A Novel Method Of Genetically Engineered K562 Cells To Significantly Expand Cord Blood (CB) NK Cells And Increase CB NK Receptor Expression And NK Cell Activation

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.

Download Full-text

Soluble HLA-G dampens CD94/NKG2A expression and function and differentially modulates chemotaxis and cytokine and chemokine secretion in CD56bright and CD56dim NK cells

Blood ◽

10.1182/blood-2011-05-352393 ◽

2011 ◽

Vol 118 (22) ◽

pp. 5840-5850 ◽

Cited By ~ 43

Author(s):

Fabio Morandi ◽

Elisa Ferretti ◽

Roberta Castriconi ◽

Alessandra Dondero ◽

Andrea Petretto ◽

...

Keyword(s):

Nk Cells ◽

Chemokine Receptor ◽

Nk Cell ◽

Receptor Expression ◽

Regulated Secretion ◽

Cell Functions ◽

Soluble Hla ◽

Ifn Γ ◽

And Function ◽

Nk Receptor

Abstract Soluble HLA-G (sHLA-G) inhibits natural killer (NK) cell functions. Here, we investigated sHLA-G–mediated modulation of (1) chemokine receptor and NK receptor expression and function and (2) cytokine and chemokine secretion in CD56bright and CD56dim NK cells. sHLA-G-treated or untreated peripheral blood (PB) and tonsil NK cells were analyzed for chemokine receptor and NK receptor expression by flow cytometry. sHLA-G down-modulated (1) CXCR3 on PB and tonsil CD56bright and CD56dim, (2) CCR2 on PB and tonsil CD56bright, (3) CX3CR1 on PB CD56dim, (4) CXCR5 on tonsil CD56dim, and (5) CD94/NKG2A on PB and tonsil CD56bright and CD56dim NK cells. Such sHLA-G–mediated down-modulations were reverted by adding anti–HLA-G or anti–ILT2 mAbs. sHLA-G inhibited chemotaxis of (1) PB NK cells toward CXCL10, CXCL11, and CX3CL1 and (2) PB CD56bright NK cells toward CCL2 and CXCL10. IFN-γ secretion induced by NKp46 engagement was inhibited by NKG2A engagement in untreated but not in sHLA-G–treated NK cells. sHLA-G up-regulated secretion of (1) CCL22 in CD56bright and CD56dim and (2) CCL2, CCL8, and CXCL2-CXCL3 in CD56dim PB NK cells. Signal transduction experiments showed sHLA-G–mediated down-modulation of Stat5 phosphorylation in PB NK cells. In conclusion, our data delineated novel mechanisms of sHLA-G–mediated inhibition of NK-cell functions.

Download Full-text

The Traffic of the NKG2D/Dap10 Receptor Complex during Natural Killer (NK) Cell Activation

Journal of Biological Chemistry ◽

10.1074/jbc.m808561200 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16463-16472 ◽

Cited By ~ 25

Author(s):

Pedro Roda-Navarro ◽

Hugh T. Reyburn

Keyword(s):

Cell Surface ◽

Nk Cells ◽

Cell Activation ◽

Cell Function ◽

Nk Cell ◽

Receptor Expression ◽

Cell Surface Expression ◽

Target Cells ◽

Receptor Complex ◽

Secretory Lysosomes

NKG2D is an important activating receptor for triggering the NK cell cytotoxic activity, although chronic engagement of specific ligands by NKG2D is also known to provoke decreased cell surface expression of the receptor and compromised NK cell function. We have studied the dynamics of surface NKG2D expression and how exposure to the specific ligand major histocompatibility complex class I chain-related molecule B (MICB) affects receptor traffic and fate. While in the NKL cell line and “resting” NK cells NKG2D was found principally at the cell surface, in activated primary NK cells an intracellular pool of receptor could also be found recycling to the plasma membrane. Exposure of NK cells to targets expressing MICB resulted in degradation of ∼50% of total NKG2D protein and lysosomal degradation of the DAP10 adaptor molecule. Consistent with these observations, confocal microscopy experiments demonstrated that DAP10 trafficked to secretory lysosomes in both transfected NKL cells and in activated primary NK cells upon interaction with MICB-expressing target cells. Interestingly, polarization to the synapse of secretory lysosomes containing DAP10 was also observed. The implications of the intracellular traffic of the NKG2D/DAP10 receptor complex for NK cell activation are discussed. We propose that the rapid degradation of NKG2D/DAP10 observed coincident with recruitment of the receptor to the cytotoxic immune synapse may explain the loss of NKG2D receptor expression after chronic exposure to NKG2D ligands.

