemia and lymphoma, which represent a nascent cellular therapy for these blood cancers. The T-box transcription factors (TFs) Eomes and T-bet are expressed by NK cells throughout their lifespan, and are required for development as evidenced by NK cell loss in Eomes and T-bet deficient mice. However, the roles of these TFs in mature human NK cell molecular programs and functions remain unclear. We hypothesized Eomes and T-bet, which are the only T-box TFs expressed in NK cells, are critical regulators of NK cell homeostasis and functionality, and are necessary for proper mature NK cell responses. To address this, we utilized the CRISPR-Cas9 system to genetically delete both Eomes and T-bet in primary human NK cells isolated from healthy donors, and investigated their role beyond guiding NK cell development, specifically in the anti-leukemia response. Gene-editing of primary human NK cells has been technically challenging, thus most reports that modified NK cells were performed with cell lines, in vitro-differentiated, or highly expanded NK cells that likely do not reflect primary human NK cell biology. Here, we introduced Cas9 mRNA and sgRNA targeting T-bet and Eomes by electroporation into unexpanded primary human NK cells isolated from healthy donors using the MaxCyte GT system. We observed highly efficient reductions of Eomes and T-bet protein expression, quantified by flow cytometry (p < 0.0001, Fig A-B) without viability differences between control (sgRNA targeting TRAC, an unexpressed locus in NK cells), and Eomes/T-bet double CRISPR-edited (DKO) cells after one week in vitro. To study Eomes and T-bet in NK cell anti-leukemia response, control or DKO primary human NK cells were engrafted into NSG mice, supported with human IL-15, and challenged with K562 leukemia cells. Utilizing bioluminescent imaging to visualize leukemia burden, we observed that NK cells lacking both TFs were unable to suppress leukemia growth in vivo. To understand the mechanism responsible for impaired leukemia control, we investigated in vivo persistence and proliferation, cytotoxic effector molecule expression, as well as ex vivo degranulation and cytokine production of DKO NK cells compared to control NK cells. DKO or control human NK cells were transferred into NSG mice and supported with human IL-15. After 2-3 weeks, significantly fewer (<30%) DKO NK cells persisted compared to control NK cells: spleen (5-fold decrease, control 240e3±65e3 vs DKO 47e3±15e3 NK cells, p<0.01, Figure C), blood (6-fold decrease, p<0.01), and liver (4-fold decrease, p<0.05). Using intracellular flow cytometry, double T-bet/Eomes CRISPR-edited NK cells that lacked both Eomes and T-bet protein after in vivo transfer were identified. A proliferative defect was evident in flow-gated DKO (62±6% undivided), compared to unedited (WT) NK cells (4±2% undivided) assessed by CellTrace Violet dilution (Figure D). In addition, there were marked reductions in granzyme B and perforin protein (p<0.001) in flow-gated DKO NK cells compared to controls. To assess DKO NK cell functional capacity, we performed an ex vivo functional assay on NK cells from spleens of the NSG mice as effectors, and K562 targets or IL-12/15/18 stimulation for 6 hours. Degranulation to K562 targets was impaired (p<0.05), and IFN-γ production was reduced (p<0.0001) after cytokine stimulation in flow-gated DKO NK cells (Figure E). Thus, CRISPR-editing of unexpanded, primary human NK cells revealed that Eomes and T-bet are required by mature human NK cells for their function and homeostasis, distinct from their role in development. This is translationally relevant, as defects in proliferation and function of human DKO NK cells manifested markedly reduced response against human leukemia cells in vivo in xenografts. These findings expand our understanding of key molecular regulators of mature NK cell homeostasis and function, with the potential to provide new avenues to enhance NK cell therapy. Figure 1 Figure 1. Disclosures Berrien-Elliott: Wugen: Consultancy, Patents & Royalties: 017001-PRO1, Research Funding. Foltz-Stringfellow: Kiadis: Patents & Royalties: TGFbeta expanded NK cells; EMD Millipore: Other: canine antibody licensing fees. Fehniger: HCW Biologics: Research Funding; Compass Therapeutics: Research Funding; Affimed: Research Funding; ImmunityBio: Research Funding; Wugen: Consultancy, Current equity holder in publicly-traded company, Patents & Royalties: related to memory like NK cells, Research Funding; Kiadis: Other; OrcaBio: Other; Indapta: Other.

