Dramatic Reduction of Chronic Lymphocytic Leukemia (CLL) Cells Following Adoptive Transfer of Cord Blood (CB) Natural Killer (NK) Cells Using CB-Engrafted NOD-SCID IL2Rγnull (NSG) Mice as a Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2370-2370 ◽  
Author(s):  
Dongxia Xing ◽  
Alan G. Ramsay ◽  
William Decker ◽  
Sufang Li ◽  
Simon Robinson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Cord Blood ◽  
Nk Cells ◽  
Adoptive Transfer ◽  
Ex Vivo ◽  
Immune Therapy ◽  
Control Group ◽  
Chimeric Mice ◽  
Post Infusion

Abstract Abstract 2370 Poster Board II-347 Adoptive transfer of NK cells in human B-CLL has been limited by the lack of suitable animal models to test the clinical efficacy of this immune therapy strategy. Primary patient B-CLL cells are difficult to engraft in NOD-SCID mice as these mice lack an immune microenvironment that provides essential accessory cells for tumor development. In the current study, we utilize a novel 2-step engraftment protocol using NOD-SCID γnull mice (NSG). Firstly, human cord blood (CB) derived CD34+ stem cells were engrafted to generate humanized chimeric mice capable of supporting B-CLL cells. By week 12, the human engraftment level reached 30% to 60% as detected in peripheral blood. Secondly, these mice were infused with human primary B-CLL cells labeled with CFSE. Our results show that following tail vein injection of 5E7 primary patient CLL cells, chimeric mice exhibited proliferation of CFSE+CD5+ B cells in the spleen and bone marrow, with disease development resembling human CLL. This allowed us to investigate the efficacy of ex vivo IL-2 expanded CB NK cell therapy using this novel mouse model system. As early as 24 hours post-infusion of IL-2 expanded human CD56+CD3- CB NK cells (10E7 per mouse) there was a detectable reduction of CD5+ leukemia cells in the peripheral circulation of CLL-engrafted mice by flow cytometry analysis. Moreover, by day 7 there was a dramatic 99.5 % reduction of CD5+ B-CLL cells in the blood, bone marrow, and spleen of experimental animals compared to the non-treated control group (P<0.001). Of note, expanded CB-NK cells exhibited high anti-leukemic specificity as healthy B cells from the original CB transplant were spared post-infusion. Overall, our studies suggest that the chimeric NSG mouse has utility as an in vivo model for testing immunotherapeutic strategies in aggressive B-CLL. Here, our results highlight the strong anti-leukemic response of infused ex vivo expanded CB-NK cells. Disclosures: No relevant conflicts of interest to declare.

Expression of CD10 and CD7 Defines Distinct In Vivo Lymphoid Reconstituting Capacity within Human Cord Blood CD34+CD38-Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3184-3184
Author(s):  
Shuro Yoshida ◽  
Fumihiko Ishikawa ◽  
Leonard D. Shultz ◽  
Noriyuki Saito ◽  
Mitsuhiro Fukata ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Cord Blood ◽  
Nk Cells ◽  
Progenitor Cells ◽  
Human Cord Blood ◽  
Cd34 Cells

