scholarly journals Role of interleukin-15 in the development of human CD56+ natural killer cells from CD34+ hematopoietic progenitor cells

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2632-2640 ◽  
Author(s):  
E Mrozek ◽  
P Anderson ◽  
MA Caligiuri
Keyword(s):  
Cell Differentiation ◽  
Nk Cells ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Nk Cell ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Ifn Gamma ◽  
Nk Cell Differentiation

Human natural killer (NK) cells are bone marrow (BM)-derived CD2+CD16+CD56+ large granular lymphocytes (LGL) that lack CD3 yet contain the T-cell receptor zeta-chain (zeta-TCR). NK cells provide requisite interferon-gamma (IFN-gamma) during the early stages of infection in several experimental animal models. A number of studies have shown that human CD3-CD56+ NK cells can be obtained from BM- derived CD34+ hematopoietic progenitor cells (HPCs) cultured in the presence of interleukin-2 (IL-2) and an allogeneic feeder cell layer, or IL-2 and other hematopoietic growth factors such as the c-kit ligand (KL). The failure to detect the IL-2 gene product within the BM stroma and the presence of NK cells in IL-2-deficient mice suggested that cytokines other than IL-2 may participate in NK cell differentiation from HPCs in vivo. IL-15 is a cytokine which, while lacking any sequence homology in IL-2, can activate cells via the IL-2 receptor. Here we show that human BM stromal cells express the IL-15 transcript, and supernatants from long-term BM stromal cell cultures contain IL-15 protein. In vitro, CD3-CD56+ NK cells can be obtained from 21-day cultures of CD34+ HPCs supplemented with IL-15 in the absence of IL-2, stromal cells, or other cytokines. The addition of the KL to these cultures had no effect on the differentiation of the CD3-CD56+ cytotoxic effector cells, but greatly enhanced their expansion. The majority of these cells lack CD2 and CD16, but do express zeta-TCR. Similar to NK cells found in peripheral blood, the CD2-CD16-CD56+ NK cells grown in the presence of IL-15 were found to be potent producers of IFN-gamma in response to monocyte-derived cytokines. Thus IL-15, like KL, appears to be produced by BM stromal cells. IL-15 can induce CD34+ HPCs to differentiate into CD3-CD56+ NK cells, and KL can amplify this. Therefore, IL-15 may be a physiologically relevant ligand for NK cell differentiation in vivo.

scholarly journals Role of interleukin-2 (IL-2), IL-7, and IL-15 in natural killer cell differentiation from cord blood hematopoietic progenitor cells and from gamma c transduced severe combined immunodeficiency X1 bone marrow cells

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3901-3909 ◽  
Author(s):  
M Cavazzana-Calvo ◽  
S Hacein-Bey ◽  
G de Saint Basile ◽  
C De Coene ◽  
F Selz ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Cord Blood ◽  
Nk Cells ◽  
Progenitor Cells ◽  
Nk Cell ◽  
Severe Combined Immunodeficiency ◽  
Interleukin 2 ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Nk Cell Differentiation

Natural killer (NK) cells are characterized by their ability to mediate spontaneous cytotoxicity against susceptible tumor cells and infected cells. They differentiate from hematopoietic progenitor cells. Patients with X-linked severe combined immunodeficiency (SCID X1) carry mutations in the gamma c cytokine receptor gene that result in lack of both T and NK cells. To assess the role of interleukin-2 (IL-2), IL-7, and IL-15 cytokines, which share gamma c receptor subunit, in NK cell differentiation, we have studied NK cell differentiation from cord blood CD34 (+) cells in the presence of either stem cell factor (SCF), IL-2, and IL-7 or SCF and IL-15. The former cytokine combination efficiently induced CD34 (+) CD7 (+) cord blood cells to proliferate and mature into NK cells, while the latter was also able to induce NK cell differentiation from more immature CD34 (+) CD7 (-) cord blood cells. NK cells expressed CD56 and efficiently killed K562 target cells. These results show that IL-15 could play an important role in the maturation of NK cell from cord blood progenitors. Following retroviral-mediated gene transfer of gamma c into SCID X1 bone marrow progenitors, it was possible to reproduce a similar pattern of NK cell differentiation in two SCID-X1 patients with SCF + IL-2 + IL-7 and more efficiently in one of them with SCF + IL-15. These results strongly suggest that the gamma c chain transduces major signal(s) involved in NK cell differentiation from hematopoietic progenitor cells and that IL-15 interaction with gamma c is involved in this process at an earlier step than IL-2/IL-7 interactions of gamma c are. It also shows that gene transfer into hematopoietic progenitor cells could potentially restore NK cell differentiation in SCID X1 patients.

