Extracellular vesicles derived from immortalized human natural killer cell line NK3.3 as a novel therapeutic for multiple myeloma

IntroductionOver the last decade, there have been many advancements in the therapeutic treatment of multiple myeloma (MM), including the use of natural killer (NK) cells. However, despite promising results from clinical trials, there are concerns over the use of NK cell-based therapy. Cells often undergo growth arrest, limiting their experimental utility; donor cells are extremely heterogeneous, resulting in content variability; and patients receiving allogeneic cells are at risk for graft-versus-host disease and/or cytokine release syndrome. Extracellular vesicles (EVs) have emerged as a new natural therapeutic tool. EVs are known to carry cargo derived from the parent cell from which they originate. NK cells play an important role in the innate immune system, targeting and killing tumor cells. This has led many researchers to isolate EVs from NK cells for their cytotoxic potential.MethodsIn this study, we isolated EVs from the NK cell line, NK3.3, which was derived from the peripheral blood of a healthy donor. Currently, it is the only normal human NK cell line reported with all the functional characteristics of healthy NK cells. To address the issue of growth arrest, we immortalized NK3.3 cells with lentivirus encoding the catalytic subunit of human telomerase htert (NK3.3-LTV). EVs from these cells were isolated using a modified polyethylene glycol (PEG)-acetate precipitation protocol to simplify processing and increase EV yield.Results and conclusionsWe demonstrated that NK3.3-LTV EVs target both sensitive and drug-resistant MM cell lines as well as primary patient MM cells in vitro, decreasing proliferation and inducing apoptotic cell death as well as or better than EVs from non-immortalized cells with no toxicity towards normal cells. This study is the first step towards developing an immunotherapeutic product designed to treat patients with relapsed/refractory MM

T-cell receptor gene rearrangement and expression in human natural killer cells: natural killer activity is not dependent on the rearrangement and expression of T-cell receptor α , β , or γ genes

To test the hypothesis that the T-cell receptor ( Tcr ) λ gene encodes a natural killer (NK) cell receptor molecule, three human NK clones and fresh peripheral blood lymphocytes with NK activity from two patients with a CD16 + lymphocytosis were analyzed for rearrangements and expression of the human Tcr α, β , and λ genes. Two of the clones displayed distinct rearrangements of their Tcr β and λ genes and expressed mature Tcr α, β , and αl RNA. However, one of the clones and both patient samples displayed marked NK activity but failed to rearrange or express any of their Tcr genes. These findings demonstrate that human natural killer activity is not dependent on Tcr λ gene rearrangement and expression. In addition, they confirm previous findings concerning the lack of Tcr α and β gene expression in some natural killer cells. Thus, they suggest the existence of additional NK-specific recognition molecules.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46745/1/251_2004_Article_BF00376117.pd

Reduced inflammation and cytokine production in NKLAM deficient mice during Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a leading cause of pneumonia and a significant economic burden. Antibiotic-resistant S. pneumoniae has become more prevalent in recent years and many pneumonia cases are caused by S. pneumoniae that is resistant to at least one antibiotic. The ubiquitin ligase natural killer lytic-associated molecule (NKLAM/RNF19b) plays a role in innate immunity and studies using NKLAM-knockout (NKLAM-KO) macrophages have demonstrated that NKLAM positively affects the transcriptional activity of STAT1. Using an inhalation infection model, we found that NKLAM-KO mice had a significantly higher lung bacterial load than WT mice but had less lung inflammation. Coincidently, NKLAM-KO mice had fewer neutrophils and NK cells in their lungs. NKLAM-KO mice also expressed less iNOS in their lungs as well as less MCP-1, MIP1α, TNFα, IL-12, and IFNγ. Both neutrophils and macrophages from NKLAM-KO mice were defective in killing S. pneumoniae as compared to wild type cells (WT). The phosphorylation of STAT1 and STAT3 in NKLAM-KO lungs was lower than in WT lungs at 24 hours post-infection. NKLAM-KO mice were afforded some protection against a lethal dose of S. pneumoniae compared to WT mice. In summary, our novel data demonstrate a role for E3 ubiquitin ligase NKLAM in modulating innate immunity via the positive regulation of inflammatory cytokine expression and bactericidal activity

Image_1_Extracellular vesicles derived from immortalized human natural killer cell line NK3.3 as a novel therapeutic for multiple myeloma.tif

IntroductionOver the last decade, there have been many advancements in the therapeutic treatment of multiple myeloma (MM), including the use of natural killer (NK) cells. However, despite promising results from clinical trials, there are concerns over the use of NK cell-based therapy. Cells often undergo growth arrest, limiting their experimental utility; donor cells are extremely heterogeneous, resulting in content variability; and patients receiving allogeneic cells are at risk for graft-versus-host disease and/or cytokine release syndrome. Extracellular vesicles (EVs) have emerged as a new natural therapeutic tool. EVs are known to carry cargo derived from the parent cell from which they originate. NK cells play an important role in the innate immune system, targeting and killing tumor cells. This has led many researchers to isolate EVs from NK cells for their cytotoxic potential.MethodsIn this study, we isolated EVs from the NK cell line, NK3.3, which was derived from the peripheral blood of a healthy donor. Currently, it is the only normal human NK cell line reported with all the functional characteristics of healthy NK cells. To address the issue of growth arrest, we immortalized NK3.3 cells with lentivirus encoding the catalytic subunit of human telomerase htert (NK3.3-LTV). EVs from these cells were isolated using a modified polyethylene glycol (PEG)-acetate precipitation protocol to simplify processing and increase EV yield.Results and conclusionsWe demonstrated that NK3.3-LTV EVs target both sensitive and drug-resistant MM cell lines as well as primary patient MM cells in vitro, decreasing proliferation and inducing apoptotic cell death as well as or better than EVs from non-immortalized cells with no toxicity towards normal cells. This study is the first step towards developing an immunotherapeutic product designed to treat patients with relapsed/refractory MM.</p

