Superior physical and mental health of healthy volunteers before and five years after mobilized stem cell donation.

BACKGROUND: Safety, tolerability and efficacy of granulocyte colony-stimulating factor (G-CSF) for mobilization of hematopoietic stem and progenitor cells (HSPCs) from healthy donors have been conclusively demonstrated. This explicitly includes, albeit for smaller cohorts and shorter observation periods, biosimilar G-CSFs. HSPC donation is non-remunerated, its sole reward being "warm glow", hence harm to donors must be avoided with maximal certitude. To ascertain, therefore, long-term physical and mental health effects of HSPC donation, a cohort of G-CSF mobilized donors was followed longitudinally. METHODS: We enrolled 245 healthy volunteers in this bi-centric long-term surveillance study. 244 healthy volunteers began mobilization with twice-daily Sandoz biosimilar filgrastim and 242 underwent apheresis after G-CSF mobilization. Physical and mental health were followed up over a period of 5-years using the validated SF-12 health questionnaire. RESULTS: Baseline physical and mental health of HSPC donors was markedly better than in a healthy reference population matched for ethnicity, sex and age. Physical, but not mental health was sharply diminished at the time of apheresis, likely due to side effects of biosimilar G-CSF, however had returned to pre-apheresis values by the next follow-up appointment after 6 months. Physical and mental health slightly deteriorated over time with kinetics reflecting the known effects of aging. Hence, superior physical and mental health compared to the general healthy non-donor population was maintained over time. CONCLUSIONS: HSPC donors are of better overall physical and mental health than the average healthy non-donor. Superior well-being is maintained over time, supporting the favorable risk-benefit assessment of volunteer HSPC donation. Trial registration National Clinical Trial NCT01766934.

Management of adults and children receiving CAR T-cell therapy: 2021 best practice recommendations of the European Society for Blood and Marrow Transplantation (EBMT) and the Joint Accreditation Committee of ISCT and EBMT (JACIE) and the European Haematology Association (EHA).

BACKGROUND: Several commercial and academic autologous chimeric antigen receptor T-cell (CAR-T) products targeting CD19 have been approved in Europe for relapsed/refractory B-cell acute lymphoblastic leukemia, high-grade B-cell lymphoma and mantle cell lymphoma. Products for other diseases such as multiple myeloma and follicular lymphoma are likely to be approved by the European Medicines Agency in the near future. DESIGN: The European Society for Blood and Marrow Transplantation (EBMT)-Joint Accreditation Committee of ISCT and EBMT (JACIE) and the European Haematology Association collaborated to draft best practice recommendations based on the current literature to support health care professionals in delivering consistent, high-quality care in this rapidly moving field. RESULTS: Thirty-six CAR-T experts (medical, nursing, pharmacy/laboratory) assembled to draft recommendations to cover all aspects of CAR-T patient care and supply chain management, from patient selection to long-term follow-up, post-authorisation safety surveillance and regulatory issues. CONCLUSIONS: We provide practical, clinically relevant recommendations on the use of these high-cost, logistically complex therapies for haematologists/oncologists, nurses and other stakeholders including pharmacists and health sector administrators involved in the delivery of CAR-T in the clinic.

Mesenchymal stromal cells for osteonecrosis.

Osteonecrosis (ON) is an acquired debilitating skeletal disorder, which is caused by a multitude of traumatic and non-traumatic etiological factors. Vascular damage, mechanical stress and increased intraosseous pressure have been discussed as contributors to ON. The optimal treatment of ON remains to be determined, since the current gold standard, core decompression, is insufficiently effective. Specific properties of mesenchymal stromal cells (MSCs) provide the rationale for their assessment in advanced stages of ON: Osteoinductive potential has been demonstrated and MSC preparations of suitable quality for use as medicinal products have been developed. Here we review the scant information on the use of allogeneic or autologous MSCs in advanced ON as well as potentially supportive data from pre-clinical studies with autologous bone marrow mononuclear cells (auto BM-MNCs), which have been studied quite extensively and the presumed therapeutic effect of which was attributed to the rare MSCs contained in these cell products. Outcomes in clinical trials with MSCs and auto-BM-MNCs remain preliminary and non-definitive, at best promising, with respect to their pharmacological effect. Clearly, though, the application of any of these cell therapies was technically feasible and safe in that it was associated with low complication rates. The heterogeneity of cell type and source, study protocols, cell manufacturing, cell properties, cell doses and surgical techniques might contribute to inconsistent results.

