Antigen Presenting Cell-Mediated Ex Vivo Expansion of Human Umbilical Cord Blood Cells Yields Log-Scale Expansion of Natural Killer Cells with Anti-Myeloma Activity

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2100-2100
Author(s):  
Nina Shah ◽  
Dongxia Xing ◽  
Dean A. Lee ◽  
Laurence J.N. Cooper ◽  
William Decker ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Cell Lines ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Research Funding ◽  
Plasma Cells ◽  
Nk Cell ◽  
Ex Vivo ◽  
Feeder Cells

Abstract Abstract 2100 Background: Multiple myeloma (MM) is the second most common hematologic malignancy in adults and, to date, is incurable. Allogeneic natural killer (NK) cells are active in various hematologic malignancies and may have a role against MM. Umbilical cord blood is a potential source for allogeneic NK cells and ex vivo expanded umbilical cord blood-derived NK (UCB-NK) cells demonstrate activity comparable to that of peripheral blood-derived NK cells. Here we demonstrate the anti-myeloma activity of UCB-NK cells expanded to clinical grade by a novel technique using artificial antigen presenting feeder cells (“K562 Clone 9” cells) modified to express IL-21 (“K562-cl9-mIL21”). Methods: Cord blood mononuclear cells (CBMCs) were cultured in 10% human serum albumin media with IL-2 (500 IU/ml) and K562-cl9-mIL21 feeder cells (2:1 feeder: CBMC ratio) for 21 days. Thereafter, cells were subjected to CD3-immunomagnetic depletion. CD3-negative cells were then used as effector cells in functional assays. Flow cytometry was used to confirm NK cell purity (C56+/CD3- cells) and a standard chromium-51 assay was performed to determine NK cell cytotoxicity. Targets included K562 cells, MM cell lines RPMI 8226, ARP-1 and U266, autologous non-neoplastic UCB cells (negative control) and bone marrow-derived CD138+ plasma cells from myeloma patients. Results: Expansion of CBMCs with K562-cl9-mIL21 yielded a >2000 fold expansion of NK cells, compared with 47 fold expansion of CD56-selected cells cultured with IL-2 alone (p <0.05). After CD3 depletion, UCB-NK cultures were comprised of 92% CD56+/CD3- cells. K562-cl9-mIL21-expanded UCB-NK cells demonstrated cytotoxicity against the classic NK cell target K562 as well as MM cell lines RPMI 8226, ARP-1 and U266 (Fig 1). In addition, these UCB-NK cells were also active against primary, bone marrow-derived CD138+ plasma cells from myeloma patients, an effect which was augmented by pre-incubation of UCB-NK cells with lenalidomide (Figure 2, NK=UCB-NK cells, NKR=UCB-NK cells+lenalidomide). Conclusions: UCB-NK cells can be expanded ex vivo to clinically relevant doses for allogeneic NK cell therapy via co-culture with K562-cl9-mIL21 feeder cells. Expanded UCB-NK cells are cytotoxic to myeloma cell lines and primary myeloma cells. Further development of UCB-NK cells as an adjunct therapy in stem cell transplantation for myeloma is warranted. Disclosures: Wang: Celgene: Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding.

Influence Of DNA Methyltransferese Inhibitors On The Anti-Leukemic Effect Of Umbilical Cord Blood Derived NK Cells Against Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4496-4496
Author(s):  
Harry Dolstra ◽  
Jeannette Cany ◽  
Anniek B. van der Waart ◽  
Marleen Tordoir ◽  
Basav Nagaraj Hangalapura ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
Dnmt Inhibitors ◽  
Nsg Mice ◽  
Drug Concentrations

