690 TIGIT blockade improves anti-tumor activity of ex vivo expanded NK cells

BackgroundNatural killer (NK) cells are innate immune cells that directly kill and coordinate responses against cancer prompting interest in using ex vivo expanded NK cells as an adoptive cell therapy for treatment of cancer. NK cells express a set of activating and inhibitory receptors that regulate their activity. Inhibitory receptor TIGIT (T cell Immunoreceptor with Ig and ITIM domain) is upregulated on intratumoral NK cells in some cancers, inhibits NK cell activity and promotes NK cell exhaustion. In this study, the effect of TIGIT blockade on the anti-tumor activities of ex vivo expanded NK cells was evaluated.MethodsNK cells were activated overnight with cytokines or ex vivo expanded with PM21-particles. Their TIGIT expression was determined with qRT-PCR and flow cytometry. Cytotoxicity was assessed by kinetic, imaging-based assay (Incucyte S3) against A549 and NCI-H1299 cells cultured in 3D. Cytotoxicity was calculated based on untreated controls at different time-points. Results from multiple donors were normalized to cytotoxicity of NK cells with isotype for individual donors and was compared to the cytotoxicity of NK cells with anti-TIGIT. Unpaired t test was used to determine statistical significance. K562 cells stably expressing Polio Virus Receptor (PVR), were used to restimulate A549 spheroid-exposed NK cells to measure IFNγ, TNFα and degranulation. Furthermore, phenotypic changes of NK cells upon TIGIT blockade were examined by analyzing a set of activating and inhibitory receptors by flow cytometry.ResultsThe effect of NK cell expansion/activation on TIGIT expression was assessed. TIGIT was upregulated on expanded and cytokine-activated NK cells both on mRNA and protein level. The effect of TIGIT blockade on NK cell cytotoxicity was examined by co-culturing PM21-NK cells with cancer cells in the presence of anti-TIGIT antibodies or respective isotypes. TIGIT blockade significantly increased cytotoxicity of PM21-NK cells against A549 (1.3 fold, P < 0.0001) and NCI-H1299 (1.3 fold, P = 0.0003) spheroids after 48 h. To access exhaustion, NK cells exposed to A549 spheroids for 7 days were restimulated with PVR+ K562 cells. TIGIT blockade prevented NK cell exhaustion resulting in increased expression of IFNγ, TNFα and surface CD107a on restimulated NK cells. TIGIT blockade did not result in changes to the surface phenotype of NK cells.ConclusionsTIGIT was highly expressed on expanded and cytokine-activated NK cells. TIGIT blockade improved anti-tumor activities of PM21-NK cells. Thus, PM21-NK cells and TIGIT antibodies have translational potential as a combination therapy to improve anti-tumor response.

Download Full-text

The Advantages and Challenges of Anticancer Dendritic Cell Vaccines and NK Cells in Adoptive Cell Immunotherapy

Vaccines ◽

10.3390/vaccines9111363 ◽

2021 ◽

Vol 9 (11) ◽

pp. 1363

Author(s):

Elena V. Abakushina ◽

Liubov I. Popova ◽

Andrey A. Zamyatnin ◽

Jens Werner ◽

Nikolay V. Mikhailovsky ◽

...

Keyword(s):

Cytotoxic Activity ◽

Nk Cells ◽

Nk Cell ◽

Therapeutic Potential ◽

Ex Vivo ◽

Adoptive Cell Therapy ◽

Immune Effector ◽

Effector Cells ◽

Cell Immunotherapy

In the last decade, an impressive advance was achieved in adoptive cell therapy (ACT), which has improved therapeutic potential and significant value in promising cancer treatment for patients. The ACT is based on the cell transfer of dendritic cells (DCs) and/or immune effector cells. DCs are often used as vaccine carriers or antigen-presenting cells (APCs) to prime naive T cells ex vivo or in vivo. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are used as major tool effector cells for ACT. Despite the fact that NK cell immunotherapy is highly effective and promising against many cancer types, there are still some limitations, including insignificant infiltration, adverse conditions of the microenvironment, the immunosuppressive cellular populations, and the low cytotoxic activity in solid tumors. To overcome these difficulties, novel methods of NK cell isolation, expansion, and stimulation of cytotoxic activity should be designed. In this review, we discuss the basic characteristics of DC vaccines and NK cells as potential adoptive cell preparations in cancer therapy.