Abstract Human cord blood (CB) CD34+ cells are known to contain both long-term hematopoietic stem cells (LT-HSCs) and lineage-restricted progenitor cells. In the past, in vitro studies suggested that CD10, CD7 or CD127 (IL7Ra) could be candidate surface markers that could enrich lymphoid-restricted progenitor cells in human CB CD34+ cells (Galy A, 1995, Immunity; Hao QL, 2001, Blood; Haddad R, 2004, Blood). However, in vivo repopulating capacity of these lymphoid progenitors has not been identified due to the lack of optimal xenogeneic transplantation system supporting development of human T cells in mice. We aim to identify progenitor activity of human CB CD34+ cells expressing CD10/CD7 by using newborn NOD-scid/IL2rgKO transplant assay that can fully support the development of human B, T, and NK cells in vivo (Ishikawa F, 2005, Blood). Although LT-HSCs exist exclusively in Lin-CD34+CD38- cells, not in Lin-CD34+CD38+ cells, CD10 and CD7 expressing cells are present in Lin-CD34+CD38- cells as well as in Lin-CD34+CD38+ cells (CD10+CD7+ cells, CD10+CD7- cells, CD10-CD7+ cells, CD10-CD7- cells accounted for 4.7+/−2.7%, 10.5+/−1.9%, 7.6+/−4.4%, and 77.1+/−5.2% in Lin-CD34+CD38- CB cells, respectively). We transplanted 500–6000 purified cells from each fraction into newborn NOD-scid/IL2rgKO mice, and analyzed the differentiative capacity. CD34+CD38-CD10-CD7- cells engrafted long-term (4–6 months) in recipient mice efficiently (%hCD45+ cells in PB: 30–70%, n=5), and gave rise to all types of human lymphoid and myeloid progeny that included granulocytes, platelets, erythroid cells, B cells, T cells, and NK cells. Successful secondary reconstitution by human CD34+ cells recovered from primary recipient bone marrow suggested that self-renewing HSCs are highly enriched in CD34+CD38–CD10–CD7- cells. CD10–CD7+ cells were present more frequently in CD34+CD38+ cells rather than in CD34+CD38- cells. Transplantation of more than 5000 CD34+CD38+CD10–CD7+ cells, however, resulted in less than 0.5% human cell engraftment in the recipients. Within CD34+CD38–CD10+ cells, the expression of CD7 clearly distinguished the distinct progenitor capacity. At 8 weeks post-transplantation, more than 70% of total human CD45+ cells were T cells in the CD10+CD7+ recipients, whereas less than 30% of engrafted human CD45+ cells were T cells in the CD10+CD7– recipients. In the CD10+CD7- recipients, instead, more CD19+ B cells and HLA–DR+CD33+ cells were present in the peripheral blood, the bone marrow and the spleen. Both CD34+CD38–CD10+CD7+ and CD34+CD38–CD10+CD7- cells highly repopulate recipient thymus, suggesting that these progenitors are possible thymic immigrants. Taken together, human stem and progenitor activity can be distinguished by the expressions of CD7 and CD10 within Lin-CD34+CD38- human CB cells. Xenotransplant model using NOD-scid/IL2rgKO newborns enable us to clarify the heterogeneity of Lin-CD34+CD38- cells in CB by analyzing the in vivo lymphoid reconstitution capacity.

Ex Vivo IL-2 Expansion of CB-NK Cells Promotes Synergistic LFA-1 and CD2 Engagement at the NK Cell Lytic Immune Synapse; Implications for Adoptive CB-NK Cell Therapy in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3029-3029
Author(s):  
Dongxia Xing ◽  
Alan G. Ramsay ◽  
William Decker ◽  
Sufang Li ◽  
Simon Robinson ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Synapse Formation ◽  
Immunological Synapse ◽  
Nk Cell ◽  
Ex Vivo ◽  
Immune Therapy ◽  
Cytolytic Activity ◽  
Lymphocyte Function ◽  
Immune Synapse

Abstract Abstract 3029 Poster Board II-1005 Donor peripheral blood (PB) natural killer (NK) cell have shown clinical promise in cancer immunotherapy. Tightly regulated receptor signaling between NK cells and susceptible tumor cells is essential for NK cell-mediated cytotoxicity. Umbilical cord blood (CB) represents an important alternative source of NK cells for adoptive immune therapy. We first demonstrated that cord blood (CB) derived NK cells have poor cytolytic activity and deficiency in the formation of the F-actin immunological synapse with HLA class I deficient target K562 cells and primary AML blasts compared to PB-NK cells. In this study, we explored the cellular mechanism of these dysfunctions. We hypothesized that adhesion and signaling molecules may be defective in unmanipulated CB NK cells. Activating receptor Both CD2 and the integrin lymphocyte function-associated antigen (LFA-1) play important roles in both T lymphocyte and NK cell immune synapse formation and their trafficking to the immune synapse regulates both T and NK cell function. We now show that unmanipulated CB NK cells exhibit reduced LFA-1 mediated adhesion to mobilized ICAM-1 compared to IL-2 expanded CB NK cells (CB NK 29.7+/- 3.2 %, vs expanded CB NK 78.5+/- 6.1%, n=6). Moreover, unmanipulated CB-NK cells demonstrated reduced surface expression of CD2, and high affintyLFA-1 detected by the specific antibody (MHM24). There was decreased recruitment of CD2 and LFA-1 to the NK cell immune synapse site as quantified by confocal microscope analysis (RRI CD2 CB NK 2.02 vs PB NK 4.98, n=3). Furthermore, defective LFA-1 trafficking lead to a decrease in downstream cytotoxic granules that traffic to the immunological synapse as demonstrated by decreased perforin trafficking to the CB-NK synapse site (> 60% reduction).We next wanted to confirm that CD2 or LFA-1 play a role in restoring the immune synapseformation for IL-2 expanded CB NK cells. We incubated expanded CB NK cells with blocking antibodies specific for LFA-1 or CD2 prior to conjugation to the K562 target cells. After CD2 or LFA-1 blocking there was decreased synapse formation, with a resultant decrease in cytotoxic function. When monoclonal antibodies against both CD2 and LFA-1 were used there was significant blockade of the formation of the immune synapse, and a marked reduction of CB NK cell cytolytic activity (Mean specific lysis of K562 targets at E:T ratio 20:1 was 81% IgG control vs 22% with anti-CD2; and 29% with anti-LFA-1, n=6, P<0.001). This data shows that CD2 and LFA-1 are defective in unmanipulated CB NK cells resulting in impaired immune synapse formation. In contrast, ex vivo IL-2 expansion of CB-NK cells enhanced lytic synapse formation with the synergistic repair of CD2 and LFA-1 localization and activity. We believe our results provide important mechanistic insights for the potential use of IL-2 expanded CB-derived NK cells for adoptive immune therapy in leukemia. Disclosures No relevant conflicts of interest to declare.

