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 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.

Murine Embryonic Liver Differentiates Human Stem Cells into a Spectrum of NK Precursors and Polyclonal KIR Expressing NK Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3317-3317
Author(s):  
Jeffrey S. Miller ◽  
Karen Brungaard ◽  
Robert A.J. Oostendorp ◽  
Valarie McCullar
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Cord Blood ◽  
Nk Cells ◽  
Direct Contact ◽  
Nk Cell ◽  
Cell Development ◽  
Nk Cell Differentiation ◽  
Blood Stem Cells ◽  
Cord Blood Stem Cells

Abstract We have shown that a murine fetal liver cell line (AFT024) and human cytokines (IL-15, IL-7, IL-3, Flt3-ligand and c-kit ligand) are needed to induce NK cell differentiation and KIR acquisition. To understand the level of maturation where these factors orchestrate NK cell development, a switch culture was designed to separate early and late events. Cord blood CD34+/Lin−/CD38− stem cells were cultured on AFT024 for 28 days. Use of IL-3 or Flt3-L alone resulted in minimal growth. In contrast, we show that NK cell differentiation can occur, albeit at low frequency, with a combination of IL-3 and Flt3-L, in the absence of IL-15. These early NK cells were negative for both CD94 and KIR. These conditions also allowed accumulation of CD56− NK cell precursors. CD34+CD7−, CD34+CD7+ and CD34−CD7+ cells were detected in cultures lacking IL-15. Each precursor was tested in secondary cultures containing AFT024 with IL-15 alone, IL-15+IL-3, or IL15+IL-3+Flt3-L. After an additional 2–4 weeks, NK cells differentiated from each distinct cell population. A few predominantly KIR negative NK cells resulted from IL-15 alone. Addition of IL-3 or IL-3+Flt3-L significantly increased the absolute number of NK cells as well as the acquisition of CD94 heterodimers and KIR. We next explored other stromal cell lines in attempt to identify novel factors important in early NK cell maturation. A novel cell line derived from murine embryonic liver (EL08-1D2), identified for its ability to support expansion of mouse stem cells, was compared to AFT024. To test the differential capacity of these microenvironments, single cord blood stem cells were plated on the two feeders supplemented with all cytokines. After 4 weeks, EL08-1D2 induced 125,852±1400 NK cells from a single stem cell, significantly more than with AFT024 (23,143±8117). KIR+ NK cells were also significantly more frequent with EL08-1D2 (3689±801 vs. 799±491), always in a polyclonal pattern. NK cell development and KIR acquisition were dependent on direct contact with EL08-1D2. Increased development could be from greater differentiation, proliferation or both. Cord blood stem cells were cultured in direct contact with EL08-1D2 under primary culture conditions with IL-3 and Flt3-L but in the absence of IL-15. All CD56− NK cell precursors developed with greater frequency on EL08-1D2 than AFT024. In conclusion, EL08-1D2, derived from a primitive microenvironment during mouse ontogeny, efficiently recapitulates NK cell development by inducing NK cell differentiation and proliferation. IL-3 and Flt3-L, but not IL-15, facilitate the isolation and study of distinct NK cell precursors. Direct contact with EL08-1D2 induces KIR acquisition, suggesting that unique environmental factors conserved between mouse and man contribute to the extrinsic signals which lead to KIR acquisition.

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.

γc Gene Transfer in the Presence of Stem Cell Factor, FLT-3L, Interleukin-7 (IL-7), IL-1, and IL-15 Cytokines Restores T-Cell Differentiation From γc(−) X-Linked Severe Combined Immunodeficiency Hematopoietic Progenitor Cells in Murine Fetal Thymic Organ Cultures

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4090-4097 ◽  
Author(s):  
S. Hacein-Bey ◽  
G. De Saint Basile ◽  
J. Lemerle ◽  
A. Fischer ◽  
M. Cavazzana-Calvo
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Stem Cell Factor ◽  
Severe Combined Immunodeficiency ◽  
T Cell Differentiation ◽  
Hematopoietic Progenitor Cells ◽  
Combined Immunodeficiency ◽  
Hematopoietic Progenitor