Progressive multifocal leukoencephalopathy in a patient post allo-HCT successfully treated with JC virus specific donor lymphocytes.

BACKGROUND: Progressive multifocal leukoencephalopathy is a demyelinating CNS disorder. Reactivation of John Cunningham virus leads to oligodendrocyte infection with lysis and consequent axonal loss due to demyelination. Patients usually present with confusion and seizures. Late diagnosis and lack of adequate therapy options persistently result in permanent impairment of brain functions. Due to profound T cell depletion, impairment of T-cell function and potent immunosuppressive factors, allogeneic hematopoietic cell transplantation recipients are at high risk for JCV reactivation. To date, PML is almost universally fatal when occurring after allo-HCT. METHODS: To optimize therapy specificity, we enriched JCV specific T-cells out of the donor T-cell repertoire from the HLA-identical, anti-JCV-antibody positive family stem cell donor by unstimulated peripheral apheresis [1]. For this, we selected T cells responsive to five JCV peptide libraries via the Cytokine Capture System technology. It enables the enrichment of JCV specific T cells via identification of stimulus-induced interferon gamma secretion. RESULTS: Despite low frequencies of responsive T cells, we succeeded in generating a product containing 20 000 JCV reactive T cells ready for patient infusion. The adoptive cell transfer was performed without complication. Consequently, the clinical course stabilized and the patient slowly went into remission of PML with JCV negative CSF and containment of PML lesion expansion. CONCLUSION: We report for the first time feasibility of generating T cells with possible anti-JCV activity from a seropositive family donor, a variation of virus specific T-cell therapies suitable for the post allo transplant setting. We also present the unusual case for successful treatment of PML after allo-HCT via virus specific T-cell therapy.

The osteo-inductive activity of bone-marrow-derived mononuclear cells resides within the CD14+ population and is independent of the CD34+ population.

Bone marrow mononuclear cells (BMC) seeded on a scaffold of ß-tricalcium phosphate (ß-TCP) promote bone healing in a critical-size femur defect model. Being BMC a mixed population of predominantly mature haematopoietic cells, which cell type(s) is(are) instrumental for healing remains elusive. Although clinical therapies using BMC are often dubbed as stem cell therapies, whether stem cells are relevant for the therapeutic effects is unclear and, at least in the context of bone repair, seems dubious. Instead, in light of the critical contribution of monocytes and macrophages to tissue development, homeostasis and injury repair, in the current study it was hypothesised that BMC-mediated bone healing derived from the stem cell population. To test this hypothesis, bone remodelling studies were performed in an established athymic rats critical-size femoral defect model, with ß-TCP scaffolds augmented with complete BMC or BMC immunomagnetically depleted of stem cells (CD34+) or monocytes/macrophages (CD14+). Bone healing was assessed 8 weeks after transplantation. Compared to BMC-augmented controls, when CD14- BMC, but not CD34- BMC were transplanted into the bone defect, femora possessed dramatically decreased biomechanical stability and new bone formation was markedly reduced, as measured by histology. The degree of vascularisation did not differ between the two groups. It was concluded that the monocyte fraction within the BMC provided critical osteo-inductive cues during fracture healing. Which factors were responsible at the molecular levels remained elusive. However, this study marked a significant progress towards elucidating the mechanisms by which BMC elicit their therapeutic effects, at least in bone regeneration.

Autologous cell-based therapy for treatment of large bone defects: from bench to bedside.

OBJECTIVES: Reconstruction of long segmental bone defects is demanding for patients and surgeons, and associated with long-term treatment periods and substantial complication rates in addition to high costs. While defects up to 4-5 cm length might be filled up with autologous bone graft, heterologous bone from cadavers, or artificial bone graft substitutes, current options to reconstruct bone defects greater than 5 cm consist of either vascularized free bone transfers, the Masquelet technique or the Ilizarov distraction osteogenesis. Alternatively, autologous cell transplantation is an encouraging treatment option for large bone defects as it eliminates problems such as limited autologous bone availability, allogenic bone immunogenicity, and donor-site morbidity, and might be used for stabilizing loose alloplastic implants. METHODS: The authors show different cell therapies without expansion in culture, with ex vivo expansion and cell therapy in local bone defects, bone healing and osteonecrosis. Different kinds of cells and scaffolds investigated in our group as well as in vivo transfer studies and BMC used in clinical phase I and IIa clinical trials of our group are shown. RESULTS: Our research history demonstrated the great potential of various stem cell species to support bone defect healing. It was clearly shown that the combination of different cell types is superior to approaches using single cell types. We further demonstrate that it is feasible to translate preclinically developed protocols from in vitro to in vivo experiments and follow positive convincing results into a clinical setting to use autologous stem cells to support bone healing.

