scholarly journals Ex Vivo Expanded and Activated Natural Killer Cells Prolong the Overall Survival of Mice with Glioblastoma-like Cell-Derived Tumors

2021 ◽  
Vol 22 (18) ◽  
pp. 9975
Author(s):  
Yoichi Shida ◽  
Tsutomu Nakazawa ◽  
Ryosuke Matsuda ◽  
Takayuki Morimoto ◽  
Fumihiko Nishimura ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
Ex Vivo ◽  
Interleukin 2 ◽  
Xenograft Model ◽  
Checkpoint Pathway ◽  
Nog Mice ◽  
Blocking Antibodies

Glioblastoma (GBM) is the leading malignant intracranial tumor and is associated with a poor prognosis. Highly purified, activated natural killer (NK) cells, designated as genuine induced NK cells (GiNKs), represent a promising immunotherapy for GBM. We evaluated the anti-tumor effect of GiNKs in association with the programmed death 1(PD-1)/PD-ligand 1 (PD-L1) immune checkpoint pathway. We determined the level of PD-1 expression, a receptor known to down-regulate the immune response against malignancy, on GiNKs. PD-L1 expression on glioma cell lines (GBM-like cell line U87MG, and GBM cell line T98G) was also determined. To evaluate the anti-tumor activity of GiNKs in vivo, we used a xenograft model of subcutaneously implanted U87MG cells in immunocompromised NOG mice. The GiNKs expressed very low levels of PD-1. Although PD-L1 was expressed on U87MG and T98G cells, the expression levels were highly variable. Our xenograft model revealed that the retro-orbital administration of GiNKs and interleukin-2 (IL-2) prolonged the survival of NOG mice bearing subcutaneous U87MG-derived tumors. PD-1 blocking antibodies did not have an additive effect with GiNKs for prolonging survival. GiNKs may represent a promising cell-based immunotherapy for patients with GBM and are minimally affected by the PD-1/PD-L1 immune evasion axis in GBM.

Interleukin-2 Gene Transduction in Human Natural Killer Cells Augments Their Survival and Anti-Leukemic Capacity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5437-5437
Author(s):  
Chihaya Imai ◽  
Takayuki Takachi ◽  
Haruko Iwabuchi ◽  
Masaru Imamura ◽  
Tae Nemoto ◽  
...  
Keyword(s):  
Cell Death ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Transduction Efficiency ◽  
Cytotoxic Activities ◽  
Hematopoietic Stem

