NK Cell Deficiency In Job Syndrome Patients With Dominant Negative STAT3 Mutations

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3476-3476
Author(s):  
Prasad V Phatarpekar ◽  
Emily M Mace ◽  
Jordan S Orange ◽  
Alexandra F Freeman ◽  
Steven M. Holland ◽  
...  
Keyword(s):  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Cell Number ◽  
Dominant Negative ◽  
Receptor Expression ◽  
Lower Percentage ◽  
Job Syndrome ◽  
Stat3 Mutations ◽  
Nkg2d Receptor

Abstract Introduction Signal transducer and activator of transcription 3 (STAT3) is an important regulator of cellular immunity, and in some animal models inhibition of STAT3 has been effective in improving natural killer (NK) cell antitumor efficacy. However, there has been little direct assessment of the STAT3 signaling role in human NK cells. In our previous work we found that (i) human NK cells stimulated with K562-based artificial antigen presenting cells (aAPC) genetically modified to express membrane bound IL-21 (mbIL21), which predominantly activates STAT3, resulted in greater proliferation, longer telomere length, and less senescence than NK cells expanded with mbIL15, which predominantly activates STAT5 (ii) NK cell proliferation, cytotoxicity and expression of the activating receptor NKG2D were enhanced by STAT3 activating cytokines and diminished by STAT3 inhibitors. Based on these results we hypothesized that activation of STAT3 plays a critical role in NK cell expansion and anti-tumor activity and tested our hypothesis by studying NK cells from Job syndrome patients. Hyper-immunoglobulin E syndrome (HIES / Job syndrome) is an immunodeficiency characterized by highly elevated serum IgE and recurrent skin and lung abscesses primarily caused by staphylococcal infection. Both, sporadic and autosomal dominant Job syndromes are caused by mutations in the gene coding for STAT3, which results in impaired cytokine signaling in immune cells. The STAT3 signaling deficiency is responsible for many lymphocyte abnormalities observed in Job syndrome patients such as impaired TH17 differentiation, defective development and maintenance of central memory T cells and reduced memory B cells. However, the effect of STAT3 deficiency on NK cell development, proliferation and function in Job syndrome patients has not been studied yet. Methods We obtained peripheral blood from healthy donors and from HIES patients with documented STAT3 mutations. NK cell proliferation was induced by stimulating peripheral blood mononuclear cells (PBMCs) with K562-based artificial antigen presenting cells genetically modified to express membrane bound cytokines. IL10 and IL21 were applied to induce STAT3 activation. Receptor expression was measured by flow-cytometry. NK cells from normal, healthy donors were used as control. Statistical comparison was performed by Student’s t test using GraphPad Prism. Results Compared to normal donors (1) Flow-cytometric analysis of PBMCs showed a significantly lower percentage of NK cells in Job syndrome patients (2) We observed impaired proliferation of Job syndrome patients’ NK cells upon stimulation of PBMCs not only with mbIL21 but also with mbIL15 in complex with its receptor α (mbIL15Rα), a physiologically relevant presentation of a physiologically relevant cytokine involved in NK cell survival and proliferation (3) Lower percentage of mature, CD56+CD16+ NK cells in Expanded NK cells from Job syndrome patients and (4) Reduced basal expression of NKG2D receptor as well as lower induction of NKG2D receptor expression upon stimulation with STAT3 activating cytokines IL10 and IL21, on Job syndrome patients’ NK cells. Conclusions Our finding of a deficient NK cell number in Job syndrome patients carrying dominant negative STAT3 mutations is the first to show an NK cell defect in this immunodeficiency. Lower percentage of NK cells in the peripheral blood and impaired ex vivo expansion of Job syndrome patient’s NK cells suggest deficient development and/or deficient proliferation and survival of NK cells in Job syndrome patients with dominant negative STAT3 mutations. Along with recurrent bacterial infections, Job syndrome patients are also more prone to viral infections and lymphoma. As NK cells perform surveillance of virally infected and tumorigenic cells through NKG2D receptor, low NK cell number with reduced NKG2D expression may explain the proclivity of Job syndrome patients to viral infections and lymphoma. Work is in progress to assess cytokine generation and anti-tumor activity of Job syndrome patients’ NK cells. Disclosures: No relevant conflicts of interest to declare.

