scholarly journals Engineering CAR-NK cells to secrete IL15 sustains their anti-AML functionality, but is associated with systemic toxicities

2021 ◽  
Author(s):  
Ilias Christodoulou ◽  
Win Jin Ho ◽  
Andrew Marple ◽  
Jonas W Ravich ◽  
Ada Tam ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Flow Analysis ◽  
Surface Expression ◽  
Myelogenous Leukemia ◽  
Cell Surface Expression ◽  
Cell Phenotype ◽  
Transgenic Expression

Background: The prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. NK cells can elicit an anti-leukemic response via a repertoire of activating receptors that bind AML surface ligands. NK cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity, and interleukin-15 (IL15) to enhance their persistence. Methods: We characterized in detail NK cell populations expressing a panel of AML (CD123)-specific CARs and/or IL15 in vitro and in AML xenograft models. Results: CARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ CAR-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK cell persistence. Transgenic expression of secretory (s)IL15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK cell subsets. 2B4.ζ/sIL15 CAR and sIL15 NK cells maintained an overall activated NK cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK cell groups. In vivo, 2B4.ζ/sIL15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL15 CAR and sIL15 NK cells induced lethal toxicity in a second model. Conclusion: Transgenic expression of CD123-CARs and sIL15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early phase clinical testing.

scholarly journals Engineering CAR-NK cells to secrete IL-15 sustains their anti-AML functionality but is associated with systemic toxicities

2021 ◽  
Vol 9 (12) ◽  
pp. e003894
Author(s):  
Ilias Christodoulou ◽  
Won Jin Ho ◽  
Andrew Marple ◽  
Jonas W Ravich ◽  
Ada Tam ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Flow Analysis ◽  
Surface Expression ◽  
Myelogenous Leukemia ◽  
Cell Surface Expression ◽  
Cell Phenotype ◽  
Transgenic Expression

BackgroundThe prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. Natural Killer (NK) cells can elicit an antileukemic response via a repertoire of activating receptors that bind AML surface ligands. NK-cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity and interleukin (IL)-15 to enhance their persistence.MethodsWe characterized in detail NK-cell populations expressing a panel of AML (CD123)-specific CARs and/or IL-15 in vitro and in AML xenograft models.ResultsCARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ Chimeric Antigen Receptor (CAR)-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK-cell persistence. Transgenic expression of secretory interleukin (sIL)-15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK-cell subsets. 2B4.ζ/sIL-15 CAR and sIL-15 NK cells maintained an overall activated NK-cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK-cell groups. In vivo, 2B4.ζ/sIL-15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL-15 CAR and sIL-15 NK cells induced lethal toxicity in a second model.ConclusionTransgenic expression of CD123-CARs and sIL-15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early-phase clinical testing.

Impact of β2 integrin deficiency on mouse natural killer cell development and function

Blood ◽  
2011 ◽  
Vol 117 (10) ◽  
pp. 2874-2882 ◽  
Author(s):  
Karine Crozat ◽  
Céline Eidenschenk ◽  
Baptiste N. Jaeger ◽  
Philippe Krebs ◽  
Sophie Guia ◽  
...  
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Surface Expression ◽  
Cell Maturation ◽  
Cell Surface Expression ◽  
Mcmv Infection ◽  
Β2 Integrin

Abstract Natural killer (NK) cells are innate immune cells that express members of the leukocyte β2 integrin family in humans and mice. These CD11/CD18 heterodimers play critical roles in leukocyte trafficking, immune synapse formation, and costimulation. The cell-surface expression of one of these integrins, CD11b/CD18, is also recognized as a major marker of mouse NK-cell maturation, but its function on NK cells has been largely ignored. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated a mouse carrying an A → T transverse mutation in the Itgb2 gene, resulting in a mutation that prevented the cell-surface expression of CD18 and its associated CD11a, CD11b, and CD11c proteins. We show that β2 integrin–deficient NK cells have a hyporesponsive phenotype in vitro, and present an alteration of their in vivo developmental program characterized by a selective accumulation of c-kit+ cells. NK-cell missing-self recognition was partially altered in vivo, whereas the early immune response to mouse cytomegalovirus (MCMV) infection occurred normally in CD18-deficient mice. Therefore, β2 integrins are required for optimal NK-cell maturation, but this deficiency is partial and can be bypassed during MCMV infection, highlighting the robustness of antiviral protective responses.