Download Full-text

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

Stem Cells and Regenerative Medicine - Biomedical and Health Research ◽

10.3233/bhr210028 ◽

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.

Download Full-text

Donor and host coexpressing KIR ligands promote NK education after allogeneic hematopoietic stem cell transplantation

Blood Advances ◽

10.1182/bloodadvances.2019000242 ◽

2019 ◽

Vol 3 (24) ◽

pp. 4312-4325 ◽

Cited By ~ 4

Author(s):

Xiang-Yu Zhao ◽

Xing-Xing Yu ◽

Zheng-Li Xu ◽

Xun-Hong Cao ◽

Ming-Rui Huo ◽

...

Keyword(s):

Stem Cell ◽

Hematopoietic Stem Cell Transplantation ◽

Stem Cell Transplantation ◽

Nk Cells ◽

Cell Transplantation ◽

Hematopoietic Stem Cell ◽

Nk Cell ◽

K562 Cells ◽

Inhibitory Receptors ◽

Hematopoietic Stem

Abstract The rate and extent of natural killer (NK)–cell education after hematopoietic cell transplantation correlates with leukemia control. To study the effect of donor and host HLA on NK-cell reconstitution, single killer-cell immunoglobulin-like receptor (KIR)+ NK cells (exhibiting KIR2DL1, KIR2DL2/KIR2DL3, or KIR3DL1 as their sole receptor) were grouped into 4 groups based on the interaction between donor/host HLA and donor inhibitory KIR in 2 cohorts (n = 114 and n = 276, respectively). On days 90 to 180 after transplantation, the absolute number and responsiveness against K562 cells (CD107a or interferon-γ expression) of single-KIR+ NK cells were higher in pairs where donor and host HLA both expressed ligands for donor inhibitory KIRs than in pairs where 1 or both of the donor and recipient HLA lacked at least 1 KIR ligand. NK-cell responsiveness was tuned commensurate with the number of inhibitory receptors from the donor. When both donor and host expressed the 3 major KIR ligands (HLA-C1, HLA-C2, and HLA-Bw4), NK cells expressing 3 inhibitory receptors (KIR2DL1/2DL3/3DL1) reached the maximum responsiveness against K562 cells compared with those NK cells expressing only 1 or 2 inhibitory receptors. When donor and host HLA both expressed all ligands for donor inhibitory KIRs, patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) showed the lowest recurrence rate after haploidentical hematopoietic stem cell transplantation (haplo-HSCT). In conclusion, this study demonstrates that when both donors and hosts present all the KIR ligands for donor KIRs, reconstituted NK cells achieve better functional education and contribute to least relapse among patients. This observation study was registered at www.clinicaltrials.gov as #NCT02978274.

Download Full-text

Development and Function of NK and T Cells in AML Patients after Allogeneic Stem Cell Transplantation (SCT).

Blood ◽

10.1182/blood.v104.11.3244.3244 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3244-3244

Author(s):

