Simulated microgravity disarms human NK-cells and inhibits anti-tumor cytotoxicity in vitro

2020 ◽  
Vol 174 ◽  
pp. 32-40 ◽  
Author(s):  
Preteesh Leo Mylabathula ◽  
Li Li ◽  
Austin B. Bigley ◽  
Melissa M. Markofski ◽  
Brian E. Crucian ◽  
...  
Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2744-2744 ◽  
Author(s):  
Maria Berg ◽  
Andreas Lundqvist ◽  
Yong Fan ◽  
J. Philip McCoy ◽  
Hisayuki Yokoyama ◽  
...  

Abstract The activation of NK cell inhibitory receptors may limit the antitumor efficacy of adoptive autologous and allogeneic NK cell infusions. Recently, we found that exposing malignant cells to the proteosome inhibitor bortezomib upregulated surface expression of death receptors for TRAIL, resulting in significant enhancement of autologous NK cell tumor cytotoxicity in vitro and in vivo. Here we show that NK cells expanded in vitro in the presence of IL-2 and EBV-LCL feeder cells upregulate surface expression of TRAIL which significantly augments bortezomib-induced tumor sensitization to NK cell killing. CD56+/CD3– NK cells were isolated from normal donors by immuno-magnetic bead selection and were co-cultured with irradiated EBV-LCL feeder cells in X-VIVO 20, 10% human AB serum, and 500 IU/ml hrIL-2 for up to 21 days. Depending on culture conditions, a 300 to 10,000 fold increase in NK cell numbers was achieved. Non-expanded and expanded NK cells were analyzed by flow cytometry for the expression of CD56, CD16, TRAIL, FasL, NKG2D, LFA-1, perforin, and granzymes A and B at baseline and ≥ 10 days following in vitro expansion. Chromium release assays were performed to assess fresh vs. expanded NK cell cytotoxicity of renal cell carcinoma (RCC) tumor targets treated with 10 nM bortezomib for 18 hr vs. untreated RCC controls. Freshly-isolated NK cells did not express TRAIL or FasL; in contrast NKG2D, LFA-1, perforin and granzymes A and B were constitutively expressed in fresh NK cells. After expansion, there was a dramatic increase in surface expression of TRAIL and NKG2D; on fresh vs. expanded NK cells from 3 different donors, TRAIL expression increased from 0% to 80.8±15.4% (mean fluorescence intensity [MFI] of TRAIL increased from 6.0±5.1 to 37.9±3.2). The MFI of NKG2D surface expression also increased following NK cell expansion (432.0±70.9 from 48.3±16.3). Expression of LFA-1 and perforin did not change, although there was a small increase in surface and intracellular expression of FasL and granzymes A and B respectively. At a 1:1 effector to target ratio, fresh NK cells lysed 3.4± 2.1% and 5.0± 2.7% of untreated and bortezomib-treated RCC tumor cells respectively. In contrast, there was a dramatic increase in bortezomib-treated tumor susceptibility to killing by expanded NK cells; NK cells expanded for 12-18 days killed 27.6± 9.3% and 55.8± 8.3% of untreated vs. bortezomib-treated RCC tumor cells respectively. Conclusion: In vitro-expanded NK cells are phenotypically and functionally different from non-expanded NK cells. Expanded cells have increased NKG2D and TRAIL expression and greatly enhanced TRAIL-mediated tumor cytotoxicity compared to non-expanded NK cells. Based on these findings, a phase I trial investigating the safety and anti-tumor effects of escalating doses of adoptively-infused ex vivo-expanded autologous NK cells following bortezomib treatment in patients with advanced metastatic tumors and hematological malignancies has recently been initiated. Figure: Freshly isolated and expanded Nk cell lysis of renal cell carcinoma cell line with and without treatment of tumor cells with bortezomib Figure:. Freshly isolated and expanded Nk cell lysis of renal cell carcinoma cell line with and without treatment of tumor cells with bortezomib


Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 463-463 ◽  
Author(s):  
Maria Berg ◽  
Andreas Lundqvist ◽  
Dawn Betters ◽  
Richard W. Childs