scholarly journals Influence of In Vitro IL-2 or IL-15 Alone or in Combination with Hsp-70-Derived 14-mer Peptide (TKD) on the Expression of NK Cell Activatory and Inhibitory Receptors

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Ilona Hromadnikova ◽  
Petra Pirkova ◽  
Lucie Sedlackova
Keyword(s):  
Cell Surface ◽  
Nk Cells ◽  
Nk Cell ◽  
Cell Activity ◽  
Surface Expression ◽  
Cell Number ◽  
Cell Surface Expression ◽  
Specific Marker ◽  
Inhibitory Receptors

NK cells represent a potential tool for adoptive immunotherapy against tumors. Membrane-bound Hsp70 acts as a tumor-specific marker enhancing NK cell activity. Using flow cytometry the effect of in vitro stimulation with IL-2 or IL-15 alone or in combination with Hsp70-derived 14-mer peptide (TKD) on cell surface expression of NK activatory receptors (CD16, NKG2D, NKG2C, NKp46, NKp44, NKp30, KIR2DL4, DNAM-1, and LAMP1) and NK inhibitory receptors (NKG2A, KIR2DL2/L3, LIR1/ILT-2, and NKR-P1A) in healthy individuals was studied. Results were expressed as the percentage of receptor expressing cells and the amount of receptor expressed by CD3−CD56+cellular population. CD94, NKG2D, NKp44, NKp30, KIR2DL4, DNAM-1, LAMP1, NKG2A, and NKR-P1A were upregulated after the stimulation with IL-2 or IL-15 alone or in combination with TKD. KIR2DL2/L3 was upregulated only by IL-15 and IL-15/TKD. Concurrently, an increase in a number of NK cells positive for CD94, NKp44, NKp30, KIR2DL4, and LAMP1 was observed. IL-15 and IL-15/TKD caused also cell number rise positive for KIR2DL2/L3 and NKR-P1A. Cell number positive for NKG2C and NKG2A was increased only by IL-2 and IL-2/TKD. The diverse effect of IL-2 or IL-15 w or w/o TKD on cell surface expression was observed in CD16, NKp46, and LIR1/ILT-2.

scholarly journals Absence of NKG2D Ligands Defines Human Acute Myeloid Leukaemia Stem Cells and Mediates Their Immune Evasion

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 769-769
Author(s):  
Anna M Paczulla ◽  
Kathrin Rothfelder ◽  
Simon Raffel ◽  
Martina Konantz ◽  
Julia Steinbacher ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nk Cells ◽  
Immune Evasion ◽  
Nk Cell ◽  
Surface Expression ◽  
Leukemic Cells ◽  
Pdx Models ◽  
Acute Myeloid