scholarly journals Monocytes control natural killer cell differentiation to effector phenotypes

Blood ◽  
2011 ◽  
Vol 117 (17) ◽  
pp. 4511-4518 ◽  
Author(s):  
Katrina Soderquest ◽  
Nick Powell ◽  
Carmelo Luci ◽  
Nico van Rooijen ◽  
Andrés Hidalgo ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Killer Cell ◽  
In Vivo Model ◽  
Dependent Manner ◽  
Nk Cell Differentiation

Abstract Natural killer (NK) cells play a major role in immunologic surveillance of cancer. Whether NK-cell subsets have specific roles during antitumor responses and what the signals are that drive their terminal maturation remain unclear. Using an in vivo model of tumor immunity, we show here that CD11bhiCD27low NK cells migrate to the tumor site to reject major histocompatibility complex class I negative tumors, a response that is severely impaired in Txb21−/− mice. The phenotypical analysis of Txb21-deficient mice shows that, in the absence of Txb21, NK-cell differentiation is arrested specifically at the CD11bhiCD27hi stage, resulting in the complete absence of terminally differentiated CD11bhiCD27low NK cells. Adoptive transfer experiments and radiation bone marrow chimera reveal that a Txb21+/+ environment rescues the CD11bhiCD27hi to CD11bhiCD27low transition of Txb21−/− NK cells. Furthermore, in vivo depletion of myeloid cells and in vitro coculture experiments demonstrate that spleen monocytes mediate the terminal differentiation of peripheral NK cells in a Txb21- and IL-15Rα–dependent manner. Together, these data reveal a novel, unrecognized role for Txb21 expression in monocytes in promoting NK-cell development and help appreciate how various NK-cell subsets are generated and participate in antitumor immunity.

Induction of NK Cell Reactivity Against Myeloid Leukemia By a Novel Fc-Optimized CD133 Antibody

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3793-3793 ◽  
Author(s):  
Kathrin Rothfelder ◽  
Samuel Koerner ◽  
Maya Andre ◽  
Julia Leibold ◽  
Philaretos Kousis ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Ex Vivo ◽  
Leukemic Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor

Abstract NK cells largely contribute to the success of monoclonal antibody (mAb) application in cancer due to their ability to mediate antibody-dependent cellular cytotoxicity (ADCC), a feature considered critical for therapeutic success. Up to now, no immunotherapeutic antibodies are available for the treatment of myeloid leukemias. Recently, we reported on the development of mAb targeting CD133, which is expressed on a wide variety of tumor cells (Koerner et al., Blood 2014 124:2309). Here we extend our analyses and provide further data on the preclinical characterization of an Fc-engineered CD133 mAb for the treatment of myeloid leukemia. Compared to two other anti-human CD133 mAb (clones AC133 and W6B3), which both bound to primary AML and CML cells in 15/25 and 7/10 cases, respectively, clone 293C3 recognized the leukemic cells in 22/25 AML cases and 7/10 CML cases. Based on these results, clone 293C3 was chosen to generate chimeric mAb with either a wildtype Fc part (293C3-WT) or a variant containing amino acid exchanges (S239D/I332E) to enhance affinity to the activating Fc receptor CD16 on NK cells (293C3-SDIE). Treatment with 293C3-SDIE resulted in significantly enhanced activation, degranulation and lysis of primary CD133-positive AML cells by NK cells in allogeneic and autologous experimental ex vivo settings as compared to its wildtype counterpart. Considering the expression of CD133 on healthy hematopoietic progenitor cells, we further performed colony forming unit assays with healthy bone marrow (BM) cells. In line with the observed lower expression levels of CD133 on healthy compared to malignant hematopoietic cells no relevant toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells was observed. Moreover, 293C3-SDIE did not induce lysis of of healthy BM cells by allogeneic or autologous NK cells. In a NOD.Cg-Prkdcscid IL2rgtmWjl/Sz (NSG) xenotransplantation model, induction of ADCC by treatment with 293C3-SDIE resulted in the elimination of patient AML cells by NK cells from a matched human donor. Thus, 293C3-SDIE constitutes an attractive immunotherapeutic compound, in particular for the elimination of minimal residual disease in CD133 bearing leukemia in the context of allogenic SCT. Disclosures No relevant conflicts of interest to declare.