Download Full-text

Significant Ex-Vivo Expansion and Functional Activation of Cord Blood (CB) Natural Killer (NK) Cells with A Concomittant Significant Decrease in CB T Cells Following Stimulation with Genetically Engineered K562 Cells (K562-mb15-41BBL).

Blood ◽

10.1182/blood.v114.22.499.499 ◽

2009 ◽

Vol 114 (22) ◽

pp. 499-499

Author(s):

Jessica Hochberg ◽

Janet Ayello ◽

Carmella VandeVen ◽

Jeremy Gold ◽

Evan Cairo ◽

...

Keyword(s):

T Cells ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

K562 Cells ◽

Fold Increase ◽

Ex Vivo Expansion ◽

Genetically Engineered ◽

Cellular Immunotherapy ◽

Adoptive Cellular Immunotherapy

Abstract Abstract 499 Introduction: CD56+ NK subsets exhibit differential NK receptors (NKR) such as cytotoxicity profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytotoxicity receptors (NCR) involved with tumor target recognition, which, in part, may play a role in adoptive cellular immunotherapy (ACI) for malignancies (Farag et al Blood, 2002). NK cell activation and NK mediated cytolysis is induced by triggering receptors such as NCR (i.e. NKp46), and NKG2 surface receptors like NKG2D (Moretta et al, Curr Opin in Immunol, 2004, Marcenaro et al, Eur J Immunol, 2003). The major limitations of the use of NK cells in ACI include lack of tumor recognition and/or limited numbers of viable and functionally active NK cells (Shereck/Cairo et al. PBC, 2007). To circumvent these limitations, methods to expand and activate PB NK cells by genetic reengineering have been developed (Imai/Campana et al. Blood, 2005). It has been demonstrated that PB NK cells expanded with modified K562 cells expressing membrane bound IL-15 and 4-1BBL (K562-mb15-41BBL; Imai et al Blood, 2005) are significantly increased in number and maintain heterogeneous KIR expression (Fusaki/Campana et al, BJH, 2009) .We have previously reported the ex-vivo expansion, activation and cytolytic activity of CB NK cells with a cocktail of antibody and cytokines (Ayello/Cairo et al, BBMT, 2006; Ayello/Cairo, Exp Hem, 2009, In Press). Objective: In this study, we compared CB NK expansion and activation following stimulation with genetically engineered K562 cells (K562-mb15-41BBL, generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, TN) with wild-type (WT) K562 cells and NK cell characterization expressing inhibiting and activating KIRs, c-lectin, NCRs and NK cytolytic activation. Methods: Following irradiation with 100Gy, K562-mb15-41BBL or WTK562 were incubated at a 1:1 ratio with fresh CB MNCs at 37C, 5% CO2 for 7 days in RPMI-1640+10IU IL-2. NKR expression (KIR2DS4, NKG2D, NKG2A, CD94, KIR3DL1, KIR2DL2, Nkp46) and LAMP-1 (CD107a) receptor expression and NK cell phenotype (CD56 dim and bright subsets) were determined by flow cytometry. Results: On Day 0, NK cells population was 3.9±1.3%. After 7 days in culture, CB NK cells were significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p<0.01). This represented a 35-fold or 3374±385% increase of the input NK cell number. This was significantly increased compared to WTK562 (1771±300%, p<0.05). Concomitantly, there was a significant decrease in CB T cells vs WTK562 or media alone (15±2 vs 36±2 vs 51±7%, p<0.001),respectively. There was a significant increase in CD56bright vs CD56dim populations (67 vs 33%, p<0.01) following stimulation with K562-mb15-41BBL. Also, there was a 10-fold increase in CB NK cells expressing KIR3DL1 following stimulation with K562-mb15-41BBL vs WTK562 (p<0.01) and a 5-fold increase in NK KIR2DS4 expression (p<0.05), respectively. There was a significant increase in the expression of NK activation marker, CD107a, compared to WTK562 (51±0.7 vs 32±1.1,p<0.05). There was no change in CB NK cell expression of the c-lectin receptor, CD94/NKG2A and CD94/NKG2D after stimulation with K562-mb15-41BBL. A standard cryopreserved CB unit (25 ml) contains approximately 750×106 MNC. By using the smaller 5-ml aliquot (20%) of a two-aliquot bag (150×106 MNCs × 3.9%=5.8×106 NK cells), this expansion method would hypothetically yield 200×106 CB NK cells after 7 days stimulation with K562-mb15-41BBL. Conclusion: These results suggest that CB MNC can be ex-vivo expanded with K562-mb15-41BBL resulting in specific expansion of CB NK cells with increased NK KIR expression (KIR2DS4 and KIR3DL1) and NK activation (CD107a), along with a significant decrease in CB T cells. This expansion provides a means to enhance specific CB NK cell expansion for possible use for adoptive cellular immunotherapy in the post UCBT setting Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Interleukin-21 Induces the Differentiation of Human Umbilical Cord Blood CD34. −lineage− Cells Into Pseudomature Lytic NK Cells.

