Effects of low molecular weight B-cell growth factor on proliferation of leukemic cells from children with B-cell precursor-acute lymphoblastic leukemia

Recently, low-molecular-weight B-cell growth factor (LMW-BCGF) has been reported to stimulate growth of leukemic cells from B-cell precursor-acute lymphoblastic leukemia (BCP-ALL). We further investigated the effects of LMW-BCGF on proliferation of leukemic clonogenic (progenitor) and nonclonogenic (progeny) cells from children with BCP-ALL (28 patients) and B-cell ALL (two patients). Patients were either at diagnosis (n = 18) or in relapse (n = 12). Response of leukemic progenitor cells was determined by culturing cells (10(5) cells/mL) in methylcellulose with 0.1 U/mL LMW-BCGF. Colonies (greater than 20 cells) were counted at day 7. The response of the leukemic progeny population was determined by DNA synthesis studies using tritiated-thymidine and by DNA quantitation with propidiumiodide for determination of cell-cycle status. LMW-BCGF supported growth of leukemic progenitor cells from 20 of 28 (71%) BCP-ALL and two of two B-cell ALL patients. Colony numbers ranged from 7 to 2,400 (mean 145, median 45). A dose-response effect in colony growth was noted, with an apparent plateau at approximately 2.0 U/mL LMW-BCGF. Colony cells were primarily of leukemic phenotype (CD19+/CD10+/-). LMW-BCGF also induced significant increases in leukemic progeny cell proliferation as measured by both thymidine incorporation (stimulation indexes of 1.6 to 34) and by cell-cycle assay (percentage S+ G2/M stimulation indexes of 1.6 to 6). LMW-BCGF was more effective in stimulating leukemic proliferation than three recombinant interleukins (rIL-2, rIL-3, rIL-4), although rIL-3 was able to support colony growth in 4 of 11 patients. These results indicate that LMW-BCGF and, to a lesser degree rIL-3, are able to stimulate proliferation of BCP-ALL progenitor and progeny cells, whereas rIL-2 and rIL-4 do not support progenitor cell proliferation and have only marginal effects on leukemic progeny cell proliferation.     

Detailed studies on expression and function of CD19 surface determinant by using B43 monoclonal antibody and the clinical potential of anti-CD19 immunotoxins

Extensive immunologic surface marker analyses and binding competition assays demonstrated that B43 monoclonal antibody (MoAb) is a new member of the CD19 cluster that recognizes the same surface epitope as several other anti-CD19 MoAbs. We used B43 MoAb to test for CD19 expression on neoplastic cells from 340 leukemia and 151 malignant lymphoma patients and on nonneoplastic cells in normal lymphohematopoietic and nonlymphohematopoietic tissues. Our study more than doubles the total number of cases with classified hematologic malignancies that have been examined for CD19 antigen expression. The data presented confirm that CD19 is the most reliable B lineage surface marker and support our view that this B lineage-restricted surface determinant may be an important functional receptor. Our findings provide unique and direct evidence that (a) CD19 is expressed on leukemic B lineage lymphoid progenitor cells freshly obtained from B lineage acute lymphoblastic leukemia patients but not on normal myeloid, erythroid, megakaryocytic, or multilineage bone marrow progenitor cells; (b) ligation of CD19 with B43 MoAb induces sustained increases in [Ca2+]i when crosslinked and inhibits high-molecular weight B cell growth factor (HMW-BCGF)-induced proliferation of activated B cells without affecting their low-molecular weight B cell growth factor (LMW-BCGF) response; therefore CD19 may be a unique signal receptor; (c) HMW-BCGF and LMW-BCGF augment expression of CD19, which suggests that CD19 and BCGF receptors may be under coordinate regulatory control; (d) approximately two million B43 MoAb molecules per cell can be bound to target B lineage lymphoma cells with a Ka of 1.9 x 10(8)/mol/L; (e) CD19 can undergo B43 MoAb-induced internalization; and (f) the opportunity is thus provided for using anti-CD19 MoAb to deliver toxins to B lineage neoplastic cells for more effective treatment of high-risk leukemia/lymphoma patients.     

