Jill Hibbin

Myeloid progenitor cells in the circulation of patients with myelofibrosis and other myeloproliferative disorders

Summary. We have measured the numbers of myeloid progenitor cells in the circulation of patients with myelofibrosis (MF) and other myeloproliferative disorders. In general, progenitor cell numbers were increased in the circulation of patients with MF compared with controls. The mean increases were 9‐fold for the multilineage progenitor cells (CFU‐GEMM), 13‐fold for the erythroid progenitor cells (BFU‐E), 37‐fold for the granulocyte‐macrophage progenitor cells (CFU‐GM) and 167‐fold for the megakaryocyte progenitors (CFU‐MK). Splenectomized patients generally had reduced numbers of circulating progenitor cells. In the CFU‐MK assay, mature megakaryocytes cultured from patients with MF regularly showed large vacuoles in the nucleus and cytoplasm, unlike control cells. The increased colony formation in patients with MK, involving especially CFU‐MK colonies, is consistent with the hypothesis that MF is a primary myeloproliferative disorder in which a megakaryocyte‐derived factor predisposes to the formation of marrow fibrosis.

Colony formation by primitive haemopoietic progenitors in cocultures of bone marrow cells and stromal cells

Human bone marrow contains a class of human haemopoietic progenitor cells that adhere to cultured marrow stromal cells and form colonies of blast cells. These progenitor cells are found in the non‐adherent mononuclear fraction of normal human bone marrow. They are not in active cell cycle and do not express Ia‐like (HLA‐DR) antigens but appear to be capable of self‐renewal in vitro. These properties indicate that they should be classified as members of the primitive haemopoietic progenitor cell compartment.

Use of minisatellite DNA probes for recognition and characterization of relapse after allogeneic bone marrow transplantation

Restriction fragment length polymorphisms can be used to distinguish blood and marrow cells from close relatives. We used two probes that recognize a series of dispersed and highly polymorphic tandem‐repetitive mini‐satellite regions in the human genome that can be detected via a shared 10–15 base pair core sequence similar to the generalized recombination sequence (χ) of E. coli. We have studied the resulting individual‐specific DNA fingerprints in 15 patients before and after allogeneic bone marrow transplantation performed for chronic myeloid leukaemia and in two patients transplanted for acute leukaemia. Early engraftment could be demonstrated at 3 weeks post‐transplant based on the recognition of cells of donor origin. One patient who failed to engraft had only recipient type marrow cells 3 months post‐transplant. Nine patients who relapsed after transplantation had only cells of recipient origin. In one patient who relapsed after transplantation with T‐cell depleted donor marrow, fractionation studies showed that his T‐cells at relapse were of recipient origin. We conclude that these minisatellite probes are valuable for characterizing the origin of different cell populations after marrow transplantation and could be useful for characterizing relapse when donor and recipient are of the same sex.

HLA-DA monoclonal antibodies inhibit the proliferation of normal and chronic granulocytic leukaemia myeloid progenitor cell

Summary. We studied the capacity of murine monoclonal antibodies with HLA‐DR specificity to inhibit the proliferation in vitro of erythroid (BFU‐E and CFU‐E) and granulocyte‐macrophage (CFU‐GM) progenitor cells in normal bone marrow and the blood of patients with chronic granulocytic leukaemia (CGL). Two IgG2 antibodies (CA 2.06 and L243) inhibited the proliferation of normal BFU‐E and CFU‐GM at relatively high dilution; a third antibody, DA2, had no effect on either progenitor cell. A complement‐king monoclonal antibody with T‐cell activity (OKT3) produced only minor reduction in progenitor cell proliferation. Further studies with L243 showed that BFU‐E and CFU‐GM from the blood of patients with CGL were inhibited to the same degree as normal marrow progenitor cells. The inhibition of progenitor

Purification of haemopoietic progenitor cells from patients with chronic granulocytic leukaemia using Percoll density gradients and elutriation

Purification of haemopoietic progenitor cells from chronic granulocytic leukaemia buffy coat preparations requires a multistep approach using complementary cell separation techniques. In this study Percoll density gradient centrifugation and centrifugal elutriation were used to isolate large numbers of immature progenitor cells. Percoll density gradients were valuable as a first separation step: CFU‐GM and CFU‐GEMM could be enriched 75‐fold in a light density fraction of d< 1mD056 g/ml and the technique could be adapted to cope with more than 1010 buffy coat leucocytes. Progenitors cells were concentrated 3‐fold by elutriation used as single method to separate buffy coat cells or when used to purify further light density Percoll fractions. When Percoll gradients and elutriation were used sequentially, undifferentiated mononuclear cells were enriched to more than 90% purity arid between 5% and 40% of these cells formed CFU‐GM or BFU‐E colonies consisting of more than 40 cells. The enriched fractions were further characterized with monoclonal antibodies. The density and elutriation profiles of these colony forming cells resembled corresponding profiles of cells that reacted with the monoclonal antibody BI‐3C5, which recognizes an antigen on primitive haemopoietic progenitor cells. Physical separation methods are a valuable first stage in the attempt to procure relatively pure myeloid progenitor cell populations, whose characteristics can then be further studied at a cellular or molecular level.