Abstract Abstract 463 IL-2 activates NK cell enhancing their capacity to lyse tumor cells. To date, most clinical studies of adoptive NK cell transfer have utilized short-term (12-16 hours) IL-2-activated NK cells. Because IL-2 alone is ineffective in expanding NK cells in vitro, the relatively low numbers of NK cells obtained for infusion following short term IL-2 activation may limit the full therapeutic impact of this approach. To obtain larger numbers of NK cells, novel ex vivo expansion protocols that utilize irradiated EBV-LCL or K562 feeder cells have recently been developed. However, concerns exist that extensive ex vivo expansion might significantly reduce the in vivo proliferative potential and long-term viability of adoptively transferred NK cells. Here we investigated for differences in phenotype, tumor cytotoxicity and in vivo persistence between short-term IL-2 activated and long-term expanded NK cells. CD56+/CD3- NK cells were isolated from normal donors by immuno-magnetic bead selection and were either activated with IL-2 (500U/ml) for 12-16 hours or were expanded in vitro over 14 days using irradiated EBV-LCL feeder cells in IL-2 containing media (500U/ml). Short-term IL-2 activated NK cells did not expand in number in contrast to EBV-LCL stimulated NK cells which expanded 400-1000 fold by culture day 14. Flow cytometry analysis revealed no differences in expression of DNAM-1, 2B4, LFA-1 or granzyme B between short-term activated and expanded NK cells. However, expanded NK cells had significantly higher expression of TRAIL, NKG2D, and the natural cytotoxicity receptors NKp30, NKp44 and NKp46 and a slight increase in KIR3DL1 and KIR2DL2/3. A 4-hour 51Cr-release assay showed expanded NK cells were significantly more cytotoxic against K562 cell compared to short-term IL-2 activated NK cells; at a 1:1 effector to target ratio, 67±6%, 26±1%, and 9±1% of K562 cells were killed by expanded, short term IL-2 activated and fresh NK cells respectively (p<0.05). Increased TRAIL expression on expanded NK cells was also associated with increased lysis of TRAIL-sensitive tumor cells (RCC tumors treated with bortezomib); at a 1:1 E:T ratio, 55±3% and 5±2% of bortezomib-treated RCC tumors were killed by expanded and short-term IL-2 activated NK cells respectively (p<0.05). We next assessed for differences in the in vivo longevity of these NK cell populations when transferred into immuno-deficient mice. Two million NK cells were labeled with a near infrared-dye (DiR; 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) and injected intra-peritoneal (i.p.) into CB.17 SCID-beige mice. Mice were administered IL-2 (100,000U/ml bid i.p.) for five days then underwent bioluminescent imaging using the IVIS100 system. Although FACS analysis of NK cells performed immediately prior to injection showed increased DiR fluorescent intensity in short-term IL-2 activated vs. expanded NK cells, fluorescence signal in vivo was slightly higher in the first 24-96 hours in mice that received expanded NK cells; fluorescence intensity was 5-41% (p=0.003) stronger in recipients of expanded NK cells compared to mice receiving short-term IL-2 activated NK cells. We next evaluated the in vivo anti-tumor effects of activated vs. expanded NK cells. CB.17 SCID-beige mice were injected i.p. with luciferase transduced 526 human melanoma cells three days prior to receiving an i.p. injection of short term IL-2 activated vs. expanded NK cells (+ bid i.p. IL-2). Bioluminescent imaging measuring tumor flux to calculate tumor burden and tumor doubling time showed no difference in tumor progression between both NK cell cohorts. In conclusion, these results demonstrate that ex vivo expanded NK cells are phenotypically and functionally different than short-term IL-2 activated NK cells. Expanded NK cells have increased expression of natural cytotoxicity receptors, NKG2D and TRAIL and have greater TRAIL-mediated tumor cytotoxicity compared to IL-2 activated NK cells. Importantly, despite extensive ex vivo proliferation, expanded NK cells appear maintain similar longevity in vivo as non-expanded short term IL-2 activated NK cells. Disclosures: No relevant conflicts of interest to declare.


