Activated Natural Killer Cells Withstand the Relatively Low Glucose Concentrations Found in the Bone Marrow of Multiple Myeloma Patients

Infusion of ex vivo expanded and cytokine-activated natural killer (NK) cells is a promising alternative way to treat multiple myeloma (MM). However, the tumor microenvironment (TME) may suppress their function. While reduced glucose availability is a TME hallmark of many solid tumors, glucose levels within the TME of hematological malignancies residing in the bone marrow (BM) remain unknown. Here, we measured glucose levels in the BM of MM patients and tested the effect of different glucose levels on NK cells. BM glucose levels were measured using a biochemical analyzer. Compared to the normal range of blood glucose, BM glucose levels were lower in 6 of 9 patients (479-1231 mg/L; mean=731.8 mg/L). The effect of different glucose levels on NK cell cytotoxicity was tested in 4-hour cytotoxicity assays with tumor cells. 500 mg/L glucose (representing low range of MM BM) during the 4-hour cytotoxicity assay did not negatively affect cytotoxicity of activated NK cells, while higher glucose concentrations (4000 mg/L) diminished NK cell cytotoxicity. Since clinical application of NK cell therapy might require ex vivo expansion, expanded NK cells were exposed to a range of glucose concentrations from 500-4000 mg/L for a longer period (4 days). This did not reduce cytotoxicity or IFN-γ secretion nor affected their phenotypic profile. In summary, low glucose concentrations, as found in BM of MM patients, by itself did not compromise the anti-tumor potential of IL-2 activated NK cells in vitro. Although follow up studies in models with a more complex TME would be relevant, our data suggest that highly activated NK cells could be used to target tumors with a reduced glucose environment.

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α-Pinene Enhances the Anticancer Activity of Natural Killer Cells via ERK/AKT Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22020656 ◽

2021 ◽

Vol 22 (2) ◽

pp. 656

Author(s):

Hantae Jo ◽

Byungsun Cha ◽

Haneul Kim ◽

Sofia Brito ◽

Byeong Mun Kwak ◽

...

Keyword(s):

Nk Cells ◽

Cancer Cells ◽

Natural Killer ◽

Nk Cell ◽

Granzyme B ◽

Akt Pathway ◽

Cell Cytotoxicity ◽

Anticancer Effects ◽

Nk Cell Cytotoxicity

Natural killer (NK) cells are lymphocytes that can directly destroy cancer cells. When NK cells are activated, CD56 and CD107a markers are able to recognize cancer cells and release perforin and granzyme B proteins that induce apoptosis in the targeted cells. In this study, we focused on the role of phytoncides in activating NK cells and promoting anticancer effects. We tested the effects of several phytoncide compounds on NK-92mi cells and demonstrated that α-pinene treatment exhibited higher anticancer effects, as observed by the increased levels of perforin, granzyme B, CD56 and CD107a. Furthermore, α-pinene treatment in NK-92mi cells increased NK cell cytotoxicity in two different cell lines, and immunoblot assays revealed that the ERK/AKT pathway is involved in NK cell cytotoxicity in response to phytoncides. Furthermore, CT-26 colon cancer cells were allografted subcutaneously into BALB/c mice, and α-pinene treatment then inhibited allografted tumor growth. Our findings demonstrate that α-pinene activates NK cells and increases NK cell cytotoxicity, suggesting it is a potential compound for cancer immunotherapy.

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Hypoxia Induced Impairment of NK Cell Cytotoxicity against Multiple Myeloma Can Be Overcome by IL-2 Activation of the NK Cells

PLoS ONE ◽

10.1371/journal.pone.0064835 ◽

2013 ◽

Vol 8 (5) ◽

pp. e64835 ◽

Cited By ~ 64

Author(s):

Subhashis Sarkar ◽

Wilfred T. V. Germeraad ◽

Kasper M. A. Rouschop ◽

Elisabeth M. P. Steeghs ◽

Michel van Gelder ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Nk Cell ◽

Cell Cytotoxicity ◽

Nk Cell Cytotoxicity

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A research-driven approach to the identification of novel natural killer cell deficiencies affecting cytotoxic function

Blood ◽

10.1182/blood.2019000925 ◽

2020 ◽

Vol 135 (9) ◽

pp. 629-637

Author(s):

Michael T. Lam ◽

Emily M. Mace ◽

Jordan S. Orange

Keyword(s):

Natural Killer Cell ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Cell Development ◽

