Activation Status Dictates the Function of Unlicensed Natural Killer Cells

Natural Killer (NK) cells are involved in innate defense against viral infection and cancer. NK cells can be divided into subsets based on the ability of different receptors to bind to major histocompatibility (MHC) class I molecules resulting in differential responses upon activation in a process called "licensing" or "arming". NK cells expressing receptors that bind self-MHC are considered licensed due to augmented effector lytic function capability compared to unlicensed subsets. However, we demonstrated unlicensed NK subsets instead positively regulate the adaptive T cell response during viral infections due to localization and cytokine production. We demonstrate here that the differential effects of the two types of NK subsets is contingent on the environment using viral infection and hematopoietic stem cell transplantation (HSCT) models. Infection of mice with high-dose (HD) MCMV leads to a loss of licensing-associated differences as compared to mice with low-dose infection, as the unlicensed NK subset no longer localized in lymph nodes (LN), but instead remained at the site of infection. Similarly, the patterns observed during HD infection paralleled with the phenotypes of both human and mouse NK cells in a HSCT setting where NK cells exhibit an activated phenotype. However, in contrast to effects of subset depletion in T-replete models, the licensed NK cell subsets still dominated anti-viral responses post-HSCT. Overall, our results highlight the intricate tuning of the NK cells and how it impacts overall immune responses with regard to licensing patterns, as it is dependent on the level of stimulation and their activation status.

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Viral Infection of Human Natural Killer Cells

Viruses ◽

10.3390/v11030243 ◽

2019 ◽

Vol 11 (3) ◽

pp. 243 ◽

Cited By ~ 9

Author(s):

Elisabeth van Erp ◽

Mirjam van Kampen ◽

Puck van Kasteren ◽

Jelle de Wit

Keyword(s):

Viral Infection ◽

Nk Cells ◽

Natural Killer ◽

Viral Entry ◽

Viral Infections ◽

Nk Cell ◽

Cell Infection ◽

Comprehensive Overview ◽

Human Natural Killer Cells ◽

Syncytial Virus

Natural killer (NK) cells are essential in the early immune response against viral infections, in particular through clearance of virus-infected cells. In return, viruses have evolved multiple mechanisms to evade NK cell-mediated viral clearance. Several unrelated viruses, including influenza virus, respiratory syncytial virus, and human immunodeficiency virus, can directly interfere with NK cell functioning through infection of these cells. Viral infection can lead to immune suppression, either by downregulation of the cytotoxic function or by triggering apoptosis, leading to depletion of NK cells. In contrast, some viruses induce proliferation or changes in the morphology of NK cells. In this review article, we provide a comprehensive overview of the viruses that have been reported to infect NK cells, we discuss their mechanisms of entry, and describe the interference with NK cell effector function and phenotype. Finally, we discuss the contribution of virus-infected NK cells to viral load. The development of specific therapeutics, such as viral entry inhibitors, could benefit from an enhanced understanding of viral infection of NK cells, opening up possibilities for the prevention of NK cell infection.

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Homeostatic proliferation generates long-lived natural killer cells that respond against viral infection

Journal of Experimental Medicine ◽

10.1084/jem.20100479 ◽

2011 ◽

Vol 208 (2) ◽

pp. 357-368 ◽

Cited By ~ 91

Author(s):

Joseph C. Sun ◽

Joshua N. Beilke ◽

Natalie A. Bezman ◽

Lewis L. Lanier

Keyword(s):

Viral Infection ◽

Nk Cells ◽

Natural Killer ◽

Viral Infections ◽

Radiation Treatment ◽

Nk Cell ◽

Clinical Importance ◽

Homeostatic Proliferation ◽

And Function ◽

Turn Over

Cells of the immune system undergo homeostatic proliferation during times of lymphopenia induced by certain viral infections or caused by chemotherapy and radiation treatment. Natural killer (NK) cells are no exception and can rapidly expand in number when placed into an environment devoid of these cells. We explored the lifespan and function of mouse NK cells that have undergone homeostatic proliferation in various settings of immunodeficiency. Adoptive transfer of mature NK cells into lymphopenic mice resulted in the generation of a long-lived population of NK cells. These homeostasis-driven NK cells reside in both lymphoid and nonlymphoid organs for >6 mo and, similar to memory T cells, self-renew and slowly turn over at steady state. Furthermore, homeostatically expanded NK cells retained their functionality many months after initial transfer and responded robustly to viral infection. These findings highlight the ability of mature NK cells to self-renew and possibly persist in the host for months or years and might be of clinical importance during NK cell adoptive immunotherapy for the treatment of certain cancers.