B8.7 antigen is present on B-cell precursor acute lymphocytic leukemia. Correlation with the low molecular weight B-cell growth factor responsiveness of these cells

The purpose of this study was to analyze the expression of B8.7 antigen and its implication in the low molecular weight B-Cell growth factor (LMW BCGF) proliferative pathway at the early stages of the human B-cell differentiation. After an overnight incubation in culture medium of B-cell precursor acute lymphoblastic leukemias (ALL), we demonstrated the presence of B8.7 antigen in 18 of 25 cases (72%). Such an incubation also induced a significant increase in the LMW BCGF responsiveness of ALL cells (P less than 0.03). In addition, we showed a significant correlation between B8.7 expression and the ability of pre-B ALL cells to respond to LMW BCGF. As previously described for normal B cells, the anti-B8.7 monoclonal antibody inhibited the LMW BCGF-dependent proliferation of pre-B ALL cells in a dose-dependent manner. These data indicate that B8.7 antigen is expressed and may be functionally related to the LMW BCGF pathway at the pre-B cell stages of differentiation. These results also suggest that human B-cell precursor ALL are not only phenotypically similar to their normal B lymphocyte counterparts, but are also sensitive to the same immunoregulatory cytokines that control normal cell growth.     

Low molecular weight B cell growth factor induces proliferation of human B cell precursor acute lymphoblastic leukemias

Experiments were conducted to determine the effect of low mol wt B cell growth factor (L-BCGF) on B cell precursor acute lymphoblastic leukemia (ALL). L-BCGF induced a significant increase in 3H-TdR incorporation in 28 of 37 bone marrow aspirates from patients with B cell precursor ALL, with stimulation indices ranging from 2 to 129. Fluorescence-activated cell sorting confirmed that in five of seven patients the common acute lymphoblastic leukemia antigen (CALLA)/CD10 positive leukemic cells were responding directly to L-BCGF. L-BCGF was capable of inducing, in some patients, an increase in absolute viable cells and could also induce colony formation in vitro. The response of B cell precursor ALL was not attributable to beta IL 1, IL 2, or gamma interferon. These results indicate that the majority of B cell precursor ALL undergo a proliferative response to L-BCGF, suggesting a regulatory role for this lymphokine in the growth of B cell precursors.     

Antigen-independent regulation of cytoplasmic calcium in B cells with a 12-kDa B-cell growth factor and anti-CD19.

Increases in cytoplasmic free calcium ([Ca2+]i) can be induced in resting B cells either by a low molecular weight (12-kDa) B-cell growth factor (LMW-BCGF) or by crosslinking the B-cell antigen CD19 with monoclonal antibody (mAb). LMW-BCGF causes a slow [Ca2+]i increase in peripheral blood and tonsillar B cells but has no effect on [Ca2+]i in resting T cells. B-cell [Ca2+]i responses mediated by anti-surface immunoglobulin (sIg) or anti-CD19 are potentiated by LMW-BCGF, but anti-sIg and anti-CD19 do not show additive [Ca2+]i responses. LMW-BCGF- and anti-CD19-induced [Ca2+]i signals are similar to the sIgM or sIgD-mediated signals in that they are inhibited by prior treatment with phorbol 12-myristate 13-acetate. However, LMW-BCGF- and CD19-mediated signals do not depend on the expression of sIg, since they were also observed on sIg-B-cell precursor acute lymphoblastic leukemia (ALL) cells. Both anti-CD19 and LMW-BCGF stimulated in vitro colony formation by ALL cells and showed additive effects when used together. [Ca2+]i responses to LMW-BCGF or CD19 cross-linking were also evident on certain pre-B-cell and lymphoma B-cell lines.     

