Refine
Language
- English (7)
Has Fulltext
- yes (7)
Is part of the Bibliography
- no (7)
Keywords
- CXCL12 (2)
- CXCR4 (2)
- G-CSF (2)
- Apheresis (1)
- CD34 (1)
- CliniMACS (1)
- Clinical trial (1)
- Mobilization (1)
- PEM-technology (1)
- Plasmacytoid dendritic cell (1)
Institute
- Medizin (7)
Background aims: Immunomagnetic enrichment of CD34+ hematopoietic “stem” cells (HSCs) using paramagnetic nanobead coupled CD34 antibody and immunomagnetic extraction with the CliniMACS plus system is the standard approach to generating T-cell-depleted stem cell grafts. Their clinical beneficence in selected indications is established. Even though CD34+ selected grafts are typically given in the context of a severely immunosuppressive conditioning with anti-thymocyte globulin or similar, the degree of T-cell depletion appears to affect clinical outcomes and thus in addition to CD34 cell recovery, the degree of T-cell depletion critically describes process quality. An automatic immunomagnetic cell processing system, CliniMACS Prodigy, including a protocol for fully automatic CD34+ cell selection from apheresis products, was recently developed. We performed a formal process validation to support submission of the protocol for CE release, a prerequisite for clinical use of Prodigy CD34+ products.
Methods: Granulocyte-colony stimulating factor–mobilized healthy-donor apheresis products were subjected to CD34+ cell selection using Prodigy with clinical reagents and consumables and advanced beta versions of the CD34 selection software. Target and non-target cells were enumerated using sensitive flow cytometry platforms.
Results: Nine successful clinical-scale CD34+ cell selections were performed. Beyond setup, no operator intervention was required. Prodigy recovered 74 ± 13% of target cells with a viability of 99.9 ± 0.05%. Per 5 × 10E6 CD34+ cells, which we consider a per-kilogram dose of HSCs, products contained 17 ± 3 × 10E3 T cells and 78 ± 22 × 10E3 B cells.
Conclusions: The process for CD34 selection with Prodigy is robust and labor-saving but not time-saving. Compared with clinical CD34+ selected products concurrently generated with the predecessor technology, product properties, importantly including CD34+ cell recovery and T-cell contents, were not significantly different. The automatic system is suitable for routine clinical application.
The genetics responsible for the inter-individually variable G-CSF responsiveness remain elusive. A single nucleotide polymorphism (SNP) in the 3’UTR of CXCL12, rs1801157, was implicated in X4-tropic HiV susceptibility and later, in two small studies, in G-CSR responsiveness in patients and donors. The position of the SNP in the 3’UTR together with in-silico predictions suggested differential binding of micro-RNA941 as an underlying mechanism. In a cohort of 515 healthy stem cell donors we attempted to reproduce the correlation of the CXCL12 3’UTR SNP and mobilization responses and tested the role of miR941 in this context. The SNP was distributed with the expected frequency. Mobilization efficiency for CD34+ cells in WT, heterozygous and homozygous SNP individuals was indistinguishable, even after controlling for gender. miR941 expression in non-hematopoietic bone marrow cells was undetectable and miR941 did not interact with the 3’ UTR of CXCL12. Proposed effects of the SNP rs1801157 on G-CSF responsiveness cannot be confirmed in a larger cohort.
Mobilized blood has supplanted bone marrow (BM) as the primary source of hematopoietic stem cells for autologous and allogeneic stem cell transplantation. Pharmacologically enforced egress of hematopoietic stem cells from BM, or mobilization, has been achieved by directly or indirectly targeting the CXCL12/CXCR4 axis. Shortcomings of the standard mobilizing agent, granulocyte colony-stimulating factor (G-CSF), administered alone or in combination with the only approved CXCR4 antagonist, Plerixafor, continue to fuel the quest for new mobilizing agents. Using Protein Epitope Mimetics technology, a novel peptidic CXCR4 antagonist, POL5551, was developed. In vitro data presented herein indicate high affinity to and specificity for CXCR4. POL5551 exhibited rapid mobilization kinetics and unprecedented efficiency in C57BL/6 mice, exceeding that of Plerixafor and at higher doses also of G-CSF. POL5551-mobilized stem cells demonstrated adequate transplantation properties. In contrast to G-CSF, POL5551 did not induce major morphological changes in the BM of mice. Moreover, we provide evidence of direct POL5551 binding to hematopoietic stem and progenitor cells (HSPCs) in vivo, strengthening the hypothesis that CXCR4 antagonists mediate mobilization by direct targeting of HSPCs. In summary, POL5551 is a potent mobilizing agent for HSPCs in mice with promising therapeutic potential if these data can be orroborated in humans.
Circadian oscillations in circulating leukocyte subsets including immature hematopoietic cells have been appreciated; the origin and nature of these alterations remain elusive. Our analysis of wild-type C57BL/6 mice under constant darkness confirmed circadian fluctuations of circulating leukocytes and clonogenic cells in blood and spleen but not bone marrow. Clock gene deficient Bmal1-/- mice lacked this regulation. Cell cycle analyses in the different hematopoietic compartments excluded circadian changes in total cell numbers, rather favoring shifting hematopoietic cell redistribution as the underlying mechanism. Transplant chimeras demonstrate that circadian rhythms within the stroma mediate the oscillations independently of hematopoietic-intrinsic cues. We provide evidence of circadian CXCL12 regulation via clock genes in vitro and were able to confirm CXCL12 oscillation in bone marrow and blood in vivo. Our studies further implicate cortisol as the conveyor of circadian input to bone marrow stroma and mediator of the circadian leukocyte oscillation. In summary, we establish hematopoietic-extrinsic cues as causal for circadian redistribution of circulating mature/immature blood cells.