Abstract Patients with acute myeloid leukaemia (AML) often achieve remission but subsequently die of relapse driven by chemotherapy resistant leukemic stem cells (LSCs). To initiate and maintain cancer, LSCs must also escape immunosurveillance. However, in vivo studies on human LSCs largely disregard lymphocyte mediated anti-tumor immunity due to the use of immunocompromised mice. Here we investigate the immunosurveillance mediated by NKG2D, a danger detector expressed by cytotoxic lymphocytes such as natural killer (NK) cells that recognizes stress-induced ligands (NKG2DL) of the MIC and ULBP protein families on AML cells. Staining of n=175 de novo AML with antibodies against MICA, MICB and ULB2/5/6 or an NKG2D-Fc chimeric protein recognizing pan-NKG2DL expression revealed NKG2DL to heterogeneously express among leukemic cells of the same patient (Fig. 1a). As expected, NKG2DLpos AML cells were efficiently cleared by natural killer (NK) cells, while NKG2DLneg leukemic cells escaped NK cell lysis. Interestingly, these NKG2DLneg AML cells also showed immature morphology, enhanced in vitro clonogenicity (39±47 colonies vs. 1±4, p<0.001, n=32 AML cases) and selective abilities to initiate leukemia in NSG mice devoid of functional NK cells (NKG2DLneg, 33/35, 94%; NKG2DLpos, 0/35, 0%; p<0.001, n=13 AML cases, Fig. 1b) and to survive chemotherapy in vivo. In mice, NKG2DLneg AML cells generated both NKG2DLpos and NKG2DLneg progeny of which again only latter induced leukemia in re-transplant assays. Even though similar leukemia-specific mutations were retrieved in NKG2DLneg and NKG2DLpos AML cells derived from the same patient (n=12 analysed patients), published LSC, HSC and 17-genes stemness score signatures were specifically enriched in NKG2DLneg fractions. Mechanistically, expression of poly-ADP-ribose polymerase 1 (PARP1) was identified as enriched in NKG2DLneg compared to NKG2DLpos leukemic subpopulations, and PARP1 inhibition (PARPi) using either siRNAs or pharmacological inhibitors such as AG-14361, veliparib, talazoparib or olaparib, increased NKG2DL mRNA transcripts between 6 and >50 fold. PARP1 binding sites were identified by in silico analysis in NKG2DL promoters and binding was confirmed by chromatin immunoprecipitation in the promoters of MICA and MICB. Importantly, treatment with PARPi induced NKG2DL surface expression on LSCs in vitro and in vivo and co-treatment with PARPi and NK cells efficiently suppressed leukemogenesis in patient derived xenograft (PDX) models (Fig. 1c). These data suggest that PARP1 inhibition sensitizes LSCs to NK cell mediated elimination. Finally, NKG2DL surface expression was found to inversely correlate with favorable molecular AML characteristics (favorable ELN risk group vs. other: p=0.034; inv(16) versus other: p=0.023), complete remission rates after induction chemotherapy (all patients: p=0.002, patients <65 years: p=0.004) and patient overall survival (patients <65 years: p=0.028). Enhanced PARP1 expression in leukemic cells furthermore associated with poor clinical outcome (TCGA data set, p=0.0038). In summary, our data link the concept of LSCs to immune escape in human AML and propose the absence of immunostimulatory NKG2DL as a novel method to identify LSCs across genetic AML subtypes (including CD34 negative AMLs). This LSC specific mechanism of immune evasion could be overcome by treatment with PARP1 inhibitors, which in conjunction with functional NK cells holds promise to eradicate LSCs and promote immune-mediated cure of AML. Fig. 1: Human AML contain NKG2DLpos as well as NKG2DLneg subpopulations but only latter display leukemia initiation capacity (a: left, analysis of n=175 AML cases using NKG2D-Fc staining, right: exemplary flow cytometry plots; b: leukemic infiltration and survival in mice transplanted with NKG2DLneg or NKG2DLpos AML cells sorted from the same AML cases). PARP1 inhibition with AG-14361 up-regulates NKG2DL on CD34+ LSCs, and in vivo co-treatment with AG-14361 and polyclonal allogeneic NK cells suppresses leukemogenesis in PDX models (c). Figure. Figure. Disclosures Salih: Several patent applications: Patents & Royalties: e.g. EP3064507A1.

Optimizing Lentiviral Transduction of Human Natural Killer Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4714-4714 ◽  
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.

scholarly journals In vitro education of human natural killer cells by KIR3DL1

2019 ◽  
Vol 2 (6) ◽  
pp. e201900434
Author(s):  
Jason Pugh ◽  
Neda Nemat-Gorgani ◽  
Zakia Djaoud ◽  
Lisbeth A Guethlein ◽  
Paul J Norman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Hla Class I ◽  
Surface Expression ◽  
Class I ◽  
Target Cells ◽  
Human Natural Killer Cells ◽  
Basic Protocol

During development, NK cells are “educated” to respond aggressively to cells with low surface expression of HLA class I, a hallmark of malignant and infected cells. The mechanism of education involves interactions between inhibitory killer immunoglobulin–like receptors (KIRs) and specific HLA epitopes, but the details of this process are unknown. Because of the genetic diversity of HLA class I genes, most people have NK cells that are incompletely educated, representing an untapped source of human immunity. We demonstrate how mature peripheral KIR3DL1+ human NK cells can be educated in vitro. To accomplish this, we trained NK cells expressing the inhibitory KIR3DL1 receptor by co-culturing them with target cells that expressed its ligand, Bw4+HLA-B. After this training, KIR3DL1+ NK cells increased their inflammatory and lytic responses toward target cells lacking Bw4+HLA-B, as though they had been educated in vivo. By varying the conditions of this basic protocol, we provide mechanistic and translational insights into the process NK cell education.