Impaired expression of cell surface receptors for B cell growth factor by chronic lymphocytic leukemia B cells

Normal human B cell proliferation is controlled by various immunoregulatory signals including the T cell-derived lymphokine B cell growth factor (BCGF). The role of BCGF in the regulation of malignant B cell proliferation is unclear. Therefore, we studied the proliferative response of purified chronic lymphocytic leukemia (CLL) B cells to BCGF. For all CLL patients studied, CLL B cells showed a decreased proliferative response as compared with control B cells for BCGF- induced B cell proliferation (patient 291 +/- 59 cpm v control 3,942 +/- 622, mean +/- SEM). This impaired proliferative response appeared to be intrinsic to CLL B cells since it was not corrected by incubation with increasing concentrations of BCGF. Attainment of normal B cell responsiveness to BCGF requires the processing of an initial activation signal which results in the expression of cell surface receptors for BCGF. Increasing concentrations of the B cell activation signal (the F(ab')2 fragment of goat anti-human mu chain) did not improve CLL B cell responsiveness to BCGF. Three-day activated CLL B cells compared with activated control B cells demonstrated a marked impairment in their ability to absorb out the BCGF activity present in the BCGF preparation (BCGF activity absorbed out, patient 12.8% v control 53%). Pretreatment of CLL B cells with neuraminidase failed to improve either the proliferative response to BCGF or the expression of cell surface receptors for BCGF by the CLL B cells. This study suggests that the impaired responsiveness to BCGF by CLL B cells is the result of impaired expression of cell surface receptors for BCGF when CLL B cells are exposed to activation signals.     

Proliferation of B cells from chronic lymphocytic leukemia is selectively promoted by B cell growth factor

B cell activation was studied in B cells from B cell chronic lymphocytic leukemia (B-CLL) patients. After in vitro stimulation, these B cells showed extensive proliferation in the presence of high-molecular-weight B cell growth factor (BCGF). In contrast, this effect was not observed upon addition of recombinant interleukin-2 (IL-2). In agreement, upon stimulation, B cells expressed Bac-1 antigen but failed to acquire the IL-2 receptor. These results demonstrate that the utilization of the BCGF pathway can be segregated from that of IL-2 in B cells from B-CLL patients.     

Establishment and characterization of human B-cell lymphoma cell lines using B-cell growth factor

B-cell non-Hodgkin's lymphomas (NHL-B) have been difficult to establish in long-term cell culture using standard techniques. We report the establishment of five representative cell lines from high grade NHL-B using B-cell growth factor (BCGF). The five NHL-B cell lines display the morphologic, immunophenotypic, genotypic, and biologic characteristics of the lymphoma cells present in the original diagnostic specimen. The cell lines showed at least a sevenfold dose-dependent increase in proliferation in vitro over background in the presence of BCGF. Other putative B-cell growth-stimulating cytokines showed no significant proliferative activity or were inhibitory in some cases. NHL-B cell lines secreted growth factor(s) into culture supernatants that mediated at least a fivefold dose-dependent increase in cell proliferation in autochthonous lymphoma cells and a 10-fold or greater stimulation in growth factor-dependent normal B cell lines in vitro. The cell lines show monoclonal rearrangements of IgH genes and nonrandom chromosomal abnormalities characteristic of NHL-B, while the expression of Epstein-Barr virus associated antigen (EBNA-I) is present in two of the five cell lines. The studies show that lineage-specific growth factors may be used to establish neoplastic B cell lines in vitro, which are important experimental systems for cellular and molecular studies in the NHL-B.     

Low molecular weight B-cell growth factor and recombinant interleukin-2 are together able to generate cytotoxic T lymphocytes with LAK activity from the bone marrow cells of children with acute lymphoblastic leukemia

We are reporting here that low-mol wt B-cell growth factor (LMW-BCGF) and recombinant interleukin-2 (rIL-2) are together able to induce CD3+ cytotoxic T lymphocytes (CTL) with lymphokine-activated killer cell (LAK) activity from the bone marrow (BM) cells of children with acute lymphoblastic leukemia (ALL). Ficoll-Hypaque (FH)-separated BM cells were obtained from patients with active disease (at diagnosis N = 13, in relapse N = 15) and in complete remission (CR; N = 12). CD3+ cells were removed by Leu-4 antibody and immunobeads. Cells were cultured (10(5) cells/mL) in semisolid media with rIL-2 (100 mu/mL), LMW-BCGF (0.1 mu/mL), and the combination of rIL-2 plus LMW-BCGF, respectively, for seven to ten days. Pooled colonies were harvested for phenotyping. LMW-BCGF plus rIL-2 induced large numbers of CD3+ colonies from CD3- precursors. rIL-2 alone did not induce colony formation. In addition, cells were cultured in liquid media with LMW-BCGF, rIL-2, and the combination of LMW-BCGF plus rIL-2, respectively, for seven to 21 days. They were harvested for phenotyping, and cytotoxicity assays were performed v K562, Raji, and autologous leukemic cells. LMW-BCGF plus rIL-2 induced significant expansion of CD3+ cells from CD3- precursors, and these cells were activated to kill autologous leukemic cells in addition to Raji and K562 cell lines. LMW-BCGF or rIL-2 alone did not induce significant expansion or activation of cytotoxic CD3- cells. Our hypothesis is that LMW-BCGF plus rIL-2 stimulates the proliferation and activation of CD3- precursors from the BM cells of children with acute leukemia to become CD3+ cells that have LAK activity. This finding may have therapeutic implications.     

