Vitamin A-Retinoic Acid Signaling Regulates Hematopoietic Stem Cell Dormancy.

Dormant hematopoietic stem cells (dHSCs) are atop the hematopoietic hierarchy. The molecular identity of dHSCs and the mechanisms regulating their maintenance or exit from dormancy remain uncertain. Here, we use single-cell RNA sequencing (RNA-seq) analysis to show that the transition from dormancy toward cell-cycle entry is a continuous developmental path associated with upregulation of biosynthetic processes rather than a stepwise progression. In addition, low Myc levels and high expression of a retinoic acid program are characteristic for dHSCs. To follow the behavior of dHSCs in situ, a Gprc5c-controlled reporter mouse was established. Treatment with all-trans retinoic acid antagonizes stress-induced activation of dHSCs by restricting protein translation and levels of reactive oxygen species (ROS) and Myc. Mice maintained on a vitamin A-free diet lose HSCs and show a disrupted re-entry into dormancy after exposure to inflammatory stress stimuli. Our results highlight the impact of dietary vitamin A on the regulation of cell-cycle-mediated stem cell plasticity. VIDEO ABSTRACT.

Enhanced c-Met activity promotes G-CSF-induced mobilization of hematopoietic progenitor cells via ROS signaling.

Mechanisms governing stress-induced hematopoietic progenitor cell mobilization are not fully deciphered. We report that during granulocyte colony-stimulating factor-induced mobilization c-Met expression and signaling are up-regulated on immature bone marrow progenitors. Interestingly, stromal cell-derived factor 1/CXC chemokine receptor-4 signaling induced hepatocyte growth factor production and c-Met activation. We found that c-Met inhibition reduced mobilization of both immature progenitors and the more primitive Sca-1(+)/c-Kit(+)/Lin(-) cells and interfered with their enhanced chemotactic migration to stromal cell-derived factor 1. c-Met activation resulted in cellular accumulation of reactive oxygen species by mammalian target of rapamycin inhibition of Forkhead Box, subclass O3a. Blockage of mammalian target of rapamycin inhibition or reactive oxygen species signaling impaired c-Met-mediated mobilization. Our data show dynamic c-Met expression and function in the bone marrow and show that enhanced c-Met signaling is crucial to facilitate stress-induced mobilization of progenitor cells as part of host defense and repair mechanisms.

Osteoclasts degrade endosteal components and promote mobilization of hematopoietic progenitor cells.

Here we investigated the potential role of bone-resorbing osteoclasts in homeostasis and stress-induced mobilization of hematopoietic progenitors. Different stress situations induced activity of osteoclasts (OCLs) along the stem cell-rich endosteum region of bone, secretion of proteolytic enzymes and mobilization of progenitors. Specific stimulation of OCLs with RANKL recruited mainly immature progenitors to the circulation in a CXCR4- and MMP-9-dependent manner; however, RANKL did not induce mobilization in young female PTPepsilon-knockout mice with defective OCL bone adhesion and resorption. Inhibition of OCLs with calcitonin reduced progenitor egress in homeostasis, G-CSF mobilization and stress situations. RANKL-stimulated bone-resorbing OCLs also reduced the stem cell niche components SDF-1, stem cell factor (SCF) and osteopontin along the endosteum, which was associated with progenitor mobilization. Finally, the major bone-resorbing proteinase, cathepsin K, also cleaved SDF-1 and SCF. Our findings indicate involvement of OCLs in selective progenitor recruitment as part of homeostasis and host defense, linking bone remodeling with regulation of hematopoiesis.

c-Met and its ligand hepatocyte growth factor/scatter factor regulate mature B cell survival in a pathway induced by CD74.

The signals regulating the survival of mature splenic B cells have become a major focus in recent studies of B cell immunology. Durable B cell persistence in the periphery is dependent on survival signals that are transduced by cell surface receptors. In this study, we describe a novel biological mechanism involved in mature B cell homeostasis, the hepatocyte growth factor/scatter factor (HGF)/c-Met pathway. We demonstrate that c-Met activation by HGF leads to a survival cascade, whereas its blockade results in induction of mature B cell death. Our results emphasize a unique and critical function for c-Met signaling in the previously described macrophage migration inhibitory factor/CD74-induced survival pathway. Macrophage migration inhibitory factor recruits c-Met to the CD74/CD44 complex and thereby enables the induction of a signaling cascade within the cell. This signal results in HGF secretion, which stimulates the survival of the mature B cell population in an autocrine manner. Thus, the CD74-HGF/c-Met axis defines a novel physiologic survival pathway in mature B cells, resulting in the control of the humoral immune response.

Novel Targeted Therapies for T-Cell Malignancies.