Abstract X-linked severe combined immunodeficiency (SCID-Xl) is a rare human inherited disorder in which early T and natural killer (NK) lymphocyte development is blocked. The genetic disorder results from mutations in the common γc chain that participates in several cytokine receptors including the interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15 receptors. We have shown in a previous report that γc gene transfer into SCID-Xl bone marrow (BM) cells restores efficient NK cell differentiation. In this study, we have focused on the introduction of the γc gene into SCID-Xl hematopoietic stem cells with the goal of obtaining differentiation into mature T cells. For this purpose, we used the in vitro hybrid fetal thymic organ culture (FTOC) system in which a combination of cytokines consisting of stem cell factor (SCF), Flt-3L, IL-7, IL-1, and IL-15 is added concomitantly. In this culture system, CD34+ marrow cells from two SCID-Xl patients were able to mature into double positive CD4+ CD8+ cells and to a lesser degree into CD4+ TCRβ+ single positive cells after retroviral-mediated γc gene transfer. In addition, examination of the output cell population at the TCR DJβ1 locus exhibited multiple rearrangements. These results indicate that restoration of the γc/JAK/STAT signaling pathway during the early developmental stages of thymocytes can correct the T-cell differentiation block in SCID-Xl hematopoietic progenitor cells and therefore establishes a basis for further clinical γc gene transfer studies.

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 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.

scholarly journals Clinical-Grade Generation of Active NK Cells from Cord Blood Hematopoietic Progenitor Cells for Immunotherapy Using a Closed-System Culture Process

PLoS ONE ◽  
2011 ◽  
Vol 6 (6) ◽  
pp. e20740 ◽  
Author(s):  
Jan Spanholtz ◽  
Frank Preijers ◽  
Marleen Tordoir ◽  
Carel Trilsbeek ◽  
Jos Paardekooper ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Progenitor Cells ◽  
Closed System ◽  
Hematopoietic Progenitor Cells ◽  
Clinical Grade ◽  
Hematopoietic Progenitor ◽  
Culture Process

A Novel Human CD34(+) Subset That Constitutively Expresses the High Affinity Interleukin-2 Receptor Traffics to Lymph Nodes and Differentiates into CD56Bright Natural Killer Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 314-314
Author(s):  
Aharon G. Freud ◽  
Brian Becknell ◽  
Sameek Roychowdhury ◽  
Hsiaoyin C. Mao ◽  
Amy K. Ferketich ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
Lymph Nodes ◽  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Constitutive Expression ◽  
High Affinity ◽  
Nk Cell Differentiation

Abstract In adult humans, T cells differentiate in the thymus and B cells develop in the bone marrow, but the site(s) of natural killer (NK) cell differentiation are unclear. Here we describe, for the first time, a unique CD34(+) population found in human lymph nodes (LN) that differentiates into NK cells. CD56bright NK cells represent <10% of NK cells in peripheral blood (PB) yet predominate in LN where they can compete for endogenous T cell-derived IL-2 during immune activation due to their unique expression of functional high affinity (HA) interleukin (IL)-2 receptors (IL-2R). We hypothesized that a subset of CD34(+) hematopoietic precursor cells (HPC) might also express functional HA IL-2R and potentially differentiate into CD56bright NK cells via activation with low dose IL-2. We first identified a novel human CD34dimCD45RA(+) HPC in PB with constitutive expression of the HA IL-2R. When cultured in picomolar concentrations of IL-2 that selectively saturate the HA IL-2R, these cells give rise to CD56bright NK cells, and this effect is blocked when IL-2 cannot bind to its HA receptor. This unique CD34(+) population expresses IL-2Rα, CD2, CD7, c-kit, L-selectin, and NKR-P1A, all of which are also expressed by CD56bright NK cells. Unique among total PB CD34(+) cells, this novel population displays high integrin α4β7 expression. This attribute, in addition to its high L-selectin expression, suggested that these cells may traffic to LN where their progeny, CD56bright NK cells, represent the major NK subset. Indeed we found a distinct CD34dimCD45RA(+)α4β7bright population that resides in the T cell rich regions of human LN, and when stimulated in vitro with 10 pM IL-2, this cell gives rise to CD56bright NK cells. This novel population represents only ~6% of all PB CD34(+) HPC yet is the major if not exclusive CD34(+) subset in LN. While murine studies strongly support the notion that most if not all NK cells require IL-15 for their development, these new human data suggest a model for development of a minor human NK subset, the CD56bright NK cells, whereby CD34dimCD45RA(+)α4β7bright HPC constitutively expressing the HA IL-2R traffic to peripheral LN where endogenous T cell-derived IL-2 can drive CD56bright NK cell differentiation in vivo.

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.

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