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IL2 infusion may benefit patients with haematological malignancies by lowering the disease burden. However, conflicting data have been reported on IL2 effects on myelopoiesis, in vitro as well as in vivo. In the present study we investigated the ability of IL2 to act on committed and primitive bone marrow progenitor cells in defined serum-free (SF) culture conditions which avoid many technical biases such as interference by exogenous stimulating or inhibiting factors. Low doses of IL2 (0.1-1000 U/ml) were studied without or in combination with recombinant IL3, GM-CSF and erythropoietin, in SF long-term marrow culture (LTMC). We report data in favour of an inhibitory activity of IL2 limited to committed progenitors and excluding more primitive haemopoietic stem cells, as shown by an alteration of CFU-GM proliferation during the first 5 weeks of LTMC, decreasing with time, unaffected BFU-E and increased nucleated cell production. Beyond week 5, no difference was observed between IL2 supplemented cultures and the SF control cultures. In parallel, IL2 induced the adherence of fibroblastic cells and their progeny. In addition to the inhibitory effect, IL2 appeared to limit the stimulating effect on granulopoiesis and erythropoiesis of myeloid growth factors (GF) such as combination of IL3, GM-CSF and EPO. Indeed, in SF-LTMC conditions, IL2 inhibitory effect is effective on CFU-GM production throughout the 7 weeks of LTMC and on BFU-E during the first 2 weeks only. These data confirm the interaction of IL2 with other GFs in the complex interplay of the cytokine network.

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 fractor 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-dependant 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.

We have tested folinic acid (FA) for ability to increase peripheral blood stem cells (PBSC) after chemotherapeutic aplasia in acute leukaemia. Five adult patients (four AML, one ALL) entered the study, each patient underwent two series of three leukapheresis, the first following induction chemotherapy and the second following the first course of consolidation. The first leukapheresis of each series was done when the white blood cell count reached 10(9)/l with subsequent leukapheresis every other day. Folinic acid (Lederle Laboratories, France) was administered at a dose of 50 mg (i.v.) per day, 15 days from initiation of chemotherapy and continuing through the third leukapheresis of the series (days 25-30). PBSC were collected on a Haemonetics V50 cell separator. In these five cases we observed an increased yield of both colony-forming units, granulocyte macrophage (CFU-GM) and burst forming units-erythroid (BFU-E) expressed per ml of cytapheresis product: CFU-GM x 18, BFU-E x 3 and if expressed per 10(4)/kg of body weight: CFU-GM x 30, BFU-E x 3 (CFU-GM P less than 0.05, BFU-E less than 0.01). Long-term blood culture (LTSC) from FA stimulated leukapheresis, in an attempt to quantitate the most primitive stem cells, demonstrated that this expansion of the PBSC was sustained in time. We found by means of LTSC that FA did not stimulate CFU-L from patients with AML (two cases tested). Finally two AML patients were grafted with FA-PBSC after Cytotoxan and total body irradiation (TBI). Haematopoietic reconstitution was rapid complete and sustained in time in both patients. This indication for folinic acid should be further studied with or as an alternative to haematopoietic growth factors.

We report a stimulating effect of leukaemic B-lymphocytes from anaemic and non-anaemic patients with CLL on the proliferation of normal peripheral blood BFU-E. Coculture of leukaemic B-cells at various concentrations (2.5 X 10(3)-10(6)) with 2.5 X 10(5) mononuclear cells from normal peripheral blood increased the number of BFU-E derived erythroid colonies. The same effect was observed when the number of target cells was varied in the presence of a fixed number of B-lymphocytes, with a clear linear relationship. B-cell conditioned medium gave a similar increase when added to the culture instead of B-cells. At high concentration of B-cells from anaemic patients, the size of the colonies was increased and a large number of macroscopic colonies was seen. The place of the B-cells in the regulation of erythroid progenitors in relation to monocytes and T-lymphocytes has still to be established.

Prolonged in vitro maintenance of human bone marrow progenitor cells was achieved using a serum-free (SF) liquid culture system. Culture medium was based on Iscove's medium supplemented with bovine serum albumin, human transferrin, bovine insulin, soybean lecithin, cholesterol, hydrocortisone and alpha-thioglycerol. Under these standardized culture conditions, CFU-GM were maintained for up to 4 weeks, as is the case when using conventional serum-dependent medium. Erythropoiesis exhibited a slower decline than that found using serum containing medium. Development of normal haematopoiesis was effective in spite of poor stromal cell development--a confluent adherent layer as classically described in serum conditions was never achieved. Our newly defined system provides a reliable technique for studying human haematopoietic stem cell proliferation and differentiation in vitro; it allows for rational utilization of currently available purified recombinant growth factors. It may be a promising tool in the clinical use of cultured haematopoietic stem cells.