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T-BET and EOMES Accelerate and Enhance Functional Differentiation of Human Natural Killer Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.732511 ◽

2021 ◽

Vol 12 ◽

Author(s):

Laura Kiekens ◽

Wouter Van Loocke ◽

Sylvie Taveirne ◽

Sigrid Wahlen ◽

Eva Persyn ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Biology ◽

Nk Cell ◽

Current Knowledge ◽

Functional Differentiation ◽

Cell Maturation ◽

Hematopoietic Stem ◽

And Function

T-bet and Eomes are transcription factors that are known to be important in maturation and function of murine natural killer (NK) cells. Reduced T-BET and EOMES expression results in dysfunctional NK cells and failure to control tumor growth. In contrast to mice, the current knowledge on the role of T-BET and EOMES in human NK cells is rudimentary. Here, we ectopically expressed either T-BET or EOMES in human hematopoietic progenitor cells. Combined transcriptome, chromatin accessibility and protein expression analyses revealed that T-BET or EOMES epigenetically represses hematopoietic stem cell quiescence and non-NK lineage differentiation genes, while activating an NK cell-specific transcriptome and thereby drastically accelerating NK cell differentiation. In this model, the effects of T-BET and EOMES are largely overlapping, yet EOMES shows a superior role in early NK cell maturation and induces faster NK receptor and enhanced CD16 expression. T-BET particularly controls transcription of terminal maturation markers and epigenetically controls strong induction of KIR expression. Finally, NK cells generated upon T-BET or EOMES overexpression display improved functionality, including increased IFN-γ production and killing, and especially EOMES overexpression NK cells have enhanced antibody-dependent cellular cytotoxicity. Our findings reveal novel insights on the regulatory role of T-BET and EOMES in human NK cell maturation and function, which is essential to further understand human NK cell biology and to optimize adoptive NK cell therapies.

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RTID-07. HUMAN PLACENTAL HEMATOPOIETIC STEM CELL DERIVED NATURAL KILLER CELLS (CYNK-001) FOR TREATMENT OF RECURRENT GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noaa215.812 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii194-ii195

Author(s):

Nazanin Majd ◽

Maha Rizk ◽

Solveig Ericson ◽

Kris Grzegorzewski ◽

Sharmila Koppisetti ◽

...

Keyword(s):

Cell Therapy ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

In Vitro Cytotoxicity ◽

Orthotopic Mouse Model ◽

Hematopoietic Stem ◽

Dismal Prognosis

Abstract Glioblastoma (GBM) is the most aggressive primary brain tumor with dismal prognosis. Recent advances of immunotherapy in cancer have sparked interest in the use of cell therapy for treatment of GBM. Active transfer of Natural Killer (NK) cells is of particular interest in GBM because NK cells are capable of exerting anti-tumor cytotoxicity without the need for antigen presentation and sensitization, processes that are impaired in GBM. CYNK-001 is an allogeneic, off-the-shelf product enriched for CD56+/CD3- NK cells expanded from placental CD34+ cells manufactured by Celularity. Here, we demonstrate in vitro cytotoxicity of CYNK-001 against several GBM lines and its in vivo anti-tumor activity in a U87MG orthotopic mouse model via intracranial administration resulting in 94.5% maximum reduction in tumor volume. We have developed a phase I window-of-opportunity trial of CYNK-001 in recurrent GBM via intravenous (IV) and intratumoral (IT) routes. In the IV cohort, subjects receive cyclophosphamide for lymphodepletion followed by 3-doses of IV CYNK-001 weekly. In the IT cohort, subjects undergo placement of an IT catheter with an ommaya reservoir followed by 3-doses of IT CYNK-001 weekly. Patients are monitored for 28-days after last infusion for toxicity. Once maximum safe dose (MSD) is determined, patients undergo IV or IT treatments at MSD followed by surgical resection and the tumor tissue will be analyzed for NK cell engraftment and persistence. We will utilize a 3 + 3 dose de-escalation design (maximum n=36). Primary endpoint is safety and feasibility. Secondary endpoints are overall response rate, duration of response, time to progression, progression free survival and overall survival. Main eligibility criteria include age ≥18, KPS ≥60, GBM at first or second relapse with a measurable lesion on ≤2mg dexamethasone. This is the first clinical trial to investigate CYNK-001 in GBM and will lay the foundation for future NK cell therapy in solid tumors.

