scholarly journals Regulation of Human NK Cell Activation by Expression of HLA Class I Molecules in Pig Endothelial Cells

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Benjamin Obando ◽  
Arthur Cross-Najafi ◽  
Kevin Lopez ◽  
Deepthi Thadasina ◽  
Wenjun Zhang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Human Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Organ Shortage ◽  
Major Barrier ◽  
Xenograft Rejection ◽  
Hla Class I Molecules

Background:   Pig-to-human xenotransplantation (XTx) is a promising solution to the organ shortage. Genetically engineered pigs lacking major xenoantigens have reduced hyperacute rejection and prolonged xenograft survival. Despite these advancements, acute xenograft rejection (AXR) remains a major barrier to clinical XTx. AXR is mediated by multiple immune cells, of which natural killer (NK) cells play a crucial role. Previous studies have shown that human HLA-E suppresses NK cell activation through the inhibitory receptor NKG2A. We seek to improve pig-to-human compatibility by expressing HLA-E in a genetically modified pig endothelial cell (pEC) line. This cell line 5GKO/ HLA-G+ has mutations in five genes encoding for xenoantigens and expresses HLA-G, an inhibitory ligand of the NK cell receptor KIR2DL4. In this study, the 5GKO/HLA-G+/HLA-E+ pEC line was established to examine whether co-expression of inhibitory ligands promotes NK cell tolerance.      Methods:   The HLA-Eα/pCDNA3.1 plasmid containing the HLA-E α-chain (HLA-Eα) cDNA driven by a CMV promoter was linearized and introduced into 106 cells of the 5GKO/HLA-G+ pEC line by electroporation. After 48 hours, HLA-E expression was analyzed by flow cytometry. HLA-E+ pECs were isolated by flow cytometry sort and co-cultured with human peripheral blood mononuclear cells (PBMCs) stimulated by IL-2. NK cell degranulation was compared between the 5GKO/HLA-G+ and 5GKO/HLA-G+/HLA-E+ pEC lines by measuring CD107a expression in the CD3- CD56+ cell population.          Results:   HLA-E molecules were successfully expressed on the pECs surface, indicating the HLA-E a chain can pair with the existing b2-microglobulin (B2M). The transfection efficiency was 38.2%. Three weeks later, the 5GKO/HLA-G+/HLA-E+ pEC was successfully established, confirming via flow cytometric analysis. The analysis of NK cell degranulation (CD107a) is underway.     Conclusion:   We established a 5GKO/HLA-G+/HLA-E+ pEC line, which is a valuable tool to study human-to-pig xenoreactive immune response in vitro, with the goal of improving pig-to-human xenograft immunotolerance. 

Role of NKG2D in Cytokine-Induced Killer (CIK) Cells Against Multiple Myeloma Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5119-5119
Author(s):  
Xu-zhang Lu ◽  
Bao-An Chen ◽  
Lin-di Ma ◽  
Xiao-hui Cai ◽  
Min Zhou
Keyword(s):  
Flow Cytometry ◽  
Mononuclear Cells ◽  
Plasma Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Human Peripheral Blood ◽  
Nkg2d Ligands ◽  
Cik Cells ◽  
Ifn Γ ◽  
Nkg2d Receptor

Abstract Abstract 5119 Cytokine-induced killer (CIK) cells are T lymphocytes enriched in CD3+CD56+ cells, which can be easily and rapidly expanded in vitro from human peripheral blood, bone marrow or cord blood mononuclear cells with the sequential addition of interferon (IFN)-{gamma}, OKT-3 and high doses of interleukin (IL)-2. The cytokine-induced killer (CIK) cells have been reported to have potent cytotoxicity against a variety of tumor cells including multiple myleoma (MM) cells. The mechanism of CIK cell recognizing MM cells remains unknow. Recent studies indicated that ligation of NKG2D on immunological cells directiy induce cytotoxicity. We suspect whether NKG2D receptor induction on CIK cells by cytokines is responsible for the killing of MM cells by CIK. We expended CIK cells from healthy controlswith interferon (IFN)-γ, CD3 monoclonal antibodies (mAb) and IL-2, and checked expression of NK cell receptors on CIK cells by flow cytometry. We found higher expression of NKG2D receptor and lower other NK receptors, such as CD158a,CD158b and NCRs on expanded CIK. These CIK cells showed higer cytotoxicity to multiple myleoma cell line U266 expressing NKG2D ligands. Interestingly, when cocultured with U266 cells, only NKG2D expressing CIK cells released IFN-γ detected by flow cytometry. We next analyzed NKG2D ligands expression on primary plasma cells in 22 MM patients by flow cytometry, the primary plasma cells in 16/22 (72.7%) MM patients expressed different levels of ULBPs or MICA/B on the cell surface. CIK cells showed higher cytotoxicity (12.5%) to NKG2D ligands expressing primary plasma cells compared to those did not express NKG2D ligands. The killing of CIK against MM cells were partially blocked by treatment of CIK with anti-NKG2D antibody. We conclude that NKG2D-NKG2D ligangd interaction may be one of the mechanisms by which CIK cells kill MM cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Rhesus rotavirus VP4 sequence-specific activation of mononuclear cells is associated with cholangiopathy in murine biliary atresia