The Aryl Hydrocarbon Receptor Antagonist Stemregenin 1 Stimulates Expression of NK Cell Related Transcription Factors, Thereby It Facilitates Generation of Highly Functional NK Cells in Vitro

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3833-3833
Author(s):  
Mieke WH Roeven ◽  
Jeanette Cany ◽  
Frans Maas ◽  
Arwa Kohela ◽  
Jansen Joop ◽  
...  
Keyword(s):  
T Cells ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
Aryl Hydrocarbon Receptor ◽  
Nk Cells ◽  
Progenitor Cells ◽  
Culture System ◽  
Nk Cell ◽  
Aryl Hydrocarbon ◽  
Nk Cell Differentiation

Abstract Introduction Allogeneic stem cell transplantation (SCT) can be a curative treatment for hematological malignancies. The therapeutic effectiveness is attributed to the graft-versus-tumor (GVT) effect, mediated by alloreactive T cells and natural killer (NK) cells. Although T cells can induce a potent GVT effect, they can also induce graft-versus-host disease (GVHD), causing high morbidity and mortality. Interestingly, after non-myeloablative allogeneic SCT, early NK cell repopulation has been associated with decreased relapse rates, without increasing GVHD incidence, illustrating a possible role for donor NK cell adoptive transfer after allogeneic SCT. However, isolation of sufficient numbers of activated NK cells from donor origin is challenging. Recently, it has been described that antagonizing the aryl hydrocarbon receptor (AhR) using the antagonist StemReginin1 (SR1) promotes expansion of human hematopoietic stem cells. Furthermore, AhR turned out to regulate differentiation of multiple immune cells like dendritic cells, regulatory T cells, γδ T cells, and also NK cells. Therefore, we investigated if SR1 could enhance NK cell generation in a cytokine-based culture system. Methods CD34+ hematopoietic progenitor cells (HPCs) were isolated using immunomagnetic beads from G-CSF mobilized aphaeresis material. These HPCs were expanded during 14 days in medium containing SCF, Flt3L, TPO, IL-7 and IL-15 and subsequently differentiated into NK cells using IL-15 and IL-2. HPC-NK cell expansions were performed with or without SR1. RNA was collected from the cultures weekly and expression of NK cell related genes was analyzed using qPCR. After 35 days, HPC-NK cells were assessed for their cytolytic functions against acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines and primary AML blasts. In addition, expression levels of typical NK-activating receptors and differentiation markers were analyzed by flow cytometry. Results Interestingly, SR1 induced expression of TOX, ID2, EOMES, GATA3 and SH2D1B, which are important factors involved in early and late NK cell differentiation. In addition, SR1 improved the expansion, differentiation, and functionality of the NK cells generated. In the presence of SR1, we were able to expand PB-derived HPCs up to 1000-fold in 5 weeks. The SR1-generated HPC-NK cell products contained 80 – 92% NK cells, which expressed high levels of activating NKG2D and natural cytotoxicity receptors. Furthermore, functional analysis showed marked degranulation and cytokine release upon co-culture with AML and MM cell lines and efficient lysis of primary AML blasts at low NK-target ratios. Conclusion Addition of the AHR antagonist SR1 in our culture system induces expression of transcription factors involved in NK cell differentiation and thereby facilitates the generation of high numbers of functional NK cells from G-CSF mobilized CD34+ progenitor cells. These NK cells hold great promise for future donor NK cell-mediated therapy after allogeneic SCT. Disclosures No relevant conflicts of interest to declare.

scholarly journals Definition of a natural killer NKR-P1A+/CD56-/CD16- functionally immature human NK cell subset that differentiates in vitro in the presence of interleukin 12.