Natural killer (NK) cell-based immunotherapy is a promising adjuvant, relatively non-toxic therapy approach for AML. However, further improvement of NK cell-based therapy is needed to increase the clinical effect. In this regard, NK cells generated ex vivo from hematopoietic progenitor cells (HPC) may have significant clinical benefits over enriched NK cells from adult donors, including the ability to choose an appropriate killer-cell immunoglobuline-like receptor (KIR)-ligand or KIR B haplotype alloreactive donor, as well as the capacity to reach high therapeutic dosages. Previously, we reported a GMP-compliant, cytokine/heparin-based culture protocol for the ex vivo generation of highly active NK cells from CD34+ HPC isolated from cryopreserved umbilical cord blood (UCB) units. Expansion in closed, large-scale bioreactors yields a clinically relevant dose of NK cells with high purity and cytolytic activity against AML cells in vitro. Currently, a clinical phase I trial with these HPC-NK cells is ongoing in our hospital. Trafficking studies in NOD/SCID/IL2Rgnull (NSG) mice demonstrated that these HPC-NK cells migrate to the bone marrow (BM) as well as to lymphoid organs where in vivo expansion and maturation can take place. Analysis of the chemokine receptor expression profile of UCB-NK cells matched in vivo findings. Particularly, a firm proportion of UCB-NK cells functionally expressed CXCR4, what could trigger BM homing in response to its ligand CXCL12. In addition, high expression of CXCR3 and CCR6 supported the capacity of UCB-NK cells to migrate to inflamed tissues via the CXCR3/CXCL10-11 and CCR6/CCL20 axis. Importantly, a single HPC-NK cell infusion combined with supportive IL-15 administration was shown to efficiently inhibit growth of K562 leukemia cells implanted in the femur of NSG mice, resulting in significant prolongation of mice survival. Furthermore, we investigated whether modulation by the DNA methyltransferase (DNMT) inhibitors Azacytidine (Aza) and Decitabine (Deci) could further potentiate the antileukemic effect of HPC-NK cells against AML cells. In concordance with previous reports, we observed a dose-dependent effect of Aza and Deci on the growth of the AML cell lines THP1 and KG1a. In subsequent NK cell killing assays, we used clinical relevant low drug concentrations to pre-treat AML cells that did not affect HPC-NK cell viability and cytolytic function. Interestingly, increased killing of pre-treated THP1 and KG1a cells by HPC-NK cells could be observed, which was correlated with an increase in the NKG2D ligand ULBP2, the DNAM-1 ligands CD112 and CD155 as well as TRAIL-R2. Notably, maintenance of low-dose DNMT inhibitors during the KG1a/NK co-culture resulted in pronounced AML growth inhibition. To examine the effect of DNMT inhibitors in vivo, THP1.LucGFP-bearing NSG mice were treated with increasing dose of both agents, which were administered according to current standard protocols applied in humans. Data indicated that treatment with Aza or Deci at dosage equivalent in human to 12.5 and 5 mg/m2 respectively was well tolerated with minimal and/or transient weight loss, and efficiently reduced the progression of THP-1.LucGFP cells in vivo. Currently, we explore whether HPC-NK cells and DNMT inhibitors can work together to combat AML in our xenograft models. These preclinical studies may provide a rationale to investigate the possible additive and/or synergistic anti-AML effects of adoptive HPC-NK cell transfer in combination with these DNMT inhibitors in AML patients. Disclosures: Tordoir: Glycostem Therapeutics: Employment. Spanholtz:Glycostem Therapeutics: Employment.

Ex vivo expansion of CD34+ and T and NK cells from umbilical cord blood for leukemic BALB/C nude mouse transplantation

2008 ◽  
Vol 87 (2) ◽  
pp. 217-224 ◽  
Author(s):  
Yaming Wei ◽  
Yinfeng Huang ◽  
Yinze Zhang ◽  
Huayou Zhou ◽  
Qiong Cao ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Nude Mouse ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion

scholarly journals CD38low Natural Killer Cells Transiently Expressing CD16F158V m-RNA Potentiates the Therapeutic Activity of Daratumumab Against Multiple Myeloma with Minimal Effector NK Cell Fratricide