NK Cell Deficiency in Job's Syndrome Patients

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3293-3293
Author(s):  
Prasad Phatarpekar ◽  
Emily Mace ◽  
Jordan Orange ◽  
Alexandra Freeman ◽  
Steven M. Holland ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Nk Cells ◽  
Viral Infections ◽  
Nk Cell ◽  
Cytolytic Activity ◽  
Dominant Negative ◽  
Receptor Expression ◽  
Lower Percentage ◽  
Job’S Syndrome ◽  
Job's Syndrome

Abstract Abstract 3293 Background: Hyper-immunoglobulin E syndrome (Job's syndrome) is an immunodeficiency characterized by highly elevated serum IgE and recurrent skin and lung abscesses from inappropriately mild immune responses to staphylococcal infection. Both sporadic and autosomal dominant Job's syndrome patients are caused by mutations in the gene coding for STAT3, which results in impaired cytokine signaling in immune cells. The STAT3 signaling deficiency is responsible for many lymphocyte abnormalities observed in Job's syndrome patients such as impaired TH17 differentiation, defective development and maintenance of central memory T cells and reduced memory B cells. However, the effect of STAT3 deficiency on NK cell development and function in Job's syndrome patients has not been studied yet. Previous work performed in our lab employing STAT3 activating cytokines and STAT3 inhibitors showed that STAT3 activation plays a role in NK cell proliferation, survival, cytolytic activity, and regulation of expression of the activating receptor NKG2D. This finding prompted us to test whether aberrant STAT3 activation affects NK cell proliferation, survival and cytolytic activity, in NK cells of Job's syndrome patients. Objective: To assess proliferation, cytolytic activity and receptor expression on NK cells from Job's syndrome patients with dominant negative STAT3 mutations. Methods: NK cell proliferation was induced by stimulating peripheral blood mononuclear cells (PBMCs) with K562-based artificial antigen presenting cells genetically modified to express membrane bound cytokines. IL10 and IL21 were applied to induce STAT3 activation. Receptor expression was measured by flow-cytometry. NK cytolytic activity was evaluated by Calcein release assay. NK cells from normal, healthy donors were used as control. Statistical comparison was performed by paired Student's t test using GraphPad Prism. Results: Flow-cytometric analysis of PBMCs showed significantly lower percentage of NK cells, identified as CD3− CD56+, in Job's syndrome patients compared to normal donors. The expansion in response to membrane bound IL21 stimulation was found to be impaired in NK cells obtained from Job's syndrome patients compared to those from normal donors. Expanded NK cells from Job's syndrome patients had lower percentage of mature, CD56+CD16+NK cells compared to those from normal donors. We also found significantly reduced cytolytic activity in NK cells from Job's syndrome patients. STAT3 activating cytokines IL10 and IL21 were also found to be impaired in their ability to induce NKG2D expression on NK cells obtained from Job's syndrome patients. Conclusions: Our finding of deficient NK cell number and cytolytic activity in Job's syndrome patients is the first to show an NK cell defect in this immunodeficiency. Lower percentage of NK cells in the peripheral blood and impaired ex vivo expansion of Job's syndrome patient's NK cells suggest deficient development and/or deficient proliferation and survival of NK cells in Job's syndrome patients with dominant negative STAT3 mutations. Along with recurrent bacterial infections, Job's syndrome patients are also more prone to viral infections and lymphoma. Given the role of NK cells in the immune surveillance of virally infected and tumorigenic cells, low NK cell function may explain the proclivity of Job's syndrome patients to viral infections and lymphoma. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Diversity of peripheral blood human NK cells identified by single-cell RNA sequencing

2020 ◽  
Vol 4 (7) ◽  
pp. 1388-1406 ◽  
Author(s):  
Samantha L. Smith ◽  
Philippa R. Kennedy ◽  
Kevin B. Stacey ◽  
Jonathan D. Worboys ◽  
Annie Yarwood ◽  
...  
Keyword(s):  
Nk Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Killer Cell ◽  
Early Response ◽  
Receptor Expression ◽  
Type I ◽  
Single Cell Rna Sequencing