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Optimizing Lentiviral Transduction of Human Natural Killer Cells

Blood ◽

10.1182/blood.v118.21.4714.4714 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4714-4714 ◽

Cited By ~ 2

Author(s):

Su Su ◽

Dawn M Betters ◽

Muthalagu Ramanathan ◽

Keyvan Keyvanfar ◽

Aleah Smith ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Line ◽

Nk Cell ◽

K562 Cells ◽

Cell Phenotype ◽

Human Renal Cell Carcinoma ◽

Hiv 1

Abstract Abstract 4714 The development of an efficient method to genetically modify natural killer (NK) cells could be used to characterize NK cell differentiation, acquisition of self-tolerance, tumor trafficking in vivo, as well as to manipulate NK cells to enhance their activity against infectious diseases and tumors. Although HIV-1 based lentiviral vectors (LVs) have been used to efficiently transfer genes into human T-cells, little data exists on LV transduction of either fresh or in vitro expanded human NK cells or its effects on NK cell phenotype and cytolytic function. In this study, we used an HIV-based LV expressing enhanced green fluorescence protein (EGFP) driven by a murine stem cell virus long terminal repeat (MSCV-LTR) promoter to transduce CD3− and CD56+ and/or CD16+ human NK cells that were either resting, IL-2 activated, or expanded in vitro using an irradiated EBV-LCL feeder cell line. We observed that resting NK cells were difficult to transduce with LVs, even at high multiplicities of infection (MOI), with transduction efficiencies (TE) in the range of only 3–14%. The efficiency of LV transduction improved when the NK cells were pre-stimulated in vitro with IL-2: TE improved to 21±0.2% in NK cells cultured for 24 hours in media containing IL-2 (200 U/mL) and 28.7±12.9% in NK cells that underwent in vitro expansion over 9 days prior to transduction using irradiated EBV-LCL feeder cells and media containing IL-2 (200U/mL). Subsequently, we evaluated incremental MOIs (3-200) to optimize LV transduction of expanded NK cells; optimal transduction was achieved using a spinoculation protocol at a MOI of 25 which resulted in the highest transduction efficiencies with the least amount of cell death. Increasing the MOI above this level resulted in a small increase in transduction, but was offset by an increase in NK cell apoptosis/death. Using a one-round, non-spinoculation protocol and an MOI of 30, we obtained a median transduction efficiency of 29% (range 16–41) with excellent retention of NK cell viability. This optimized protocol was used to transduce expanded NK cells with a LV vector encoding an shRNA targeting a region of the NK cell inhibitory receptor transcript NKG2A. Following transduction, surface expression of NKG2A decreased significantly on expanded NK cells compared to non-transduced expanded NK cells and “scramble transduced” LV controls; at a MOI of 10, the MFI of NKG2A on expanded human NK cells decreased 35% compared to non-transduced and LV transduced scramble controls (median MFI 428, 673, 659 in shRNA, non-transduced and scramble LV control transduced NK cells respectively). A comparison of transduction efficiencies using LVs expressing EGFP driven by MSCV-LTR, EF1a, and Ubi promoters showed MSCV-LTR mediated the highest level of gene expression in expanded NK cells. Transduced NK cells maintained stable EGFP transgene expression in vitro, which peaked 5 days following LV transduction and remained stable for an additional 9 days. The phenotype of lentiviral transduced NK cells was similar to non-transduced NK cells. Specifically, expression of CD56, CD16, granzyme A and B, perforin, the inhibitory receptors NKG2A, KIR3DL1, KIR3DL2, and KIR2DL1/DL2, and the activating receptors NKG2D, NCRs NKp46, and NKp30 were not altered in either fresh or expanded NK cells following LV transduction, although we did observe a significant reduction in NKp44 expression in LV transduced cells (22% compared to 50% on untransduced NK cells; 0.02). Furthermore, NK cell function, as assessed by cytokine production and cytotoxicity vs tumor targets was not altered in LV transduced NK cells. A 51Cr release cytotoxicity assay showed GFP+ NK cells, flow sorted following LV transduction of expanded NK cells, had similar cytotoxicity against K562 cells and human renal cell carcinoma cells (RCC) compared to non-transduced expanded NK cell controls (figures). In conclusion, we show that an HIV-1 based lentiviral vector driven by a MSCV-LTR, mediated efficient and stable gene transfer in IL-2 activated and in vitro expanded human NK cells. This study provides valuable insights for methods to optimize the long-term expression of LV transduced genes in human NK cells which could be used to improve their anti-tumor function in vivo. Target: K562 cells Target: RCC cell line Disclosures: No relevant conflicts of interest to declare.