Abstract Novel therapies are required to circumvent the resistance of leukemic cells to chemotherapy and improve cure rates. Infusions of natural killer (NK) cells might enhance the anti-leukemic effects of chemotherapy and hematopoietic stem cell transplantation. Indeed, haploidentical NK cells were shown to induce remission in patients with high-risk acute myeloid leukemia (AML; Miller at al. Blood105: 3051, 2005). Current NK-cell therapy protocols include administration of interleukin (IL)-2 to sustain the survival and activation of the infused NK cells in vivo. In this study, we tested whether enforcing the expression of the IL-2 gene in NK cells would abrogate their requirement for exogenous IL-2, prolong their survival and augment their anti-leukemic capacity., We previously reported a method that induces NK cells to proliferate and reliably allows their genetic modification (Imai et al, Blood106: 376, 2005). We used this method to expand primary NK cell from healthy individuals for 7 days. After depletion of residual T cells, NK cells were transduced with a retroviral vector containing human IL-2 cDNA and GFP. Fourteen days after transduction, more than 99% of cells were CD3− CD56+ NK cells; median transduction efficiency, assessed by GFP positivity, was 70.0% (40.4%–91.4%, n=9). NK cells expressing IL-2 cDNA (NK-gIL2) showed intracellular expression of IL-2 and secreted IL-2 (1.3 ± 0.5 U/106 cells/24 hours). Karyotyping of NK-gIL2 cells propagated for more than 6 weeks revealed no chromosomal aberration. The cytotoxicity of NK-gIL2 cells against the AML cell line K562 was similar to that of NK cells transduced with a vector containing GFP only when the cytotoxicity assay was performed with 4-hour coculture. However, NK-gIL2 exerted a much more potent cytotoxicity if the cultures were prolonged to 7 days. For example, residual leukemia was 58.4% ± 1.3% versus 97.3% ± 2.1% in control culture at a 0.5:1 E:T ratio. The superiority of NK-gIL2 was particularly evident in experiments where exogenous IL-2 was withdrawn for 72 hours before testing. In 4-hour cytotoxicity assays performed this way, cytotoxicity against K562 cells, as well as the T-ALL cell line MOLT4 and the Burkitt lymphoma cell line Daudi was significantly higher than that exerted by control NK cells (e.g., K562 killing: 82.9% ± 1.1% versus 30.9% ± 4.1% at a 1:1 ratio). NK-gIL2 showed significantly lower apoptotic rates than control NK cells after 72 hours of culture without exogenous IL-2 by Annexin V/7AAD staining. Survival after 7 days of culture in the absence of exogenous cytokines was also much higher in NK-gIL2 than NK cells transduced with GFP only. Cell death of NK cells may occur after killing of K562. We found that this was significantly lower in NK-gIL2 than in control NK cells, thus explaining the more potent anti-leukemic activity of NK-gIL2 in long-term culture. The addition of more than 100 U/ml IL-2 was needed to suppress cell death in control NK cells to a degree similar to that measured for NK-gIL2 cells in cultures without exogenous IL-2, suggesting that secretion of low amounts of IL-2 in an autocrine/paracrine fashion could sustain NK cell survival better than exogenous IL-2. We also found that the expression of the natural cytotoxicity receptor p30 and p44 was higher in NK-gIL2 cells than in control NK cells after 72-hour of IL-2 depletion, in line with their higher cytotoxic activities after IL-2 withdrawal. Our results indicate that endogenous IL-2 production can replace the requirement for exogenous IL-2 in NK cells and significantly improve their survival and cytotoxicity. An in vivo infusion of 1010 NK-gIL2 cells would result in the secretion of 1 × 104 U IL-2, an amount much lower than that administered in most immunotherapy protocols (1–2 × 106 U/sqm/day). These data suggest that infusion of NK-gIL2 could be more effective than that of unmodified NK cells while minimizing adverse effects of IL-2 administration.

scholarly journals A cloned cell line mediating natural killer cell function inhibits immunoglobulin secretion.

1982 ◽  
Vol 156 (2) ◽  
pp. 658-663 ◽  
Author(s):  
G Nabel ◽  
W J Allard ◽  
H Cantor
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
B Lymphocytes ◽  
Cell Function ◽  
Killer Cell ◽  
Major Histocompatibility Complex Gene ◽  
Cell Surface Antigens

We previously described a cloned cell line that combines information for a unique display of cell surface antigens and specialized function similar to activated natural killer (NK) cells. In addition to conventional cellular targets such as the YAC-1 and MBL-2 lymphomas, this cloned line also lysed lipopolysaccharide-activated B lymphocytes. To determine whether some NK cells can inhibit B cell function, we tested the ability of NK-like clones to suppress Ig secretion in vitro and in vivo. These cloned cells suppressed Ig secretion when they constituted as few as 0.2% of the total cell population and inhibition did not require identity at the H-2 locus. We suggest that some NK cells might recognize non-major histocompatibility complex gene products on activated B lymphocytes and lyse these cells, and this might represent a fundamental cell-cell interaction that regulates antibody secretion by activated B cells.

scholarly journals Stable Transduction of the Interleukin-2 Gene Into Human Natural Killer Cell Lines and Their Phenotypic and Functional Characterization In Vitro and In Vivo

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3850-3861 ◽  
Author(s):  
Shigeki Nagashima ◽  
Robbie Mailliard ◽  
Yoshiro Kashii ◽  
Torsten E. Reichert ◽  
Ronald B. Herberman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Cell Lines ◽  
Killer Cell ◽  
Interleukin 2 ◽  
Human Natural Killer Cell ◽  
Antitumor Effects ◽  
Packaging Cell Line