1996 ◽  
Vol 184 (5) ◽  
pp. 1845-1856 ◽  
Author(s):  
I M Bennett ◽  
O Zatsepina ◽  
L Zamai ◽  
L Azzoni ◽  
T Mikheeva ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Cell Subset ◽  
Interleukin 12 ◽  
Ifn Gamma ◽  
Differentiation Antigens ◽  
Nk Cell Differentiation

Human natural killer (NK) cell differentiation from immature lineage negative (Lin-) umbilical cord blood cells was examined in vitro. Cells expressing differentiation antigens of mature NK cells (CD56, CD16, CD2, CD8, NKR-P1A) were generated from Lin- cells cultured with interleukin (IL)-2 and a murine bone marrow stromal cell line expressing the human membrane-bound form of stem cell factor. Two subsets of NK cells were identified in these cultures: one expressed both NKR-P1A and CD56 and, in variable proportions, all other NK cell differentiation antigens; the second subset expressed only NKR-P1A and, unlike the former, was not cytotoxic. Neither subset expressed interferon (IFN)-gamma mRNA even after stimulation with phorbol di-ester and Ca2+ ionophore, but both expressed tumor necrosis factor alpha mRNA and the cytotoxic granule-associated proteins TIA-1, perforin, and serine esterase-1. After 10-d culture with IL-2, IL-12, and irradiated B lymphoblastoid cells, approximately 45% of the NKR-P1A+/ CD56- cells became CD56+, and the same cultures contained cells capable of cytotoxicity and of IFN-gamma production. These results indicate that NKR-P1A expression in the absence of other NK cell markers defines an intermediate, functionally immature stage of NK cell differentiation, and that effector functions develop in these cells, concomitantly with CD56 expression, in the presence of IL-12. These cells likely represent the counterpart of a CD3-/NKR-P1A+/ CD56-/CD16- cell subset that, as shown here, is present both in adult and neonatal circulating lymphocytes.

scholarly journals Aryl Hydrocarbon Receptor Antagonism Promotes Hematoendothelial Development from Human Pluripotent Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3585-3585
Author(s):  
Mathew G. Angelos ◽  
Anna Kim ◽  
Dan S. Kaufman
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Progenitor Cells ◽  
Nk Cell ◽  
Fold Change ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Aryl Hydrocarbon ◽  
Nk Cell Differentiation ◽  
Early Human

Abstract The aryl hydrocarbon receptor (AHR) is an evolutionarily conserved transcription factor originally characterized for its role in mediating biological responses to carcinogenic environmental agents. Recent studies have elucidated the importance of AHR-dependent signaling for normal physiological function in the absence of environmental ligands, most notably the development of Th17 cells, regulatory T-cells, and natural killer (NK) cells from human peripheral blood and umbilical cord blood. Additionally, AHR is highly expressed in hematopoietic stem/progenitor cells (HSPCs), and antagonism of AHR using small molecules results in a marked expansion of umbilical cord blood-derived HSPCs suitable for clinical transplantation. It remains unclear what role, if any, AHR plays during early human hematoendothelial development. We hypothesized inhibition of AHR-mediated cell signaling could promote early human hematopoietic cell development. To model human hematopoiesis, we employed a xenogeneic-free and chemically defined in vitro method to differentiate human embryonic stem cells (hESCs) into endothelial and hematopoietic cells. qRT-PCR analysis demonstrated a significant increase in AHR (13.36±5.52 fold change, p<0.05, n=3) by Day 11 of differentiation relative to undifferentiated hESCs. CYP1A1 and CYP1B1, two downstream targets of AHR-mediated signaling, were similarly upregulated on Day 11 (27.90±6.17 fold change, p<0.05, n=3; 134.28±10.06 fold change, n=3, respectively). Increase in AHR expression mirrored the onset of early hematopoietic progenitor cell differentiation; CD34+ CD43+ and CD34+ CD41a+ cells were markedly increased by Day 12 of hematopoietic differentiation as assessed by flow cytometry (18.9%±3.22, p<0.01, n=7; 8.23±2.00, p<0.05, n=7, respectively). We next modified the relative activity of AHR signaling by differentiating hESCs in the presence of 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD), a prototypical AHR agonist, or StemReginin-1 (SR-1), an AHR antagonist, and assessed its effects on hematopoietic progenitor cell production. Interestingly, we observed a significant increase in the appearance of both CD34+ CD31+ hematoendothelial cells in SR-1 treated hESCs relative to DMSO treated controls (17.63%±1.25, p<0.05, n=3 vs. 11.21±0.63, p<0.05, n=3) at Day 9. Later by Day 12, we also found approximately a two-fold expansion of CD34+ CD45+ hematopoietic progenitor cells in SR-1 treated hESCs relative to DMSO treated controls (16.35%±4.05, p<0.05, n=3 vs. 7.53±0.19, p<0.05, n=3). Treatment with TCDD reciprocally attenuated the development of CD34+ CD45+ progenitor cells at Day 15 relative to DMSO treated controls (3.99%±0.80 vs. 11.79%±1.41, p<0.05, n=3) and resulted in an expansion of terminally differentiated hematopoietic cells (CD34- CD43+: 84.5%±2.78 vs. 70.9±1.58, p<0.05, n=3; CD34- CD45+: 81.75%±1.75 vs. 71.95±2.35, p<0.05, n=3). We confirmed the functionality of the hematopoietic progenitor cells in each group by harvesting non-adherent cells at Day 12 and performing standard colony-forming assays. SR-1 treated cells yielded a 4-fold increase in the total number of colonies generated relative to DMSO treated control cells along with an increased proportion of CFU-M and CFU-GM. We also evaluated whether AHR antagonism could be used to promote NK cell differentiation from hESCs. Using previously optimized and defined NK cell differentiation conditions, we found SR-1 treatment caused an increase in CD56+ CD45+ NK cells relative to DMSO treated controls (26.4% vs. 19.7%, n=2) whereas TCDD treatment caused a decrease (6.7%, n=2). Work assessing how hematopoiesis from hESCs is affected using AHR gene knockouts developed from CRISPR/Cas9-mediated gene deletion is ongoing. Collectively, our results demonstrate AHR antagonism promotes early human hematoendothelial development and may be used as a potential molecular target to enhance hematopoietic cell production from human pluripotent stem cells for clinical applications. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stimulation by a dipeptidyl peptidase inhibitor reveals a novel regulatory mechanism and therapeutic treatment for blood cell deficiencies