Improved Homing To Bone Marrow, Spleen and Lung Of Adoptively Infused NK Cells Expanded Ex Vivo With The Small Molecule Nicotinamide Using Feeder-Free Conditions

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 897-897 ◽  
Author(s):  
Gabi M. Frei ◽  
Maria Berg ◽  
Tony Peled ◽  
Robert N. Reger ◽  
Ritesh Kotecha ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Nk Cells ◽  
Adoptive Transfer ◽  
Hematological Malignancies ◽  
Nk Cell ◽  
Ex Vivo ◽  
Feeder Cells ◽  
Nsg Mice ◽  
Cells Cultured

Abstract Clinical results with NK cells in investigational tumor immunotherapy protocols have at best resulted in partial responses only. The inability of ex vivo expanded NK cells to proliferate in vivo, as well as to home to and be retained in the tumor micro-environment, likely plays a role in their limited efficacy to date. Clinical grade NK cells expanded using EBV-LCL feeder cells (FC) have recently been evaluated in the clinic (NHLBI) for hematological malignancies and metastatic cancers with only minor responses being observed to date. We observed that CD62L expression was down-regulated on EBV-LCL NK cells compared to fresh NK cells, potentially limiting their clinical activity. Since NAM up-regulates CD62L on NK cells cultured in feeder-free (FF) conditions, we compared the ability of FF NK cells with EBV-LCL NK cells to home and persist in vivo following adoptive transfer into NSG mice. FF NK cultures were initiated with CD3 depleted PB TNC while EBV-LCL NK cell cultures were initiated by co-culturing 100 cGy-irradiated SMI-EBV-LCL FC with CD3 depleted, CD56 enriched cells. NSG mice received 200 cGy of TBI followed 24 hours later by 10 million IV NK cells. Three cohorts were studied: a) NK cells expanded using EBV-LCL FC; b) FF expanded NK cells without NAM and c) FF expanded NK cells with NAM (5mM). All NK cell cohorts were expanded from the same human donor. Cells were harvested from the blood, lungs, spleen, and BM of mice 4 days after infusion (n=5). NK cells expanded with NAM in FF conditions were detectable in all mouse organs including the PB at significantly higher levels than NK cells expanded in FF conditions without NAM or using EBV-LCL FC (Fig.1a). Subsequent experiments transferring CFSE-labeled NK cells expanded with NAM into irradiated NSG mice showed a marked reduction in CFSE intensity and the presence of multiple peaks after 4 days, indicative of in vivo proliferation. We next evaluated the impact of daily exogenous IL-2 or IL-15 administration on the homing potential of expanded NK cells 4 days post-infusion into irradiated NSG mice (n=5). We observed that both IL-2 and IL-15 enhanced homing of NK cells expanded with NAM in FF conditions, but failed to enhance the homing of NK cells expanded with EBV-LCL FC (Fig. 1b). With the exception of CD62L, no consistent differences in the phenotype of NK cells between the various expansion methods were observed. NK cells expanded in all groups maintained similar levels of cytotoxicity against multiple different targets including K562 cells, myeloma and renal cell carcinoma tumor cell lines. The number of NK cells expanded using FF conditions was lower than NK cells expanded with EBV-LCL. Nevertheless, NK cells cultured in NAM without feeder cells still expanded a median 50 (37-87) fold, yielding a total of 140x108 NK cells (purity >98%) from a single aphaeresis collection. Further, cytokine levels measured from the supernatants of NK cells cultured with tumor targets showed significantly higher levels of IFNγ, TNFα and FAS-L secretion from FF NK cells expanded with NAM in comparison to the other two groups (Fig1c). Conclusion These data show human NK cells expanded ex vivo in NAM utilizing FF conditions substantially up-regulate CD62L, have enhanced inflammatory cytokine secretion against tumors, and have improved in vivo proliferation and homing to multiple organs including the bone marrow compared to EBV-LCL expanded NK cells. These differences suggest NAM expanded NK cells could have superior clinical efficacy compared to EBV-LCL expanded NK cells following adoptive transfer into patients with hematological malignancies and metastatic cancers. Frei: Gamida Cell: Employment. Peled:Gamida Cell: Employment. Persi:Gamida Cell: Employment. Lador:Gamida Cell: Employment. Peled:Gamida Cell: Consultancy. Nicotinamide (NAM) is a small molecule form of Vitamin B3 and a potent inhibitor of enzymes that use NAD for their activity and thus is involved in the control of redox-sensitive enzymes, mitochondrial functions, cell metabolism and production of energy and cell motility. NAM, when used as an epigenetic modulator has been shown to increase the homing and engraftment efficacy to the BM of ex vivo expanded CD34+ cells. Recently, we found (Gamida-Cell) that NAM also enhances the in-vivo homing and retention of peripheral blood (PB) derived NK cells expanded over two weeks in feeder-free culture conditions stimulated with IL-2 or IL-15. Immunophenotype studies demonstrated NAM-treated cultures had a substantial increase in CD62L (L-selectin), pivotal for NK cell trafficking and homeostatic proliferation.