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Generation of human natural killer cells from immature progenitors does not require marrow stromal cells

Blood ◽

10.1182/blood.v84.3.841.841 ◽

1994 ◽

Vol 84 (3) ◽

pp. 841-846 ◽

Cited By ~ 37

Author(s):

MR Silva ◽

R Hoffman ◽

EF Srour ◽

JL Ascensao

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Mononuclear Cells ◽

Viral Infections ◽

Nk Cell ◽

Interleukin 2 ◽

Peripheral Blood Mononuclear ◽

Specific Lysis ◽

Human Natural Killer Cells

Abstract Human natural killer (NK) cells comprise 10% to 15% of peripheral blood mononuclear cells and have an important role in immune responses against tumors, viral infections, and graft rejection. NK cells originate in bone marrow (BM), but their progenitors and lineage development have not been completely characterized. We studied the generation of NK cells from purified CD34+HLADR- and CD34+HLADR+ BM progenitors and the influence of various cytokines on their production. We show that CD3-CD56+ cytotoxic NK cells can develop from both progenitors populations when interleukin-2 (IL-2) is present in an in vitro suspension culture system containing IL-1 alpha and stem cell factor. Up to 83.8% and 98.6% CD3-CD56+ cells were detected in CD34+HLADR- and CD34+DR+ cultures, respectively, after 5 weeks of culture; significant numbers of NK cells were first detected after 2 weeks. Cytotoxic activity paralleled NK cell numbers; up to 70% specific lysis at an effector:target ratio of 10:1 was observed at 5 weeks. IL-7 also triggered development of CD3-CD56+ cells from these immature progenitors (up to 24% and 55% appeared in CD34+HLADR- and CD34+HLADR+ cultures, respectively). Our data suggest that BM stromas are not necessary for NK cell development and that IL-2 remains essential for this lineage development and differentiation.

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Dysregulation of Natural Killer Cells in Obesity

Cancers ◽

10.3390/cancers11040573 ◽

2019 ◽

Vol 11 (4) ◽

pp. 573 ◽

Cited By ~ 15

Author(s):

Donal O’Shea ◽

Andrew E. Hogan

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Biology ◽

Viral Infections ◽

Nk Cell ◽

Viral Immunity ◽

Host Protection ◽

Cell Functions ◽

Innate Lymphocytes ◽

The Impact

Natural killer (NK) cells are a population of lymphocytes which classically form part of the innate immune system. They are defined as innate lymphocytes, due to their ability to kill infected or transformed cells without prior activation. In addition to their cytotoxic abilities, NK cells are also rapid producers of inflammatory cytokines such as interferon gamma (IFN-γ) and are therefore a critical component of early immune responses. Due to these unique abilities, NK cells are a very important component of host protection, especially anti-tumour and anti-viral immunity. Obesity is a worldwide epidemic, with over 600 million adults and 124 million children now classified as obese. It is well established that individuals who are obese are at a higher risk of many acute and chronic conditions, including cancer and viral infections. Over the past 10 years, many studies have investigated the impact of obesity on NK cell biology, detailing systemic dysregulation of NK cell functions. More recently, several studies have investigated the role of NK cells in the homeostasis of adipose tissue and the pathophysiology of obesity. In this review, we will discuss in detail these studies and focus on emerging data detailing the metabolic mechanisms altering NK cells in obesity.

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Evidence for Killing of Mesenchymal Stem Cells (MSC) by Autologous Natural Killer Lymphocytes.

Blood ◽

10.1182/blood.v104.11.1290.1290 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1290-1290 ◽

Cited By ~ 1

Author(s):