Blood ◽  
2003 ◽  
Vol 102 (5) ◽  
pp. 1641-1648 ◽  
Author(s):  
Barry Jones ◽  
Sharlene Adams ◽  
Glenn T. Miller ◽  
Michael I. Jesson ◽  
Takeshi Watanabe ◽  
...  
Keyword(s):  
Blood Cell ◽  
Progenitor Cells ◽  
Stromal Cells ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Amino Terminal ◽  
Cytokine Levels ◽  
Dipeptidyl Peptidases

Abstract In hematopoiesis, cytokine levels modulate blood cell replacement, self-renewal of stem cells, and responses to disease. Feedback pathways regulating cytokine levels and targets for therapeutic intervention remain to be determined. Amino boronic dipeptides are orally bioavailable inhibitors of dipeptidyl peptidases. Here we show that the high-affinity inhibitor Val-boro-Pro (PT-100) can stimulate the growth of hematopoietic progenitor cells in vivo and can accelerate neutrophil and erythrocyte regeneration in mouse models of neutropenia and acute anemia. Hematopoietic stimulation by PT-100 correlated with increased cytokine levels in vivo. In vitro, PT-100 promoted the growth of primitive hematopoietic progenitor cells by increasing granulocyte–colony-stimulating factor (G-CSF), interleukin-6 (IL-6), and IL-11 production by bone marrow stromal cells. Two molecular targets of PT-100 are expressed by stromal cells— CD26/DPP-IV and the closely related fibroblast activation protein (FAP). Because PT-100 was active in the absence of CD26, FAP appears to be the hematopoietic target for PT-100. Interaction of PT-100 with the catalytic site seems to be required because amino-terminal acetylation of PT-100 abrogated enzyme inhibition and hematopoietic stimulation. PT-100 is a therapeutic candidate for the treatment of neutropenia and anemia. The data support increasing evidence that dipeptidyl peptidases can regulate complex biologic systems by the proteolysis of signaling peptides.

scholarly journals Preclinical Evaluation of CSL362/JNJ-56022473 in Combination with Decitabine or Azacitidine in in Vitro Assays

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1370-1370 ◽  
Author(s):  
Khaja Syed ◽  
Christine Pietsch ◽  
Amy Axel ◽  
Ann Forslund ◽  
Kate Sasser ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Day Treatment ◽  
Hematopoietic Progenitor Cells ◽  
Inhibitory Receptor ◽  
Adcc Activity ◽  
Hematopoietic Progenitor ◽  
Ifn Gamma ◽  
Activating Receptors