Abstract A variety of strategies have been attempted in the past to stably transduce natural killer (NK) cells with cytokine or other cellular genes. Here, we demonstrate the successful delivery of the interleukin-2 (IL-2) gene into two human NK cell lines, IL-2–dependent NK-92 and IL-2–independent YT, by retroviral transduction. An MuLV-based retroviral vector expressing human IL-2 andneor markers from a polycistronic message was constructed and transduced into a CRIP packaging cell line. By coincubation of NK cells with monolayers of CRIP cells or by using retrovirus-containing supernatants in a flow-through method, 10% to 20% of NK cells were stably transduced. Upon selection in the presence of increasing G418 concentrations, transduced NK cells were able to proliferate independently of IL-2 for more than 5 months and to secrete up to 5.5 ng/106 cells/24 h of IL-2. IL-2 gene-transduced NK-92 cells had an in vitro cytotoxicity against tumor targets that was significantly higher than that of parental cells and secreted interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) in addition to IL-2. Moreover, the in vivo antitumor activity of IL-2 gene-transduced NK-92 cells against established 3-day liver metastases in mice was greater than that of parental nontransduced NK cells. Stable expression of the IL-2 transgene in NK cells improved their therapeutic potential in tumor-bearing hosts. Thus, transduced NK cells secreted sufficient quantities of bioactive IL-2 to proliferate in vitro and mediated the antitumor effects both in vitro and in vivo in the absence of exogenous IL-2. These results suggest that genetic modification of NK cells ex vivo could be useful for clinical cancer therapy in the future.

scholarly journals Cord Blood Derived Natural Killer Cells Loaded with a Tetravalent Bispecific Antibody Construct (AFM13) As Off-the-Shelf Cell Therapy for CD30+ Malignancies

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 341-341
Author(s):  
Lucila Kerbauy ◽  
Mecit Kaplan ◽  
Pinaki P Banerjee ◽  
Francesca Lorraine Wei Inng Lim ◽  
Ana Karen Nunes Cortes ◽  
...  
Keyword(s):  
T Cell ◽  
Cord Blood ◽  
Nk Cells ◽  
Natural Killer ◽  
Research Funding ◽  
Bispecific Antibody ◽  
Nk Cell ◽  
Ex Vivo

Abstract Chimeric antigen receptors to redirect T cell specificity against tumor antigens have shown remarkable clinical responses against CD19+ malignancies. However, the manufacture of an engineered autologous T cell product is expensive and cumbersome. Natural killer (NK) cells provide an alternative source of immune effectors for the treatment of cancer. NK cell cytolytic function can be directed towards specific targets by exploiting their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) through the NK cell Fc receptor, CD16 (FcγRIIIa). AFM13 is a tetravalent bispecific antibody construct based on Affimed's ROCK™ platform. AFM13 is bispecific for CD30 and CD16A, designed for the treatment of CD30 expressing malignancies. It binds CD16A on the surface of NK cells, thus activating and recruiting them to CD30 expressing tumor cells and mediating subsequent tumor cell killing. Since autologous NK effector function is impaired in many patients with malignancies, we propose to overcome this by the use of allogeneic NK cells in combination with AFM13. Cord blood (CB) is a readily available ("off-the-shelf") source of allogeneic NK cells that can be expanded to large, highly functional therapeutic doses. The feasibility and safety of therapy with allogeneic ex vivo expanded CB-derived NK cells have been shown by our group and others. In this study, we hypothesized that we can redirect the specificity of NK cells against CD30+ malignancies by preloading ex vivo activated and expanded CB-derived NK cells with AFM13 prior to adoptive infusion. Briefly, mononuclear cells were isolated from fresh or frozen CB units by ficoll density gradient centrifugation. CD56+ NK cells were cultured with rhIL-12, rhIL-18 and rhIL-15 for 16 hrs, followed by ex vivo expansion with rhIL-2 and irradiated (100 Gy) K562-based feeder cells expressing membrane-bound IL-21 and CD137-ligand (2:1 feeder cell:NK ratio). After 14 days, NK cells were loaded with serial dilutions of AFM13 (0.1, 1, 10 and 100 mg/ml). After washing twice with PBS, we tested the effector function of AFM13-loaded NK-cells (AFM13-NK) compared to expanded CB-NK cells without AFM13 against Karpas-299 (CD30 positive) and Daudi (CD30 negative) lymphoma cell lines by 51Cr release and intracellular cytokine production assays. AFM13-NK cells killed Karpas-299 cells more effectively at all effector:target ratios tested than unloaded NK cells (Figure 1) and produced statistically more INFγ and CD107a (P=0.0034; P=0.0031 respectively, n=4). In contrast, AFM13-NK cells and unloaded NK cells exerted similar cytotoxicity against Daudi cells. Next, we established the optimal concentration of AFM13 for loading (determined to be 100 μg/ml) and the optimal incubation time to obtain maximal activity (1 h) in a series of in vitro experiments. We also confirmed that the activity of AFM13-NK cells against Karpas-299 cells remains stable for at least 72h post-wash (Figure 2). Additionally, we characterized the phenotype of AFM13-NK vs. unloaded NK cells by flow cytometry using monoclonal antibodies against 22 markers, including markers of activation, inhibitory receptors, exhaustion markers and transcription factors. Compared to unloaded NK cells, AFM13-NK cells expressed higher levels of CD25, CD69, TRAIL, NKp44, granzyme B and CD57, consistent with an activated phenotype. We next tested the in vivo anti-tumor efficacy of AFM13-NK cells in an immunodeficient mouse model of FFluc-Karpas-299. Briefly, six groups of NOD/SCID/IL2Rγc null mice (n=5 per group) were transplanted by tail-vein injection with 1 x 10e5 FFluc-transduced Karpas cells. Group 1 and 6 received tumor alone or tumor + AFM13 and served as a control. Groups 2-4 receive Karpas FFLuc with either expanded NK cells or AFM13-NK cells (NK cells loaded with AFM13) or expanded NK cells and AFM13 injected separately. Group 5 received AFM13-NK cells without tumor. Initial studies confirm the antitumor activity of AFM13-NK cells. In summary, we have developed a novel premixed product, comprised of expanded CB-NK cells loaded with AFM13 to 'redirect' their specificity against CD30+ malignancies. The encouraging in vitro and in vivo data observed in this study, provide a strong rationale for a clinical trial to test the strategy of an off-the-shelf adoptive immunotherapy with AFM13-loaded CB-NK cells in patients with relapsed/refractory CD30+ malignancies. Disclosures Champlin: Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