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3199-3199 ◽  
Author(s):  
Subhashis Sarkar ◽  
Sachin Chauhan ◽  
Arwen Stikvoort ◽  
Alessandro Natoni ◽  
John Daly ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Advisory Committees ◽  
Cd38 Expression ◽  
Rpmi 8226

Abstract Introduction: Multiple Myeloma (MM) is a clonal plasma cell malignancy typically associated with the high and uniform expression of CD38 transmembrane glycoprotein. Daratumumab is a humanized IgG1κ CD38 monoclonal antibody (moAb) which has demonstrated impressive single agent activity even in relapsed refractory MM patients as well as strong synergy with other anti-MM drugs. Natural Killer (NK) cells are cytotoxic immune effector cells mediating tumour immunosurveillance in vivo. NK cells also play an important role during moAb therapy by inducing antibody dependent cellular cytotoxicity (ADCC) via their Fcγ RIII (CD16) receptor. Furthermore, 15% of the population express a naturally occurring high affinity variant of CD16 harbouring a single point polymorphism (F158V), and this variant has been linked to improved ADCC. However, the contribution of NK cells to the efficacy of Daratumumab remains debatable as clinical data clearly indicate rapid depletion of CD38high peripheral blood NK cells in patients upon Daratumumab administration. Therefore, we hypothesize that transiently expressing the CD16F158V receptor using a "safe" mRNA electroporation-based approach, on CD38low NK cells could significantly enhance therapeutic efficacy of Daratumumab in MM patients. In the present study, we investigate the optimal NK cell platform for generating CD38low CD16F158V NK cells which can be administered as an "off-the-shelf"cell therapy product to target both CD38high and CD38low expressing MM patients in combination with Daratumumab. Methods: MM cell lines (n=5) (MM.1S, RPMI-8226, JJN3, H929, and U266) and NK cells (n=3) (primary expanded, NK-92, and KHYG1) were immunophenotyped for CD38 expression. CD16F158V coding m-RNA transcripts were synthesized using in-vitro transcription (IVT). CD16F158V expression was determined by flow cytometry over a period of 120 hours (n=5). 24-hours post electroporation, CD16F158V expressing KHYG1 cells were co-cultured with MM cell lines (n=4; RPMI-8226, JJN3, H929, and U266) either alone or in combination with Daratumumab in a 14-hour assay. Daratumumab induced NK cell fratricide and cytokine production (IFN-γ and TNF-α) were investigated at an E:T ratio of 1:1 in a 14-hour assay (n=3). CD38+CD138+ primary MM cells from newly diagnosed or relapsed-refractory MM patients were isolated by positive selection (n=5), and co-cultured with mock electroporated or CD16F158V m-RNA electroporated KHYG1 cells. CD16F158V KHYG1 were also co-cultured with primary MM cells from Daratumumab relapsed-refractory (RR) patients. Results: MM cell lines were classified as CD38hi (RPMI-8226, H929), and CD38lo (JJN3, U266) based on immunophenotyping (n=4). KHYG1 NK cell line had significantly lower CD38 expression as compared to primary expanded NK cells and NK-92 cell line (Figure 1a). KHYG1 electroporated with CD16F158V m-RNA expressed CD16 over a period of 120-hours post-transfection (n=5) (Figure 1b). CD16F158V KHYG1 in-combination with Daratumumab were significantly more cytotoxic towards both CD38hi and CD38lo MM cell lines as compared to CD16F158V KHYG1 alone at multiple E:T ratios (n=4) (Figure 1c, 1d). More importantly, Daratumumab had no significant effect on the viability of CD38low CD16F158V KHYG1. Moreover, CD16F158V KHYG1 in combination with Daratumumab produced significantly higher levels of IFN-γ (p=0.01) upon co-culture with CD38hi H929 cell line as compared to co-culture with mock KHYG1 and Daratumumab. The combination of CD16F158V KHYG1 with Daratumumab was also significantly more cytotoxic to primary MM cell ex vivo as compared to mock KHYG1 with Daratumumab at E:T ratio of 0.5:1 (p=0.01), 1:1 (p=0.005), 2.5:1 (p=0.003) and 5:1 (p=0.004) (Figure 1e). Preliminary data (n=2) also suggests that CD16F158V expressing KHYG1 can eliminate 15-17% of primary MM cells from Daratumumab RR patients ex vivo. Analysis of more Daratumumab RR samples are currently ongoing. Conclusions: Our study provides the proof-of-concept for combination therapy of Daratumumab with "off-the-shelf" CD38low NK cells transiently expressing CD16F158V for treatment of MM. Notably, this approach was effective against MM cell lines even with low CD38 expression (JJN3) and primary MM cells cultured ex vivo. Moreover, the enhanced cytokine production by CD16F158V KHYG1 cells has the potential to improve immunosurveillance and stimulate adaptive immune responses in vivo. Disclosures Sarkar: Onkimmune: Research Funding. Chauhan:Onkimmune: Research Funding. Stikvoort:Onkimmune: Research Funding. Mutis:Genmab: Research Funding; OnkImmune: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Celgene: Research Funding; Novartis: Research Funding. O'Dwyer:Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; BMS: Research Funding; Glycomimetics: Research Funding; Onkimmune: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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.