Impact Testing ◽

Primary Immune Deficiency ◽

Cell Cytotoxicity ◽

Nk Cell Cytotoxicity

Abstract Natural killer cell deficiencies (NKDs) are an emerging phenotypic subtype of primary immune deficiency. NK cells provide a defense against virally infected cells using a variety of cytotoxic mechanisms, and patients who have defective NK cell development or function can present with atypical, recurrent, or severe herpesviral infections. The current pipeline for investigating NKDs involves the acquisition and clinical assessment of patients with a suspected NKD followed by subsequent in silico, in vitro, and in vivo laboratory research. Evaluation involves initially quantifying NK cells and measuring NK cell cytotoxicity and expression of certain NK cell receptors involved in NK cell development and function. Subsequent studies using genomic methods to identify the potential causative variant are conducted along with variant impact testing to make genotype-phenotype connections. Identification of novel genes contributing to the NKD phenotype can also be facilitated by applying the expanding knowledge of NK cell biology. In this review, we discuss how NKDs that affect NK cell cytotoxicity can be approached in the clinic and laboratory for the discovery of novel gene variants.

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Killer Cell Inhibitory Receptor Recognition of Human Leukocyte Antigen (HLA) Class I Blocks Formation of a pp36/PLC-γ Signaling Complex in Human Natural Killer (NK) Cells

Journal of Experimental Medicine ◽

10.1084/jem.184.6.2243 ◽

1996 ◽

Vol 184 (6) ◽

pp. 2243-2250 ◽

Cited By ~ 96

Author(s):

Nicholas M. Valiante ◽

Joseph H. Phillips ◽

Lewis L. Lanier ◽

Peter Parham

Keyword(s):

Human Leukocyte Antigen ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Human Leukocyte ◽

Class I ◽

Cell Cytotoxicity ◽

Leukocyte Antigen ◽

Nk Cell Cytotoxicity

The killer cell inhibitory receptors (KIR) of human natural killer (NK) cells recognize human leukocyte antigen class I molecules and inhibit NK cell cytotoxicity through their interaction with protein tyrosine phosphatases (PTP). Here, we report that KIR recognition of class I ligands inhibits distal signaling events and ultimately NK cell cytotoxicity by blocking the association of an adaptor protein (pp36) with phospholipase C-γ in NK cells. In addition, we demonstrate that pp36 can serve as a substrate in vitro for the KIR-associated PTP, PTP-1C (also called SHP-1), and that recognition of class I partially disrupts tyrosine phosphorylation of NK cell proteins, providing evidence for KIR-induced phosphatase activity.

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Pseudomonas aeruginosa Infection Impairs NKG2D-Dependent NK Cell Cytotoxicity through Regulatory T-Cell Activation

Infection and Immunity ◽

10.1128/iai.00363-20 ◽

2020 ◽

Vol 88 (12) ◽

Author(s):

Mickael Vourc’h ◽

Gaelle David ◽

Benjamin Gaborit ◽

Alexis Broquet ◽

Cedric Jacqueline ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Nk Cells ◽

Natural Killer ◽

Antitumor Immunity ◽

Nk Cell ◽

Dependent Manner ◽

Cell Cytotoxicity ◽

Content Type ◽

Cytotoxic Response ◽

Nk Cell Cytotoxicity

ABSTRACT Natural killer (NK) cells play a key role in both antibacterial and antitumor immunity. Pseudomonas aeruginosa infection has already been reported to alter NK cell functions. We studied in vitro the effect of P. aeruginosa on NK cell cytotoxic response (CD107a membrane expression) to a lymphoma cell line. Through positive and negative cell sorting and adoptive transfer, we determined the influence of monocytes, lymphocytes, and regulatory T cells (Treg) on NK cell function during P. aeruginosa infection. We also studied the role of the activating receptor natural killer group 2D (NKG2D) in NK cell response to B221. We determined that P. aeruginosa significantly altered both cytotoxic response to B221 and NKG2D expression on NK cells in a Treg-dependent manner and that the NKG2D receptor was involved in NK cell cytotoxic response to B221. Our results also suggested that during P. aeruginosa infection, monocytes participated in Treg-mediated NK cell alteration. In conclusion, P. aeruginosa infection impairs NK cell cytotoxicity and alters antitumor immunity. These results highlight the strong interaction between bacterial infection and immunity against cancer.