Ex Vivo Induction of Megakaryocytic Differentiation of Umbilical Cord Blood CD34+ Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 318-318
Author(s):  
Xin Guan ◽  
Meng Qin ◽  
Bin Shen ◽  
Yu Zhang ◽  
Wenhong Jiang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cord Blood ◽  
Ex Vivo ◽  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
Mouse Bone Marrow ◽  
Post Transplantation ◽  
Cd34 Cells

Abstract Background and Objectives: Due to platelet shortage, megakaryocytes have been regarded as an effective substitute to the alleviation of frequent thrombocytopenia after stem cell transplantation. However, ex vivo expansion of megakaryoblasts and their subsequent differentiation into mature megakaryocytes for clinical applications remains a challenge. Here, we describe the development of a two-stage culture system for producing megakaryocytes from cord blood CD34+ cells. Design and Methods: Firstly, we expanded CD34+ hematopoietic progenitor/stem cells for 6 days in a serum-free culture system (IMDM basal medium with the addition of biotin, putrescine, insulin, human serum albumin, selenium, and some other nutrients) supplemented with stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), interleukin 3 (IL-3), low density lipoprotein (LDL), StemRegenin 1 (SR1), and DMSO. CD34+ cells expansion was monitored by flow cytometric analysis of cell surface markers coupled with cell counting. Subsequently, these expanded cells were induced toward the megakaryocytic lineage for additional 7 days in the same serum-free medium as above supplemented with SCF, TPO, IL-3, IL-6, IL-11, granulocyte-macrophage colony-stimulating factor (GM-CSF), and LDL. Megakaryocytes were detected by flow cytometry using antibodies against specific cell surface markers including CD41a and CD42b. Differentiated megakaryocytes were also confirmed by morphological criteria such as cell size and DNA polyploidy. To functionally evaluate induced megakaryocytes, these cells were transplanted into sublethally irradiated NOD/SCID mice. Viability and cell being of these mice were monitored after injection. Mice of the negative control group (n=3) were injected with saline. In the experimental group (n=6), each mouse was injected with 1.0×107 cells from the second stage of culture. Results: After the first stage culture, proliferation folds of total cells and CD34+ cells were 85.65±7.03 and 62.91±4.36, respectively. The calculated yield from each CD34+ cell was between 1.0×104 to 1.5×104 CD41+ megakaryocytes with a purity of CD41+ and CD42+ cells reaching 93.7%±2.8% and 80.3%±5.8%, respectively. Differentiated cells were morphologically discernible as they were much larger than starting CD34+ cells with apparent lobular nuclei and numerous α-granules. In addition, about 32.67%±7.43% of induced megakaryocytes exhibited 4N or larger DNA content (4N 17.6%±4.12%; 8N 10.93%±2.48%; >8N 3.23%±1.34%). In mouse studies, samples collected from the negative control group contained no cells positive for human CD41a and CD42b markers. In the experimental group, human platelets were detected in mouse peripheral blood 3 days post-transplantation. At day 14 post-transplantation, the percentage of platelets derived from injected human megakaryocytes reached 13.6%±6.2%. Human megakaryocytes were also detected in mouse bone marrow 7 days post-transplantation, peaking at day 14 (~2.38% of total bone marrow megakaryocytes). Conclusions: We have established a stem cell expansion and differentiation platform that can be adapted to large-scale production of mature megakaryocytes from umbilical cord blood cells. Significantly, induced megakaryocytes are capable of engrafting in mouse bone marrow and producing platelets after transplantation into irradiated NOD/SCID mice. Therefore, our experimental platform is capable of producing a sufficient number of functional megakaryocytes for various clinical applications in the future. Disclosures Qin: Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment. Ren:Biopharmagen corp: Employment. Jiang:Biopharmagen.corp: Employment.