Assessment of PD-1 expression in human resting and activated natural killer cells and murine tumor models.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 36-36
Author(s):  
Sean J. Judge ◽  
Cordelia Dunai ◽  
Ian R. Sturgill ◽  
Kevin M. Stoffel ◽  
William J. Murphy ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Ex Vivo ◽  
Fold Increase ◽  
Wild Type ◽  
Immunodeficient Mice

36 Background: Blockade of the PD-1/PD-L1/2 axis has revolutionized cancer therapy. Although reinvigorated PD-1+ T cells are the main effectors in the response to checkpoint blockade, the contribution of Natural Killer (NK) cells to PD-1/PD-L1 inhibition is under debate. While PD-1 has been identified on NK cells, this appears to be restricted to small populations under limited conditions. We sought to evaluate the extent of PD-1 expression in mouse and human resting and activated NK cells. Methods: Human NK cells were isolated from healthy donor PBMCs and cancer patients. Ex vivo activation and proliferation techniques included recombinant human cytokine and feeder line co-culture. Murine NK cells were isolated from splenocytes, and PBMCs from wild type and immunodeficient mice. We assessed NK cell surface markers and intracellular cytokine by flow cytometry, and gene expression by quantitative RT-PCR. Results: Over 21-days of ex vivo expansion, expression of PD-1 or PD-L1 on human NK cells was < 1% at all time points, while TIGIT+ expression increased to > 85%. Conversely, ConA stimulation of T cells increased PD-1 expression with no change in TIGIT expression. QRT-PCR demonstrated absent PD-1 expression in purified NK cells compared to a 5-fold increase in PD-1 gene expression in ConA stimulated PBMCs. PD-1/PD-L1 was also < 1% in the NK92 cell line and < 2.5% in peripheral CD56+CD3- NK cells from patients with soft tissue sarcoma (STS). NK cells from digested freshly resected STS show variable PD-1 ( < 10%) and minimal PD-L1 ( < 1%) expression with a small, but measurable population of intra-tumoral NK cells (1% of immune cells). In vivo mouse studies showed < 5% PD-1+ NK cells in spleen and tumor of CT26 tumor-bearing mice, while PD-L1+ NK cells increased in frequency from spleen (5-35%) to tumor (40-95%) in both wild type BALB/C and SCID mice. Conclusions: In contrast to prior studies, we did not observe a substantial PD-1+ population on human or murine NK cells after multiple activation strategies compared to T cells. Contrary to its application in T cells, our data suggest that PD-1 is not a useful marker for NK cell exhaustion/dysfunction. PD-L1 on NK cells may represent an important link between NK and T cell immunotherapy.