Analysis of natural killer (NK) cell activity and adhesion molecules on NK cells from umbilical cord blood

2003 ◽  
Vol 71 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Hidehisa Tanaka ◽  
Shunro Kai ◽  
Masao Yamaguchi ◽  
Mahito Misawa ◽  
Yoshihiro Fujimori ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Natural Killer ◽  
Adhesion Molecules ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Nk Cell ◽  
Cell Activity ◽  
Nk Cell Activity

scholarly journals Umbilical cord blood-derived ILC1-like cells constitute a novel precursor for mature KIR+NKG2A- NK cells

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Sabrina Bianca Bennstein ◽  
Sandra Weinhold ◽  
Angela Riccarda Manser ◽  
Nadine Scherenschlich ◽  
Angela Noll ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Nk Cell ◽  
Functional Characterization ◽  
Lymphoid Tissues ◽  
The Novel ◽  
Effector Cells ◽  
Notch Ligands

Despite their identification several years ago, molecular identity and developmental relation between human ILC1 and NK cells, comprising group 1 ILCs, is still elusive. To unravel their connection, thorough transcriptional, epigenetic, and functional characterization was performed from umbilical cord blood (CB). Unexpectedly, ILC1-like cells lacked Tbet expression and failed to produce IFNγ. Moreover, in contrast to previously described ILC1 subsets they could be efficiently differentiated into NK cells. These were characterized by highly diversified KIR repertoires including late stage NKG2A-KIR+ effector cells that are commonly not generated from previously known NK cell progenitor sources. This property was dependent on stroma cell-derived Notch ligands. The frequency of the novel ILC1-like NK cell progenitor (NKP) significantly declined in CB from early to late gestational age. The study supports a model in which circulating fetal ILC1-like NKPs travel to secondary lymphoid tissues to initiate the formation of diversified NK cell repertoires after birth.

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

Blood ◽  
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.

Early NK Cell Proliferation After Umbilical Cord Blood Transplantation Is Associated With Superior Disease-Free Survival Due To Reduced Leukemia Relapse

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4610-4610
Author(s):  
Rachel Joy Bergerson ◽  
Sarah Cooley ◽  
Julie Curtsinger ◽  
Ryan Shanley ◽  
Claudio Brunstein ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cord Blood ◽  
Nk Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Nk Cell ◽  
Disease Free Survival ◽  
Free Survival ◽  
Disease Free