Blood ◽

10.1182/blood.v114.22.1476.1476 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1476-1476

Author(s):

Giuseppina Bonanno ◽

Maria Corallo ◽

Annabella Procoli ◽

Andrea Mariotti ◽

Luca Pierelli ◽

...

Keyword(s):

Cell Surface ◽

Cord Blood ◽

Nk Cells ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Stromal Cell ◽

Nk Cell ◽

K562 Cells ◽

Gm Csf ◽

Ifn Γ

Abstract Abstract 1476 Poster Board I-499 Background: Umbilical cord blood (UCB) is increasingly used as an alternative source of transplantable CD34+ haematopoietic stem cells (HSC) for neoplastic and non-neoplastic diseases. In addition to CD34-expressing HSC, human UCB contains a rare population of CD34−lineage− cells endowed with the ability to differentiate along the T/NK pathway in response to interleukin (IL)-15 and in the presence of a stromal cell support. IL-21 is a four-helix bundle cytokine released by activated CD4+ T cells and by NKT cells. IL-21 is a crucial regulator of NK cell function, whose influence on IL-15-induced differentiation of CD34−lineage− cells has not been investigated previously. The present study was designed and conducted to address whether IL-21 might replace the stromal cell requirements and foster the IL-15-induced NK differentiation of human UCB CD34−lineage− cells. Methods: CD34−lineage− cells were maintained in liquid culture with 10−6M hydrocortisone, 20 ng/ml Flt3-L and 20 ng/ml SCF, with the addition of 50 ng/ml IL-15 and 20 ng/ml IL-21, either alone or in combination. Cultures were established in the absence of feeder cells or serum supplementation. Cytokine-treated cells were used to evaluate the following parameters: a) cell surface phenotype; b) expression of molecular determinants of lymphoid/NK cell differentiation; c) secretion of IFN-γ, GM-CSF, TNF-α and CCL3/MIP-1α; d) cytolytic activity against NK-sensitive tumour cell targets and e) relative amount of Stat1 (Tyr701), Stat3 (Tyr705) and Stat5 (Tyr694) phosphorylation in response to IL-21. For all the above detailed experiments, control cultures were established with UCB-derived CD34+ HSC. Results: Freshly isolated CD34−lineage− cells stained negatively for stem cell-associated (CD34, CD133) and NK/lymphoid surface antigens (CD7, CD56, CD16, CD3, TCRαβ), and comprised 0.22% on average of UCB mononuclear cells (samples analyzed = 8). CD34−lineage− cells proliferated vigorously in response to IL-15 and IL-21 (average fold expansion at week +4 of culture = 42.5) but not to IL-21 alone, and up-regulated phosphorylated Stat1 and Stat3 proteins, in good agreement with previously published reports on the IL-21-induced activation of Stat signaling. CD34−lineage− cells expanded by IL-21 in combination with IL-15 acquired a peculiar lymphoid morphology with heavy cytoplasmic granules. When compared with CD34-derived NK cells, CD34−lineage− cells emerging from IL-15+IL-21-containing cultures expressed very low levels of CD16 and killer-cell immunoglobulin-like receptor (KIR), but high levels of CD56, NKG2D and IL-21 receptor, consistent with pseudo-mature NK cells. IL-21/IL-15-differentiated cells up-regulated mRNA signals for Bcl-2, GATA-3 and Id2, a master switch required for NK-cell development, and harboured un-rearranged TCRγ genes, suggesting that NK commitment under the experimental conditions here established occurs through a pathway that does not include TCR rearrangement. From a functional standpoint, IL-21/IL-15-treated cells secreted copious amounts of IFN-γ, GM-CSF and CCL3/MIP-1α, and expressed cell surface CD107a upon contact with NK-sensitive tumour targets, a measure of exocytosis of NK secretory granules. Specifically, an average 65±11% of CD56+ NK cells differentiated with IL-15+IL-21 stained positively for CD107a in co-cultures established with NK-sensitive K562 cells. NK cell degranulation occurred at significantly lower levels in co-cultures containing K562 cells and IL-15-differentiated CD34−lineage− cells (mean percentage of CD107a+CD56+ NK cells equal to 35±6 at E:T ratio = 1; p < 0.01 compared with cultures containing IL-15+IL-21-matured NK cells), suggesting that IL-15 and IL-21 exerted synergistic effects on NK activity. Finally, NK cells differentiated from CD34+ HSC with either IL-15 alone or IL-15+IL-21 manifested a similar cytotoxic activity to that of cytokine-differentiated CD34−lineage− cells. Conclusions: This study suggests that considerable numbers of highly pure, lytic CD56+CD16−/+ NK cells for adoptive immunotherapy can be obtained from UCB CD34−lineage− cells using a serum-free, feeder cell-free culture system. The findings highlighted herein also shed some light into the developmental intermediates of NK cells that can be differentiated after the exposure of CD34−lineage− cells to IL-21. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