Abstract Human natural killer (NK) cells in peripheral blood perform many functions, and classification of specific subsets has been a longstanding goal. We report single-cell RNA sequencing of NK cells, comparing gene expression in unstimulated and interleukin (IL)-2–activated cells from healthy cytomegalovirus (CMV)-negative donors. Three NK cell subsets resembled well-described populations; CD56brightCD16−, CD56dimCD16+CD57−, and CD56dimCD16+CD57+. CD56dimCD16+CD57− cells subdivided to include a population with higher chemokine mRNA and increased frequency of killer-cell immunoglobulin-like receptor expression. Three novel human blood NK cell populations were identified: a population of type I interferon–responding NK cells that were CD56neg; a population exhibiting a cytokine-induced memory-like phenotype, including increased granzyme B mRNA in response to IL-2; and finally, a small population, with low ribosomal expression, downregulation of oxidative phosphorylation, and high levels of immediate early response genes indicative of cellular activation. Analysis of CMV+ donors established that CMV altered the proportion of NK cells in each subset, especially an increase in adaptive NK cells, as well as gene regulation within each subset. Together, these data establish an unexpected diversity in blood NK cells and provide a new framework for analyzing NK cell responses in health and disease.

Resveratrol Enhances NKG2D-Mediated Cytotoxicity Against Leukemia Cells by Upregulating Both NKG2D Receptor on NK Cells As Well As NKG2D Ligands on Target Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3236-3236
Author(s):  
Luis J. Espinoza ◽  
Akiyoshi Takami ◽  
Katsuya Nakata ◽  
Ly Quoc Trung ◽  
Kayoko Yamada ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Specific Inhibitor ◽  
Receptor Expression ◽  
Leukemia Cells ◽  
Target Cells ◽  
Lymphoid Leukemia ◽  
Nkg2d Ligands ◽  
Nkg2d Receptor

Abstract Abstract 3236 NKG2D is a powerful activating receptor expressed by natural killer (NK) cells that promotes cytotoxic lysis of cancer cells expressing NKG2D ligands (NKG2D-Ls). Pharmacological induction of NKG2D-Ls in malignant cells has been an attractive therapeutic approach but has gained poor clinical utility because currently available NKG2D-Ls inducers have been hampered either by their limited efficacy or by their associated toxicity. Resveratrol (RVT), a compound derived from several natural sources, has proved in vivo and in vitro potent anti-tumor effects against various cancers. Extensive research in the last decade has shown that such effects are mediated by targeting various molecules involved in the regulation of proliferation and cell survival and those include, NFκB, STAT3, ATM/ATR and ERK1/2. To date, it is unknown whether RVT has any effect on NKG2D-Ls expression. We report here NKG2D-Ls induction by RVT in a broad range of leukemia cells. RVT upregulated the NKG2D-Ls MICA/B, ULBP1, ULBP2 and ULBP3 in the myeloid leukemia cells OUN-1, NB4, THP-1 and KG1 and upregulated MICA/B, ULBP-1 and ULBP3 ligands in the lymphoid leukemia cells Jurkat and Molt-4. The upregulation of NKG2D-Ls by RVT was associated with increased transcription of each NKG2D-L gene. Ligand upregulation induced by RVT was prevented by cell pretreatment with caffeine, and inhibitor of ATM/ATR, which is the main signal regulator of NKG2D-Ls expression. Leukemia cells treated with RVT were more susceptible to killing by NK cells than untreated cells and the enhanced cytotoxicity of NK cells was blocked by the treatment of NK cells with anti-NKG2D monoclonal antibodies. Interestingly, the same concentration of RVT that effectively induced NKG2D-Ls in tumor cells, consistently upregulated NKG2D receptor expression in primary NK cells from healthy individuals and in the NK cell lines NKL and NK-92 and this effect was also associated with enhanced NKG2D-mediated NK cells cytotoxicity. RVT-induced NKG2D receptor enhancement in NK cells associated with the activation of the MAP kinase ERK1/2 and was prevented by the ERK1/2 specific inhibitor PD98059. Thus, RVT represents the first identified agent capable of activating both arms of the NKG2D axis. Since several clinical trials on RVT are ongoing, these previously unrecognized properties of this non toxic compound have an attractive immunotherapeutic potential. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Immunophenotypic, cytotoxic, proteomic and genomic characterization of human cord blood vs. peripheral blood CD56Dim NK cells