Gabriele Multhoff ◽

Catharina Gross ◽

Anne Dickinson ◽

Ernst Holler

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Stem Cell ◽

Nk Cells ◽

Nk Cell ◽

Granzyme B ◽

Hla Class I ◽

K562 Cells ◽

Cytolytic Response

Abstract Purpose: Hsp70 was frequently found on the plasma membrane of bone marrow-derived leukemic blasts, but not on normal bone marrow cells. Hsp70 membrane expression could be correlated with protection against therapy-induced apoptosis (Nylandsted et al 2004). In contrast, these tumor cells have been found to be highly sensitive to the cytolytic attack mediated by NK cells. In vitro, Hsp70-activated NK cells efficiently lysed autologous Hsp70 membrane-positive leukemic blasts (Gehrmann et al 2003). Granzyme B release served as a surrogate marker for estimating the cytolytic response of NK cells against Hsp70 membrane-positive tumor target cells (Gross et al 2003). Here, we studied the development of NK and T cells in AML patients (n=6) after allogeneic SCT at different time points (days 14–20, 45, 90, 180, 1 year) after allogeneic stem cell transplantation (SCT). Methods: HLA class I, HLA-E and Hsp70 surface expression was determined on all patient-derived leukemic blasts of the bone marrow by flow cytometry. The amount of NK and T cells was investigated by multicolor flow cytometry using CD3/ CD16 and CD56 and CD94/ CD56 antibody-combinations detecting NK cell specific markers. Effector cell function was tested in a granzyme B ELISPOT assay against patient-derived leukemic blasts and K562 cells. Results: All tested leukemic blasts were positive for HLA class I, HLA-E, and Hsp70. After induction therapy the amount of CD3-negative, CD56/CD94-positive NK cells was 28±16%, that of CD3-positive T cells was 58±3%. On days 14–21 after allogeneic SCT, 58±9% of the donor-derived peripheral blood lymphocytes (PBL) were CD3-negative, CD56/CD94-positive NK cells; the amount of CD3-positive T cells was 26±7.5%. On day 45, the amount of NK cells further increased up to 68±7.9%; that of T cells further decreased down to 16±5.6%. On day 90 and day 180 the amount of NK cells was still 41±10%; that of T cells was 29±12%. Interestingly, high NK cell counts correlated with an increased cytolytic response against leukemic blast and K562 cells. One year after allogeneic SCT, NK (20±1%) and T cell (52±18%) ratios were comparable to that of healthy human individuals. Conclusions: Between days 14 and 180 after allogeneic SCT, the amount of NK cells was significantly elevated if compared to that of T cells. Concomitantly, cytolytic function against leukemic blasts was significantly elevated. Normal levels, in the composition of NK and T cells were reached 1 year after SCT. Project funded by EU-TRANS-EUROPE grant QLK3-CT-2002-01936.

Download Full-text

Ex Vivo Activated Natural Killer (NK) Cells from Myeloma Patients Kill Autologous Myeloma and Killing Is Enhanced by Elotuzumab

Blood ◽

10.1182/blood.v112.11.3666.3666 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3666-3666

Author(s):

Tarun K. Garg ◽

Susann Szmania ◽

Jumei Shi ◽

Katie Stone ◽

Amberly Moreno-Bost ◽

...

Keyword(s):

Bone Marrow ◽

Cell Therapy ◽

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

K562 Cells ◽

Scid Mice ◽

Target Cells ◽

Recent Trial ◽

Nk Cell Therapy

Abstract Immune-based therapies may improve outcome for multiple myeloma (MM) by eradicating chemo-resistant disease. Our recent trial utilizing IL2 activated, killer immunoglobulin-like receptor-ligand mismatched NK cell transfusions from haplo-identical donors yielded (n) CR in 50% of patients. Unfortunately, after NK cell therapy, 2/10 patients had progressive disease, and the median duration of response for the other 8/10 patients was only 105 days (range 58–593). This may have been due to an insufficient dose of alloreactive NK cells and early rejection. Furthermore, appropriate donors were identified for only 30% of otherwise eligible patients. We therefore investigated whether NK cells from MM patients could be expanded and activated to kill autologous MM. We then examined whether pre-treatment of MM cell targets with elotuzumab, a humanized antibody to the MM tumor antigen CS1, could further enhance NK cell-mediated lysis. PBMC from 5 MM patients were co-cultured for 14 days with irradiated K562 cells transfected with 4-1BBL and membrane bound IL15 in the presence of IL2 (300U/ml) as previously described (Imai et al, Blood2005;106:376–383). The degree of NK cell expansion, NK immunophenotype, and ability to kill MM (4 hour 51Cr release assays) were assessed. To determine the ability of ex vivo expanded NK cells to traffic to bone marrow, activated NK cells were injected into the tail vein of NK cell depleted NOD-SCID mice, which were then sacrificed after 48 hours. Flow cytometry for human CD45, CD3, and CD56 was performed on cells from blood, marrow and spleen. There was an average 64-fold expansion of NK cells (range: 8–200) after 2 weeks of co-culture with K562 transfectants. Expansion of T cells was not observed. The NK cell activating receptor NKG2D, and natural cytotoxicity receptors NKp30, NKp44, and NKp46 were up-regulated following the expansion. Expanded NK cells were able to kill autologous MM (E:T ratio 10:1, average 31%, range 22–41%), whereas resting NK cells did not. Pretreatment of autologous MM cells with elotuzumab increased the activated NK cell-mediated killing by 1.7-fold over target cells pretreated with an isotype control antibody. This level of killing was similar to that of the highly NK kill-sensitive cell line K562 (Figure). Autologous PHA blasts and CD34+ stem cells were not killed. Activated human NK cells were detectable in the bone marrow of NOD-SCID mice 48 hours after injection. Ex vivo activation of NK cells from MM patients with K562 transfectants can induce killing of autologous MM and produce large numbers of NK cells for potential therapy. The addition of elotuzumab to activated NK cell therapy enhances anti-MM effects by ADCC thus invoking an additional NK cell-mediated mechanism of MM killing. Importantly, ex vivo activated NK cells traffic to the bone marrow in mice. Autologous NK cell therapy eliminates the issues related to allo-donor availability and early NK cell rejection, and could provide an option for patients refractory to chemotherapy agents. Figure Figure