GMP-Compliant Universal Antigen Presenting Cells (uAPC) Promote the Metabolic Fitness and Antitumor Activity of Armored Cord Blood CAR-NK Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.626098 ◽

2021 ◽

Vol 12 ◽

Author(s):

Enli Liu ◽

Sonny O. T. Ang ◽

Lucila Kerbauy ◽

Rafet Basar ◽

Indreshpal Kaur ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Cellular Therapy ◽

Nk Cell ◽

Human Leukocyte ◽

Genetically Engineered ◽

Effector Function ◽

Antigen Presenting ◽

Metabolic Fitness

Natural killer (NK) cells are innate lymphocytes recognized for their important role against tumor cells. NK cells expressing chimeric antigen receptors (CARs) have enhanced effector function against various type of cancer and are attractive contenders for the next generation of cancer immunotherapies. However, a number of factors have hindered the application of NK cells for cellular therapy, including their poor in vitro growth kinetics and relatively low starting percentages within the mononuclear cell fraction of peripheral blood or cord blood (CB). To overcome these limitations, we genetically-engineered human leukocyte antigen (HLA)-A− and HLA-B− K562 cells to enforce the expression of CD48, 4-1BBL, and membrane-bound IL-21 (mbIL21), creating a universal antigen presenting cell (uAPC) capable of stimulating their cognate receptors on NK cells. We have shown that uAPC can drive the expansion of both non-transduced (NT) and CAR-transduced CB derived NK cells by >900-fold in 2 weeks of co-culture with excellent purity (>99.9%) and without indications of senescence/exhaustion. We confirmed that uAPC-expanded research- and clinical-grade NT and CAR-transduced NK cells have higher metabolic fitness and display enhanced effector function against tumor targets compared to the corresponding cell fractions cultured without uAPCs. This novel approach allowed the expansion of highly pure GMP-grade CAR NK cells at optimal cell numbers to be used for adoptive CAR NK cell-based cancer immunotherapy.