2019 ◽  
Vol 25 (5) ◽  
pp. 294-304 ◽  
Author(s):  
Evan Shereck ◽  
Nancy S Day ◽  
Aradhana Awasthi ◽  
Janet Ayello ◽  
Yaya Chu ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Cell Function ◽  
Nk Cell ◽  
Chronic Gvhd ◽  
Cell Subset ◽  
Receptor Expression ◽  
Unrelated Donor ◽  
Human Cord Blood

Unrelated cord blood (CB) is an excellent alternative as an allogeneic donor source for stem cell transplantation. CB transplantation is associated with lower incidence of severe acute graft versus host disease (GVHD) and chronic GVHD but similar rates of malignant relapse compared with other unrelated donor cell transplants. NK cells are critical innate immune components and the comparison of CB vs. peripheral blood (PB) NK cells is relatively unknown. NK cell receptor expression, cell function, and maturation may play a role in the risk of relapse after CB transplant. We investigated CB vs. PB NK cell subset cytotoxicity, function, receptor expression, and genomic and proteomic signatures. The CB CD56dim compared with PB CD56dim demonstrated significantly increased expression of NKG2A and NKG2D, respectively. CB vs. PB CD56dim NK cells had significantly decreased in vitro cytotoxicity against a variety of non-Hodgkin lymphoma targets. Various proteins were significantly under- and over-expressed in CB vs. PB CD56dim NK cells. Microarray analyses and qRT-PCR in CB vs. PB CD56dim demonstrated significantly increased expression of genes in cell regulation and development of apoptosis, respectively. In summary, CB vs. PB CD56dim NK cells appear to be earlier in development, have decreased functional activity, and increased capacity for programmed cell death, suggesting that CB NK cells require functional and maturational stimulation to achieve similar functional levels as PB CD56dim NK cells.

Acquired immune deficiency of cancer: Evaluation of NK cell number as a predictor of overall survival in breast cancer.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e13003-e13003
Author(s):  
R. Michael Williams ◽  
Edmond J Yunis
Keyword(s):  
Breast Cancer ◽  
Immune Deficiency ◽  
Immune System ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Cell Number ◽  
Breast Cancer Patients ◽  
Treatment Protocols ◽  
Acquired Immune Deficiency

e13003 Background: Cancer specialists, be they molecular geneticists or oncology clinicians, now accept the fact that a cancer results after a deleterious mutation in the apoptosis program AND failure of the immune system, innate (non-specific) and adaptive (specific), to eliminate the now abnormal transformed cell and it's progeny. Just as the acquired immune deficiency syndrome existed as a syndrome BEFORE the HIV virus was described, the disease AIDS/HIV was designated because the virus explained the syndrome and made HIV/AIDS a disease. We believe cancer works the same way. Cancer is a failure of surveillance by the immune system which leads to a disease, one we have described as AIDS/cancer (J Immunol May 1, 2019, 202, 1S, 182.79). Methods: Commercially available laboratory analysis of peripheral blood (Quest Diagnostics) and standard treatment protocols for breast cancer. Results: The immune system can be evaluated with samples of peripheral blood where cell subsets, antibody levels, etc. are measurable. Here we present results from over 200 unselected breast cancer patients, independent of Stage. All patients were treated with standard protocols. The initial number of NK cells in peripheral blood, but not other cell subsets or immunoglobulin levels, are a, and likely the, most significant predictor for survival. Kaplan Meier survival analysis, done when 20% of the entire population had died, showed that none of the patients in the upper quartile for NK cell number had died. Survival differences, comparing the top 50% vs. bottom 50% in initial NK number. were also highly significant (P < 0.05). Conclusions: We conclude that the innate immune system, reflected as numbers of NK cells in the blood are the most significant predictor of survival.