Transfer of minimally manipulated CMV-specific T cells from stem cell or third-party donors to treat CMV infection after allo-HSCT.

Cytomegalovirus (CMV) infection is a common, potentially life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We assessed prospectively the safety and efficacy of stem cell-donor- or third-party-donor-derived CMV-specific T cells for the treatment of persistent CMV infections after allo-HSCT in a phase I/IIa trial. Allo-HSCT patients with drug-refractory CMV infection and lacking virus-specific T cells were treated with a single dose of ex vivo major histocompatibility complex-Streptamer-isolated CMV epitope-specific donor T cells. Forty-four allo-HSCT patients receiving a T-cell-replete (D+ repl; n=28) or T-cell-depleted (D+ depl; n=16) graft from a CMV-seropositive donor were screened for CMV-specific T-cell immunity. Eight D+ depl recipients received adoptive T-cell therapy from their stem cell donor. CMV epitope-specific T cells were well supported and became detectable in all treated patients. Complete and partial virological response rates were 62.5% and 25%, respectively. Owing to longsome third-party donor (TPD) identification, only 8 of the 57 CMV patients transplanted from CMV-seronegative donors (D-) received antigen-specific T cells from partially human leukocyte antigen (HLA)-matched TPDs. In all but one, TPD-derived CMV-specific T cells remained undetectable. In summary, adoptive transfer correlated with functional virus-specific T-cell reconstitution in D+ depl patients. Suboptimal HLA match may counteract expansion of TPD-derived virus-specific T cells in D- patients.

Automated isolation of primary antigen-specific T cells from donor lymphocyte concentrates: results of a feasibility exercise.

BACKGROUND: The safety and clinical efficacy of adoptive transfer of prospectively isolated antigen-specific T cells are well established. Several competing selection methods are available, one of which is based on immunomagnetic enrichment of T cells secreting IFNγ after incubation with the relevant antigen. The proprietary, GMP-conforming selection technology, called 'cytokine capture system' (CCS) is established in many laboratories for the CliniMACS Plus system. It is robust and efficient, but labour-intensive and incompatible with a single-shift working schedule. An automatic immunomagnetic cell processing system, CliniMACS Prodigy ('Prodigy'), including a protocol for fully automatic CCS execution was recently released. MATERIAL AND METHODS: Feasibility of clinical-scale CMV-specific T-cell selection using Prodigy was evaluated using leukoapheresis products from five healthy CMV sero-positive volunteers. Clinical reagents and consumables were used throughout. RESULTS: The process required no operator input beyond set-up and QC-sample collection, that is, feasibility was given. An IFNγ-secreting target T-cell population was detectable after stimulation, and >2 log-scale relative depletion of not CMV-reactive T cells in the target population was achieved. Purity, that is the frequency of CMV-reactive T cells among all CD3(+) cells ranged between 64 and 93%. CONCLUSION: The CCS protocol on Prodigy is unrestrictedly functional. It runs fully automatically beyond set-up and thus markedly reduces labour. The quality of the products generated is similar to products generated with CliniMACS Plus. The automatic system is thus suitable for routine clinical application.

NF-κB-dependent DNA damage-signaling differentially regulates DNA double-strand break repair mechanisms in immature and mature human hematopoietic cells.

Hematopoietic stem and progenitor cells (HSPC), that is, the cell population giving rise not only to all mature hematopoietic lineages but also the presumed target for leukemic transformation, can transmit (adverse) genetic events, such as are acquired from chemotherapy or ionizing radiation. Data on the repair of DNA double-strand-breaks (DSB) and its accuracy in HSPC are scarce, in part contradictory, and mostly obtained in murine models. We explored the activity, quality and molecular components of DSB repair in human HSPC as compared with mature peripheral blood lymphocytes (PBL). To consider chemotherapy/radiation-induced compensatory proliferation, we established cycling HSPC cultures. Comparison of pathway-specific repair activities using reporter systems revealed that HSPC were severely compromised in non-homologous end joining and homologous recombination but not microhomology-mediated end joining. We observed a more pronounced radiation-induced accumulation of nuclear 53BP1 in HSPC relative to PBL, despite evidence for comparable DSB formation from cytogenetic analysis and γH2AX signal quantification, supporting differential pathway usage. Functional screening excluded a major influence of phosphatidylinositol-3-OH-kinase (ATM/ATR/DNA-PK)- and p53-signaling as well as chromatin remodeling. We identified diminished NF-κB signaling as the molecular component underlying the observed differences between HSPC and PBL, limiting the expression of DSB repair genes and bearing the risk of an inaccurate repair.