Download Full-text

Abstract 49: Over-activation Of Natural Killer Cells Due To Dysfunction Of Cd16-mediated Signaling In Adamts13-deficient Ttp Patients With A History Of Relapse

Circulation Research ◽

10.1161/res.115.suppl_1.49 ◽

2014 ◽

Vol 115 (suppl_1) ◽

Author(s):

FANG ZHOU ◽

Darise Farris

Keyword(s):

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

K562 Cells ◽

Antibody Treatment ◽

History Of ◽

Relapse Group

Thrombotic thrombocytopenic purpura (TTP) is a rare life-threaten vascular autoimmune disease. There is no effective method to treat it in clinical trials since pathogenesis of TTP has not been fully elucidated. Here we investigate the role of NK cells in relapse development of TTP. Our results showed that the frequencies of CD3 − CD56 dim CD16 − and CD3 − CD56 bri CD16 − NK cells are increased in TTP patients with a history of relapse. Expression of CD107a, granzyme A and IFN-γ by CD3 − CD56 dim NK cells following in vitro stimulation with PMA/ionomycin / monensin is improved in the relapse group, compared with those on NK cells derived from TTP patients without relapse development. NK cells isolated from TTP patients with a history of relapse indicated stronger cytotoxicity to target K562 cells than those of NK cells derived from TTP patients without relapse development, suggesting prior activation of NK cells in vivo . Treatment with anti-human CD16 antibody up-regulates cytotoxicity of NK cells derived from TTP patients without relapse development. However, Anti-human CD16 antibody treatment does not affect cytotoxicity of NK cells isolated from TTP patients with a history of relapse, suggesting inability of CD16-mediated signaling in NK cells derived from TTP patients with relapse development. These data provide evidence of altered NK cell activation and/or licensing in TTP patients with a history of relapse modulated by CD16-mediated signaling and a new avenue of investigation into mechanisms of TTP immunopathogenesis.

Download Full-text

Optimal Ex-Vivo Expansion (EvE) of Cord Blood (CB) Natural Killer (NK) Cells, NK Cytotoxicity and NK Receptor (NKR) Expression: Potential for CB Adoptive Cellular Immunotherapy (ACI).

Blood ◽

10.1182/blood.v106.11.34.34 ◽

2005 ◽

Vol 106 (11) ◽

pp. 34-34

Author(s):

Janet Ayello ◽

Prakash Satwani ◽

Carmella Van de Ven ◽

Elizabeth Roman ◽

Laxmi Baxi ◽

...

Keyword(s):

Cell Activation ◽

Nk Cell ◽

Ex Vivo ◽

Granzyme B ◽

Receptor Expression ◽

Cellular Immunotherapy ◽

Short Term ◽

Effector Cells ◽

Perforin Expression ◽

Nk Receptor

Abstract CD56+ NK subsets exhibit differential receptor profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytotoxicity receptors (NCR) involved with tumor target recognition which may play a role in ACI for malignancies (Farag et al Blood, 2002). NK cell activation and NK mediated cytolysis is induced by several triggering receptors such as NCR (i.e. NKp44, NKp46) and NKG2 surface receptors like NKG2D (Moretta, et al, Curr Opinion in Immunol, 2004; Marcenaro et al, Eur J Immunol, 2003). UCB is limited by the absence of available donor effector cells (NK, CTL, LAK and NKT cells) for infusion after transplantation for the treatment of minimal residual resistant hematological relapse and/or PTLD (Barker et al, Blood, 2001; Locatelli, et al Blood,1999). We demonstrated the ability to EvE CB in short term culture (48 hrs) with IL-2, IL-7, IL-12 and anti-CD3 (ABCY) cryopreserved, thawed, recryopreserved, rethawed and ex vivo expanded (CTCTE) with significant increase in CD3−/16+/56+ bright/dim subsets expressing KIR3DL1, KIR2DL1/S1, KIR2DL2 and CD94/NKG2A (Ayello/Cairo et al, BBMT, 25a, 2004). In this study, we compared short-term cultures (48 hrs) with prolonged cultures (4 to 10 days) on expansion, maturation and expression of NCR, NKG2, KIR and cytolytic mechanisms in previously cryopreserved CB that were TCTE. CB was cryopreserved and thawed by the NHLBI/COBLT method. (Kurtzberg/Cairo, Transfuison, 2005). Rethawed nonadherent CB cells were cultured (2–10 days) in serum-free medium alone or with anti-CD3 (50 ng/ml, IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) (ABCY). NK receptor expression (CD94, NKG2C, NKG2D, NKp44, NKp46, KIR2DS4) and intracellular perforin and granzyme B activity were determined by flow cytometry. NK and LAK cytotoxicity was measured by europium release assay. Significant increases were seen in NK activating KIR2DS4 at day 10 vs 2 in ABCY both in CD3−/16+/56+ dim and bright subsets (16.9±0.4 vs 2.1±0.2% and 22.3±0.3 vs 0.9±0.2%, p<0.001, respectively). C-lectin activating receptor CD94/NKG2D was increased at day 7 vs 2 following ABCY EvE (41.4 ±0.43 vs 23.7±2.0 %, p<0.001). A significant increase was seen in NK (CD3−/16+/56+dim) KIR3DL1 subset at day 10 vs 2 (38.3±2.8 vs 18.9±6.3 7%, p<0.05). In contrast, NCR expression in CD3−/16+/56+ dim NKp44 subset was significantly decreasedat day 10 vs 2 of EvE with ABCY (15.2±0.7 vs 27.2±0.7%, p<0.001). A significant decrease was seen in CD3−/16+/56+ dim NKp46 expression following day 10 vs 2 (8.5±0.2 vs 23.5±1.2 %, p<0.001). Perforin expression demonstrated a significant decrease in ABCY at day 10 vs 2 (55.7±1.8 vs 84.3±1.3%, p<0.001) yet increasing levels of granzyme B from day 2 to 10 (25.8±1.8 vs 45.1±1.7%, p<0.0001). A significant increase in CB NK and LAK cytotoxicity was seen in ABCY on day 10 vs 2 (NK: 71.5 ±1.6 vs 53.8±10.3%, p<0.001; LAK: 63.2±0.24 vs 31.8±1.8%, p<0.001). In summary, CB MNC may be thawed at time of CB transplantation, recryopreserved, rethawed and at a later date EvE and activated for 7–10 days to yield viable NK subsets. EvE in ABCY at 10 days yielded increased expression of NK KAR (CD56+dim and bright) and granzyme B expression but decreased NK C-lectin CD94/NKG2D, NCR NKp46 and NKp48 and perforin expression.