scholarly journals NK Cells Generated Ex Vivo from Cord Blood CD34+ Cells Are Viable and Retain Cytotoxic Potency Following Cryopreservation

2021 ◽  
Vol 27 (3) ◽  
pp. S215
Author(s):  
Joseph Blake ◽  
Erin Massey ◽  
Carrie A Stoltzman ◽  
Jody Cook ◽  
Devikha Chandrasekaran ◽  
...  
Keyword(s):  
Cord Blood ◽  
Nk Cells ◽  
Ex Vivo ◽  
Cd34 Cells ◽  
Cord Blood Cd34

scholarly journals Superior ex vivo cord blood expansion following co-culture with bone marrow-derived mesenchymal stem cells

2006 ◽  
Vol 37 (4) ◽  
pp. 359-366 ◽  
Author(s):  
S N Robinson ◽  
J Ng ◽  
T Niu ◽  
H Yang ◽  
J D McMannis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Ex Vivo

scholarly journals B-cell differentiation following autologous, conventional, or T-cell depleted bone marrow transplantation: a recapitulation of normal B-cell ontogeny

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1647-1656 ◽  
Author(s):  
TN Small ◽  
CA Keever ◽  
S Weiner-Fedus ◽  
G Heller ◽  
RJ O'Reilly ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
Bone Marrow Transplantation ◽  
B Cells ◽  
Cord Blood ◽  
B Cell ◽  
First Year ◽  
B Cell Differentiation ◽  
And Function ◽  
Cell Ontogeny

Abstract The circulating lymphocytes of 88 consecutive patients following autologous, conventional, or T-cell depleted bone marrow transplantation were serially analyzed for B-cell surface antigen expression and function. In the majority of patients, except for those who developed chronic graft-versus-host disease, the number of circulating CD20+ B cell normalized by the fourth posttransplant month. The earliest detectable B cells normally expressed HLA-DR, CD19, surface immunoglobulin (slg), CD21, Leu-8, and lacked expression of CD10 (CALLA). In addition, the circulating B cells expressed CD1c, CD38, CD5, and CD23 for the first year following transplant, antigens that are normally expressed on a small percentage of circulating B cells in normal adults, but highly expressed on cord blood B cells. Similar to cord blood B cells, patient B cells isolated during the first year following transplant, proliferated normally to Staphylococcus aureus Cowan strain I (SAC), and produced IgM, but minimal or no IgG when stimulated with pokeweed mitogen and SAC, unlike normal adult B cells that produce both. The similar phenotype and function of posttransplant and cord blood B cells, and their similar rate of decline in patients and normal children adds further evidence to support the hypothesis that B-cell differentiation posttransplant is recapitulating normal B-cell ontogeny.

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
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