Alessandro Poggi ◽

Anna-Maria Massaro ◽

Simone Negrini ◽

Ivana Pierri ◽

Manuela Balocco ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

T Lymphocytes ◽

Nk Cells ◽

Natural Killer ◽

Viral Infections ◽

Nk Cell ◽

Cell Lineage ◽

Culture Conditions ◽

Inhibitory Receptors

Abstract In this study, Mesenchymal Stem Cells (MSC) were obtained from bone marrow of 10 patients suffering from acute myeloid leukemia (AML), six M0/1 two M2, and two M5 (according to the FAB classification), 8 out of 10 in post-chemotherapy complete remission. These cells differentiated into adipocytes or osteoblasts under appropriate culture conditions. MSC were CD44+, CD73a+ CD73b+ CD105+, beta1 integrin+, ICAM1+, HLA-I+, HLA-II+ (variable proportions), CD45−, CD31−, CD34− and they constitutively expressed the stress-inducible MHC-related molecules MIC-A and the UL16 (induced at the surface of cells infected by cytomegalovirus) binding protein ULBP3. These molecules are reported ligands for the NKG2D receptor expressed by natural killer (NK) and CD8+ T lymphocytes, effector cells that are thought to play a role in host defence against tumors. NK cells have also been shown to regulate normal differentiation of hemopoietic precursor into the myeloid or lymphoid cell lineage. Moreover, it has been stated that NK cells are not able to damage autologous cells, as they receive negative signals through inhibitory receptors, including killer Ig-like receptors (KIR) or C-type lectin inhibitory receptors (CLIR), which bind to HLA-I discrete alleles. Surprisingly, we found that autologous IL2-activated, but not freshly isolated, NK cells lysed MSC, while T lymphocytes did not kill self or non-self MSC. Binding of ICAM-1 expressed by MSC to its receptor, the integrin LFA-1, expressed by NK cells plays a key role in MSC/NK interaction. More importantly, NKG2D/MICA and/or NKG2D/ULBP3 engagement is responsible for the delivery of lethal hit. Conversely, it appears that HLA-I molecules do not protect MSC from NK cell-mediated injury. Taken together, these data suggest that NK cells, when activated as it may occur during the first response to viral infections, are able to eliminate MSC, thus altering the normal interactions with hemopoietic precursors and possibly affecting their differentiation. This mechanism might also contribute to the development of aberrant precursors as observed in acute leukaemias.

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Early Evaluation Of Natural Killer Cell Reconstitution Following Unmanipulated Haploidentical Transplantation Compared With HLA-Identical Sibling Transplantation

Blood ◽

10.1182/blood.v122.21.5480.5480 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5480-5480

Author(s):

Isabel Gonzalez-Gascon y Marin ◽

Ana María Pérez-Corral ◽

Jorge Gayoso ◽

Javier Anguita ◽

Ana Carolina Franco ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cyclosporine A ◽