Defective Triggering of NK Cells Results in Primary CLL Cells Resistance to Cytotoxicity,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3876-3876
Author(s):  
Caroline Veuillen ◽  
Jerome Rey ◽  
Rémy Castellano ◽  
Florence Orlanducci ◽  
Françoise Mallet ◽  
...  
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
K562 Cells ◽  
Surface Expression ◽  
Resting Cells ◽  
Nk Receptors ◽  
Healthy Donors

Abstract Abstract 3876 Chronic lymphocytic leukemia (CLL) remains an incurable disease except after allogenic transplantation. Natural killer (NK) cells are one of the main effectors of immune surveillance involved in tumor control. Alterations of NK cells functions have been well characterized in myeloid malignancies. However the role of NK cells in immune escape of CLL in less known and controversial. Here we describe extensive phenotypic and functional characterization of NK cells and primary CLL cells and their interactions in vitro and in vivo. Twenty eight untreated CLL patients, twenty four age-matched healthy donors and ten AML patients were enrolled in the study. We have previously shown that expression and function of NK cell-triggering receptors is defective in AML. We then assessed the phenotypic and functional properties of NK cells from CLL patients. Unlike the results found in AML, no significant differences were observed in term of activating receptors, NKp46, DNAM-1, NKG2D, 2B4 and CD16. Only the natural cytotoxicity receptor (NCR) NKp30 was weakly decreased compared to healthy donors (p=0.0107). There wasn't any difference in the expression of inhibitory receptors CD158a, b, e, ILT2 and NKG2A. Looking at the spontaneous NK-mediated cytotoxicity, CLL NK cells displayed a cytolytic activity similar to that of healthy donors against K562 cell line. To further evaluate the functional consequences of the decreased expression of NKp30, mAb redirected killing assays was performed against P815 cell lines. The NK cells killing was slightly lower in CLL patients compared to healthy donors when anti-NKp30 was used although no difference could be observed with anti-NKp46 and anti-CD16. All these results supported that NK cells cytotoxicity should be effective in CLL. We then studied the susceptibility of CLL B cells to allogenic NK killing both in vitro and in vivo. Unlike AML cells and K562 cells, CLL cells were resistant to NK cytotoxicity mediated by resting cells. Exogenous stimulation of allogenic NK cells with IL2 and IL15 restored partially CLL killing, which was nevertheless still lower than AML blasts and K562 cells killing (p=0.0288 and <0.0001 respectively). Murine xenotransplantation model using NOD/SCID g null (NSG) mice allowed us to study the anti-leukemic capacity of purified NK cells after activation with IL2. We didn't observe any clearance of CLL cells after allogenic NK cell injection while CLL and NK cells were checked to be present in blood, bone marrow, spleen and liver. These experiments confirmed the CLL resistance to NK-mediated killing. To investigate the potential mechanisms of this resistance, we analyzed the surface expression of ligands for activating and inhibitory NK receptors on CLL cells. CLL cells displayed poor expression of ligands for activating NK receptors MICA/B, ULBP1-3, PVR, nectin-2 and CD54. Interestingly, this profile of surface expression was similar to that of normal B cells except a slight increase of ULBP3 expression on CLL cells. Regarding ligands for inhibitory NK receptors, HLA-class I molecules were significantly down-regulated while HLA-E tended to be up-regulated on CLL cells compared to normal B cells. Finally, we tested ADCC in order to overcome the resistance of CLL cells to NK killing: the presence of rituximab increased significantly CLL lysis. Of note, priming of NK cells with IL2+IL15 still increased CLL cytotoxicity (p<0.0001). Our findings demonstrate that primary CLL cells are resistant to NK mediated killing. This defect is mainly due to the lack of ligands for NK receptors on CLL cells surface leading to deficient triggering of NK cells. However NK cells of CLL patients are fully competent. Attempts to optimize NK cell therapy for treatment of CLL will require overcoming the low immunogenicity of B-CLL cells. Our xenograft model provides the tools for such preclinical development. Disclosures: No relevant conflicts of interest to declare.

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

2017 ◽  
Author(s):  
Alexander Vargas-Hernandez ◽  
Emily M. Mace ◽  
Ofer Zimmerman ◽  
Christa S. Zerbe ◽  
Alexandra F. Freeman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Function ◽  
Viral Infections ◽  
Nk Cell ◽  
Cell Phenotype ◽  
Effector Cells ◽  
Viral Susceptibility ◽  
And Function

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.

scholarly journals Glucose Oligosaccharide and Long-Chain Glucomannan Feed Additives Induce Enhanced Activation of Intraepithelial NK Cells and Relative Abundance of Commensal Lactic Acid Bacteria in Broiler Chickens