2015 ◽  
Vol 309 (6) ◽  
pp. G466-G474 ◽  
Author(s):  
Ashley Walther ◽  
Sujit K. Mohanty ◽  
Bryan Donnelly ◽  
Abigail Coots ◽  
Celine S. Lages ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Biliary Atresia ◽  
Nk Cells ◽  
Bile Ducts ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
The United States ◽  
Newborn Mice

Biliary atresia (BA), a neonatal obstructive cholangiopathy, remains the most common indication for pediatric liver transplantation in the United States. In the murine model of BA, Rhesus rotavirus (RRV) VP4 surface protein determines biliary duct tropism. In this study, we investigated how VP4 governs induction of murine BA. Newborn mice were injected with 16 strains of rotavirus and observed for clinical symptoms of BA and mortality. Cholangiograms were performed to confirm bile duct obstruction. Livers and bile ducts were harvested 7 days postinfection for virus titers and histology. Flow cytometry assessed mononuclear cell activation in harvested cell populations from the liver. Cytotoxic NK cell activity was determined by the ability of NK cells to kill noninfected cholangiocytes. Of the 16 strains investigated, the 6 with the highest homology to the RRV VP4 (>87%) were capable of infecting bile ducts in vivo. Although the strain Ro1845 replicated to a titer similar to RRV in vivo, it caused no symptoms or mortality. A Ro1845 reassortant containing the RRV VP4 induced all BA symptoms, with a mortality rate of 89%. Flow cytometry revealed that NK cell activation was significantly increased in the disease-inducing strains and these NK cells demonstrated a significantly higher percentage of cytotoxicity against noninfected cholangiocytes. Rotavirus strains with >87% homology to RRV's VP4 were capable of infecting murine bile ducts in vivo. Development of murine BA was mediated by RRV VP4-specific activation of mononuclear cells, independent of viral titers.

scholarly journals Optimizing the Procedure to Manufacture Clinical-Grade NK Cells for Adoptive Immunotherapy

Cancers ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 577
Author(s):  
Adrián Fernández ◽  
Alfonso Navarro-Zapata ◽  
Adela Escudero ◽  
Nerea Matamala ◽  
Beatriz Ruz-Caracuel ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Fold Increase ◽  
Clinical Grade ◽  
Transcriptome Profile ◽  
Expansion Process ◽  
Peripheral Blood Mononuclear ◽  
Activation Status

Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation–expansion process and its validation on clinical-scale. Methods: RPMI-1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA+ cells with irradiated K562mbIL15-41BBL or K562mbIL21-41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical-grade NKAE cells were manufactured in CliniMACS Prodigy. Results: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA+ cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA+ cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21-41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21-41BBL or K562mbIL15-41BBL. Clinical-grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. Conclusions: GMP-grade NK cells for clinical use can be obtained by using different starting cells and aAPC.

PBMC-07- Stimulation of human peripheral blood mononuclear cells with immobilized anti-CD3 and soluble anti-CD28 antibodies. v1

2021 ◽  
Author(s):  
Alessia Furgiuele ◽  
Massimilano Legnaro ◽  
Alessandra Luini ◽  
Marco Ferrari ◽  
Emanuela Rasini ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Functional Responses ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