Download Full-text

Genetically Reengineered K562 Cells Significantly Expand Cord Blood (CB) Natural Killer (NK) Cells Ex-Vivo Expanded with Increased NK Cell Activation and Cytolytic Activity Both In-Vitro and In-Vivo: Potential Use In Adoptive Cellular Immunotherapy (ACI)

Blood ◽

10.1182/blood.v116.21.3928.3928 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3928-3928

Author(s):

Michele Levin ◽

Janet Ayello ◽

Frances Zhao ◽

Andrew Stier ◽

Lauren Tiffen ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Ex Vivo ◽

Granzyme B ◽

K562 Cells ◽

Cytolytic Activity ◽

Scid Mice ◽

Activation Marker

Abstract Abstract 3928 Background: NK cells play a role in reducing relapse in hematological malignancy following AlloSCT (Dunbar et al, Haematologica, 2008). NK cell limitations include lack of tumor recognition and/or limited numbers of viable and functional NK cells (Shereck/Cairo et al, Ped Bld Can, 2007). NK ACI provide safe and effective therapy against tumor relapse; yet NK cells are limited to specific cancer types and not all patients demonstrate optimal response (Ruggieri et al. Science, 2002; Ljunggren et al. Nat Rev Immuno, 2007). To circumvent these limitations, methods to expand and activate PBMNCs with genetically engineered K562 cells expressing membrane bound IL-15 and 41BB ligand (K562-mbIL15-41BBL [modK562]; Imai/Campana et al, Blood, 2005) have shown to significantly increase NK cells in number and maintain heterogeneous KIR expression (Fusaki/Campana et al BJH, 2009). We have shown that CB NK cells can be activated/expanded and exhibit enhanced cytolytic activity when cultured in a cytokines/antibody cocktail (Ayello/Cairo et al, BBMT, 2006; Exp Heme, 2009). Objective: To evaluate CBNK expansion, activation, cytolytic mechanism and function against Burkitt lymphoma (BL) tumor target and its influence on NK cell mediated in-vitro and in-vivo cytotoxicity in NOD-SCID mice following stimulation with modK562 cells (generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, Tx). Methods: Following 100GY irradiation, modK562cells were incubated 1:1 with CBMNCs in RPMI+IL-2 (10IU/ml) for 7 days in 5%CO2, 37°C. NK activation marker (LAMP-1), perforin and granzyme B were determined by flow cytometry. Cytotoxicty was determined via europium assay at 20:1 E:T ratio with Ramos (BL) tumor targets (ATCC). The mammalian expression construct (ffLucZeo-pcDNA (generously supplied by L.Cooper, MD, PhD) was transfected to BL cells using lipofectin and selected by zeocin for stable transfection. Six week old NOD-SCID mice received 5×106 BL cells subcutaneously. Upon engraftment, xenografted NOD-SCID mice were divided in 5 groups: injected with PBS (control), BL only, 5×106 wildtype (WT) K562 expanded (E) CBNK cells, modK562 expanded (E) CB NK cells (5×106) and modK562 expanded (E) CBNK cells (5×107). Ex-vivo ECBNK cells were injected weekly for 5 weeks and xenografted NOD-SCID mice were monitored by volumetric measurement of tumor size (Tomayko/Reynolds, Can Chemother Pharmac, 1989), bioluminescent imaging (Inoue et al Exp Heme, 2007) and survival. The survival distribution for each group was estimated using the Fisher exact test. Results: On Day 0, NK cells (CD56+/3-) population was 3.9±1.3%. After 7 days, modK562 expanded CBNK cells was 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). ModK562 ECBNK cells demonstrated increased perforin and granzyme B expression compared to WTK562 (42±1.5 vs 15±0.5%,p<0.001; 22±0.5 vs 11±0.3%,p<0.001, respectively). Cytotoxicity was against BL tumor targets was significantly increased (42±3 vs 18±2%,p<0.01), along with NK activation marker expression, CD107a (p<0.05). At 5 weeks, in-vivo studies demonstrated increased survival of NOD-SCID mice receiving both 5×106 and 5×107 modK562 ECBNK cells when compared to those with no treatment (p=0.05, p=0.0007, respectively). There was no difference in survival when comparing mice that received 5×106 vs 5×107 modK562 ECBNK cells (p=0.0894) at 5 weeks. Tumor volume of mice receiving either dose of modK562 ECBNK cells was significantly less than those receiving WTK562 ECBNK cells (1.92±0.57 and 0.37±0.05 vs 3.41±0.25, p=0.0096 and p=0.0001, respectively). Conclusions: CBMNCs stimulated and expanded with modK562 cells results in significant expansion of CBNK cells with enhanced in-vitro cytotoxicity, significant receptor expression of NK activation marker (LAMP-1), and perforin and granzyme B. Furthermore, modK562 ECBNK cells leads to increased survival and lower tumor burden of NOD-SCID mice xenografted with BL. Future directions include modK562 ECBNK cells to be genetically modified to express chimeric antigen receptor CD20 (MSCV-antiCD20-41BB-CD3 ζ) against CD20+ hematologic malignancies for future studies to evaluate whether targeting enhances in-vitro and in-vivo cytotoxicity. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽

10.1182/blood.v126.23.3090.3090 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3090-3090 ◽

Cited By ~ 1

Author(s):

Folashade Otegbeye ◽

Nathan Mackowski ◽

Evelyn Ojo ◽

Marcos De Lima ◽

David N. Wald

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Ex Vivo ◽

Leukemia Cells ◽

Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

Download Full-text

Immunostimulating and Antimetastatic Effects of Polysaccharides Purified from Ginseng Berry

The American Journal of Chinese Medicine ◽

10.1142/s0192415x19500435 ◽

2019 ◽

Vol 47 (04) ◽

pp. 823-839 ◽

Cited By ~ 5

Author(s):

Dae-Young Lee ◽

Chan Woong Park ◽

Sue Jung Lee ◽

Hye-Ryung Park ◽

Dae Bang Seo ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cancer Metastasis ◽

Nk Cell ◽

Ex Vivo ◽

Cell Activity ◽

Gel Filtration ◽

Inhibitory Effect ◽

Ginseng Root ◽

High Dose

Ginseng root has been used in traditional oriental medicine for the enhancement of immune system function. The immunostimulatory effects of ginseng berry polysaccharides, however, remain unclear. Effects of polysaccharides from ginseng berry on the activation of natural killer (NK) cells and inhibition of tumors are reported. A crude polysaccharide was isolated from ginseng berry as a ginseng berry polysaccharide portion (GBPP) and was further fractionated using gel filtration chromatography to obtain the three polysaccharide fractions GBPP-I, -II and -III. GBPP-I consisted of mainly galactose (46.9%) and arabinose (27.5%). GBPP-I showed a high dose-dependent anticomplementary activity. Stimulation of murine peritoneal macrophages by GBPP-I showed the greatest enhancement of interleukin (IL)-6 and IL-12 and tumor necrosis factor (TNF)-[Formula: see text] production. In addition, an ex vivo assay of natural killer (NK) cell activity showed that oral ([Formula: see text] administration of GBPP-I significantly increased NK cell cytotoxicity in YAC-1 tumor cells and production of granzyme B. Prophylactic intravenous ([Formula: see text] and [Formula: see text] administration of GBPP-I significantly and dose-dependently inhibited lung metastatic activity in B16BL6 melanoma cells. Depletion of NK cells after injection of rabbit anti-asialo GM1 partially abolished the inhibitory effect of GBPP-I on lung metastasis, indicating that NK cells play an important role in anticancer effects. GBPP-I exerts a strong immune-enhancing activity and can prevent cancer metastasis through activation of NK cells and other immune-related cells.