Peripheral Blood ◽

Nk Cell ◽

Lymphoid Cells ◽

High Dose ◽

Color Flow ◽

Activating Receptors ◽

Activating Receptor

Abstract Background The main functions of Natural Killer (NK) cells are early protection against viruses or tumour cells and production of cytokines that regulate immune functions. NK cells are the first lymphoid cells to repopulate the marrow after Stem Cell Transplantation (SCT) and reach normal levels within 1 month after transplant. Acquisition of both, inhibiting and activating receptors on developing NK cells is an important step in their functional maturation. Previous studies showed the beneficial effect of NK alloreactivity in prevention of relapse, especially in the setting of haploidentical SCT. The aim of this study is to compare the reconstitution of the NK cell compartment during the first 3 months after unmanipulated haploidentical peripheral blood SCT (Haplo) and HLA-identical sibling peripheral blood SCT (HLA-id). Patients and Methods 11 adult patients received SCT (7 Haplo and 4 HLA-id) at Gregorio Marañón Hospital (Madrid-Spain) from November 2012 to April 2013. Conditioning regimen comprised fludarabine, cyclophosphamide and busulfan for Haplo SCT and fludarabine and busulfan or fludaribine and melphalan for HLA-id SCT. Prophylaxis for acute graft-versus-host disease consisted of high dose cyclophosphamide on days +3 and +4, cyclosporine A and mycophenolate mofetil for Haplo and Cyclosporine A and methotrexate for HLA-id. Patient´s characteristics and transplant outcomes are shown in table 1. We analysed reconstitution patterns and phenotype of NK at day +15, +30, +60, and +90 after transplantation by multi-color flow cytometry on FC500 Beckman Coulter® cytometer using the following anti-human monoclonal antibodies: CD3 FITC, CD56 ECD, CD45 PC7, NKG2A PC7, NKp30 PC5, NKp44 PE, Nkp46 PC5, and NKG2D PE (Beckman Coulter®). For comparison between the two groups Mann–Whitney U-test was used. Results 2/7 patients who received Haplo SCT died early in the post-transplantation period (day +50 and +66), and were excluded of the analysis because NK cells were not recovered by those days. NK cells reached normal levels by day +30: median 71 cells/µl (21-1089)) after Haplo; median 213.5 cells/µl (113-499) after HLA-id, and remained at high levels through follow up, with no significant differences between the two groups. Similarly to previous studies, a large percentage of NKbright cells was observed at day +30 after Haplo (median 89% of NK cells (55-97%)), a percentage that tended to decrease at day +60 (30% (7-38%)) and +90 (35% (10-45%)). Interestingly the percentage of NKbright cells after HLA-id SCT at day +30 (median 14.5% of NK cells (6-30%)) compared with Haplo, was significantly lower (p=0.016). This was accompanied by a significantly lower expression of inhibitory receptor NKG2A after HLA-id SCT than after Haplo: 59.5% (50-62%) versus 92.5% (50-62%) at day +30; 54% (38-61%) versus 86% (70-88%) versus at day +60 (p=0.016). Activating receptors NKp44 and NKp30 showed a low expression after both types of SCT throughout the first 3 months after transplantation. By contrast, activating receptor NKp46 levels were significantly higher at day +30 after Haplo than after HLA-id SCT (93% (87-98%) versus 50% (37-51%)) (p=0.016). Finally, high and similar proportions of activating receptor NKG2D were observed in both types of SCT. Figure 1 illustrates the recovery of the NK cell receptor phenotype for each type of SCT. Conclusions Our data showed an early and fast recovery of NK cells after Haplo and HLA-id SCT. However, phenotypic maturation of NK cells appears to be different for each type of transplant. NK cells generated after Haplo exhibit a more immature phenotype, characterized by a higher proportion of NKbright cells, and a higher expression of NKG2A at day +30. Interestingly expression of NKp46 was significantly higher after Haplo than after HLA-id SCT. Other authors have reported cytotoxic activity of these NK cells with high expression of NKp46, suggesting that cytotoxicity may be preserved in these immature NK cells. NKp30, NKG2D and NKp44 expression is less affected by the type of SCT. Acknowledgments This work has been partially supported by Project “Evaluación de la reconstitución inmune después del trasplante haploidéntico de progenitores hemopoyéticos sin depleción T” from Fundación Mutua Madrileña. Disclosures: No relevant conflicts of interest to declare.

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The reactivity of Bw4+ HLA-B and HLA-A alleles with KIR3DL1: implications for patient and donor suitability for haploidentical stem cell transplantations

Blood ◽

10.1182/blood-2008-01-132902 ◽

2008 ◽

Vol 112 (2) ◽

pp. 435-443 ◽

Cited By ~ 66

Author(s):

Bree A. Foley ◽

Dianne De Santis ◽

Els Van Beelen ◽

Louise J. Lathbury ◽

Frank T. Christiansen ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Target Cells ◽

Systematic Analysis ◽

Hematopoietic Stem ◽

Hla Type

Abstract Natural killer (NK)–cell alloreactivity can be exploited in haploidentical hematopoietic stem cell transplantation (HSCT). NK cells from donors whose HLA type includes Bw4, a public epitope present on a subset of HLA-B alleles, can be alloreactive toward recipients whose cells lack Bw4. Serologically detectable epitopes related to Bw4 also exist on a subset of HLA-A alleles, but the interaction of these alleles with KIR3DL1 is controversial. We therefore undertook a systematic analysis of the ability of most common HLA-B alleles and HLA-A alleles with Bw4 serologic reactivity to protect target cells from lysis by KIR3DL1-dependent NK cells. All Bw4− HLA-B alleles failed to protect target cells from lysis. All Bw4+ HLA-B alleles with the exception of HLA-B*1301 and -B*1302 protected targets from lysis. HLA-A*2402 and HLA-A*3201 unequivocally protected target cells from lysis, whereas HLA-A*2501 and HLA-A*2301 provided only weak protection from lysis. KIR3DL1-dependent alloreactive NK clones were identified in donors with HLA-A*2402 but not in donors with HLA-B*1301 or -B*1302. These findings clarify the HLA types that donors and recipients need in haploidentical HSCT and other NK allotherapies in order to benefit from NK alloreactivity.