Abstract Acute myeloid leukemia is a heterogeneous clonal disorder characterized by abnormal differentiation and unregulated proliferation of hematopoietic progenitor cells (blast), leading to defects in hematopoiesis. Accumulating evidence has implicated leukemic stem cells (LSCs) as the main cause for perpetuation of the disease based on their profile to rapidly give rise to proliferating blasts, resistance to chemotherapeutics, and ability to self-renew. CD123 (IL-3α) has been demonstrated to be expressed at high levels on LSCs and AML blasts compared to normal hematopoietic progenitor cells (Jordon et al Leukemia 2000), providing a potential strategy to target and eliminate AML cells. CSL362, a humanized monoclonal antibody that specifically targets CD123, was engineered to have increased affinity for CD16 on natural killer (NK) cells, leading to an enhanced ability to activate NK cell antibody-dependent cellular cytotoxicity (ADCC) against CD123-positive cells (Busfield et al Leukemia 2014). JNJ-56022473 is a CSL362 variant generated from a new line and has highly similar biochemical, biophysical, and biological properties to CSL362. NK cells are an integral part of the innate immune response to AML cells, and their activity is determined by the balance of activating and inhibitory receptor stimulation (Lanier Ann Rev Immunol 2005). Decitabine and azacitidine, DNA-hypomethylating agents, have been shown to differentially modulate the expression of activating and inhibitory receptors on NK cells at physiologically used doses (Kopp et al Mol Immunol 2013, Schonefeldt et al Blood Cancer J 2013). Decitabine and azacitidine can also activate key epigenetically silenced pathways in AML cells, leading to an arrest of AML cell proliferation. We evaluated the effects of decitabine and azacitidine on CD123 expression and CSL362-mediated cytotoxicity in the TF-1 AML cell line and on NK cell activation in human peripheral blood mononuclear cells (PBMCs). To assess the effect of decitabine and azacitidine on CD123 expression and NK cell activation, 200nM of each compound was incubated with cells under various conditions. CD123 expression in treated TF-1 cells was quantitated by fluorescence-activated cell sorting, where a significant increase in CD123 expression was detected with the 3 and 5 day treatments for decitabine (p<0.001) and with the 5 day treatment for azacitidine (p<0.05). Decitabine and azacitidine treatment increased expression of the NK cell activating receptors NKG2D+ (p<0.001[decitabine] and p<0.005 [azacitidine]) and NKp46+ (p<0.001 [decitabine] and p=0.6 [azacitidine]) on PBMCs with the 5 day treatment schedule but did not affect the expression of the NK cell inhibitory receptor, KIR. Treatment with azacitidine (200nM and 1µM) reduced NK cell viability (p<0.001) compared to decitabine and also significantly downregulated IFN-gamma production (p<0.001). The ability of decitabine and azacitidine to increase expression of CD123 and NK cell activating receptors suggests that the compounds may potentiate the ADCC profile of CSL362/JNJ-56022473. TF-1 cells and PBMCs were pretreated with 200nM of each compound under different dosing regimens followed by ADCC assay with CSL362/JNJ-56022473. The percentage of specific lysis was significantly enhanced in the 5 day treatment with decitabine (66.45% lysis compared to control [32.80%]; p<0.005). In contrast, azacitidine reduced JNJ-56022473-mediated cell lysis (22.71% compared to control [30.96%]; p<0.005) with the 5 day treatment. In summary, decitabine and azacitidine pretreatment stimulated the expression of CD123 in TF-1 cells and NK cell activating receptors, NKG2D+ and NKp46+. The combination of decitabine and CSL362 increased CSL362-induced ADCC activity in TF-1 cells. In contrast, azacitidine inhibited CSL362-mediated ADCC activity, downregulated IFN-gamma production, and reduced NK cell viability. Disclosures Syed: Janssen Research & Development at Johnson & Johnson: Employment. Pietsch:Janssen Research & Development at Johnson & Johnson: Employment. Axel:Janssen: Employment. Forslund:Janssen Research & Development at Johnson & Johnson: Employment. Sasser:Janssen Pharmaceuticals: Employment. Salvati:Janssen Research & Development at Johnson & Johnson: Employment.