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Lentiviral Transduction of Ex Vivo Expanded Natural Killer Cells with a CD19 Chimeric Antigen Receptor Induces Cytotoxicity against Resistant B Cell Malignancies

Blood ◽

10.1182/blood.v112.11.3540.3540 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3540-3540

Author(s):

Muthalagu Ramanathan ◽

Su Su ◽

Andreas Lundqvist ◽

Maria Berg ◽

Aleah Smith ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Cell Cytotoxicity ◽

Gfp Expression ◽

B Cell Malignancies ◽

Activated T Cells ◽

Nk Cell Cytotoxicity

Abstract NK cells play an important role in innate immunity against tumors and viral infection. NK cell cytotoxicity is suppressed by self-HLA molecules that bind and activate inhibitory killer immunoglobulin like receptors (KIRs). Expression of a CD19 chimeric receptor on NK cells could induce target specific activating signals that overcome KIR-mediated inhibition, enhancing autologous NK cell cytotoxicity against B-cell malignancies. Although HIV-1 based lentiviral vectors (LVs) have been used to efficiently transfer genes into human T-cells, little data exists on the use of LV vectors to transduce NK cells. In this study, we designed a HIV-based LV vector encoding both a CD19 chimeric antigen receptor (CAR) and green fluorescence protein (GFP) transgenes controlled by a MSCV-LTR promoter (CD19CAR LV vector) to transduce CD3−CD56+ ex vivo expanded human NK cells. The CAR consists of a single chain Fv portion of a mouse mAb against human CD19 fused to the signaling intracellular domain of a CD3 zeta subunit. CD3−CD56+NK cells were expanded ex vivo using irradiated EBV-LCL feeder cells and IL-2 containing media for 7 to 10 days. NK92 cells or expanded NK cells underwent 2 rounds of transduction with the CD19CAR LV vector in the presence of protamine sulfate using retronectin-coated plates. GFP expression measured by flow cytometry 3–4 days following LV transduction was used to assess transduction efficiencies (TE). GFP expression was detected in a mean 41% (range 27–56%) of NK92 cells and a mean 15% (range 6–40%) of ex vivo expanded NK cells. NK cell viability assessed up to 1 week following LV transduction was similar to non transduced NK cells. Following transduction, NK cells continued to expand in culture similar to non-transduced NK cells; seven days following their transduction, transduced NK cells expanded a median 30 fold while non transduced NK cells expanded a median 27 fold (p=n.s.). Cytotoxicity assays showed EBV-LCLs were resistant to killing by IL-2 activated T cells and in vitro expanded NK cells. In contrast, CD19CAR LV vector transduced NK cells were highly cytotoxic against EBV-LCLs; at 10:1 effector to target ratio (E:T), 43% of EBV-LCLs were killed by CD19CAR LV transduced NK cells versus 6% killing by non transduced NK cells (p=0.0002). NK cytotoxicity of K562 targets was not altered by CD19CAR LV transduction; at a 10:1 E: T ratio, LV transduced NK cells lysed 80% of K562 cells vs. 84% lysis by non transduced NK cells (p=n.s.). We next transduced IL-2 activated T-cells with the CD19CAR LV vector to compare their cytotoxicity to transduced NK cells against CD19+ LCLs. At a 10:1 E: T ratio, 11 % vs 1% of LCLs were killed by transduced vs non transduced T cells respectively (p=0.002). Although the TE of IL-2 activated T-cells was higher than NK cells (mean TE of 38 % vs 15% in T-cells and NK cells respectively, p=0.02), LV transduced NK cells were more cytotoxic to EBV-LCLs than transduced T-cells at the same E: T ratios. In conclusion, we show successful transduction of ex vivo expanded NK cells with a CD19CAR can be achieved using a LV vector, with CD19CAR transduced NK cells exhibiting enhanced antigen specific cytotoxicity. These findings provide both a method and rationale for clinical trials exploring the antitumor effects of adoptively infused CD19CAR LV transduced NK cells in patients with refractory B cell malignancies.

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The PP2A inhibitor SET regulates granzyme B expression in human natural killer cells

Blood ◽

10.1182/blood-2010-05-285130 ◽

2011 ◽

Vol 117 (8) ◽

pp. 2378-2384 ◽

Cited By ~ 22

Author(s):

Rossana Trotta ◽

David Ciarlariello ◽

Jessica Dal Col ◽

Hsiaoyin Mao ◽

Li Chen ◽

...

Keyword(s):

Gene Expression ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Granzyme B ◽