2021 ◽  
Vol 8 (6) ◽  
pp. 110
Author(s):  
Nathalie Meijerink ◽  
Jean E. de Oliveira ◽  
Daphne A. van Haarlem ◽  
Guilherme Hosotani ◽  
David M. Lamot ◽  
...  
Keyword(s):  
Immune System ◽  
Nk Cells ◽  
Relative Abundance ◽  
Nk Cell ◽  
Feed Additives ◽  
Microbiota Composition ◽  
In Ovo ◽  
Long Chain

Restrictions on the use of antibiotics in the poultry industry stimulate the development of alternative nutritional solutions to maintain or improve poultry health. This requires more insight in the modulatory effects of feed additives on the immune system and microbiota composition. Compounds known to influence the innate immune system and microbiota composition were selected and screened in vitro, in ovo, and in vivo. Among all compounds, 57 enhanced NK cell activation, 56 increased phagocytosis, and 22 increased NO production of the macrophage cell line HD11 in vitro. Based on these results, availability and regulatory status, six compounds were selected for further analysis. None of these compounds showed negative effects on growth, hatchability, and feed conversion in in ovo and in vivo studies. Based on the most interesting numerical results and highest future potential feasibility, two compounds were analyzed further. Administration of glucose oligosaccharide and long-chain glucomannan in vivo both enhanced activation of intraepithelial NK cells and led to increased relative abundance of lactic acid bacteria (LAB) amongst ileum and ceca microbiota after seven days of supplementation. Positive correlations between NK cell subsets and activation, and relative abundance of LAB suggest the involvement of microbiota in the modulation of the function of intraepithelial NK cells. This study identifies glucose oligosaccharide and long-chain glucomannan supplementation as effective nutritional strategies to modulate the intestinal microbiota composition and strengthen the intraepithelial innate immune system.

scholarly journals Direct Binding of Human NK Cell Natural Cytotoxicity Receptor NKp44 to the Surfaces of Mycobacteria and Other Bacteria

2008 ◽  
Vol 76 (4) ◽  
pp. 1719-1727 ◽  
Author(s):  
Semih Esin ◽  
Giovanna Batoni ◽  
Claudio Counoupas ◽  
Annarita Stringaro ◽  
Franca Lisa Brancatisano ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Cell Subset ◽  
Surface Expression ◽  
Binding Assays ◽  
Igg Antibody ◽  
Direct Stimulation ◽  
Activating Receptors ◽  
Accessory Cells

ABSTRACT Our previous studies demonstrated that Mycobacterium bovis bacillus Calmette-Guérin (BCG) can directly interact with human NK cells and induce the proliferation, gamma interferon production, and cytotoxic activity of such cells without the need for accessory cells. Thus, the aim of the present study was to identify the putative receptor(s) responsible for the recognition of BCG by human NK cells and potentially involved in the activation of NK cells. To this end, we first investigated the surface expression of three NK cell-activating receptors belonging to the natural cytoxicity receptor (NCR) family on highly purified human NK cells upon in vitro direct stimulation with BCG. An induction of the surface expression of NKp44, but not of NKp30 or NKp46, was observed after 3 and 4 days of in vitro stimulation with live BCG. The NKp44 induction involved mainly a particular NK cell subset expressing the CD56 marker at high density, CD56bright. In order to establish whether NKp44 could directly bind to BCG, whole BCG cells were stained with soluble forms of the three NCRs chimeric for the human immunoglobulin G (IgG) Fc fragment (NKp30-Fc, NKp44-Fc, NKp46-Fc), followed by incubation with a phycoerythrin (PE)-conjugated goat anti-human IgG antibody. Analysis by flow cytometry of the complexes revealed a higher PE fluorescence intensity for BCG incubated with NKp44-Fc than for BCG incubated with NKp30-Fc, NKp46-Fc, or negative controls. The binding of NKp44-Fc to the BCG surface was confirmed with immunogold labeling using transmission electron microscopy, suggesting the presence of a putative ligand(s) for human NKp44 on the BCG cell wall. Similar binding assays performed on a number of gram-positive and gram-negative bacteria revealed a pattern of NKp44-Fc binding restricted to members of the genus Mycobacterium, to the mycobacterium-related species Nocardia farcinica, and to Pseudomonas aeruginosa. Altogether, the results obtained indicate, for the first time, that at least one member of the NCR family (NKp44) may be involved in the direct recognition of bacterial pathogens by human NK cells.

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