Compared to traditional chemotherapy, allogeneic hematopoietic cell transplantation (allo-HCT) has the potential of triggering graft vs. leukemia (GVL) reactions that make this procedure uniquely curative. Despite this, relapse remains the most common cause of treatment failure. Early after allo-HCT the donor immune system reconstitutes within the host and natural killer (NK) cells are the earliest lymphocyte population to recover. Previous studies by us and other investigators have linked rapid lymphocyte recovery and/or high NK cell numbers early after transplant with less relapse. Mechanistically, the reasons for this are unknown. We hypothesized that NK cell proliferation would be associated with allo-HCT outcomes. In a large data set with short-term follow-up we compared stem cell source to NK cell proliferation at Day 28 after transplantation (using Ki67+ staining). In patients undergoing autologous (n=117), sibling (n=57), single umbilical cord blood (sUCB) (n=62) or double umbilical cord blood (dUCB) (n=50) transplantation there were significant differences in the median NK cell proliferation (2.1%, 3.3%, 3.8%, and 19.2%, respectively, p<0.0001). These results suggest that NK cell proliferation is increased when patients are transplanted with stem cell sources that have an increased number of HLA mismatches (dUCB). We next examined factors extrinsic to the NK cells to determine whether differences in the four patient groups were due to these factors. Regulatory T cells (Tregs) are known to negatively regulate the proliferation and activation of a variety of cell types, including NK cells. We utilized the percentage and absolute number of Tregs (defined as CD4+CD25highFoxP3+CD127low) at Day 28 (available for a subset of the above patient samples) and correlated it with the percentage of proliferating NK cells at Day 28 after allogeneic transplant (n=212). The median percentage of Tregs within the lymphocyte fraction was 1.49%. In patients with higher than 1.49% Tregs, the mean NK cell proliferation was 10.8% +/-16.4. In contrast, patients with lower than 1.49% Tregs had a mean NK cell proliferation of 21.2% +/-24.3 (p=0.0004). A nearly identical trend was observed for the absolute numbers of Tregs, suggesting that Tregs may play an extrinsic role in NK cell proliferation after allo-HCT. To further address differences in lymphocyte proliferation and clinical outcomes, we used Ki67 staining to assess T cell (CD4+ and CD8+) and NK cell (CD3-CD56+) proliferation at Day 28 in a different subset of patients undergoing dUCB transplant with acute leukemia or MDS (80%) or other malignant disorders using either myeloablative (42%) or reduced intensity conditioning (58%) and a median of one year follow-up (n=53). There was no association of transplant outcomes with T cell (CD4 or CD8) proliferation. In the NK cell compartment there was a wide range in the percentage of proliferating NK cells (0-86%), with a median of 20%. Patients were segregated into high (>20%) and low (<20%) NK cell proliferating groups and assessed for cytokine levels and transplant outcomes. At Day 28 soluble IL15 levels were not different between high and low NK cell proliferating patients. There was no significant association between NK cell proliferation and the probability or time to neutrophil recovery (p=0.15) or treatment related mortality (p=0.88). Excluding the 14 patients who developed aGVHD prior to day of NK cell assessment (Day 28), the high NK proliferating group had a trend toward a higher cumulative incidence of aGVHD (46% vs. 25%, p=0.17). Using multivariable analysis to control for a variety of patient and disease-associated variables, patients with high NK cell proliferation had significantly better disease-free survival (HR=0.29, 95%CI=0.12-0.71, p=0.01) (Figure 1). Strikingly, patients with high NK cell proliferation were 4-times less likely to relapse (HR=0.24, 95%CI=0.08-0.77, p=0.02). Collectively, these studies show that early NK cell proliferation is associated with superior outcomes after dUCB transplantation, due to reduced relapse, and that this is partly modulated by Tregs. Prospective strategies to increase NK cell proliferation may be of therapeutic benefit to improve transplant outcomes.Figure 1Disease Free Survival for patients undergoing dUCB transplant (n=53) based on low (blue) vs high (red) NK cell proliferation at Day 28 post transplant.Figure 1. Disease Free Survival for patients undergoing dUCB transplant (n=53) based on low (blue) vs high (red) NK cell proliferation at Day 28 post transplant. Disclosures: Wagner: Novartis: Research Funding. Miller:Coronado Biosciences: Scientific Advisory Board Other.