Autologous Expanded Natural Killer Cells As a New Therapeutic Option for High-Risk Myeloma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2918-2918
Author(s):  
Tarun K. Garg ◽  
Junaid Khan ◽  
Susann Szmania ◽  
Amy D Greenway ◽  
Joshuah D Lingo ◽  
...  
Keyword(s):  
High Risk ◽  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Cell Line ◽  
Nk Cell ◽  
Tumor Burden ◽  
Cytolytic Activity ◽  
Killer Cells

Abstract Abstract 2918 Natural killer cells (NK) have the unique ability to kill target cells without priming. While their therapeutic potential against various malignancies is becoming more apparent, it has been restricted to the allogeneic setting; NK cells are inhibited by autologous targets by engaging killer immunoglobulin-like receptors with their ligands. Another major challenge to the clinical utility of NK cells is obtaining a sufficient number of NK cells for infusion. Co-culture of blood mononuclear cells (PBMNC) with the leukemic cell line K562, genetically modified to express membrane-bound IL15 and the co-stimulatory molecule 41BBL (K562mbIL15-41BBL) in the presence of IL2 results in robust expansion and activation of NK cells. To determine if NK cells derived from myeloma (MM) patients can be used therapeutically in the autologous setting, we explored the expansion of NK cells from MM patients, their gene expression profiles (GEP), and their ability to kill autologous and allogeneic MM cells from high-risk patients in vitro and in vivo, and compared these to NK cells from healthy donors (HD). PBMNC from MM patients (N=30) co-cultured with irradiated K562mbIL15-41BBL cells expanded a median of 351 fold (range20–10, 430), comparable to the expansion of HD-derived NK cells (N=15, median 803, range 127–1, 727; p=0.5). GEP of MM non-exp-NK differed from HD non-exp-NK in the expression of only one gene (PRKCi), underexpessed in MM (false discovery rate (FDR) <0.05, p-value <3×10−10). GEP of exp-NK cells from both MM patients and HD was very different from non-exp-NK cells (8 pairs each, 10, 639 differentially overexpressed and 26, 057 underexpressed probe sets, FDR <0.05). Genes associated with proliferation, cytolytic activity, activation, adhesion, migration and cell cycle regulation were highly up-regulated in exp-NK cells. Standard chromium release assays demonstrated that MM exp-NK cells killed both allogeneic and autologous primary MM cells more efficiently compared to non-exp-NK cells, via a perforin mediated mechanism. Blocking studies revealed that the natural cytotoxicity receptors, activating receptors, and DNAX accessory molecule (DNAM-1) played a central role in target cell lysis. The killing ability of MM patient and HD derived exp-NK cells was very similar against allogeneic targets, while primary MM targets were more resistant to killing by autologous exp-NK. The anti-MM activity of allogeneic and autologous exp-NK cells was further examined in vivo. NOD/SCID/IL2R γ-null mice were implanted subcutaneously with a human fetal bone, and primary MM cells or luciferase-transfected OPM2 MM cell line were engrafted into the bone. The tumor burden was determined by ELISA for human Ig and/or bio-imaging. The mice were randomized to control and exp-NK treatment groups. A total of 160 ×106 exp-NK cells, in 4 doses 48 hrs apart, were injected in the exp-NK treatment group via tail vein injection. The mice were administered 1000U of IL2 subcu daily to support the NK cells. The mice were bled on days 7, 14, 21 & 28 for the assessment of human Ig by ELISA and enumerating circulating NK cells by flow cytometry. Exp-NK treated mice had a significantly reduced MM burden by ELISA (p<0.04) on day 21, and exp-NK could be detected in the murine blood up to day 28 post-administration in both primary MM and OPM2 tumor bearing mice. The mice were sacrificed and the tumors were harvested after 4 weeks. A noticeable reduction in tumor burden in the exp-NK cell treated mice was confirmed by histology. NK cells were detected by immunohistochemistry (CD57 or CD16) in the hu-bone implants harvested 28 days after infusion. In conclusion, MM patient-derived NK cells have a similar expansion potential, and MM exp-NK cells have cytolytic activity against allogeneic targets similar to those of HD exp-NK cells, and somewhat reduced activity against autologous targets. These exp-NK cells have significant activity against the aggressive cell line OPM2 and high-risk autologous primary MM cells in vivo. Exp-NK cells trafficked to MM tumors and persisted in the myelomatous hu bone microenvironment for 4 weeks. The anti-MM activity of autologous exp-NK cells is exciting and avails a new therapeutic avenue for patients with GEP-defined high-risk disease. A phase II clinical trial of allogeneic and autologous exp-NK cell therapy for relapsed/refractory high-risk MM is in progress at our institution. Disclosures: No relevant conflicts of interest to declare.