2005 ◽  
Vol 113 (S 1) ◽  
Author(s):  
M Penna-Martinez ◽  
K Andric ◽  
RA Wahl ◽  
KH Usadel ◽  
PM Schumm-Draeger
Keyword(s):  
Nk Cells ◽  

2021 ◽  
Vol 8 (6) ◽  
pp. 110
Author(s):  
Nathalie Meijerink ◽  
Jean E. de Oliveira ◽  
Daphne A. van Haarlem ◽  
Guilherme Hosotani ◽  
David M. Lamot ◽  
...  

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.


2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii115-ii115
Author(s):  
Rongze Olivia Lu ◽  
Winson Ho ◽  
Brandon Chiou

Abstract Checkpoint immunotherapy (ICB) thus far has shown limited efficacy against brain tumors, such as medulloblastoma (MB). Its low mutational burden is thought to result in a paucity of neoantigen to trigger an effective T-cell response. Natural killer (NK) cells, can recognize tumor cells independently of neoantigens, making them appealing against MBs. Modulation of NK cells to enhance cytotoxicity against MBs could be a novel treatment strategy. Protein Phosphatase 2A (PP2A), a ubiquitous serine/threonine phosphatase, has been shown to inhibit IFNg and Granzyme B production by NK cells. We hypothesize that NK92, a transformed human NK cell line, has intrinsic activity against human MB cells and that inhibiting PP2A pharmacologically can enhance cytotoxicity of NK92 cells. We performed NK cytotoxicity assay and granulation assay against human MB cell line D425. We also used a small molecular inhibitor, LB100, to modulate PP2A activity in NK92. NK92 cells were co-cultured with D425, in increasing E:T (Effector:Target) ratio for 4 hours. D425 cells were pre-labeled with CellTrace Violet dye. The percentage of D425 (Violet+) cells in apoptosis (Cas3/7+) or necrosis (AAD+) were compared with different ET ratios to quantify NK mediated cell cytotoxicity. We also measured CD107a expression in NK92 to assess granulation with LB100 treatment. D425 cells were sensitive to NK92 killing. Percentage of D425 cells either apoptotic or necrotic increased with increasing ET ratio, suggesting that there was NK92 mediated cytotoxicity. Percentage of killed D425 cells ranged from 18% at baseline (without NK92) to 80% at ET ratio of 20. Inhibition of PP2A using LB100, enhanced NK92 degranulation. CD107a+ NK92 cells increased from 19% to 28% with 8uM of LB100. NK92 cells are cytotoxic against MB cells in vitro and inhibition of PP2A in NK cells can enhance their activity against MB cells.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Katherine E. Harris ◽  
Kyle J. Lorentsen ◽  
Harbani K. Malik-Chaudhry ◽  
Kaitlyn Loughlin ◽  
Harish Medlari Basappa ◽  
...  

AbstractThe use of recombinant interleukin-2 (IL-2) as a therapeutic protein has been limited by significant toxicities despite its demonstrated ability to induce durable tumor-regression in cancer patients. The adverse events and limited efficacy of IL-2 treatment are due to the preferential binding of IL-2 to cells that express the high-affinity, trimeric receptor, IL-2Rαβγ such as endothelial cells and T-regulatory cells, respectively. Here, we describe a novel bispecific heavy-chain only antibody which binds to and activates signaling through the heterodimeric IL-2Rβγ receptor complex that is expressed on resting T-cells and NK cells. By avoiding binding to IL-2Rα, this molecule circumvents the preferential T-reg activation of native IL-2, while maintaining the robust stimulatory effects on T-cells and NK-cells in vitro. In vivo studies in both mice and cynomolgus monkeys confirm the molecule’s in vivo biological activity, extended pharmacodynamics due to the Fc portion of the molecule, and enhanced safety profile. Together, these results demonstrate that the bispecific antibody is a safe and effective IL-2R agonist that harnesses the benefits of the IL-2 signaling pathway as a potential anti-cancer therapy.


2021 ◽  
Vol 7 (8) ◽  
pp. eabc2331 ◽  
Author(s):  
Jose M. Ayuso ◽  
Shujah Rehman ◽  
Maria Virumbrales-Munoz ◽  
Patrick H. McMinn ◽  
Peter Geiger ◽  
...  