Interleukin-7 is a growth factor of precursor B and T acute lymphoblastic leukemia

We investigated the proliferation-inducing effects of human recombinant interleukin-7 (IL-7) on acute lymphoblastic leukemia (ALL) cells. It is shown that IL-7 stimulates DNA synthesis in ALL cells of B-cell precursor (n = 5) as well as immature T-cell origin (n = 2). Cytogenetic analysis of the cells of four patients proliferating in IL7-supplemented cultures established the leukemic descendence of the IL-7-responsive cells. 125I-IL-7 binding experiments with the cells of one patient and with two ALL cell lines showed the presence of two types of IL-7 receptors: one with a high affinity (kd 29 to 51 pmol/L) and one with a low affinity (kd 2.3 to 76 nmol/L) for the ligand. We conclude that IL-7 is one of the cytokines involved in the complex regulation of ALL cell proliferation.     

B-cell growth factor: distinction from T-cell growth factor and B-cell maturation factor.

A T-cell hybridoma was derived by somatic cell hybridization between concanavalin A-activated BALB/c spleen cells and the AKR thymoma BW 5147. Media conditioned by hybridoma cells, even at high dilutions (1:1,000) support the growth of lipopolysaccharide-stimulated B-cell blasts but not that of T-cell growth factor (TCGF)-reactive T-cells. This activity, herein designated B-cell growth factor (BCGF), has a Mr of approximately equal to 20,000 and it can readily be separated from TCGF (Mr approximately equal to 30,000) by gel filtration. BCGF is constitutively produced by the hybridoma cells, it is removed from conditioned media by incubation with target cells at +4 degrees C, and it is equally effective on B-cell blasts carrying different major histocompatibility complex and Ig haplotypes. BCGF shows no T-cell replacing factor (TRF) activity, and it is poor in supporting the development of Ig-secreting plaque-forming cells in B-cell blast cultures. Terminal maturation, however, can be induced in BCGF-dependent blasts by addition of conditioned media from normal helper T cell cultures, suggesting that two distinct factors are involved in the helper cell-dependent growth and maturation of B lymphocytes.     

Immunoglobulin and T cell receptor gene configuration in acute lymphoblastic leukemia of infancy

We examined immunoglobulin (Ig) heavy chain, K light chain, and T cell receptor (TCR) gamma and beta gene configuration in the leukemic cells from a series of infants aged less than 1 year with acute lymphoblastic leukemia (ALL). Each of these 11 cases demonstrated leukemic cell surface antigens that have been correlated with a B cell precursor phenotype. Of the 11, lymphoblasts of 4 retained the germline configuration of both Ig and TCR loci, whereas 7 had rearranged the Ig heavy chain gene. Two of these seven showed light chain gene rearrangement. TCB beta chain rearrangement had occurred in only one of the 11 patients' tumors. No TCR gamma chain rearrangements were identified. These results are in contrast to earlier studies of B cell precursor ALL in children in which Ig heavy chain gene rearrangements were evident in every case and approximately 40% showed Ig light chain rearrangement as well. In addition, 45% of cases of B cell precursor ALL of children had rearranged their gamma TCR genes, and 20% had rearranged beta. These data suggest that ALL in infancy represents an earlier stage of B cell development than is found in B cell precursor ALL of children. ALL in the infant age group has been associated with the worst prognosis of all patients with ALL. This study suggests that the disease in infants differs not only clinically, but also at the molecular genetic level, from the disease in children.     