T-cell malignancies comprise a heterogeneous group of cancers resulting from the clonal expansion of T-cells at different developmental stages [...].

Blueprint of human thymopoiesis reveals molecular mechanisms of stage-specific TCR enhancer activation.

Cell differentiation is accompanied by epigenetic changes leading to precise lineage definition and cell identity. Here we present a comprehensive resource of epigenomic data of human T cell precursors along with an integrative analysis of other hematopoietic populations. Although T cell commitment is accompanied by large scale epigenetic changes, we observed that the majority of distal regulatory elements are constitutively unmethylated throughout T cell differentiation, irrespective of their activation status. Among these, the TCRA gene enhancer (Eα) is in an open and unmethylated chromatin structure well before activation. Integrative analyses revealed that the HOXA5-9 transcription factors repress the Eα enhancer at early stages of T cell differentiation, while their decommission is required for TCRA locus activation and enforced αß T lineage differentiation. Remarkably, the HOXA-mediated repression of Eα is paralleled by the ectopic expression of homeodomain-related oncogenes in T cell acute lymphoblastic leukemia. These results highlight an analogous enhancer repression mechanism at play in normal and cancer conditions, but imposing distinct developmental constraints.

Sustained long-term engraftment and transgene expression of peripheral blood CD34+ cells transduced with third-generation lentiviral vectors.

As mobilized peripheral blood (MPB) represents an attractive cell source for gene therapy, we investigated the ability of third-generation lentiviral vectors (LVs) to transfer the enhanced green fluorescent protein gene into MPB CD34(+) cells in culture conditions allowing expansion of transplantable human hematopoietic stem cells. To date, few studies have reported transduction of MPB cells with vesicular stomatitis virus G pseudotyped LVs. The critical issue remains whether primitive, hematopoietic repopulating cells have, indeed, been transduced. In vitro (5 weeks' culture in FLT3 ligand + thrombopoietin + stem cell factor + interleukin 6) and in vivo (serial transplantation in NOD/SCID mice) experiments show that MPB CD34(+) cells can be effectively long-term transduced by LV and maintain their proliferation, self-renewal, and multilineage differentiation potentials. We show that expansion following transduction improves the engraftment of transduced MPB CD34(+) (4.6-fold expansion of SCID repopulating cells by limiting dilution studies). We propose ex vivo expansion after transduction as an effective tool to improve gene therapy protocols with MPB. Disclosure of potential conflicts of interest is found at the end of this article.

Functional CXCR4-expressing microparticles and SDF-1 correlate with circulating acute myelogenous leukemia cells.

Stromal cell-derived factor-1 (SDF-1/CXCL12) and its receptor CXCR4 are implicated in the pathogenesis and prognosis of acute myelogenous leukemia (AML). Cellular microparticles, submicron vesicles shed from the plasma membrane of various cells, are also associated with human pathology. In the present study, we investigated the putative relationships between the SDF-1/CXCR4 axis and microparticles in AML. We detected CXCR4-expressing microparticles (CXCR4(+) microparticles) in the peripheral blood and bone marrow plasma samples of normal donors and newly diagnosed adult AML patients. In samples from AML patients, levels of CXCR4(+) microparticles and total SDF-1 were elevated compared with normal individuals. The majority of CXCR4(+) microparticles in AML patients were CD45(+), whereas in normal individuals, they were mostly CD41(+). Importantly, we found a strong correlation between the levels of CXCR4(+) microparticle and WBC count in the peripheral blood and bone marrow plasma obtained from the AML patients. Of interest, levels of functional, noncleaved SDF-1 were reduced in these patients compared with normal individuals and also strongly correlated with the WBC count. Furthermore, our data indicate NH(2)-terminal truncation of the CXCR4 molecule in the microparticles of AML patients. However, such microparticles were capable of transferring the CXCR4 molecule to AML-derived HL-60 cells, enhancing their migration to SDF-1 in vitro and increasing their homing to the bone marrow of irradiated NOD/SCID/beta2m(null) mice. The CXCR4 antagonist AMD3100 reduced these effects. Our findings suggest that functional CXCR4(+) microparticles and SDF-1 are involved in the progression of AML. We propose that their levels are potentially valuable as an additional diagnostic AML variable.

CAR T-cells for T-cell malignancies: challenges in distinguishing between therapeutic, normal, and neoplastic T-cells.

Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrated remarkable efficacy for the treatment of B-cell malignancies. The development of CAR T-cells against T-cell malignancies appears more challenging due to the similarities between the therapeutic, normal and malignant T-cells. The obstacles include CAR T-cell fratricide, T-cell aplasia, and contamination of CAR T-cell products with malignant T-cells. Here, we review these challenges and propose solutions to overcome these limitations.