Solid tumors generate a suppressive environment that imposes an overwhelming burden on the immune system. Nutrient depletion, waste product accumulation, hypoxia, and pH acidification severely compromise the capacity of effector immune cells such as T and natural killer (NK) cells to destroy cancer cells. However, the specific molecular mechanisms driving immune suppression, as well as the capacity of immune cells to adapt to the suppressive environment, are not completely understood. Thus, here, we used an in vitro microfluidic tumor-on-a-chip platform to evaluate how NK cells respond to the tumor-induced suppressive environment. The results demonstrated that the suppressive environment created by the tumor gradually eroded NK cell cytotoxic capacity, leading to compromised NK cell surveillance and tumor tolerance. Further, NK cell exhaustion persisted for an extended period of time after removing NK cells from the microfluidic platform. Last, the addition of checkpoint inhibitors and immunomodulatory agents alleviated NK cell exhaustion.


Author(s):  
Elena Pánisová ◽  
Anna Lünemann ◽  
Simone Bürgler ◽  
Monika Kotur ◽  
Julien Lazarovici ◽  
...  

AbstractAround 30–50% of classical Hodgkin lymphoma (cHL) cases in immunocompetent individuals from industrialized countries are associated with the B-lymphotropic Epstein-Barr virus (EBV). Although natural killer (NK) cells exhibit anti-viral and anti-tumoral functions, virtually nothing is known about quantitative and qualitative differences in NK cells in patients with EBV+ cHL vs. EBV- cHL. Here, we prospectively investigated 36 cHL patients without known immune suppression or overt immunodeficiency at diagnosis. All 10 EBV+ cHL patients and 25 out 26 EBV- cHL were seropositive for EBV antibodies, and EBV+ cHL patients presented with higher plasma EBV DNA levels compared to EBV- cHL patients. We show that the CD56dim CD16+ NK cell subset was decreased in frequency in EBV+ cHL patients compared to EBV- cHL patients. This quantitative deficiency translates into an impaired CD56dim NK cell mediated degranulation toward rituximab-coated HLA class 1 negative lymphoblastoid cells in EBV+ compared to EBV- cHL patients. We finally observed a trend to a decrease in the rituximab-associated degranulation and ADCC of in vitro expanded NK cells of EBV+ cHL compared to healthy controls. Our findings may impact on the design of adjunctive treatment targeting antibody-dependent cellular cytotoxicity in EBV+ cHL.


Cancers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1577
Author(s):  
Matteo Tanzi ◽  
Michela Consonni ◽  
Michela Falco ◽  
Federica Ferulli ◽  
Enrica Montini ◽  
...  

The limited efficacy of Natural Killer (NK) cell-based immunotherapy results in part from the suboptimal expansion and persistence of the infused cells. Recent reports suggest that the generation of NK cells with memory-like properties upon in vitro activation with defined cytokines might be an effective way of ensuring long-lasting NK cell function in vivo. Here, we demonstrate that activation with IL-12, IL-15 and IL-18 followed by a one-week culture with optimal doses of Interleukin (IL-2) and IL-15 generates substantial numbers of memory-like NK cells able to persist for at least three weeks when injected into NOD scid gamma (NSG) mice. This approach induces haploidentical donor-derived memory-like NK cells that are highly lytic against patients’ myeloid or lymphoid leukemia blasts, independent of the presence of alloreactive cell populations in the donor and with negligible reactivity against patients’ non-malignant cells. Memory-like NK cells able to lyse autologous tumor cells can also be generated from patients with solid malignancies. The anti-tumor activity of allogenic and autologous memory-like NK cells is significantly greater than that displayed by NK cells stimulated overnight with IL-2, supporting their potential therapeutic value both in patients affected by high-risk acute leukemia after haploidentical hematopoietic stem cell transplantation and in patients with advanced solid malignancies.


2008 ◽  
Vol 76 (4) ◽  
pp. 1719-1727 ◽  
Author(s):  
Semih Esin ◽  
Giovanna Batoni ◽  
Claudio Counoupas ◽  
Annarita Stringaro ◽  
Franca Lisa Brancatisano ◽  
...  

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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