Production of B cell growth factor(s) by neoplastic B cells from hairy cell leukemia patients

Recent studies have shown that normal human T cells contain a high- molecular-weight (mol wt) protein exhibiting B cell growth factor (BCGF) activity. Other studies have shown that virally transformed human B cells also secrete a high-mol-wt BCGF-like molecule in vitro. We have studied neoplastic B cells from patients with untreated hairy cell leukemia (HCL) to ascertain whether such cytoplasmic BCGF activity is present in the tumor cells. Studies on HCL cells from four patients indicated that BCGF-like activity was in fact present in the cytosolic extracts when tested on autochthonous HCL cells as well as on normal BCGF-dependent human B cell lines. Chromatographic analysis indicated that the BCGF activity from HCL cells was similar in mol wt as well as function to the normal T cell-derived cytosolic BCGF activity. These studies suggest that HCL cells contain and, in some cases, secrete a high-mol-wt growth factor that can be autostimulatory and appears to resemble a similar growth factor molecule found in normal human T cells.     

Cellular and molecular studies on infant null acute lymphoblastic leukemia

We have studied the cellular and molecular basis of eight cases of infant null acute lymphoblastic leukemia (ALL). All eight patients were under 9 months of age and presented with leukocyte counts in excess of 60 X 10(9)/L, organomegaly, and in two cases CNS infiltration. Although seven cases were morphologically classified as ALL, one patient had both lymphoid and myeloid features. Phenotypic analysis of leukemic blasts from all patients showed a typical null ALL pattern, ie, CD10 (common ALL antigen)-negative, strongly HLA-DR-positive, and CD19 (B4)-positive. The presence of terminal deoxynucleotidyl transferase (TdT) at presentation was positive in six patients' cells and negative in two. Two patients also expressed the myeloid-associated markers CD33 (MY9) and CD15 (TG1), and coexpression of CD19 and CD33 was confirmed in these two by using dual marker flow cytometry (fluorescence-activated cell sorting). Electron microscopic examination of the same two patients' cells showed the presence of monocytoid blasts that labeled with the pan-B cell antibody B4 (CD19). Short-term culture of one of these patients cells in the presence of phorbol ester resulted in the majority of the cells exhibiting myeloid markers, strong nonspecific esterase positivity, and phagocytic properties. Cytogenetic analysis showed the common feature in 7 of 8 cases to be a break in band 11q23. Molecular analysis of DNA from the blast cells of all eight patients showed rearrangement of the immunoglobulin heavy-chain genes in all cases without, however, any evidence of kappa light-chain rearrangement. T cell receptor genes were present in the germline configuration in all cases. Rearrangements of the c-ets 1 oncogene, which maps to band 11q23, were not detected, thus providing no evidence for involvement of this oncogene in the common disease process. Our data indicate that although infant null ALL may present as a heterogeneous disease the similarity of many features between cases suggests a common derivation from a precursor cell sharing phenotypic and genotypic features of both B and myeloid progenitor cells.     

Leukemic B-cell precursors constitutively express functional receptors for human interleukin-1