Background: Certain disadvantages of the standard hematopoietic stem and progenitor cell (HSPC) mobilizing agent G-CSF fuel the quest for alternatives. We herein report results of a Phase I dose escalation trial comparing mobilization with a peptidic CXCR4 antagonist POL6326 (balixafortide) vs. G-CSF.
Methods: Healthy male volunteer donors with a documented average mobilization response to G-CSF received, following ≥6 weeks wash-out, a 1–2 h infusion of 500–2500 µg/kg of balixafortide. Safety, tolerability, pharmacokinetics and pharmacodynamics were assessed.
Results: Balixafortide was well tolerated and rated favorably over G-CSF by subjects. At all doses tested balixafortide mobilized HSPC. In the dose range between 1500 and 2500 µg/kg mobilization was similar, reaching 38.2 ± 2.8 CD34 + cells/µL (mean ± SEM). Balixafortide caused mixed leukocytosis in the mid-20 K/µL range. B-lymphocytosis was more pronounced, whereas neutrophilia and monocytosis were markedly less accentuated with balixafortide compared to G-CSF. At the 24 h time point, leukocytes had largely normalized.
Conclusions: Balixafortide is safe, well tolerated, and induces efficient mobilization of HSPCs in healthy male volunteers. Based on experience with current apheresis technology, the observed mobilization at doses ≥1500 µg/kg of balixafortide is predicted to yield in a single apheresis a standard dose of 4× 10E6 CD34+ cells/kg from most individuals donating for an approximately weight-matched recipient. Exploration of alternative dosing regimens may provide even higher mobilization responses.
Trial Registration European Medicines Agency (EudraCT-Nr. 2011-003316-23) and clinicaltrials.gov (NCT01841476)
Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.
Background: Blood donation saves lives. Provided they are in good health, male volunteers can donate as often as six times per year from the age of 18 into their late sixties. The burden of blood donation is very unevenly distributed, with a small minority of altruistic individuals providing this critical resource. While the consequences of persistent iron depletion in blood donors have been studied in the context of cancer and coronary heart disease, potential effects of the erythropoietic stress from repetitive large-volume phlebotomy remain unexplored. We sought to investigate if and how repeated blood donations affect the clonal composition of the hematopoietic stem and progenitor cell (HSPC) compartment.
Methods: 105 healthy, male individuals with an extensive blood donation history (median of 120 donations per donor; median age of 66 yrs.) were screened for the presence of clonal hematopoiesis (CH) using a sequencing panel covering 141 genes commonly mutated in human myeloid neoplasms. The control cohort consisted of 103 healthy, male donors with a median of 5 donations per donor and a median age of 63. Donors positive for CH were subsequently studied longitudinally. The pathogenicity of detected variants was compared using established scoring systems. Finally, to assess the functional consequences of blood-donation induced CH, selected CH mutations were introduced by CRISPR-mediated editing into HSPCs from human cord blood (CB) or bone marrow (BM). The effect of these mutations was tested under different stress stimuli using functional ex vivo long-term culture initiating cells (LTC-IC) assays.
Results: Compared to the control cohort, frequent donors were significantly more likely to have mutations in genes encoding for epigenetic modifiers (44.7 vs. 22.3 %), most specifically in the two genes most commonly mutated in CH, DNMT3A and TET2 (35.2 vs. 20.3 %). However, no difference in the variant allele frequency (VAF) of detected mutations was found between the groups. Longitudinal analysis revealed that the majority of the mutations remained at a stable VAF over an observation period of approximately one year. Three DNMT3A variants from the frequent donor cohort were introduced into healthy HSPCs and functionally analyzed: All expanded in response to EPO, but none responded to LPS or IFNγ stimulation. This contrasted with the leukemogenic DNMT3A R882H mutation, which did not expand in the presence of EPO but instead responded strongly to inflammatory stimuli.
Conclusions: Frequent whole blood donation is associated with a higher prevalence of CH driven by mutations in genes encoding for epigenetic modifiers, with DNMT3A and TET2 being the most common. This increased CH prevalence is not associated with a higher pathogenicity of the associated variants and is likely a result of the selection of clones with improved responsiveness to EPO under the condition of bleeding stress. Our data show that even highly frequent blood donations over many years is not increasing the risk for malignant clones further underscoring the safety of repetitive blood donations. To our knowledge, this is the first CH study analyzing a cohort of individuals known for their superior health and survival, able to donate blood until advanced age. Thus, our analysis possibly identified mutations associated with beneficial outcomes, rather than a disease condition, such as mutations in DNMT3A that mediated the improved expansion of HSPCs in EPO enriched environments. Our data support the notion of ongoing Darwinian evolution in humans at the somatic stem cell level and present EPO as one of the environmental factors to which HSPCs with specific mutations may respond with superior fitness.