542 Expansion of cytotoxic NK Cells from PBMCs using individualized cytokine combination

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A578-A578
Author(s):  
Andreia Maia ◽  
Joana Lerias ◽  
Markus Maeurer ◽  
Mireia Castillo-Martin
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Blood Donors ◽  
Nk Cell ◽  
Tumour Cells ◽  
List Type ◽  
Mhc I ◽  
Cytokine Combination

BackgroundAdoptive immunotherapy relies on the use of T-cells to target tumour cells, through Major Histocompatibility Complex (MHC) Class I recognition(1). However, many tumours display alterations in the MHC-I pathway, a well-described immune evasion mechanism(2). Natural Killer (NK) cells recognize transformed cells independently from the presence of MHC-I and may be a reliable therapeutic option for patients with altered tumour MHC-I expression. The source of NK cells may be autologous or allogeneic and NK cells are also clinically relevant recipients of transgenic receptors (TCRs or antibodies) targeting tumour cells. NK cells have been categorized according to their CD56 and CD16 surface expression into different subpopulations: cytotoxic (CD56+CD16+) and regulatory (CD56brightCD16-)(3). Expanding cytotoxic NK cells is challenging, since the frequency of NK cells is low in peripheral blood(4) and there is also – at this point – not an optimal expansion protocol available.The goal of this project is to determine the best cytokine combination that facilitates expansion of cytotoxic NK cells that either target tumor cells directly or serve as recipients for transgenic receptors.MethodsPeripheral Blood Mononuclear Cells (PBMCs) were extracted using Ficoll methodology from blood donors and cultured in T25 flasks with Cell Genix Medium supplemented with 10% human serum and antibiotics. NK cells were expanded supplemented with feeder cells (ratio 1:1) and different cytokine combinations (1000 U/mL of IL-2, 10 U/ml of IL-12, 180 U/mL of IL-15 and/or 1 U/mL of IL-21) during 20 days. The immunophenotype of expanded NK cells was analyzed at days 0, 5, 10, 15 and 20 by flow cytometry. The cytotoxicity of NK cells was measured by a CD107a Assay or by a Total Cytotoxicity and Apoptosis Assay at days 10 and 20. Thirteen different cytokine combinations were tested.Results4/13 cytokine combinations produced a statistically significant increase of the absolute number of NK cells with a higher percentage of cytotoxic NK cells (figure 1). However, induction of cytotoxicity was not associated with a strong NK cell expansion. The regulatory NK cells subset (CD56brightCD16-) showed the highest percentage of CD107a-expressing cells, more than the CD56+CD16+, the most cytotoxic subpopulation of NK cells.Abstract 542 Figure 1Representative percentage of NK cells in total lymphocytes (A), CD56+CD16+ subpopulation in total NK cells (B), and CD56brightCD16- subpopulation amongst total NK cells (C) at different time points (5, 10, 15 and 20 days) expanded from PBMCs* p-value < 0.05ConclusionsThis work shows that we are able to grow and efficiently expand NK cells from PBMCs with different cytokine combinations leading to clinically relevant NK cell numbers as well as cytotoxic functions. This enables to produce NK cell products for therapy and as recipients for transgenic tumor antigen-specific receptors.AcknowledgementsThe authors would like to thank the Champalimaud Foundation Biobank, the Vivarium Facility and the Flow Cytometry Platform of the Champalimaud Centre for the Unknown.Ethics ApprovalThis study was approved by the Champalimaud Foundation Ethics Committee and by the Ethics Research Committee of NOVA Medical School of NOVA University of Lisbon.ConsentWritten informed consent was obtained from the blood donors to use their samples for research purposes.ReferencesRosenberg SA, Restifo NP, Yang JC, Morgan RA, Mark E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 2008;8(4):299–308.Aptsiauri N, Ruiz-Cabello F, Garrido F. The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses. Curr Opin Immunol 2018;51:123–32.Di Vito C, Mikulak J, Mavilio D. On the way to become a natural killer cell. Front Immunol. 2019;10(August):1–15.Zotto G Del, Antonini F, Pesce S, Moretta F, Moretta L. Comprehensive phenotyping of human PB NK Cells by Flow Cytometry. 2020;1–9.

scholarly journals Medulloblastoma rendered susceptible to NK-cell attack by TGFβ neutralization

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Allison B. Powell ◽  
Sridevi Yadavilli ◽  
Devin Saunders ◽  
Stacey Van Pelt ◽  
Elizabeth Chorvinsky ◽  
...  
Keyword(s):  
Nk Cells ◽  
Transforming Growth Factor ◽  
Nk Cell ◽  
Transforming Growth Factor Β ◽  
In Vitro Models ◽  
Dominant Negative ◽  
Conditioned Media ◽  
Retroviral Transduction ◽  
Effector Cells