The Unique Ontogeny Of Natural Killer Cells As Revealed By Genetic Barcoding In The Nonhuman Primate Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 15-15
Author(s):  
Chuanfeng Wu ◽  
Jan K. Davidson-Moncada ◽  
Rong Lu ◽  
Brian Li ◽  
Allen E. Krouse ◽  
...  
Keyword(s):  
Lymph Node ◽  
Nk Cells ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Ex Vivo ◽  
Clonal Diversity ◽  
Hematopoietic Stem ◽  
Over Time

Abstract Natural killer (NK) cells are cytotoxic leukocytes defined in humans and rhesus macaques by the expression of CD56 and/or CD16, and the absence of T, B, and myeloid markers. Functionally, they are defined by lysis of tumor targets in a major histocompatibility complex (MHC)–unrestricted fashion, and production of cytokines critical to innate immunity. Ex vivo expanded NK cells are being developed as adoptive transfer therapeutics for a variety of malignancies. However, the ontogeny and hematopoietic lineage relationships of human NK cells have been difficult to study, given differences in phenotype and function between human and murine NK cells, poor NK engraftment and development in human xenografts, and restricted development of NK cells from hematopoietic stem and progenitor cells (HSPC) in vitro. In addition, several phenotypic subsets of NK cells have been described, and their relationships and relative locations within the hematopoietic hierarchy are supported by limited in vivo data. We “barcoded” individual CD34+ HSPC from 3 macaques using lentiviral vectors carrying highly diverse oligonucleotides, allowing precise quantitation of clonal contributions via low cycle PCR amplification followed by high-throughput sequencing and computational analysis (Lu et al, Nat Biotech). We quantitated barcode levels, and thus individual HSPC clonal contributions, over time and between lineages, following TBI and infusion of autologous barcoded CD34+ cells. Reconstitution of the NK compartment was clonally distinct from T, B and myeloid (My) lineages. Following reconstitution of NK, T, B and My from uni-lineage progenitors at 1-2 months(m), by 3-4 m, as expected from murine and in vitro models, multi-lineage clones began to contribute and dominate, first B/My, then B/T/My. However, NK cells remained clonally distinct through 9 m, despite overall clonal diversity and marking levels in NK lineage similar to B/T and My. Even the most abundant clones in the NK lineage were not found contributing in any significant way to B/T or My lineages. Shared B/T/My/NK clones finally began to contribute more extensively at 9-14 m. We fractionated peripheral blood NK cells into 3 NK subsets and compared them to overall PB and lymph node NK cells at 4.5-6.5 m. The majority (75-90%) of PB rhesus NK cells are found in the CD16+/CD56- PB subset (corresponding to human CD16+/CD56dim cytotoxic NK), and this subset accounted for the clonal pattern in overall NK cells (Pearson r=0.98 vs overall NK cells), and the disparity from B/T/My, with r values all <0.17 for CD16+/CD56- vs B/T or My lineages at the same time point. The minor PB (but dominant lymph node) cytokine-producing CD16-/CD56+ or CD16+/CD56+ NK subsets, previously hypothesized to be precursors for CD16+/CD56- NK cells, had clonal patterns that were more closely correlated with T/B/My lineages (r=0.37-0.62) than the CD16+/CD56- cells. The clonal correlations between the putative precursor CD16-/CD56+ cells and mature cytotoxic CD16+/CD56- cells was very low, only r=0.08. Furthermore, we expanded purified NK cells from barcoded macaques in vitro in the presence of IL-2 and irradiated EBV-LCL cells and assessed NK phenotype, function, and clonal diversity over time. 40-90X expansion was achieved by 15 days and was highly polyclonal, with 90% of the starting number of barcodes still present at the end of expansion. Clonal contribution levels between pre and post-expanded NK were highly correlated (r=0.73). CD16+/CD56+ cells became more dominant in post-exp NK cells, in contrast to the majority CD16+CD56- pre-exp. The clonal contributions in the post-exp CD16+/CD56- and CD16+/CD56+ cells correlated with each other (r=0.66), and with the starting CD16+/CD56- cells (r=0.75), but not with the starting CD16-/CD56+ (r=0.15) nor the post-exp CD16-/CD56+ cells (r=0.18). Our in vivo and in vitro results call into question the hypothesis that CD16-/CD56+ NK cells are precursors of circulating cytotoxic CD16+/CD56- NK cells, as does the complete deficiency of CD16-/CD56+ but not CD16+/CD56dim cells in GATA2 mutant patients. Our results also reveal that the dominant blood rhesus NK cell population has a distinct ontogeny in macaques, and thus potentially in humans. NK cells expanded ex vivo using EBV-LCL cells are polyclonal, and barcoding allows dissection of events during expansion, and will permit tracking studies of expanded cells following adoptive transfer. Disclosures: No relevant conflicts of interest to declare.