This study analyzes the expression of functional interleukin-1 (IL-1) receptors on leukemic B-cell precursors (BCPs) from B-cell precursor acute lymphoblastic leukemia (BCP ALL) patients. We first investigated the specific binding of 125I-labeled recombinant IL-1 (125I-rIL-1) (4 x 10(17) cpm/mol) to fresh marrow blasts from 11 BCP ALL patients. In five of 11 cases, the binding of 125I-rIL-1 was significantly blocked by excess cold rIL-1. In these five cases, the cell-bound radioactivity ranged from 146 cpm/10(6) cells to 2,412 cpm/10(6) cells (mean +/- SE = 782 +/- 414 cpm/10(6) cells), indicating that 4 to 60 femtomols (mean +/- SE = 20 +/- 10 femtomols) of 125I-rIL-1 specifically bound per 10(7) cells. The estimated number of 125I-rIL-1 molecules bound per cell ranged from 219 to 3,618 (mean +/- SE = 1173 +/- 621). In all five cases, BCP colony formation was stimulated by 10 ng/mL (570 femtomolar) rIL-1, and the background-subtracted colony numbers ranged from 130 to 298 (mean +/- SE = 226 +/- 31). In contrast, no stimulation was observed in six cases that showed no significant 125I-rIL-1 binding. Hence, there was a high correlation between 125I-rIL-1 binding and IL-1 responsiveness, indicating that functional IL-1 receptors were detected in ligand binding assays. Scatchard plot analysis of the specific equilibrium binding data for leukemic BCPs from two IL-1-responsive BCP ALL cases yielded straight linear regression lines, indicating the existence of a single class of 132 to 154 high affinity IL-1 receptors/cell. The apparent affinity constants (Ka) values ranged from 5.2 x 10(9) mol/L-1 to 1.2 x 10(10) mol/L-1. Notably, the concentrations of IL-1 required for half-maximal receptor occupancy (kd = 83 pmol/L to 190 pmol/L) were approximately three orders of magnitude higher than those needed to elicit a half-maximal proliferative response of leukemic BCPs in colony assays (0.1 to 1.0 ng/mL = 5.7 to 57 femtomolar), indicating that only a small fraction of IL-1 receptors need to be occupied to stimulate leukemic BCPs. Notably, IL-1 unresponsive leukemic BCPs from one BCP ALL patient and two BCP ALL cell lines (REH, KM-3) did not exhibit any significant IL-1 binding (less than 10 IL-1 binding sites/cell), and two additional IL-1 unresponsive BCP ALL cell lines (NALM-6, HPB-NULL) expressed only 24 to 54 IL-1 binding sites/cell with a Ka of 7.8 to 9.8 x 10(9) mol/L-1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spontaneously increased B cell growth factor and B cell differentiation factor activities in the synovial fluid of patients with rheumatoid arthritis.

The presence of factors implicated in B cell proliferation and differentiation was studied in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and from patients with ankylosing spondylitis (AS) and traumatic joint injury. Culture with Staphylococcus aureus Cowan I B cell blasts showed strong B cell growth factor (BCGF) activity in the SF from patients with RA. This BCGF activity was significantly greater than that found in SF from patients with traumatic joint injury and similar to that of patients with AS. The presence of B cell differentiation factor (BCDF) for IgM(mu) in the SF from patients with RA was also demonstrated and was significantly greater than that found in SF from patients with AS and traumatic joint injury. Moreover, a significantly increased BCDF for IgG(gamma) was also found in the SF from patients with RA compared with that observed in those patients with traumatic joint injury, which, however, was similar to that of patients with AS.     

Leukemic B-cell precursors express functional receptors for human interleukin-3

The purpose of this study was to analyze the expression of functional interleukin-3 (IL-3) receptors on leukemic B-cell precursors (BCPs) from 12 BCP acute lymphoblastic leukemia (ALL) patients and five BCP ALL cell lines. The specific binding of biosynthetically labeled 35S-recombinant (r) IL-3 to freshly obtained leukemic marrow blasts was initially investigated. In five of 12 BCP ALL cases, the binding of 35S-rIL-3 was markedly blocked by excess cold rIL-3, and the percentage of inhibitable binding ranged from 53% to 78% (mean +/- SE = 65% +/- 4%). In these cases, the cell-bound radioactivity ranged from 146 cpm/10(7) cells to 1,433 cpm/10(7) cells (mean +/- SE = 627 +/- 250 cpm/10(7) cells), indicating that 1 to 14 femtomole (mean +/- SE = 6 +/- 2 fms) of [35S]rIL-3/10(7) cells were specifically bound (= 60 to 840 molecules per cell). rIL-3 stimulated the proliferative activity of leukemic BCPs in a dose-dependent fashion without inducing differentiation, and the half-maximal stimulatory activity was observed at a concentration of 17 to 34 pmol/L. Fluorescence-activated cell sorter (FACS)-isolated virtually pure populations of CD10+CD19+ leukemic BCPs from two BCP ALL patients, as well as from two of five BCP ALL cell lines, showed a marked proliferative response to highly purified rIL-3, providing formal evidence that the observed IL-3 responses were not mediated by accessory cells. There was a high correlation between [35S]rIL-3 binding and proliferative response in colony assays, indicating that functional IL-3 receptors were detected in ligand binding assays. Scatchard plot analysis of the specific equilibrium binding data for IL-3-responsive leukemic BCPs from one BCP ALL patient and two BCP ALL cell lines yielded a straight linear regression line, indicating the existence of a single class of 60 to 210 high-affinity IL-3 binding sites/cell. The calculated apparent affinity constant (Ka) values ranged from 3.6 x 10(9) to 5.9 x 10(9) mol/L-1. Hence, the concentration of IL-3 required to produce 50% maximal receptor occupancy (Kd) was in the range of 168 to 280 pmol/L. These concentrations are approximately tenfold higher than those required to induce 50% maximal proliferative response from leukemic BCPs in colony assays, indicating that low receptor occupancy is sufficient for growth stimulation of leukemic BCPs by rIL-3. In comparison, less than 10 to 20 IL-3 molecules/cell were bound to IL-3 unresponsive leukemic BCPs even when the concentration of free-[35S]rIL-3 was as high as 2 nmol/L.(ABSTRACT TRUNCATED AT 400 WORDS)     