Download Full-text

A Highly Efficient Method to Expand CD3-CD56+ NK Cells from Cord Blood Segments

Blood ◽

10.1182/blood.v112.11.3902.3902 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3902-3902

Author(s):

Sumithira Vasu ◽

Maria Berg ◽

Andreas Lundqvist ◽

Muthalagu Ramanathan ◽

Rebecca Lopez ◽

...

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Nk Cell ◽

K562 Cells ◽

Surface Expression ◽

Feeder Cells ◽

Cell Numbers ◽

In Vitro Expansion ◽

Cytotoxic Function

Abstract Background: The infusion of in-vitro expanded cord blood derived CD3−CD56+ NK cells could potentially be used to enhance graft-vs-tumor effects following cord blood transplantation. In order to infuse NK cells derived from the same cord unit used during allogeneic transplantation, we sought to develop a highly efficient culture method to expand large numbers of NK cells in vitro from < 1 ml of cord blood. Methods: Immunomagnetic beads were used to deplete CD3+ T-cells from thawed cord blood. CD3 depleted mononuclear cells (<0.1% CD56+) were co-cultured with either irradiated EBV-LCL feeder cells or 41BB transduced K562 cells in X-VIVO 20, 10% human AB serum, and 500 IU/ml hrIL-2 for up to 47 days. Results: Day 12 EBV-LCL expanded NK cell cultures contained up to 90% CD3−CD56+ NK cells with less than 0.5% CD3+CD56+ cells. Expanded cord blood derived CD56+NK cells had similar expression of CD16, NKG2D, LFA-1, perforin, and granzymes A and B and had similar cytotoxic function as NK cells expanded from adult PBMC. Surface expression of NK cell TRAIL increased dramatically with in vitro expansion. By day 12, TRAIL surface expression by FACS was at similar levels observed on expanded adult NK cells, although expression gradually declined with prolonged cell culture; on days 12, 20, and 34, 89%, 57% and 11% of cord derived NK cells expressed TRAIL respectively. NK cell cultures expanded with 41BB-transduced APCs had a similar phenotype and cytotoxic function against K562 cells and renal cell carcinoma (RCC) cells as EBV-LCL expanded cells. Furthermore, NK cell cytotoxicity against RCC tumor targets treated with 10 nM bortezomib for 18 hrs (bortezomib upregulates RCC surface expression of DR5) was higher than untreated RCC cells confirming the functional cytolytic activity of TRAIL expressed on cord blood derived NK cells. We next evaluated the feasibility of expanding CD56+ NK cells from thawed segments attached to the umbilical cord blood units. TNC numbers of each segment ranged from 3–9 × 106 cells. Unmanipulated thawed segments were co-cultured with irradiated EBV-LCL feeder cells as described above. On Day 12, 43% of viable cells were CD3+, 21.5% were CD3+CD56+ and 36% were CD3− CD56+. CD3−CD56+ NK cells from thawed segments increased 200 – 300 fold with in vitro expansion when maintained in culture for 2–3 weeks (Table). To enrich for pure NK cell populations, subsequent expansions were performed on CD3 depleted cells pooled from three thawed segments stimulated with EBV-LCL; cultures on day 12 contained a pure population of CD3−CD56+ NK cells with virtually an identical phenotypic and cytotoxicity profile as NK cells expanded from larger aliquots taken directly from the