Deferrals of volunteer stem cell donors referred for evaluation for matched-unrelated stem cell donation.

To minimize donor risk and maintain public support, volunteer donor stem cell donation, whether by mobilized leukapheresis or marrow aspiration, requires careful donor eligibility assessment. Many contraindications to stem cell donation exist, yet analyses of donor deferral rates are not available. In a 36-month series encompassing 2493 potential stem cell donors, we analyzed frequencies and reasons for deferrals. All were presumed eligible by their registries because of previously submitted structured health questionnaire and formal telephone interviews. After assessment by our center's physicians, 3.3% of donors proved ineligible, but 5.6% more were eligible for only one of the collection methods. Higher deferral rates were associated with female sex, increasing age and mobilized stem cell donation vs marrow. Exclusion criteria were identified with approximately similar frequency by medical history, physical examination and laboratory testing. Reasons for deferrals almost exclusively served to protect donor safety; the rare recipient-directed safety concerns could be, and often were, overridden in agreement with the transplant center. As formal analyses have shown, with careful assessment, stem cell donation is acceptably safe, but the plethora of deferral reasons mandate that only physicians with specific experience should evaluate stem cell donors, that is, this task should not be delegated to paramedical personnel.

Effects of storage temperature on hematopoietic stability and microbial safety of BM aspirates.

Bone marrow (BM) remains a common source for hematopoietic SCT. Due to the transcutaneous approach, contamination with skin bacteria is common. The delay between harvest and transfusion can be considerable, potentially allowing for bacterial proliferation. The optimal transportation temperature, specifically with respect to bacterial growth and consequences thereof for hematopoietic quality, remain undefined. For 72 h, 66 individual BM samples, non-spiked/spiked with different bacteria, stored at 20-24 °C room temperature (RT) or 3-5 °C (cold), were serially analyzed for hematopoietic quality and microbial burden. Under most conditions, hematopoietic quality of BM was equal or better at RT: Typical BM contaminants (P. acnes and S. epidermidis) and E. coli were killed or bacterial proliferation was arrested at RT; hematopoietic quality was not impacted by the contamination. However, several pathogenic bacteria not typically found in BM (S. aureus and K. pneumoniae) proliferated dramatically at RT and impaired hematopoietic quality. Bacterial proliferation was arrested in the cold. The overwhelming majority of BM samples, that is, those that are sterile or contaminated only with skin commensals, will benefit from transportation at RT. Those bacteria that proliferate and perturb hematopoietic quality are not typically found in BM. Our data support recommendations for RT transportation and storage of BM.

The novel CXCR4 antagonist POL5551 mobilizes hematopoietic stem and progenitor cells with greater efficiency than Plerixafor.

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 corroborated in humans.

Delayed onset of graft-versus-host disease in immunodeficent human leucocyte antigen-DQ8 transgenic, murine major histocompatibility complex class II-deficient mice repopulated by human peripheral blood mononuclear cells.

Haematopoietic humanization of mice is used frequently to study the human immune system and its reaction upon experimental intervention. Immunocompromised non-obese diabetic (NOD)-Rag1(-/-) mice, additionally deficient for the common gamma chain of cytokine receptors (γc) (NOD-Rag1(-/-) γc(-/-) mice), lack B, T and natural killer (NK) cells and allow for efficient human peripheral mononuclear cell (PBMC) engraftment. However, a major experimental drawback for studies using these mice is the rapid onset of graft-versus-host disease (GVHD). In order to elucidate the contribution of the xenogenic murine major histocompatibility complex (MHC) class II in this context, we generated immunodeficient mice expressing human MHC class II [human leucocyte antigen (HLA)-DQ8] on a mouse class II-deficient background (Aß(-/-) ). We studied repopulation and onset of GVHD in these mouse strains following transplantation of DQ8 haplotype-matched human PBMCs. The presence of HLA class II promoted the repopulation rates significantly in these mice. Virtually all the engrafted cells were CD3(+) T cells. The presence of HLA class II did not advance B cell engraftment, such that humoral immune responses were undetectable. However, the overall survival of DQ8-expressing mice was prolonged significantly compared to mice expressing mouse MHC class II molecules, and correlated with an increased time span until onset of GVHD. Our data thus demonstrate that this new mouse strain is useful to study GVHD, and the prolonged animal survival and engraftment rates make it superior for experimental intervention following PBMC engraftment.