41BBL-Based Activation and Expansion of Autologous Natural Killer Cells Results in Enhanced Activity Against Leukemia Including ALL

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2293-2293
Author(s):  
Ekta Kapadia ◽  
Elad Jacoby ◽  
Mark Kohler ◽  
Waleed Haso ◽  
Christopher Daniel Chien ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Enhanced Activity

Abstract Childhood leukemia is the most common pediatric malignancy. There are now excellent cure rates for these patients, however outcomes remain poor for those with refractory disease and for those who relapse after standard salvage therapies, with a disease recurrence of approximately 50%. Therefore, development of novel cellular therapies is essential to treat these refractory patients. Natural Killer (NK) cells generated from an allograft contribute to improved disease free survival after Hematopoietic Stem Cell Transplantation for leukemia when there is a KIR mismatch. This effect appears to be particularly potent in the setting of Acute Myelogenous Leukemia (AML) with less benefit demonstrated in Acute Lymphoblastic Leukemia (ALL). Preclinical studies have also suggested that activation and expansion of resting NK cells can enhance NK cell cytotoxicity and eliminate the need for KIR mismatch due to up-regulation of activating receptors. We are currently testing this approach in the clinic following a fully matched allogeneic transplant platform for leukemia. Our aim is to explore whether 41BB ligand (41BBL) and recombinant IL-15 (rIL-15) mediated ex vivo expansion of autologous NK cells results in enhanced activity against AML and ALL. The activation/expansion process may allow for the use of autologous NK cell infusions, thus eliminating the need for allogeneic NK cell donors. To test this hypothesis, we ex vivo expanded and activated NK cells derived from C57BL/6J (B6) mice using artificial Antigen Presenting Cells (aAPCs) containing 41BBL and rIL-15 for 7-14 days. NK cells were co-cultured with murine AML cells (C1498) and murine ALL cells (E2A-PBX) – both on B6 background. Controls included YAC cells (murine T-cell lymphoma cell line sensitive to NK cell killing) as well as Phorbol Myristate Acetate (PMA)/ionomycin. All cells were co-cultured for 5 hours prior to functional assessment of NK cells via CD107a degranulation. NK cells cultured with 41BBL aAPCs and rIL-15 had a 30-fold expansion in numbers (Figure 1) and an increase in purity to approximately 95-98% (NK1.1+, CD3–) by Day 7. In the absence of cytokine or aAPCs, cultured NK cells underwent rapid apoptosis. Functionally, although resting NK cells (harvested prior to assessment) expressed CD107a when cultured with YAC cells and PMA, only minimal degranulation was observed in the presence of autologous AML cells or ALL cells. In contrast, activated and expanded autologous NK cells displayed enhanced activity against ALL, AML, as well as YAC cells, while only minimal levels of CD107a were seen in the absence of targets (Figure 2). In vivo experiments with a single injection of activated and expanded NK cells did not result in prolonged survival of mice bearing either AML or ALL. Assessment of adoptively transferred NK cells demonstrated very transient persistence (<2 days) with no in vivo expansion, suggesting that repeated injections may be necessary for leukemia eradication. Future murine experiments will investigate the effect repeated injections of activated/expanded NK cells and/or the administration of rIL-15 will have on survival and leukemia eradication. In addition, the ability to activate and expand NK cells in culture provides an opportunity for lentiviral-based transduction with chimeric antigen receptor (CAR) vectors. We are currently testing this with a murine CD19 CAR. These experiments suggest that autologous activated and expanded NK cells may serve as a viable cellular therapy for pediatric patients with refractory/relapsed leukemia. As demonstrated in these in vitro experiments, autologous activated/expanded NK cells still show increased targeting of mouse AML and ALL cell lines despite the lack of KIR mismatch. Thus, they may serve as a potential platform for leukemia therapy, including ALL, which appear to be poor targets for resting NK cells. In addition, these cells demonstrate transient persistence in vivo, a potential advantage in the context of redirected cytotoxicity using CAR constructs that target antigens with broader expression in the hematopoietic compartment. Figure 1: <![if !vml]><![endif]> Figure 1:. <![if !vml]><![endif]> Figure 2: Figure 2:. Disclosures No relevant conflicts of interest to declare.