Abstract Background Medulloblastoma (MB), the most common pediatric brain cancer, presents with a poor prognosis in a subset of patients with high risk disease, or at recurrence, where current therapies are ineffective. Cord blood (CB) natural killer (NK) cells may be promising off-the-shelf effector cells for immunotherapy due to their recognition of malignant cells without the need for a known target, ready availability from multiple banks, and their potential to expand exponentially. However, they are currently limited by immune suppressive cytokines secreted in the MB tumor microenvironment including Transforming Growth Factor β (TGF-β). Here, we address this challenge in in vitro models of MB. Methods CB-derived NK cells were modified to express a dominant negative TGF-β receptor II (DNRII) using retroviral transduction. The ability of transduced CB cells to maintain function in the presence of medulloblastoma-conditioned media was then assessed. Results We observed that the cytotoxic ability of nontransduced CB-NK cells was reduced in the presence of TGF-β-rich, medulloblastoma-conditioned media (21.21 ± 1.19% killing at E:T 5:1 in the absence vs. 14.98 ± 2.11% in the presence of medulloblastoma-conditioned media, n = 8, p = 0.02), but was unaffected in CB-derived DNRII-transduced NK cells (21.11 ± 1.84% killing at E:T 5:1 in the absence vs. 21.81 ± 3.37 in the presence of medulloblastoma-conditioned media, n = 8, p = 0.85. We also observed decreased expression of CCR2 in untransduced NK cells (mean CCR2 MFI 826 ± 117 in untransduced NK + MB supernatant from mean CCR2 MFI 1639.29 ± 215 in no MB supernatant, n = 7, p = 0.0156), but not in the transduced cells. Finally, we observed that CB-derived DNRII-transduced NK cells may protect surrounding immune cells by providing a cytokine sink for TGF-β (decreased TGF-β levels of 610 ± 265 pg/mL in CB-derived DNRII-transduced NK cells vs. 1817 ± 342 pg/mL in untransduced cells; p = 0.008). Conclusions CB NK cells expressing a TGF-β DNRII may have a functional advantage over unmodified NK cells in the presence of TGF-β-rich MB, warranting further investigation on its potential applications for patients with medulloblastoma.

scholarly journals Production of colony-stimulating activity by human natural killer cells: analysis of the conditions that influence the release and detection of colony-stimulating activity

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 156-164
Author(s):  
V Pistoia ◽  
S Zupo ◽  
A Corcione ◽  
S Roncella ◽  
L Matera ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Nk Cell ◽  
K562 Cells ◽  
Cell Suspensions ◽  
Normal Peripheral Blood ◽  
Colony Stimulating Activity ◽  
Culture Supernatants

Highly purified natural killer (NK) cell suspensions were tested for their capacity to release colony-stimulating activity (CSA) in vitro. NK cell suspensions comprised primarily CD16+ cells and were devoid of CD3+ T cells, CD15+ monocytes, and of B cells. CSA was detected in the NK cell supernatants and sustained the growth of myeloid colonies from both normal peripheral blood and bone marrow. CSA could be in part inhibited by pretreating NK cell culture supernatants with a specific goat anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antiserum. The inhibition, however, was never complete, a finding that suggests that additional factors were responsible for CSA. Incubation of NK cells with K562 cells (an NK-sensitive target) or with normal bone marrow cells resulted in the appearance of a strong colony- inhibiting activity (CIA) in the culture supernatants. Such CIA was demonstrable in an experimental system where bone marrow or peripheral blood progenitors were induced to form myeloid colonies in the presence of conditioned medium by CSA-producing giant cell tumor (GCT) cells. Stimulation of NK cells with NK-insensitive targets failed to induce CIA production. Neutralizing antitumor necrosis factor (TNF) monoclonal antibodies (MoAbs) were found capable of inhibiting CIA present in the supernatants of NK cells stimulated with K562 cells. Following treatment with anti-TNF antibodies, CSA was again detectable in the same supernatants. This finding indicates that induction of TNF production did not concomitantly switch off CSA production by NK cells. Pretreatment of NK cells with recombinant interleukin-2 (rIL-2) or gamma interferon (r gamma IFN) did not change the amount of CSA released. However, treatment with rIL-2 caused the appearance of a factor in the NK cell supernatants capable of sustaining the formation of colonies of a larger size.