Leucodepletion for hyperleucocytosis--first report on a novel technology featuring electronic interphase management.

BACKGROUND AND OBJECTIVES: Therapeutic leucodepletion plays an established role in the initial treatment of patients with acute myeloid leukaemia (AML) and possibly other leukaemias presenting with leucostasis. Recently, a new leucodepletion technology, Spectra Optia IDL, has become available that differs from its predecessor, COBE Spectra MNC, by a variety of electronic supports, including by electronic adjustment of buffy coat positioning at the collection port. Given the paucity of patients in need of leucodepletions and marked differences in clinical presentation as well as blast properties (e.g. size, density), formal clinical trials comparing leucodepletion technologies have never been executed. MATERIALS AND METHODS: Here, we present aggregate data from eight leucodepletions performed in AML patients with clinical signs of leucostasis between 11/2011 and 07/2012 with the new device and compare the apheresis outcomes with those from fifteen leucodepletions performed with the old technology between 06/2010 and 10/2011. RESULTS: Patients did not differ with respect to epidemiological data. Pre-apheresis leucocyte count (WBC) was significantly higher in Spectra Optia IDL patients. Tolerability was excellent with both devices. Basic apheresis denominators such as duration, processed volume, inlet pump rate, ACD-A consumption and product volume were very similar. A negative correlation between pre-apheresis WBC and collection efficiency was noted. Mean collection efficiency for leucocytes with Spectra Optia IDL (47·3%) was similar to that with COBE Spectra MNC (50·5%). Platelet attrition was similar with both devices, approximately 30%. CONCLUSION: The novel, electronically guided leukapheresis system is suitable for leucodepletion.

Safety and efficacy of healthy volunteer stem cell mobilization with filgrastim G-CSF and mobilized stem cell apheresis: results of a prospective longitudinal 5-year follow-up study.

BACKGROUND AND OBJECTIVES: G-CSF-mobilized peripheral blood stem cells have long replaced marrow as the major source for allogeneic transplants. Conclusive evidence questioning the long-term safety of G-CSF for donors has not been provided, but the cumulative number of followed donors remains insufficient to rule out rare adverse events. A long-term active follow-up study of G-CSF-mobilized healthy volunteer donors was therefore performed. PATIENTS AND METHODS: Two hundred and three successive donors were evaluated pre-apheresis, subjected to G-CSF-mobilization/apheresis, and actively followed for 5 years by the same physicians and laboratories. Follow-up laboratory work included standard biochemical/haematological tests and T-cell phenotyping. RESULTS: Donor epidemiology was typical for reported stem cell donor cohorts. Acute adverse effects of G-CSF and apheresis were mild and transient, consistent with the previous reports. Mean circulating CD34(+) cells after nine doses of G-CSF were 124 per µl. Other biochemical/haematological parameters were also altered, consistent with G-CSF treatment. Spleen enlargement was modest. At first follow-up, all clinical and laboratory parameters had normalized. Leucocyte/lymphocyte counts and CD4/CD8 ratios were the same as during premobilization work-up and remained unchanged throughout. A single severe but likely unrelated adverse event, a case of papillary thyroid carcinoma, was reported. CONCLUSION: The studies add an observation time of almost 500 donor years to the growing body of evidence of the long-term safety of G-CSF for allogeneic donor stem cell mobilization.

Hematopoietic stem cell mobilization: updated conceptual renditions.

Despite its specific clinical relevance, the field of hematopoietic stem cell mobilization has received broad attention, owing mainly to the belief that pharmacologic stem cell mobilization might provide clues as to how stem cells are retained in their natural environment, the bone marrow 'niche'. Inherent to this knowledge is also the desire to optimally engineer stem cells to interact with their target niche (such as after transplantation), or to lure malignant stem cells out of their protective niches (in order to kill them), and in general to decipher the niche's structural components and its organization. Whereas, with the exception of the recent addition of CXCR4 antagonists to the armamentarium for mobilization of patients refractory to granulocyte colony-stimulating factor alone, clinical stem cell mobilization has not changed significantly over the last decade or so, much effort has been made trying to explain the complex mechanism(s) by which hematopoietic stem and progenitor cells leave the marrow. This brief review will report some of the more recent advances about mobilization, with an attempt to reconcile some of the seemingly inconsistent data in mobilization and to interject some commonalities among different mobilization regimes.