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Evidence That Novel NK-NK Cell Subset Regulation Exists With Regard To Effects In Tumor and Viral Models

Blood ◽

10.1182/blood.v122.21.1038.1038 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1038-1038

Author(s):

Ethan G Aguilar ◽

Can M Sungur ◽

Anthony E Zamora ◽

William J Murphy

Keyword(s):

Immune Response ◽

Nk Cells ◽

Mhc Class I ◽

Viral Infections ◽

Nk Cell ◽

Class I ◽

Inhibitory Receptors ◽

The Individual

Abstract Natural killer (NK) cells are lymphocytes of the innate immune system and are classically associated with cytotoxic responses to both virally infected as well as neoplastic cells. Activation of NK cells to exhibit their cytotoxicity is dependent on signaling through a number of activating and inhibitory receptors. In mice, one such family of inhibitory receptors is the C-type lectin-like Ly49 family. In humans, the killer immunoglobulin-like receptors (KIRs) serve as the primary family of inhibitory receptors and are functional analogs of the Ly49s. Despite markedly different structures, the Ly49s and KIRs display similar binding capabilities and bind primarily to distinct MHC class I haplotypes, which plays an important role in regulating NK cell function. NK cells that express inhibitory receptors that are specific for the MHC class I haplotype of the individual are termed “licensed” and have been shown to have increased functionality in terms of cytotoxicity and cytokine production. In contrast, NK cells that express inhibitory receptors that are unable to bind to the MHC class I haplotype of the individual are termed “unlicensed” and have been shown to be hyporesponsive. We have recently reported on the role of NK licensing on the immune response to viral infections such as MCMV. In addition, we have previously described how regulatory T cells can regulate NK cell activity in vivo. However, there are limited data examining the interaction and regulation between the different NK subsets based on differences in licensing. We hypothesized that different NK cell subsets, based on licensing, can regulate each other in the context of anti-tumor and anti-viral responses. Here we first provide in vitro data providing evidence to support the hypothesis of NK-NK regulation based on licensing. In vitro killing assays using MCMV infected fibroblasts, or C1498 (murine acute myeloid leukemia) cells as targets and using different combinations of murine NK Ly49 subsets as effectors were used to assess this NK-NK regulation. To further test our hypothesis, in vivo experiments were also performed using a mouse leukemia model as well as an MCMV model. Mice were injected with C1498 cells and then given hematopoietic stem cell transplantation (HSCT). The mice were then depleted of all NK cells or either licensed or unlicensed subsets by antibody depletion once a week, and monitored for survival. Mice that were depleted of the unlicensed population survived significantly longer compared with the other depleted groups, suggesting a negative regulation of the anti-tumor response by the unlicensed population resulting in greater tumor burden and death in the presence of the unlicensed population. This negative regulation by the unlicensed population is further supported by another experiment where mice were infected with MCMC following total NK or subset depletion and monitored for ten days throughout the course of the immune response to MCMV. Mice that were depleted of their unlicensed population displayed a significantly larger expansion of the licensed population of NK cells, without reciprocal greater expansion of the unlicensed population upon licensed NK cell depletion. More specifically, depletion of the unlicensed population resulted in an expansion of the Ly49H+NK cells which have previously been shown to be the primary effector population during MCMV infection. Thus, the unlicensed NK cells are playing a role in down-regulating the anti-viral response by limiting the expansion of the effector licensed population. Our data highlight a role for the murine NK subsets to negatively regulate the immune response of the effector licensed NK population in the context of anti-tumor and anti-viral responses. This new insight into the regulatory role of NK cells may have clinical benefit for patients receiving bone marrow transplants during cancer treatment to enhance graft vs. tumor effects, and to combat opportunistic viral infections that may manifest in the immune compromised environment of the BMT patient. Disclosures: No relevant conflicts of interest to declare.

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