Image_2_Extracellular vesicles derived from immortalized human natural killer cell line NK3.3 as a novel therapeutic for multiple myeloma.tif

IntroductionOver the last decade, there have been many advancements in the therapeutic treatment of multiple myeloma (MM), including the use of natural killer (NK) cells. However, despite promising results from clinical trials, there are concerns over the use of NK cell-based therapy. Cells often undergo growth arrest, limiting their experimental utility; donor cells are extremely heterogeneous, resulting in content variability; and patients receiving allogeneic cells are at risk for graft-versus-host disease and/or cytokine release syndrome. Extracellular vesicles (EVs) have emerged as a new natural therapeutic tool. EVs are known to carry cargo derived from the parent cell from which they originate. NK cells play an important role in the innate immune system, targeting and killing tumor cells. This has led many researchers to isolate EVs from NK cells for their cytotoxic potential.MethodsIn this study, we isolated EVs from the NK cell line, NK3.3, which was derived from the peripheral blood of a healthy donor. Currently, it is the only normal human NK cell line reported with all the functional characteristics of healthy NK cells. To address the issue of growth arrest, we immortalized NK3.3 cells with lentivirus encoding the catalytic subunit of human telomerase htert (NK3.3-LTV). EVs from these cells were isolated using a modified polyethylene glycol (PEG)-acetate precipitation protocol to simplify processing and increase EV yield.Results and conclusionsWe demonstrated that NK3.3-LTV EVs target both sensitive and drug-resistant MM cell lines as well as primary patient MM cells in vitro, decreasing proliferation and inducing apoptotic cell death as well as or better than EVs from non-immortalized cells with no toxicity towards normal cells. This study is the first step towards developing an immunotherapeutic product designed to treat patients with relapsed/refractory MM.</p

Neutrophil and NK cell lung infiltration and bone marrow-derived macrophage iNOS expression.

<p>Cells were isolated from infected lungs at 24 and 48h post infection and stained for CD45, CD3, NK1.1, CD11b and Ly-6G. CD45⁺ cells were gated and the percentage of CD11b⁺/Ly-6G⁺ (A) or CD3^-/NK1.1⁺ (B) cells within the CD45⁺ population was determined. A representative histogram is shown for each group (n = 3–4 mice per group). (C) BMDM were treated with 100 μg/mL lipoteichoic acid (LTA) or formalin-fixed <i>S</i>. <i>pneumoniae</i> (SP) at an MOI of 10 for 18 hr at 37°C and protein lysates were immunoblotted for iNOS protein. Beta actin was used as a loading control. Immunoblots represent 1 of 3 identical experiments, (D) Graphical representation of C. n = 3; *p < 0.05.</p

<i>S</i>. <i>pneumoniae</i> high-dose survival experiment.

<p>WT and NKLAM-KO mice were infected via intranasal route with 5 x 10⁷ <i>S</i>. <i>pneumoniae</i> CFU in 30 μL sterile PBS and monitored for signs of severe illness for 5 days. WT, n = 11; NKLAM-KO, n = 12. p = not significant.</p

<i>S</i>. <i>pneumoniae</i>-induced phosphorylation of STAT1 and STAT3 is diminished in NKLAM-KO mice.

<p>Lung homogenates from <i>S</i>. <i>pneumoniae</i>-infected WT and NKLAM-KO mice were immunoblotted for (A) STAT1 and pSTAT1 (Tyr701) and (B) STAT3 and pSTAT3 (Tyr705). Each lane represents an individual mouse and is representative of the overall experiments. Immunoblots were used to calculate the ratio of pSTAT1 (Tyr701)/STAT1 (C) and pSTAT3 (Tyr705)/STAT3 (D). Data represent the mean ± SD of 1 of 3 representative experiments (n = 3–5 mice per group per experiment); * p ≤ 0.05. (E) Sections from paraffin-embedded lungs from <i>S</i>. <i>pneumoniae</i>-infected (24h) WT and NKLAM-KO mice were stained for pSTAT1 (Tyr701) (red) and DAPI (blue). Colocalization is depicted in pink (arrows). (n = 2–3 mice per group). (F) Aliquots (50 μg) of lung homogenate from PBS control mice and <i>S</i>. <i>pneumoniae</i>-infected (24h) WT and NKLAM-KO mice were separated by native gel electrophoresis then incubated with 4-methylumbelliferyl phosphate to visualize phosphatase activity. Each lane represents an individual mouse. Graph represents the mean band intensity ± SD. Results are representative of two experiments with 3–5 mice per group per experiment; * p ≤ 0.05.</p

Mouse inflammatory cytokine profile from <i>S</i>. <i>pneumoniae</i>-infected WT and NKLAM-KO mice.

<p>Mouse inflammatory cytokines (MCP-1, TNΦα, IFNγ and IL-12p70) were evaluated in lung homogenates (A) and plasma (B) from infected (24 and 48hr) WT and NKLAM-KO mice by cytometric bead array. Data shown were pooled from 3 independent experiments (n = 8–11 mice per group). * p ≤ 0.05.</p

Lack of NKLAM is associated with reduced lung inflammation.

<p>(A) Lungs from 24h-infected or (B) 48h-infected <i>S</i>. <i>pneumoniae</i>-infected (10 x 10⁶ CFU/mouse) WT and NKLAM-KO or PBS-treated mice were formalin-fixed, embedded in paraffin and sections were stained with H&E or anti-mouse MPO. Magnification, x 200 for H&E, x 400 for MPO. Black bar equals 50 μm. Data are representative of two experiments, with n = 2 mice per group per experiment.</p