Therapeutic applications: natural killer cells in the clinic

Hematology ◽  
2013 ◽  
Vol 2013 (1) ◽  
pp. 247-253 ◽  
Author(s):  
Jeffrey S. Miller
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Adoptive Transfer ◽  
Cell Activation ◽  
Tumor Antigens ◽  
Nk Cell ◽  
Ex Vivo ◽  
T Cell Stimulation ◽  
Refractory Acute Myeloid Leukemia

Abstract Natural killer (NK) cells recognize targets stressed by malignant transformation or infection (particularly CMV). We now know that NK cells can be long-lived and remember past exposures. They become educated by interaction with MHC class I molecules to gain potent function to kill targets and produce cytokines. In the clinical setting, haploidentical NK cells can be transferred adoptively to treat cancer. Persistence and in vivo expansion of NK cells depends on lymphodepleting chemotherapy to make space for the release of endogenous IL-15. In vivo expansion is also enhanced by cytokine administration. IL-2 has been used at low doses to stimulate NK cells in vivo, but has the down side of stimulating CD25hi regulatory T cells. IL-15 is now being tested and has the advantage of avoiding inhibitory regulatory T cell stimulation. In refractory acute myeloid leukemia, leukemia clearance is correlated with the persistence and in vivo expansion of NK cells after adoptive transfer. Limitations to NK cell therapy include poor in vivo survival and lack of specificity. Monoclonal antibodies and bispecific or trispecific killer engagers to target CD16 on NK cells to enhance recognition of various tumor antigens and ADAM17 inhibition to prevent CD16 shedding after NK cell activation should promote enhanced killing of cancer with specificity. Future strategies to exploit favorable donor immunogenetics or to expand NK cells ex vivo from blood, progenitors, or pluripotent progenitors may overcome immune barriers of adoptive transfer and comparative clinical trials will be needed to test these approaches.

Influence of in vivo hypobaric hypoxia on function of lymphocytes, neutrocytes, natural killer cells, and cytokines

1993 ◽  
Vol 74 (3) ◽  
pp. 1100-1106 ◽  
Author(s):  
M. Klokker ◽  
A. Kharazmi ◽  
H. Galbo ◽  
I. Bygbjerg ◽  
B. K. Pedersen
Keyword(s):  
Immune System ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Cell Activity ◽  
Nk Cell Activity ◽  
Cd8 Cells ◽  
The Absolute

We have investigated the effects of short-term hypoxia in vivo on the human cellular immune system. Seven young healthy volunteers were placed in a decompression chamber (380 Torr) for 20 min with or without supplemental O2. The leukocyte concentration increased during hypobaric conditions because of an increased concentration of lymphocytes. The absolute and relative concentration of CD16+ natural killer (NK) cells increased markedly during hypoxia and returned to pretest values after 2 h of recovery. The NK cell activity of blood mononuclear cells (BMNC, %lysis/fixed no. of BMNC) boosted with interferon-alpha, interleukin-2 (IL-2), and indomethacin rose in parallel with unboosted NK cell activity during hypoxia. The percentage of CD4+ and CD8+ cells declined during hypoxia, whereas the absolute concentration of both CD8+ cells and CD14+ monocytes increased. Although the BMNC composition varied, the proliferative responses of BMNC after stimulation with phytohemagglutinin, purified derivative of tuberculin, and IL-2 did not change significantly. The in vitro production of interleukin-1 beta and IL-2 in supernatants obtained after stimulation of BMNC with phytohemagglutinin or lipopolysaccharide was not affected. The chemiluminescence response of neutrocytes increased 2 h after hypoxia. It was concluded that acute hypoxia induced marked alterations in the immune system and that the NK cells are especially sensitive to the hypoxic stimulus.

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