thawed cord blood unit. By day 33, CD3−CD56+ NK cells expanded by up to 30,000 fold; in one experiment, 391 ×106 CD3-CD56+ NK cells were expanded from only 13,000 NK cells obtained from a pool of three thawed cord segments. Conclusions: In vitro-expansion of a pure population of CD3−CD56+ cells derived from cord blood can be achieved using EBV-LCL or 41BB transduced feeder cells. Expanded cells have increased NKG2D and TRAIL expression and enhanced TRAIL-mediated tumor cytotoxicity. Even with very low starting numbers of TNCs, substantial numbers of CD3+, CD3+56+ and CD3−CD56+ cells can be expanded in vitro from thawed segments using EBV-LCL feeder cells in advance of thawing of the cord unit. These methods are being optimized to allow for clinical scale expansion of NK cells from the same cord unit used for hematopoietic cell transplantation. Cell numbers after expansion of individual segments with EBV – LCL feeder cells Day 0 (×10e6) Day 16 (× 10e6) Fold expansion TNC 3–6 78–112 20–30 CD3+ 0.42–0.91 33–47 50–70 CD3+CD56- 0.015–0.032 16–24 700–1000 CD3-CD56+ 0.081–0.175 28–40 200–300 Cell numbers after CD3+ depletion of pooled segments and expansion with EBV – LCL feeder cells Day 0 (×10e6) Day 33 (× 10e6) Fold expansion TNC 4.16 401 96 CD3+ 0 0 - CD3+CD56- 0 0 - CD3-CD56+ 0.0132 391 29621

Download Full-text

Neutrophils Induced Licensing of Natural Killer Cells

Mediators of Inflammation ◽

10.1155/2015/747680 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 10

Author(s):

Keishiro Amano ◽

Masahiro Hirayama ◽

Eiichi Azuma ◽

Shotaro Iwamoto ◽

Yoshitaka Keida ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Activation ◽

Mononuclear Cells ◽

Nk Cell ◽

Peripheral Blood Mononuclear ◽

Ligand Interaction ◽

Receptor Ligand ◽

Hematopoietic Stem ◽

Nk Receptor

Natural killer (NK) cells acquire effector function through a licensing process and exert anti-leukemia/tumor effect. However, there is no means to promote a licensing effect of allogeneic NK cells other than cytomegalovirus reactivation-induced licensing in allogeneic hematopoietic stem cell transplantation in human. In mice, a licensing process is mediated by Ly49 receptors which recognize self-major histocompatibility complex class I. The distribution of four Ly49 receptors showed similar pattern in congenic mice, B10, B10.BR, and B10.D2, which have B10 background. Forty Gy-irradiated2×106B10.D2 cells including splenocytes, peripheral blood mononuclear cells in untreated mice, or granulocyte colony-stimulating factor treated mice were injected intraperitoneally into B10 mice. We found that murine NK cells were effectively licensed by intraperitoneal injection of donor neutrophils with its corresponding NK receptor ligand in B10 mice as a recipient and B10.D2 as a donor. Mechanistic studies revealed that NK cells showed the upregulation of intracellular interferon-γand CD107a expression as markers of NK cell activation. Moreover, enriched neutrophils enhanced licensing effect of NK cells; meanwhile, licensing effect was diminished by depletion of neutrophils. Collectively, injection of neutrophils induced NK cell licensing (activation) via NK receptor ligand interaction.

Download Full-text