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The possibility to modulate ex vivo human NK cell differentiation towards specific phenotypes will contribute to a better understanding of NK cell differentiation and facilitate tailored production of NK cells for immunotherapy. In this study, we show that addition of a specific low dose of IL-12 to an ex vivo NK cell differentiation system from cord blood CD34+ stem cells will result in significantly increased proportions of cells with expression of CD62L as well as KIRs and CD16 which are preferentially expressed on mature CD56dim peripheral blood NK cells. In addition, the cells displayed decreased expression of receptors such as CCR6 and CXCR3, which are typically expressed to a lower extent by CD56dim than CD56bright peripheral blood NK cells. The increased number of CD62L and KIR positive cells prevailed in a population of CD33+NKG2A+ NK cells, supporting that maturation occurs via this subtype. Among a series of transcription factors tested we found Gata3 and TOX to be significantly downregulated, whereas ID3 was upregulated in the IL-12-modulated ex vivo NK cells, implicating these factors in the observed changes. Importantly, the cells differentiated in the presence of IL-12 showed enhanced cytokine production and cytolytic activity against MHC class I negative and positive targets. Moreover, in line with the enhanced CD16 expression, these cells exhibited improved antibody-dependent cellular cytotoxicity for B-cell leukemia target cells in the presence of the clinically applied antibody rituximab. Altogether, these data provide evidence that IL-12 directs human ex vivo NK cell differentiation towards more mature NK cells with improved properties for potential cancer therapies. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cell-based adoptive immunotherapy is an attractive adjuvant treatment option for patients with acute myeloid leukemia. Recently, we reported a clinical-grade, cytokine-based culture method for the generation of NK cells from umbilical cord blood (UCB) CD34+ hematopoietic progenitor cells with high yield, purity and in vitro functionality. The present study was designed to evaluate the in vivo anti-leukemic potential of UCB-NK cells generated with our GMP-compliant culture system in terms of biodistribution, survival and cytolytic activity following adoptive transfer in immunodeficient NOD/SCID/IL2Rgnull mice. Using single photon emission computed tomography, we first demonstrated active migration of UCB-NK cells to bone marrow, spleen and liver within 24 h after infusion. 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. Thereafter, we showed that low dose IL-15 mediates efficient survival, expansion and maturation of UCB-NK cells in vivo. Most importantly, we demonstrate that a single UCB-NK cell infusion combined with supportive IL-15 administration efficiently inhibited growth of human leukemia cells implanted in the femur of mice, resulting in significant prolongation of mice survival. These preclinical studies strongly support the therapeutic potential of ex vivo-generated UCB-NK cells in the treatment of myeloid leukemia after immunosuppressive chemotherapy. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cells are the most investigated mammalian stem cells. Like other stem cells they are undifferentiated cells that can self-renew over a long period of time and give rise to progenitor cells that will reconstitute the whole immune and blood system. Although the mechanisms regulating the decision process self-renewal versus differentiation remain largely unknown, there is good evidence that a combination of both extrinsic and intrinsic factors as well as the Notch signaling pathway are involved in controlling the cell fates of primitive hematopoietic cells and their arising daughter cells. Similarly, the Notch signaling pathway, its extrinsic ligands and its intrinsic modulators specify the cell fates of the four cells of developing external sensory organs of Drosophila melanogaster. In this system, the so-called sensory organ precursor cells divide asymmetrically to give rise to IIa and IIb daughter cells. Mechanistically, the cell fate determinant Numb, an antagonistic protein of Notch, segregates mainly into IIa daughter cells and inhibits the signal transduction of Notch, being activated by its ligands Delta and Serrate (the homolog of mammalian Jagged). Depending on the transduction of the Notch signal, daughter cells are either specified as IIa (no transduced signal) or as IIb (transduced signal). Since Numb is conserved during evolution and Notch activity seems to be required to maintain primitive hematopoietic cell fates, we wondered whether Numb is involved in cell fate specification during early hematopoiesis as well. GeneChip analyzes performed in collaboration with N Ivanova and I Lemischka (Princeton University) or with L Klein-Hitpass, T Moritz and J Thomale (Universitätklinikum Essen) together with numb specific RT-PCR analyzes revealed that numb is expressed in primitive hematopoietic cells of the lin-CD34+CD38low/- and lin-CD34+CD38+ cell fractions. By immunohistochemical and flow cytometric analyzes we could further show that even on the protein level Numb is expressed in all CD34+ cells. To functionally analyze the impact of Numb on the biology of primitive hematopoietic cells, in other words on CD34+ cells, we performed over-expression experiments and realized that enforced Numb expression inhibits the maintenance of primitive hematopoietic cell fates. In contrast to this and in agreement with published data, CD34+ cells expressing a constitutive active variant of Notch-1 contained a higher rate of more primitive cells than CD34+ cells of corresponding controls. [ABSTRACT FROM AUTHOR]