Interleukin 2 ◽

Mrna Levels ◽

Cell Cytotoxicity ◽

Nk Cell Cytotoxicity ◽

Pp2a Inhibitor

Abstract The ability of natural killer (NK) cells to kill malignant or infected cells depends on the integration of signals from different families of cell surface receptors, including cytokine receptors. How such signals then regulate NK-cell cytotoxicity is incompletely understood. Here we analyzed an endogenous inhibitor of protein phosphatase 2A (PP2A) activity called SET, and its role in regulating human NK-cell cytotoxicity and its mechanism of action in human NK cells. RNAi-mediated suppression of SET down-modulates NK-cell cytotoxicity, whereas ectopic overexpression of SET enhances cytotoxicity. SET knockdown inhibits both mRNA and protein granzyme B expression, as well as perforin expression, whereas SET overexpression enhances granzyme B expression. Treatment of NK cells with the PP2A activator 1,9-dideoxy-forskolin also inhibits both granzyme B expression and cytotoxicity. In addition, pretreatment with the PP2A inhibitor okadaic acid rescues declining granzyme B mRNA levels in SET knockdown cells. Down-modulation of SET expression or activation of PP2A also decreases human NK-cell antibody-dependent cellular cytotoxicity. Finally, the induction of granzyme B gene expression by interleukin-2 and interleukin-15 is inhibited by SET knockdown. These data provide evidence that granzyme B gene expression and therefore human NK-cell cytotoxicity can be regulated by the PP2A-SET interplay.

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Identification of the earliest natural killer cell–committed progenitor in murine bone marrow

Blood ◽

10.1182/blood-2011-04-348912 ◽

2011 ◽

Vol 118 (20) ◽

pp. 5439-5447 ◽

Cited By ~ 126

Author(s):

John W. Fathman ◽

Deepta Bhattacharya ◽

Matthew A. Inlay ◽

Jun Seita ◽

Holger Karsunky ◽

...

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Natural Killer ◽

Cell Fate ◽

Nk Cell ◽

Ex Vivo ◽

Killer Cell ◽

Lineage Commitment ◽

Color Flow ◽

Cell Commitment

Abstract Natural killer (NK) cells develop in the bone marrow and are known to gradually acquire the ability to eliminate infected and malignant cells, yet the cellular stages of NK lineage commitment and maturation are incompletely understood. Using 12-color flow cytometry, we identified a novel NK-committed progenitor (pre-NKP) that is a developmental intermediate between the upstream common lymphoid progenitor and the downstream NKP, previously assumed to represent the first stage of NK lineage commitment. Our analysis also refined the purity of NKPs (rNKP) by 6-fold such that 50% of both pre-NKP and rNKP cells gave rise to NKp46+ NK cells at the single-cell level. On transplantation into unconditioned Rag2−/−Il2rγc−/− recipients, both pre-NKPs and rNKPs generated mature NK cells expressing a repertoire of Ly49 family members that degranulated on stimulation ex vivo. Intrathymic injection of these progenitors, however, yielded no NK cells, suggesting a separate origin of thymic NK cells. Unlike the rNKP, the pre-NKP does not express IL-2Rβ (CD122), yet it is lineage committed toward the NK cell fate, adding support to the theory that IL-15 signaling is not required for NK commitment. Taken together, our data provide a high-resolution in vivo analysis of the earliest steps of NK cell commitment and maturation.

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Natural killer cell lytic activity and CD56dim and CD56bright cell distributions during and after intensive training

Journal of Applied Physiology ◽

10.1152/japplphysiol.00513.2003 ◽

2004 ◽

Vol 96 (6) ◽

pp. 2167-2173 ◽

Cited By ~ 39

Author(s):

Masatoshi Suzui ◽

Takeshi Kawai ◽

Hiroko Kimura ◽

Kazuyoshi Takeda ◽

Hideo Yagita ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Lytic Activity ◽

Cell Cytotoxicity ◽

Intensive Training ◽

Nk Cell Cytotoxicity ◽

The Impact ◽

Heavy Training

The purpose of this study was to examine the impact of intensive training for competitive sports on natural killer (NK) cell lytic activity and subset distribution. Eight female college-level volleyball players undertook 1 mo of heavy preseason training. Volleyball drills were performed 5 h/day, 6 days/wk. Morning resting blood samples were collected before training (Pre), on the 10th day of training (During), 1 day before the end of training (End), and 1 wk after intensive training had ceased (Post). CD3-CD16brightCD56dim (CD56dim NK), CD3-CD16dim/-CD56bright NK (CD56bright NK), and CD3+CD16-CD56dim (CD56dim T) cells in peripheral blood were determined by flow cytometry. The circulating count of CD56dim NK cells (the predominant population, with a high cytotoxicity) did not change, nor did the counts for other leukocyte subsets. However, counts for CD56bright NK and CD56dim T cells (subsets with a lower cytotoxicity) increased significantly ( P < 0.01) in response to the heavy training. Overall NK cell cytotoxicity decreased from Pre to End ( P = 0.002), with a return to initial values at Post. Lytic units per NK cell followed a similar pattern ( P = 0.008). Circulating levels of interleukin-6, interferon-γ, and tumor necrosis factor-α remained unchanged. These results suggest that heavy training can decrease total NK cell cytotoxicity as well as lytic units per NK cell. Such effects may reflect in part an increase in the proportion of circulating NK cells with a low cytotoxicity.

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