Antigenic characteristics of circulating CFU-GM in chronic granulocytic leukaemia resemble those of CFU-GM in normal marrow and differ from those in normal blood

We studied the surface antigenic determinants of myeloid progenitor cells (Day 7 CFU-GM, Day 14 CFU-GM and BFU-E) in the peripheral blood and bone marrow of patients with chronic granulocytic leukaemia (CGL) and normal subjects by complement-mediated cytotoxicity with a panel of 8 selected murine monoclonal antibodies (McAbs) followed by culture in methyl cellulose. All classes of progenitor cell studied expressed HLA-DR antigens and also expressed other antigens recognized by two of the McAbs (S3-13 and S17-25) with myeloid specificity. Two other McAbs (R1.B19 and WGHS.29.1). Recognized antigens on Day 14 CFU-GM derived from normal marrow but not on those from normal blood. The pattern of reactivity of Day 14 CFU-GM from the blood of patients with CGL resembled to a considerable extent that of CFU-GM from normal marrow and differed from that of CFU-GM from normal blood. BFU-E from the blood of patients with CGL reacted with these McAbs in a manner very similar to that of BFU-E from normal blood; however the same two McAbs (R1.B19 and WGHS.19.1) reacted with a much higher proportion of the BFU-E from the marrows of CGL patients than of normal subjects. Our data are consistent with the hypothesis that normal blood-derived CFU-GM are more primitive than marrow-derived CFU-GM; however the CFU-GM in the circulation in CGL differ from those in normal blood, perhaps because they reflect overflow from or exchange with a hyperplastic marrow population.

Antigenic determinants on myeloid leukaemia colony‐forming cells resemble those of normal myeloid progenitor cells and differ from those of circulating blast cells

Summary. We studied the antigenic characteristics of leukaemic colony‐forming cells (CFU‐L) from the blood of patients with chronic granulocytic leukaemia (CGL) in blastic transformation (BT) and acute myeloid leukaemia (AML) by in vitro culture techniques after complement‐mediated lysis with one anti‐DR and 10 selected myeloid monoclonal antibodies (McAbs), all of which were cytotoxic in the presence of complement. At the same time we studied the antigenic characteristics of the circulating blast cells from the same patients using in addition one non‐complement fixing antibody (BI.3C5) with standard immunofluorescence and immunoalkaline phosphatase techniques. We also used myeloid progenitor cell assays in conjunction with cytotoxic McAbs to investigate the antigenic determinants on Day 7 CFU‐GM, Day 14 CFU‐GM and BFU‐E from the blood of patients with CGL in chronic phase (CP) and from normal bone marrow. We found that two of the McAbs, S4–7 and WGHS29.1, recognized a higher proportion of CFU‐L from the blood of AML patients than from patients with CGL‐BT. However, the patterns of reactivity for CFU‐L from CGL‐BT and AML patients with the other McAbs quite closely resembled those observed in CFU‐GM and BFU‐E from normal individuals and patients with CGL in CP. A McAb with DR specificity and one of the myeloid McAbs, 54/39, recognized both CFU‐L from CGL‐BT and AML and reacted also with circulating blast cells from the same patients. In contrast, six of the other myeloid McAbs that recognized CFU‐L failed to label the corresponding blast cells. We conclude

Proliferation in liquid culture of megakaryocytes from the blood of patients with primary myelofibrosis and other myeloproliferative disorders

Using a short term liquid system we have shown that blood from some patients with primary myelofibrosis (PMF) and chronic granulocytic leukaemia (CGL) in megakaryoblastic transformation (CGL-Mk) gives rise to large numbers of progenitor cells committed to the megakaryocyte (Mk) lineage. As assessed by indirect immunofluorescence the number of cells reacting with three antiplatelet monoclonal antibodies, C17, J15 and AN51, increases during the culture period. There is no equivalent increase in cultures from the blood of normal individuals or patients with essential thrombocythaemia (ET). Furthermore plasma-free supernatants from cultures of the cells from patients with PMF and CGL-Mk stimulate the rate of proliferation of fibroblasts from normal bone marrow. These data provide further evidence for the involvement of the Mk lineage in PMF and CGL and suggest that the excess fibrosis seen in these conditions may be caused by a factor emanating from Mks.