This protocol was designed to activate the lymphocytes T of a population of peripheral blood mononuclear cells (PBMCs), simulating their physiological response to antigen/MHC complex acting on T Cell Receptors-TCR , in order to test their functional responses including cell proliferation and cytokine production. The co-stimulation protocol include: i)anti-CD3 antibody a polyclonal activator specific for invariant framework epitopes on TCR complex (in particular, we use UCHT1 clone an anti-human CD3 antibody that recognizes the ε-chain of CD3 which is used for immobilized option of activation) (http://static.bdbiosciences.com/documents/BD_Tcell_Human_CD3_Activation_Protocol.pdf) ii) anti-CD28 antibody used to cooperate with TCR signals promoting activation of T cells The procedure has been reproduced following the indications contained in the protocol of "EBiooscience" (https://tools.thermofisher.com/content/sfs/manuals/t-cell-activation-in-vitro.pdf). Pilot experiments on PBMC were carried out to determine the best concentrations of anti-CD3 and anti-CD28 to induce optimal proliferation of PBMC and production of cytokines TNF-α and IFN-γ. We found a dose dependent correlation between immobilized anti-CD3 and cells functional responses. The selected amount was 2 µg/mL for both anti-CD3 and anti-CD28 that was the concentration below the maximum response which allows also to test possible modulations by therapeutic agents. References http://static.bdbiosciences.com/documents/BD_Tcell_Human_CD3_Activation_Protocol.pdf https://tools.thermofisher.com/content/sfs/manuals/t-cell-activation-in-vitro.pdf https://www.bdbiosciences.com/ds/pm/tds/555330.pdf https://www.bdbiosciences.com/ds/pm/tds/555726.pdf BEFORE STARTING with this procedure Moreover, work under laminar flow hood when you are processing samples from the beginning to the end of the culture. Make sure you are using,sterile culture mediumand sterile plastic disposable as well.

Umbilical Mesenchymal Stem Cell-Derived Extracellular Vesicle Conditioning Has an Immunosuppressive Effect on NK Cells

2021 ◽  
Author(s):  
G. Dostert ◽  
V. Jouan-Hureaux ◽  
H. Louis ◽  
É. Velot
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Cell Communication ◽  
K562 Cells ◽  
Cell Cytotoxicity ◽  
Nk Cell Cytotoxicity

Background: In peripheral blood, human natural killer (NK) cells are immunological cells that nearly don’t express the ectonucleotidase CD73 on their plasma membrane. When exposed to mesenchymal stem cells (MSCs), NK cells are able to acquire CD73. MSCs are known to be CD73-positive (CD73+) and also to modulate the immune system, e.g. through adenosynergic pathway by ectonucleosidases, such as CD73. Extracellular vesicles (EVs) are involved in cell-to-cell communication. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as paracrine mediators that are part of MSC immunomodulatory effects including immunosuppressive properties and immune privilege. Objective: The aim of our work was to study if CD73 could be acquired by NK cells through cell-to-cell communication with MSC-EVs as cell culture additives. We also hypothesised that MSC-EVs would act as tolerance inducers to attenuate NK cell cytotoxicity. Methods: Cell isolation was made from human umbilical cords for MSCs and from human peripheral blood for NK cells. MSC-EVs were isolated by ultracentrifugation and filtration, then characterized by nanoparticle tracking assay and flow cytometry (CD9, 63, 81 and 73). MSC-EV interaction with NK cells was monitored by PKH67 staining. NK cell activation was followed by measuring the expression of CD73 and NK-activating receptor natural-killer group 2, member D (NKG2D) by flow cytometry. The cytotoxicity of NK cells or EV-conditioned NK cells was evaluated after co-culture with K562 cells. Results: We showed that MSC-EVs are nanoparticles able to express CD73 and interact with NK cells. MSC-EV conditioned NK cells seem to increase CD73 and decrease NKG2D through an EV-mediated mechanism. MSC-EVs have an immunosuppressive effect on NK cells by preventing NK cell activation and NK cell cytotoxicity towards K562 cells. Conclusions: Our results demonstrate that MSC-EVs could influence NK cell behaviour and act as immunosuppressant cell-based products.

scholarly journals Aberrant anti-viral response of natural killer cells in severe asthma

2020 ◽  
Vol 55 (5) ◽  
pp. 1802422
Author(s):  
Justine Devulder ◽  
Cécile Chenivesse ◽  
Valérie Ledroit ◽  
Stéphanie Fry ◽  
Pierre-Emmanuel Lobert ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Severe Asthma ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Asthma Patients ◽  
Ifn Γ