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Potently Cytotoxic Natural Killer Cell Potential Initially Emerges from Erythro-Myeloid Progenitors during Mammalian Development

Blood ◽

10.1182/blood-2019-130347 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2464-2464

Author(s):

Carissa Dege ◽

Katherine H Fegan ◽

J Philip Creamer ◽

Melissa M Berrien-Elliott ◽

Stephanie A. Luff ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Fetal Liver ◽

Killer Cell ◽

Hematopoietic Progenitors ◽

Cell Potential ◽

Hematopoietic Stem ◽

Myeloid Progenitor

Natural killer (NK) cells are innate immune cells that target and kill virally infected and malignant cells, making them an attractive target for adoptive immunotherapies. An alternative to donor-derived NK cells is the use of human pluripotent stem cell (hPSC)-derived NK cells, as a renewable "off the shelf" product. Previous studies have identified hPSC-derived NK cells as potently cytotoxic, compared to donor-derived NK cells. As the differentiation of hPSCs mimics early embryonic development, this raises the possibility that hPSC-derived NK cells are ontogenically distinct from adult NK cells. NK cells are present during embryonic hematopoiesis, but their ontogenic origins are poorly understood. NK cells are thought to arise from a common lymphoid progenitor (CLP), lying downstream of hematopoietic stem cells (HSCs), but evidence exists that NK cells may arise from HSC-independent progenitors as NK cells are found in the early murine fetal liver, and NK cell progenitors are found in the early human yolk sac (YS). In this study, we investigated the emergence of NK cells during murine and human embryonic hematopoietic development. During murine embryogenesis, overlapping HSC-independent waves of hematopoietic progenitors occur in the YS that give rise to hematopoietic cells prior to HSC emergence at E10.5. The "primitive" wave occurs at E7.5, followed by an "erythro-myeloid progenitor" (EMP) wave at E8.5. To study NK cell potential during murine YS hematopoiesis, we cultured total YS and sorted hematopoietic progenitors under NK cell promoting conditions. Strikingly, we found that the YS contains NK cell potential. Further, sorted E8.5 kit+CD41+CD16/32+ EMP progenitors, but not primitive hematopoietic progenitors, contain robust NK cell potential. EMP-derived NK (EMP-NK) cells were larger and more granular than adult CLP-derived NK cells. Additionally, NK cells from the E15.5 fetal liver were larger and more granular than NK cells from the adult spleen. Both EMP-NK cells and E15.5 fetal liver NK cells had a more robust degranulation response than their HSC-derived counterparts. Together, these data support the concept that EMP in the YS serve as an initial source of physiologically relevant, functional embryonic NK cells that are phenotypically and functionally distinct from adult NK cells. As hPSC-derived NK cells were described as potently cytotoxic, and we observed that murine HSC-independent NK cells robustly degranulate, we next asked whether NK cell development from hPSCs recapitulates that found in the murine embryo. We have demonstrated previously, using a stage-specific WNT signal manipulation approach that specifies ontogenically distinct hematopoietic progenitors, that hPSC-derived NK cell progenitors can be obtained from two distinct progenitors in vitro. In this study, we sought to better understand the development and function of these two NK cell populations. Stage-specific WNT inhibition (WNTi) during hPSC mesodermal patterning yielded extra-embryonic-like HOXA-/low CD34+ populations that possessed erythroid, myeloid and NK cell potential, but lacked T cell potential. The CD56+ NK cells in these cultures co-emerged with CD15+ granulocytes, indicating that these NK cells may arise from a committed myeloid progenitor. In contrast, HOXA+ CD34+ cells, obtained in a WNT-dependent (WNTd) manner, harbored erythro-myelo-lymphoid multi-lineage potential, including NK cell potential. Phenotypically, WNTi-NK cells were larger, more granular and more mature, compared to WNTd-NK and cord blood (CB)-derived NK cells, reminiscent of murine EMP-NK cells. Further, following multiple stimulation assays, WNTi-NK and WNTd-NK cells had different effector biases. WNTi-NK cells are biased for potent cytotoxic degranulation and exhibited superior cell killing in an ADCC assay. In contrast, WNTd-NK and CB-NK had an attenuated degranulation response, but robustly produced inflammatory cytokines. Finally, RNA-seq analysis demonstrated that WNTd-NK cells were most similar to CB-NK cells. Collectively, these studies identify for the first time that the murine EMP harbor NK cell potential, and these NK cells are functionally unique. These observations raise new questions regarding which ontogenic origin of NK cells should be used in future hPSC-derived adoptive immunotherapy strategies. Disclosures Fehniger: Cyto-Sen Therapeutics: Consultancy; Horizon Pharma PLC: Other: Consultancy (Spouse). Palis:Rubius Therapeutics: Consultancy.

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