Generation Of Clinical Grade, Highly Cytotoxic NK Cells Using Feeder Cell Free, Particle-Based Technology

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4503-4503
Author(s):  
Jerremiah Oyer ◽  
Igarashi Y Robert ◽  
Colosimo Dominic ◽  
Melhem M. Solh ◽  
Yasser Khaled ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Peripheral Blood ◽  
Nk Cell ◽  
Ex Vivo ◽  
Free Particle ◽  
Leukemia Cell ◽  
High Dose ◽  
Feeder Cells ◽  
Peripheral Blood Mononuclear

NK cell immunotherapy shows exciting promise, but inconsistency and variability remain as a significant challenge. Since NK cells comprise a small fraction (∼5%) of the peripheral blood mononuclear cell fraction, expansion of NK cells in vivo or ex vivo is a critical requirement to attain therapeutically effective dosages and to observe consistent positive clinical outcomes. Most of currently developed ex vivo expansion protocols depend on co-culture with various engineered and/or cancer derived stimulator/feeder cells to induce the proliferation of NK cells. The use of accessory cells poses significant challenges to clinical transfer. Our laboratory has developed a nanoparticle-based expansion technology that utilizes particles, few hundred nanometers in size, derived from the plasma membrane (PM) of K562 feeder cells expressing IL-15 and 41BBL on their surface (PM-mb15-41BBL). These particles in combination with low concentration of IL-2 induce selective and efficient expansion of NK cells within human peripheral blood mononuclear cells (PBMC). When PBMC are stimulated with PM-mb15-41BBL over 21 days the NK cell numbers increase exponentially between days 6 and 18 of culture. The numbers of NK cell increased on average 200 fold (range 104-557, n=11, 4 donors) after 12-13 days of culture in the presence of PM-mb15-41BBL particles (at 200 µg of membrane protein/mL). The expansions with the PM particles are comparable to those in the presence of live feeder cells that gave ∼200 fold (79-895, n=11, 4 donors). The PM-particle based NK expansion is far better in comparison to NK stimulation with soluble purified 41BBL, IL-15 and IL-2, at matching concentrations, that yielded only 3 fold (1-4, n=6, 3 donors) increase in NK cells. Furthermore, the NK cells expand selectively under these conditions where they initially consisted only about 10% of the population of PBMC isolated from fresh peripheral blood, but increased to more than 95% of the cell suspension after 14 days in culture. The extent of expansion and NK cell content on day 12 of culture was dependent on the concentration of PM particles used with 200 µg of PM protein/mL being the optimal dose. Thus, PM nanoparticles can expand NK cells as efficiently and selectively as feeder cells. Furthermore, the PM-particle based expansion is more reproducible between trials and with different donors as compared to NK cell expansion induced with feeder cells (coefficient of variation 63% vs. 88%, respectively). The NK cells expanded in presence of PM-particles were highly cytotoxic against several leukemia cell lines and also against patient derived AML blasts. Expanded NK cells were 4 to 9 times more potent against AML cell lines K562, KG1 and HL-60 as compared to freshly isolated NK cells that were pre-activated with a high dose of IL-2. The PM-particle expanded NK cells also were selectively cytotoxic where they efficiently killed patient derived CD34+ leukemia blasts while sparing healthy CD34- peripheral blood cells. The expanded NK cells were observed to have an increase in the expression of major activating receptors such as NKG2D, NKp44, NKp30 and of the death receptor ligand FasL. This expression difference corresponds well with the activated cytotoxic phenotype and is likely responsible for their increased cytotoxicity against AML cells. Pilot trials in NSG mice are currently ongoing. Disclosures: Solh: Celgene: Speakers Bureau.

Sign in / Sign up

Export Citation Format

Share Document