scholarly journals 151 Potentiating the Large-Scale Expansion and Engineering of Peripheral Blood-Derived CAR NK Cells for Off-the-Shelf Application

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A159-A159
Author(s):  
Michael Whang ◽  
Ming-Hong Xie ◽  
Kate Jamboretz ◽  
Hadia Lemar ◽  
Chao Guo ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Therapy ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Adoptive Cell Therapy ◽  
Therapeutic Window ◽  
Target Cells ◽  
Healthy Donors ◽  
Custom Made

BackgroundPeripheral blood natural killer (NK) cells are mature cytotoxic innate lymphocytes possessing an inherent capacity for tumor cell killing, thus making them attractive candidates for adoptive cell therapy. These NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for off-the-shelf therapy since they are not known to cause graft-versus-host disease, unlike T cells. Presently, approved CAR cell therapy is custom-made from each patient‘s own T cells, a process that can limit patient pool, narrow therapeutic window, and contribute to product variability. In this study, we investigate whether peripheral blood NK cells from a selected donor can be edited, engineered, and expanded sufficiently for off-the-shelf use in a wide patient population.MethodsUsing the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from 3 healthy donors. Subsequently, we expanded edited NK cells by using IL-2 and sequential stimulations using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added twice during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD19-targeted CAR and mbIL-15 during an interval between the first and second NKSTIM pulses. We assessed NK cell cytotoxicity against Nalm6 target cells by IncuCyte.ResultsIsolated peripheral blood NK cells from 3 healthy donors were successfully edited using CRISPR/Cas9, engineered to express high levels of CAR, extensively expanded using a series of NKSTIM pulses in the presence of IL-2, and differentiated into memory-like NK cells using IL-12 and IL-18. Interestingly, NK cells from the 3 donors exhibited distinct outcomes. NK cells from one donor reached a peak expansion limit of approximately 7-million-fold before undergoing contraction whereas NK cells from two donors continued to expand over the length of the study surpassing 100-million-fold expansion, without appearing to have reached a terminal expansion limit. At the end of the study, NK cells from one donor exceeded 1-billion-fold expansion and maintained 88% cytolytic activity compared to Nkarta’s standard process control in a 72-hour IncuCyte assay.ConclusionsIn this study, we demonstrate that healthy donor-derived peripheral blood NK cells are capable of expanding over billion-fold while maintaining potency. These results provide a rationale for the development of off-the-shelf CAR NK cell therapies using NK cells from donors selected to provide optimal product characteristics.Ethics ApprovalHuman samples were collected with written informed consent by an approved vendor.

scholarly journals Tissue Trafficking Kinetics of Rhesus Macaque Natural Killer Cells Measured by Serial Intravascular Staining

2022 ◽  
Vol 12 ◽  
Author(s):  
Ryland D. Mortlock ◽  
Chuanfeng Wu ◽  
E. Lake Potter ◽  
Diana M. Abraham ◽  
David S. J. Allan ◽  
...  
Keyword(s):  
Steady State ◽  
Nk Cells ◽  
Tissue Distribution ◽  
Natural Killer ◽  
Rhesus Macaque ◽  
Peripheral Blood ◽  
Cell Biology ◽  
Nk Cell ◽  
Lymphoid Tissues ◽  
Intravascular Staining

The in vivo tissue distribution and trafficking patterns of natural killer (NK) cells remain understudied. Animal models can help bridge the gap, and rhesus macaque (RM) primates faithfully recapitulate key elements of human NK cell biology. Here, we profiled the tissue distribution and localization patterns of three NK cell subsets across various RM tissues. We utilized serial intravascular staining (SIVS) to investigate the tissue trafficking kinetics at steady state and during recovery from CD16 depletion. We found that at steady state, CD16+ NK cells were selectively retained in the vasculature while CD56+ NK cells had a shorter residence time in peripheral blood. We also found that different subsets of NK cells had distinct trafficking kinetics to and from the lymph node as well as other lymphoid and non-lymphoid tissues. Lastly, we found that following administration of CD16-depleting antibody, CD16+ NK cells and their putative precursors retained a high proportion of continuously circulating cells, suggesting that regeneration of the CD16 NK compartment may take place in peripheral blood or the perivascular compartments of tissues.

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