Download Full-text

Preclinical Ex-Vivo Generation of Clinical Relevant Amounts of NK Cells from CD34+ Progenitor Cells for Immunotherapy

Blood ◽

10.1182/blood.v112.11.2916.2916 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2916-2916

Author(s):

Jan Spanholtz ◽

T. M. de Witte ◽

Marleen Tordoir ◽

Harry Dolstra

Keyword(s):

Flow Cytometry ◽

Stem Cell ◽

Nk Cells ◽

Progenitor Cells ◽

Nk Cell ◽

Ex Vivo ◽

Cell Product ◽

Cytotoxicity Assays ◽

Cd34 Cells ◽

Specific Antigens

Abstract Alloreactive donor Natural Killer (NK) cells, displaying a KIR-ligand mismatch with the recipient play a pivotal role in graft-versus-leukemia (GVL) reactivity without significant graft-versus-host disease (GVHD) following haploidentical stem cell transplantation. Therefore, infusions of haploidentical NK cells are suggested to become an attractive approach for cancer immunotherapy. So far, difficulties in isolation and expansion of peripheral NK cells resulted in only limited data about safety and clinical efficacy of purified NK cell infusions. Therefore, we have developed a novel culture system without the use of feeder cells for the ex vivo generation of NK cells from CD34+ hematopoietic progenitor cells (HPCs) isolated from cord blood (CB) or bone marrow (BM). It is based on a two-step procedure using an expansion and a differentiation step. The NK cell generation system uses mainly cytokines such as SCF, TPO, Flt3-L, IL-2, IL-7 and IL-15 and specific modified glycosaminoglycans (GAGs) to direct and control the two phases. The developmental phase and the final NK cell product is controlled and characterized by immunophenotyping using multi-colour flow cytometry and CFSE-based cytotoxicity assays against various tumor cells. Our system generates a homogeneous final cell product of CD56+/CD3- cells with a purity of >99%. A total cell expansion of more than 5×10^4 fold allows to generate 5×10^10 NK cells from 1×10^6 CB CD34+ stem and progenitor cells within 4–5 weeks of culture. For BM cells an expansion rate of more than 1×10^4 fold was detected after a 5–6 week cell culture period. During the two week expansion phase step,we expand UCB CD34+ cells more than 100 fold. Phenotypic analysis showed a decrease of stem cell-specific antigens such as CD34 and CD117 during the first three weeks, whereas antigens specific for NK cell progenitors and mature NK cells such as CD56, CD94 and CD161 are up-regulated after initiating differentiation at day 14. Furthermore, distinct cell populations can be detected reflecting NK cell developmental stages in vitro. Effective differentiation of the expanded progenitor cells into mature NK cells is characterized by the expression of NK cell-specific antigens including CD56, CD94, NKG2A, NKG2D and NCRs as well as homing receptors such as CD62L, CXCR4 and CCR7. The final NK cell product shows high expression levels of inhibitory and activating receptors as well the intrinsic capability to express KIR, which is detected by flow cytometry after 6–7 weeks of culture. Cytotoxicity assays demonstrated robust lysis of more than 90% against AML as well as melanoma tumor cell lines. This system, with its huge expansion potential to generate highly activated NK cells with homing capability, is the basis for a first clinical trial in 2009, to infuse haploidentical NK cells generated from CD34+ cells in poor-risk AML patients. The use of our defined culture conditions enables new prospects in NK cell research, regarding NK cell development and NK cell maturation, as well as new aspects for the clinical use of NK cell products derived from HPCs.

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