Functional heterogeneity of B-CLL lymphocytes: dissociated responsiveness to growth factors and distinct requirements for a first activation signal

We studied the effects of B cell directed growth factors on B lymphocytes from 11 patients with chronic lymphocytic leukemia (B-CLL). B-CLL lymphocytes were costimulated with anti-mu antibody (Ab) and with three growth factor preparations: recombinant IL2, B cell growth factor (BCGF) (20 kiloDalton (kD) BCGF) and a high molecular weight BCGF (50 kD BCGF). IL2 was the more active factor (in six of 11 patients). The effect of IL2 was dependent on a costimulation with anti-mu Ab or occurred independently of anti-mu Ab, according to the patients. This pattern of reactivity did not correlate with the presence or absence of the IL2 receptor (IL2-R) molecule on fresh B-CLL lymphocytes. Five patients responded to the 20 kD BCGF. Although four of them were also strong responders to IL2, one strongly responded to the 20 kD BCGF and did not respond to IL2. Only one patient responded to the 50 kD BCGF. When an anti-IL2-R Ab was introduced into the culture, only the responsiveness to IL2 was abolished: thus both 20 kD and 50 kD BCGFs activate B-CLL lymphocytes independently of the IL2-R. These results show that several B cell directed growth factors can act independently to support the proliferation of B-CLL lymphocytes.     

Effects of human FLT3 ligand on myeloid leukemia cell growth: heterogeneity in response and synergy with other hematopoietic growth factors

A novel hematopoietic growth factor for primitive hematopoietic progenitor cells, the ligand for the flt3/flk2 receptor, (FL), has been recently purified and its gene has been cloned. In the present study, we investigated the effects of FL on the proliferation and differentiation of normal and leukemic myeloid progenitor cells. We demonstrate that FL is a potent stimulator of the in vitro growth of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 3 (IL-3), or G-CSF-dependent granulocyte-macrophage committed precursors from Lin- CD34+ bone marrow cells of normal donors. By contrast, FL does not affect the growth of erythroid-committed progenitors even in the presence of erythropoietin. The effect of FL on the proliferation and on the in vitro growth of clonogenic leukemic precursor cells was studied in 54 acute myeloid leukemia (AML) cases. Fresh leukemia blasts from 36 of 45 patients with AML significantly responded to FL without any relation to the French-American-British (FAB) subtype. FL stimulated the proliferation of leukemic blasts in a dose-dependent fashion. Synergistic activities were seen when FL was combined with G-CSF, GM-CSF, IL-3, or stem cell factor (SCF). FL as a single factor induced or increased significantly colony formation by clonogenic precursor cells from 21 of 24 patients with AML. In the presence of suboptimal and optimal concentrations of G-CSF, GM-CSF, IL3, SCF, or a combination of all factors, FL strongly enhanced the number of leukemic colonies (up to 18-fold). We also evaluated the induction of tyrosine phosphorylated protein on FL stimulation in fresh AML cells. We demonstrate that, on FL stimulation, a band of phosphorylated protein(s) of about 90 kD can be detected in FL- responsive, but not in FL-unresponsive cases. This study suggests that FL may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor with other cytokines.