Rhinovirus infections are the main cause of asthma exacerbations. As natural killer (NK) cells are important actors of the antiviral innate response, we aimed at evaluating the functions of NK cells from severe asthma patients in response to rhinovirus-like molecules or rhinoviruses.Peripheral blood mononuclear cells from patients with severe asthma and healthy donors were stimulated with pathogen-like molecules or with the rhinoviruses (RV)-A9 and RV-2. NK cell activation, degranulation and interferon (IFN)-γ expression were analysed.NK cells from severe asthma patients were less cytotoxic than those from healthy donors in response to toll-like receptor (TLR)3, TLR7/8 or RV-A9 but not in response to RV-2 stimulation. Furthermore, when cultured with interleukin (IL)-12+IL-15, cytokines which are produced during viral infections, NK cells from patients with severe asthma were less cytotoxic and expressed less IFN-γ than NK cells from healthy donors. NK cells from severe asthmatics exhibited an exhausted phenotype, with an increased expression of the checkpoint molecule Tim-3.Together, our findings indicate that the activation of NK cells from patients with severe asthma may be insufficient during some but not all respiratory infections. The exhausted phenotype may participate in NK cell impairment and aggravation of viral-induced asthma exacerbation in these patients.

scholarly journals Optimizing recovery of frozen human peripheral blood mononuclear cells for flow cytometry

PLoS ONE ◽  
2017 ◽  
Vol 12 (11) ◽  
pp. e0187440 ◽  
Author(s):  
Bo Langhoff Hønge ◽  
Mikkel Steen Petersen ◽  
Rikke Olesen ◽  
Bjarne Kuno Møller ◽  
Christian Erikstrup
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Inactivation of Human Leukocytes in Platelet Products After Pathogen Reduction Technology Treatment in the Presence of Platelet Additive Solution.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2116-2116
Author(s):  
Loren D. Fast ◽  
Susanne Marschner ◽  
Gilbert DiLeone ◽  
Raymond Goodrich
Keyword(s):  
T Cell Activation ◽  
Cell Activation ◽  
Mononuclear Cells ◽  
Uv Light ◽  
Human Peripheral Blood ◽  
Control Sample ◽  
Blood Products ◽  
Peripheral Blood Mononuclear ◽  
Pathogen Reduction ◽  
Additive Solution

Abstract Abstract 2116 Poster Board II-93 During the transfusion of blood or blood products, a recipient can receive a large number of allogeneic leukocytes. This can lead to leukocyte-mediated adverse reactions in the recipient and include donor anti-recipient responses such as the life-threatening transfusion-associated graft versus host disease (TA-GVHD) and cytokine production; or recipient anti-donor responses that are induced by direct presentation of foreign antigen by donor leukocytes or indirectly after processing of the donor cells by recipient antigen-presenting cells. To avoid or minimize leukocyte mediated reactions, the leukocytes present in blood products are inactivated or depleted prior to administration. Nucleic acid targeted pathogen reduction processes (PRT) are well suited for leukocyte inactivation. The Mirasol® PRT System uses riboflavin (Vitamin B2) and ultraviolet (UV) light to reduce the active pathogen load and inactivate residual leukocytes in blood products used for transfusion. To make the PRT System more widely applicable, the effect of treating leukocytes in the presence of platelet additive solution (PAS) was tested. Human peripheral blood mononuclear cells (PBMNC) were purified by Ficoll-Hypaque discontinuous centrifugation and placed in 350 ml of storage solution consisting of 65% PAS (SSP+) and 35% plasma. An untreated control sample was removed before addition of 35 ml of riboflavin (500 μM) and exposure to UV light (9.1 J/ml). PBMNC were recovered after treatment and tested for their ability to proliferate in response to polyclonal stimulators such as phytohemagglutinin, and anti-CD3/CD28 or to allogeneic stimulator cells in a mixed lymphocyte culture (MLC). Treatment was found to inhibit proliferation as well as T cell activation as measured by the upregulation of CD69 expression when incubated with phorbol 12-myristate 13-acetate. Treated PBMNC were unable to produce inflammatory or TH1/TH2 cytokines when stimulated with lipopolysaccharide for 24 hours or anti-CD3/CD28 for 72 hours. Levels of cytokines that are released in the absence of activation, such as IL-6, IL-8 and IL1β, were reduced below levels of detection of the assay after PRT-treatment. Quantitation of the degree of inactivation using limiting dilution assays showed that 5.2 log inactivation could be achieved at the specified energy doses. These treatment conditions resulted in acceptable platelet cell quality over 8 days in storage. In summary, PRT treatment was able to functionally inactivate leukocytes in the presence of PAS to the levels seen with gamma-irradiation without adversely affecting the quality of the platelets. Disclosures: Fast: CaridianBCT Biotechnologies: Research Funding. Marschner:CaridianBCT Biotechnologies: Employment. Goodrich:CaridianBCT Biotechnologies: Employment.

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