Purpose: Older acute myeloid leukemia (AML) patients have a poor prognosis; therefore, novel therapies are needed. Allogeneic natural killer (NK) cells have been adoptively transferred with promising clinical results. Here, we report the first-in-human study exploiting a unique scalable NK-cell product generated ex vivo from CD34 + hematopoietic stem and progenitor cells (HSPC) from partially HLA-matched umbilical cord blood units. Experimental Design: Ten older AML patients in morphologic complete remission received an escalating HSPC-NK cell dose (between 3 and 30 × 10 6 /kg body weight) after lymphodepleting chemotherapy without cytokine boosting. Results: HSPC-NK cell products contained a median of 75% highly activated NK cells, with <1 × 10 4 T cells/kg and <3 × 10 5 B cells/kg body weight. HSPC-NK cells were well tolerated, and neither graft-versus-host disease nor toxicity was observed. Despite no cytokine boosting being given, transient HSPC-NK cell persistence was clearly found in peripheral blood up to 21% until day 8, which was accompanied by augmented IL15 plasma levels. Moreover, donor chimerism up to 3.5% was found in bone marrow. Interestingly, in vivo HSPC-NK cell maturation was observed, indicated by the rapid acquisition of CD16 and KIR expression, while expression of most activating receptors was sustained. Notably, 2 of 4 patients with minimal residual disease (MRD) in bone marrow before infusion became MRD negative (<0.1%), which lasted for 6 months. Conclusions: These findings indicate that HSPC-NK cell adoptive transfer is a promising, potential "off-the-shelf" translational immunotherapy approach in AML. Clin Cancer Res; 23(15); 4107-18. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Adoptive transfer of allogeneic natural killer (NK) cells represents a promising treatment approach against cancer, including acute myeloid leukemia (AML). Previously, we reported a cytokine-based culture method for the generation of NK cell products with high cell number and purity. In this system, CD34 + hematopoietic progenitor cells (HPC) were expanded and differentiated into NK cells under stroma-free conditions in the presence of IL-15 and IL-2. We show that combining IL-15 with IL-12 drives the generation of more mature and highly functional NK cells. In particular, replacement of IL-2 by IL-12 enhanced the cytolytic activity and IFNγ production of HPC-NK cells toward cultured and primary AML cells in vitro , and improved antileukemic responses in NOD/SCID-IL2Rγnull (NSG) mice bearing human AML cells. Phenotypically, IL-12 increased the frequency of HPC-NK cells expressing NKG2A and killer immunoglobulin-like receptor (KIR), which were more responsive to target cell stimulation. In addition, NK15/12 cell products demonstrated superior maturation potential, resulting in >70% positivity for CD16 and/or KIR within 2 weeks after infusion into NSG mice. We predict that higher functionality and faster in vivo maturation will favor HPC-NK cell alloreactivity toward malignant cells in patients, making this cytokine combination an attractive strategy to generate clinical HPC-NK cell products for cancer adoptive immunotherapy.

The targeted manipulation of the genetic program of single cells as well as of complete organisms has strongly enhanced our understanding of cellular and developmental processes and should also help to increase our knowledge of primary human stem cells, e.g., hematopoietic stem cells (HSCs), within the next few years. An essential requirement for such genetic approaches is the existence of a reliable and efficient method to introduce genetic elements into living cells. Retro- and lentiviral techniques are efficient in transducing primary human HSCs, but remain labor and time consuming and require special safety conditions, which do not exist in many laboratories. In our study, we have optimized the nucleofection technology, a modified electroporation strategy, to introduce plasmid DNA into freshly isolated human HSC-enriched CD34(+) cells. Using enhanced green fluorescent protein (eGFP)-encoding plasmids, we obtained transfection efficiencies of approximately 80% and a mean survival rate of 50%. Performing functional assays using GFU-GEMM and long-term culture initiating cells (LTC-IC), we demonstrate that apart from a reduction in the survival rate the nucleofection method itself does not recognizably change the short- or long-term cell fate of primitive hematopoietic cells. Therefore, we conclude, the nucleofection method is a reliable and efficient method to manipulate primitive hematopoietic cells genetically.

For the lymphohematopoietic system, maturation-dependent alterations in DNA repair function have been demonstrated. Because little information is available on the regulatory mechanisms underlying these changes, we have correlated the expression of DNA damage response genes and the functional repair capacity of cells at distinct stages of human hematopoietic differentiation. Comparing fractions of mature (CD34-), progenitor (CD34+ 38+), and stem cells (CD34+ 38-) isolated from umbilical cord blood, we observed: 1) stringently regulated differentiation-dependent shifts in both the cellular processing of DNA lesions and the expression profiles of related genes and 2) considerable interindividual variability of DNA repair at transcriptional and functional levels. The respective repair phenotype was found to be constitutively regulated and not dominated by adaptive response to acute DNA damage. During blood cell development, the removal of DNA adducts, the resealing of repair gaps, the resistance to DNA-reactive drugs clearly increased in stem or mature compared with progenitor cells of the same individual. On the other hand, the vast majority of differentially expressed repair genes was consistently upregulated in the progenitor fraction. A positive correlation of repair function and transcript levels was found for a small number of genes such as RAD23 or ATM, which may serve as key regulators for DNA damage processing via specific pathways. These data indicate that the organism might aim to protect the small number of valuable slow dividing stem cells by extensive DNA repair, whereas fast-proliferating progenitor cells, once damaged, are rather eliminated by apoptosis.