IL-13 induces CD34+ cells isolated from G-CSF mobilized blood to differentiate in vitro into potent antigen presenting cells.

Dendritic cells (DCs), which are antigen presenting cells of potential use in human antitumor vaccination trials, are presently the subject of intense investigation. Many recent studies have reported the possibility of generating ex vivo large numbers of DCs with high antigen presenting capacity by the culture of bone marrow or blood progenitors. In this study, we examined the differentiation into DCs of CD34+ progenitors isolated from the G-CSF mobilized blood of 3 healthy donors and 5 patients with breast cancer and cultured in the presence of GM-CSF + IL-13. The characteristics of the cells were compared to those of cells obtained in the presence of GM-CSF + TNF alpha. By day 15, one third of the bulk cells cultured with IL-13 were CD1a+/CD14- and strongly expressed CD1c, CD40, CD80 and HLA-DR. In contrast, cells obtained with TNF alpha expressed CD1a on one in three cells but with a considerably lower fluorescence intensity than on IL-13-cultured cells and strongly expressed CD14 on more than 50% of cells. CD1a+/CD14- cells emerged in IL-13 cultures at day 5, while in TNF alpha cultures CD14+ cells appeared before CD1a+ cells. Cells grown in the presence of IL-13 had an increased capacity to present antigens to autologous lymphocytes and to stimulate allogeneic T-lymphocytes. This effect was greater than that of cells grown in the presence of TNF alpha. These cells should therefore have greater effector potential in any therapeutic applications in humans.

Characterization of late and early hematopoietic progenitor/stem cell sensitivity to mafosfamide.

The preservation of the hematopoietic value of the graft is essential in the context of bone marrow purging for autologous transplantation. In the present study we have investigated mafosfamide toxicity to transplantable hematopoietic stem cells using combinations of recombinant growth factors (GF) in semi-solid assays and by measurement of Long-Term Culture Initiating Cells (LTC-IC). Our data show that various subtypes of progenitor/stem cells were inhibited in a dose-dependent manner by mafosfamide. A hierarchy appeared clearly regarding sensitivity to the drug in the following order of increasing resistance: PCM CFU-GM (grown in presence of placenta-conditioned medium), 4R CFU-GM (grown in presence of GM-CSF + IL-3 + G-CSF + EPO), 5R CFU-GM (grown in presence GM-CSF + IL-3 + G-CSF + EPO + SCF) and LTC-IC with respective lethal dose 95 (LD95) levels of 40 micrograms, 55 micrograms, 90 micrograms and 140 micrograms/10(7) MNC (P = 0.05 to P = 0.018). Even the primitive stem cells treated with very high doses of mafosfamide (2 to 4 times the usually recommended dose) responded to a combination of SCF + GM-CSF + G-CSF + IL-3 in liquid marrow culture, suggesting that they were functionally spared by the treatment. We also observed a higher sensitivity of 5R CFU-GM and LTC-IC from patients with hematological malignancies, compared with normal donors, when marrow was treated with the dose chosen for clinical purging.(ABSTRACT TRUNCATED AT 250 WORDS)

[Towards a standardization of the culture of hematopoietic progenitor cells CFU-GM applied to the transplantation of hematopoietic stem cells in France]. / Vers une standardisation de la culture des progéniteurs hématopoïétiques CFU-GM appliquée à la greffe de cellules souches hématopoïétiques en France.

The CFU-GM (granulo-macrophagic colony forming unit) content of 130 bone marrow samples and 105 cytapheresis blood samples was evaluated by two culture techniques, one in agar with human placenta conditioned medium as stimulating factor, the second in methyl cellulose with a cocktail of recombinant G-CSF, GM-CSF, IL-3 and erythropoietin as stimulating factors. The aim of the study was to evaluate in methyl cellulose the CFU-GM threshold doses necessary to ensure hemopoietic reconstitution in autologous transplantation that we previously determined in the agar technique. Thirty-one out of the 130 BM samples were also tested after incubation with mafosfamide, and 33 after freezing and thawing. Results showed that the numbers of CFU-GM in the 2 techniques were significantly correlated (p < 0.0001). The strongest correlation was found with the formula "log CFU-GM methyl = a log CFU-GM agar + b" with a slight variation in the values of a and b among the 4 settings, i.e. unmanipulated BM, mafosfamide-treated BM, frozen-thawed BM and unmanipulated blood samples. According to the formulas, the threshold doses of CFU-GM for BM and blood, previously determined in the agar technique (respectively 10(4)/kg and 5 x 10(4)/kg) were calculated for CFU-GM grown in methyl and corresponded respectively to 2.3 x 10(4)/kg and 2.1 x 10(5)/kg.

In vivo effects of GM-CSF and IL-3 on hematopoietic cell recovery in bone marrow and blood after autologous transplantation with mafosfamide-purged marrow in lymphoid malignancies.

This retrospective study evaluates the impact of GM-CSF and interleukin 3 (IL-3) on bone marrow (BM) and peripheral blood (PB) cell recovery following autologous bone marrow transplantation (ABMT) with mafosfamide-purged BM in patients with lymphoid malignancies compared with a control group receiving no colony-stimulating factor. GM-CSF was administered at 250 micrograms/m2/day (8 patients) as a continuous infusion from day of autologous BMT until the absolute neutrophil count (ANC) reached 0.5 x 10(9)/l for 7 days or until day 30, whichever was first. IL-3 was administered daily starting on the first day of transplant at a dose of 1 microgram/kg/day (6 patients) and 5 micrograms/kg/day (6 patients) for 30 days. CFU-GM and BFU-E were sequentially evaluated in BM and PB at days 7, 14, 21, 28, and 56 post-graft. The neutrophil recovery (ANC > 0.5 x 10(9)/l) was significantly faster in the GM-CSF group compared with IL-3 5 micrograms, IL-3 1 microgram and control group (respectively, days 15, 21, 22, 24) (p < 0.05 to p < 0.01). Similarly, leukocyte recovery was faster in the GM-CSF group compared with control and IL-3 1 microgram groups (p < 0.01 and p < 0.05). No difference was noticed between the two IL-3 groups. Although no difference was observed in platelet recoveries (> 50 x 10(9)/l), it appeared that the GM-CSF group required more units of platelets than either the IL-3 1 microgram or 5 micrograms groups (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The role of recombinant haematopoietic growth factors in human long-term bone marrow culture in serum-free medium.

We have previously reported prolonged in vitro maintenance of human bone marrow progenitor cells using a serum-free (SF) liquid culture system. The present study was undertaken to determine recombinant growth factor (rGF) requirement of long-term marrow culture (LTMC) in absence of exogenous serum, to avoid interference of any undefined components. Our data clearly show that the presence of serum is a major obstacle to the correct evaluation of rGF activity. However, in SF conditions the sequential analysis of these rGFs, alone or in combination, clearly showed a stimulating activity of interleukin 3 (IL3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) on granulopoiesis and erythropoiesis. Indeed, the cumulative number of progenitors recovered during an 8-week period exceeded the initial input by a factor of 1.7 for granulocyte-macrophage (CFU-GM), of 3 for erythroid blast-forming units (BFU-E) and of 5.45 for CFU-E when EPO, GM-CSF and IL3 were combined. This study has confirmed that the system is able to sustain haematopoiesis for 8 weeks in a way similar to that in serum-dependent LTMC, despite diminished stromal adherent layer development which never covered more than 50% of the flask surface. We conclude that this defined SF-LTMC system provides a reproducible technique for studying human haematopoiesis.