scholarly journals Development and Preclinical Characterization of an Fc-Optimized CD133 Antibody for Induction of NK Cell Reactivity Against Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2309-2309 ◽  
Author(s):  
Samuel Koerner ◽  
Julia Leibold ◽  
Ludger Grosse-Hovest ◽  
Hans-Joerg Buehring ◽  
Gundram Jung ◽  
...  
Keyword(s):  
Nk Cells ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Fc Receptor ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Allogenic Stem Cell Transplantation

Abstract NK cells are cytotoxic lymphocytes that play a major role in anti-tumor immunity and largely contribute to the efficacy of allogenic stem cell transplantation (SCT) in leukemia. Another clinically important feature of NK cells is their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of monoclonal antibodies (mAb) like Rituximab, a feature considered critical for the therapeutic success of antibody treament. Modifications of the human IgG1 Fc-part in anti-tumor antibodies lead to markedly improved capability to recruit Fc-receptor bearing effector cells as highlighted by the improved clinical efficacy of the Fc-engineered CD20 antibody Ofatumumab as compared to its unmodified counterpart Rituximab in CLL. So far, no immunotherapeutic antibodies are available for the treatment of myeloid leukemias. Here we report on the development and preclinical characterization of an Fc-optimized mAb directed towards CD133, which is expressed on a wide variety of malignant cell types. As a first step we evaluated binding of three different mouse anti-human CD133 mAbs (clones AC133, W6B3 and 293C3) to 20 primary AML and 6 primary CML samples in order to identify a clone with optimal binding characteristics. AC133 and W6B3 comparably bound to the leukemic cells in 11/20 AML and 5/6 CML samples. In contrast, binding of 293C3 was observed in 18/20 AML cases and 5/6 CML cases. Thus, 293C3 recognizes a different epitope than the other two antibody clones, which is expressed in a high proportion of myeloid leukemia cases. Accordingly, 293C3 was selected for generating chimeric mAbs with either a wildtype Fc part (293C3-WT) or a variant containing distinct modifications (S239D/I332E) to enhance its affinity to the activating Fc receptor CD16 (293C3-SDIE). The binding specificity of 293C3-WT and 293C3-SDIE was validated by FACS in analyses with CD133 transfectants and mock controls. When comparing 293C3-WT and 293C3-SDIE with regard to their immunostimulatory properties, we found that already 293C3-WT induced NK cell ADCC against primary leukemia cells as revealed by analyses of degranulation and target cell lysis. These effects were by far exceeded by treatment with 293C3-SDIE, confirming the functional relevance of the SDIE modification in its Fc part. Notably, treatment with 293C3-SDIE also enhanced the reactivity of NK cells against CD133-positive AML cells in an autologous setting. Considering the expression of CD133, among others, on healthy hematopoietic progenitor cells, we further performed colony forming unit assays with healthy bone marrow cells, which did not reveal any toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells. Thus, 293C3-SDIE constitutes an attractive immunotherapeutic compound which we envisage in particular for the elimination of minimal residual disease in CD133 bearing leukemia, especially in the context of allogenic SCT. Disclosures No relevant conflicts of interest to declare.

The receptor tyrosine kinase c-kit provides a critical signal for survival, expansion, and maturation of mouse natural killer cells

Blood ◽  
2000 ◽  
Vol 95 (3) ◽  
pp. 984-991 ◽  
Author(s):  
Francesco Colucci ◽  
James P. Di Santo
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Fetal Liver ◽  
Lower Percentage ◽  
Nk Cell Differentiation

Fetal liver kinase ligands (flk2L/flt3L) and stem cell factor (SCF) have been shown to promote natural killer (NK) cell differentiation from hematopoietic stem cell (HSC) precursors in vitro. However, the contribution of signaling through the receptors for these growth factors for in vivo NK cell development remains ill-defined. We have analyzed the role of the SCF receptor c-kit in NK cell differentiation by reconstituting NK-deficient mice with fetal liver (FL) HSCs of c-kit−/− (W/W) mice. Although c-kit−/−NK cells were generated inW/W chimeras, they were reduced in number, contained a lower percentage of CD45R (B220)+ cells, and were poorly cytolytic. In vitro experiments showed that generation of NK cells from FL precursors was reduced in the absence of c-kit signaling and that SCF promoted the survival of peripheral c-kit+ NK cells. We conclude that c-kit/SCF interactions in vivo are dispensable for the commitment of HSC to the NK lineage, but they provide essential signals for generating normal numbers of fully mature NK cells.

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