Background: Human multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity. Results: After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G₁ phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation. Conclusion: Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.

Key Points: KIR haplotype B donors and high KIR B content score confer better protection against relapse after HLA-haploidentical transplantation in pediatric acute lymphoblastic leukemia. Haploidentical donor selection criteria for childhood acute lymphoblastic leukemia should include KIR haplotype and KIR B-content score.

The treatment of high-risk neuroblastoma patients consists of multimodal induction therapy to achieve remission followed by consolidation therapy to prevent relapses. However, the type of consolidation therapy is still discussed controversial. We applied metronomic chemotherapy in the prospective NB90 trial and monoclonal anti-GD2-antibody (MAB) ch14.18 in the NB97 trial. Here, we present the long term outcome data of the patient cohort. A total of 334 stage 4 neuroblastoma patients one year or older were included. All patients successfully completed the induction therapy. In the NB90 trial, 99 patients received at least one cycle of the oral maintenance chemotherapy (NB90 MT, 12 alternating cycles of oral melphalan/etoposide and vincristine/cyclophosphamide). In the NB97 trial, 166 patients commenced the MAB ch14.18 consolidation therapy (six cycles over 12 months). Patients who received no maintenance therapy according to the NB90 protocol or by refusal in NB97 (n = 69) served as controls. The median observation time was 11.11 years. The nine-year event-free survival rates were 41 ± 4%, 31 ± 5%, and 32 ± 6% for MAB ch14.18, NB90 MT, and no consolidation, respectively (p = 0.098). In contrast to earlier reports, MAB ch14.18 treatment improved the long-term outcome compared to no additional therapy (p = 0.038). The overall survival was better in the MAB ch14.18-treated group (9-y-OS 46 ± 4%) compared to NB90 MT (34 ± 5%, p = 0.026) and to no consolidation (35 ± 6%, p = 0.019). Multivariable Cox regression analysis revealed ch14.18 consolidation to improve outcome compared to no consolidation, however, no difference between NB90 MT and MAB ch14.18-treated patients was found. Follow-up analysis of the patient cohort indicated that immunotherapy with MAB ch14.18 may prevent late relapses. Finally, metronomic oral maintenance chemotherapy also appeared effective.

We sought to develop a method for the clinical large-scale depletion of αβ T lymphocytes from mobilized peripheral stem cells, which would allow the allogeneic transplantation of a graft enriched for stem cells, natural killer (NK) cells and γδ T lymphocytes. Therefore, we obtained mononuclear cells from either mobilized or non-mobilized healthy adult volunteer donors and incubated the cells with a biotinylated anti-αβ T-cell Ab and subsequently with an anti-biotin Ab conjugated with magnetic microbeads. The depletion was then performed using a CliniMACS^® device. The median T-cell depletion was 3.9 log (range 3.5–4.1 log). The recovery of the γδ and NK cells was 92% and 80%, respectively. The recovery of CD34⁺ stem cells from the mobilized donors was 66%. This method had no negative influence on the in vitro colony formation of stem cells, and transplantation of αβ-depleted cells into NOD-SCID IL-2 common gamma chain knockout (NOD-scid IL2r ^null) mice resulted in a rapid engraftment of human myeloid and lymphoid cells. This method will allow large-scale depletion of αβ T cells from mobilized peripheral blood in clinical trials.

In contrast to CD34(+) positive selection, negative depletion strategies retain large numbers of effector cells in allogeneic peripheral stem cell grafts, such as natural killer (NK) and other cells. This review summarizes the clinical experience obtained using negative depletion approaches of CD3(+) and T-cell receptor (TcR)αβ(+) T lymphocytes. Attempts to improve immune reconstitution and to better exploit the graft-versus-malignancy effect after transplantation of T-cell-depleted grafts through the preservation of immune effector cells led to the development of CD3-, CD3/CD19- and more recently TcRαβ/CD19-negative depletion strategies of mobilized peripheral stem cell grafts. A faster immune reconstitution has been observed in patients with negatively depleted grafts after haploidentical transplantation, although no prospective randomized trials have been reported to date. In a randomized study of matched sibling and matched unrelated transplantation, CD3/CD19-depleted peripheral stem cell grafts led to a faster recovery of NK cells compared with the CD34(+)-positive selection group. New technologies allow the large-scale graft engineering of peripheral stem cells for clinical use in matched and mismatched stem cell transplantation. Further clinical trials are necessary to decide which of these methods is associated with a faster immune reconstitution and a better outcome after transplantation.