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Background: Defects in phagocytic nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) function cause chronic granulomatous disease (CGD), a primary immunodeficiency characterized by dysfunctional microbicidal activity and chronic inflammation., Objective: We sought to study the effect of chronic inflammation on the hematopoietic compartment in patients and mice with X-linked chronic granulomatous disease (X-CGD)., Methods: We used immunostaining and functional analyses to study the hematopoietic compartment in patients with CGD., Results: An analysis of bone marrow cells from patients and mice with X-CGD revealed a dysregulated hematopoiesis characterized by increased numbers of hematopoietic progenitor cells (HPCs) at the expense of repopulating hematopoietic stem cells (HSCs). In patients with X-CGD, there was a clear reduction in the proportion of HSCs in bone marrow and peripheral blood, and they were also more rapidly exhausted after in vitro culture. In mice with X-CGD, increased cycling of HSCs, expansion of HPCs, and impaired long-term engraftment capacity were found to be associated with high concentrations of proinflammatory cytokines, including IL-1β. Treatment of wild-type mice with IL-1β induced enhanced cell-cycle entry of HSCs, expansion of HPCs, and defects in long-term engraftment, mimicking the effects observed in mice with X-CGD. Inhibition of cytokine signaling in mice with X-CGD reduced HPC numbers but had only minor effects on the repopulating ability of HSCs., Conclusions: Persistent chronic inflammation in patients with CGD is associated with hematopoietic proliferative stress, leading to a decrease in the functional activity of HSCs. Our observations have clinical implications for the development of successful autologous cell therapy approaches., (Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The RUNX1/ETO (RE) fusion protein, which originates from the t(8;21) chromosomal rearrangement, is one of the most frequent translocation products found in de novo acute myeloid leukemia (AML). In RE leukemias, activated forms of the c-KIT tyrosine kinase receptor are frequently found, thereby suggesting oncogenic cooperativity between these oncoproteins in the development and maintenance of t(8;21) malignancies. In this report, we show that activated c-KIT cooperates with a C-terminal truncated variant of RE, REtr, to expand human CD34+ hematopoietic progenitors ex vivo. CD34+ cells expressing both oncogenes resemble the AML-M2 myeloblastic cell phenotype, in contrast to REtr-expressing cells which largely undergo granulocytic differentiation. Oncogenic c-KIT amplifies REtr-depended clonogenic growth and protects cells from exhaustion. Activated c-KIT reverts REtr-induced DNA damage and apoptosis. In the presence of activated c-KIT, REtr-downregulated DNA-repair genes are re-expressed leading to an enhancement of DNA-repair efficiency via homologous recombination. Together, our results provide new mechanistic insight into REtr and c-KIT oncogenic cooperativity and suggest that augmented DNA repair accounts for the increased chemoresistance observed in t(8;21)-positive AML patients with activated c-KIT mutations. This cell-protective mechanism might represent a new therapeutic target, as REtr cells with activated c-KIT are highly sensitive to pharmacological inhibitors of DNA repair.

Gene therapy for hematological disorders relies on the genetic modification of CD34(+) cells, a heterogeneous cell population containing about 0.01% long-term repopulating cells. Here, we show that the lentiviral vector CD133-LV, which uses a surface marker on human primitive hematopoietic stem cells (HSCs) as entry receptor, transfers genes preferentially into cells with high engraftment capability. Transduction of unstimulated CD34(+) cells with CD133-LV resulted in gene marking of cells with competitive proliferative advantage in vitro and in immunodeficient mice. The CD133-LV-transduced population contained significantly more cells with repopulating capacity than cells transduced with vesicular stomatitis virus (VSV)-LV, a lentiviral vector pseudotyped with the vesicular stomatitis virus G protein. Upon transfer of a barcode library, CD133-LV-transduced cells sustained gene marking in vivo for a prolonged period of time with a 6.7-fold higher recovery of barcodes compared to transduced control cells. Moreover, CD133-LV-transduced cells were capable of repopulating secondary recipients. Lastly, we show that this targeting strategy can be used for transfer of a therapeutic gene into CD34(+) cells obtained from patients suffering of X-linked chronic granulomatous disease. In conclusion, direct gene transfer into CD133(+) cells allows for sustained long-term engraftment of gene corrected cells.

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells (HSPCs) remains challenging due to induction of rapid cellular differentiation after detachment from the supporting bone marrow stem cell niche. However, a few leukemia-related chimeric transcription factors, including MLL fusion proteins, confer remarkable CD34+ ex vivo expansion capacity. In this study, we fused the coding sequence of a FKBP12-derived destabilization domain (DD) to an MLL-fusion gene (MLL-X) and then retrovirally expressed the protein switch in human CD34+ progenitors derived from healthy donors. In the presence of Shield1, a chemical inhibitor of DD fusion protein turnover, the cytokines FLT3-L and IL-3 were necessary and sufficient to yield massive and long-term expansion of DD-MLL-X engineered HSPC-derived late monocytic precursors, with normal karyotype preserved. Upon removal of Shield1, the cells completely lost self-renewal capacity, lost colony-forming properties and spontaneously differentiated, even after 150 days of ex vivo expansion. In the absence of Shield1, a five-day stimulation with IFNγ, LPS, and GM-CSF triggered robust monocytic differentiation. Immunophenotypic characterization revealed upregulation of the monocytic cell surface markers CD14, CD68, CD80, CCR2, and MHC class I and II, in well-accordance with cellular morphology as judged by cytospin preparations. Upregulation of inflammatory markers such as IL-6, IL-10, and CCL2 on mRNA level was detected by qRT-PCR. In functional assays, we finally demonstrated the propensity of the obtained cells to migrate towards the chemokine CCL2, attach to tissue-culture treated plastic surfaces and VCAM-1 under flow and shear stress, to produce reactive oxygen species and engulf both bacterial and cellular particles. Moreover, we demonstrated Fc receptor recognition and phagocytosis of Raji lymphoblastic tumor cells in a daratumumab antibody-dependent manner, proving their identity as monocytes. Taken together, we demonstrate the conversion of a harmful transcription factor to a useful tool for ex vivo blood cell production. Using this engineered protein switch, we were able to produce HSPC-derived late monocytic progenitors in large-scale and differentiate those towards functional monocytes under well-defined ex vivo conditions, both efficiently and quantitatively. This controllable system could set the stage for directed generation of patient-derived monocytes for cell-based immunotherapeutic approaches. [ABSTRACT FROM AUTHOR]