Low Molecular Weight B Cell Growth Factor Research Articles

Role in growth regulation of cytokines and cytokine receptors in acute lymphoblastic leukaemia expressing myeloid markers

The biological roles of cytokines and cytokine receptors were examined in acute lymphoblastic leukaemia cells expressing myeloid antigens (My+ ALL). Interleukin-3 (IL-3), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), and low molecular-weight B-cell growth factor (LW-BCGF) could induce DNA synthesis in certain cases of My+ ALL. Whereas in My- ALL the stimulatory effects were shown only with LW-BCGF. Acute myelogenous leukaemia (AML) cells were activated with multiple cytokines including interleukin-1 (IL-1), IL-3, G-CSF and GM-CSF. Specific receptors for IL-1 and IL-3 were strongly expressed on both My+ and My- ALL cells. These receptors, however, were weakly detectable on AML cells. Additionally we studied the production of tumour necrosis factor-alpha and IL-1 by leukaemic blasts and found that distinct amounts of both cytokines were released from My+ ALL cells and AML cells, but not from My- ALL cells. The profiles of cytokines and cytokine receptors expressed by My+ ALL showed both similarities and differences to those in My- ALL or AML. The proliferation of My+ ALL cells was dependent on multiple cytokines that would regulate growth and maturation in a lineage-restricted fashion. These data suggested that My+ ALL cells might originate from uncommitted haematopoietic precursor cells coexpressing features of both lymphoid and myeloid lineages.

Complement and virus-specific antibody-dependent infection of normal B lymphocytes by human immunodeficiency virus type 1

We tested the susceptibility of human purified, normal B lymphocytes to human immunodeficiency virus type 1 (HIV-1) infection, in the presence or absence of complement-sufficient serum and of virus-specific antibodies. Virus replication was detected when cells were infected in the presence of both complement and anti-HIV antibodies (C'-ADE conditions), by day 2 postinfection. Similar results were obtained when B lymphocytes were purified either from peripheral blood (three healthy donors) or from tonsils (four individuals with chronic tonsillitis). HIV infection was shown by polymerase chain reaction (PCR) detection of proviral sequences (gag and pol genes), by p24 antigen synthesis, and by cocultivation assay with MT2 cells. The higher p24 production was obtained when B cells were preactivated for 2 days by phorbol 12-myristate 13-acetate (PMA) before infection and then cultured in the presence of low-molecular weight B-cell growth factor (LMW-BCGF). Expression of virus envelope glycoprotein (gp) 120 could also be detected on a subpopulation of B cells (CD19+, CD22+) by flow cytometry. Blocking experiments with monoclonal antibodies (MoAbs) against CD4, CD21 (complement receptor 2 [CR2]), CD35 (CR1), CD19, and CD5 surface molecules indicated that infection of B cells involves CD4, CD21, and CD35 antigens. Indeed, blocking of CD4 receptor inhibited 10% of p24 production, and blocking of both CD21 and CD35 led to extinction of p24 signal. CR-dependent pathway is thus a major route for C'-ADE of HIV infection in normal B cells. Our results emphasize the importance of studying interactions between HIV and the complement system for better understanding infection mechanisms and the major dysfunctions of B cells in HIV-infected individuals.

Complement and Virus-Specific Antibody-Dependent Infection of Normal B Lymphocytes by Human Immunodeficiency Virus Type 1

Abstract We tested the susceptibility of human purified, normal B lymphocytes to human immunodeficiency virus type 1 (HIV-1) infection, in the presence or absence of complement-sufficient serum and of virus-specific antibodies. Virus replication was detected when cells were infected in the presence of both complement and anti-HIV antibodies (C'-ADE conditions), by day 2 postinfection. Similar results were obtained when B lymphocytes were purified either from peripheral blood (three healthy donors) or from tonsils (four individuals with chronic tonsillitis). HIV infection was shown by polymerase chain reaction (PCR) detection of proviral sequences (gag and pol genes), by p24 antigen synthesis, and by cocultivation assay with MT2 cells. The higher p24 production was obtained when B cells were preactivated for 2 days by phorbol 12- myristate 13-acetate (PMA) before infection and then cultured in the presence of low-molecular weight B-cell growth factor (LMW-BCGF). Expression of virus envelope glycoprotein (gp) 120 could also be detected on a subpopulation of B cells (CD19+, CD22+) by flow cytometry. Blocking experiments with monoclonal antibodies (MoAbs) against CD4, CD21 (complement receptor 2 [CR2]), CD35 (CR1), CD19, and CD5 surface molecules indicated that infection of B cells involves CD4, CD21, and CD35 antigens. Indeed, blocking of CD4 receptor inhibited 10% of p24 production, and blocking of both CD21 and CD35 led to extinction of p24 signal. CR-dependent pathway is thus a major route for C'-ADE of HIV infection in normal B cells. Our results emphasize the importance of studying interactions between HIV and the complement system for better understanding infection mechanisms and the major dysfunctions of B cells in HIV-infected individuals.

Intracellular Signaling Events Associated with the Induction of DNA Synthesis in Human B Lymphocytes: I. Stimulation of PKC-Dependent and -Independent Pathways by LMW-BCGF

The low molecular weight B cell growth factor (LMW-BCGF) induces the G1 → S transition in human B lymphocytes activated by a first signal, Stāphylococcus aureus Cowan (SAC) or anti-μ antibody. It also stimulates proliferation of normal long-term B cell lines and some B cell tumors. We have previously reported that LMW-BCGF induces the hydrolysis of polyphospho-inositides (PI) and a rise in intracellular free calcium concentration, through the generation of inositol trisphosphate (InsP 3) (Renard et al., Eur, J. Immunol. 18, 1705, 1988). In the present work we have analyzed the possible association between early signaling events elicited by LMW-BCGF in SAC-activated B cells and its ability to provoke DNA synthesis, notably at the level of phospholipase C (PLC) and protein kinase C (PK-C) activation. Inhibitors of PLC and of InsP 3-induced calcium release were found to block LMW-BCGF-dependent DNA synthesis. An increase in membrane-associated protein kinase C (PK-C) activity was detected after the addition of the growth factor and the mitogenic effect of LMW-BCGF was partially suppressed when B cell blasts were incubated with staurosporine or H-7, two inhibitors of PK-C activity. In addition, the mitogenic effect due to the addition of LMW-BCGF was not modified by the incubation of B cell blasts with high concentrations of TPA, even if this treatment inhibited cellular response to a low concentration of TPA. LMW-BCGF also increased intracellular pH in B cell blasts and lymphokine-induced mitogenic activity was reduced when the Na +/H + amiloride or ethylisopropyl amiloride (EIPA) antiport blockers were added. These results suggest that (i) LMW-BCGF-induced PI breakdown and Ca 2+ mobilization and cell alkalinization are associated with the induction of cell proliferation, and (ii) the activation of PK-C does not appear to be the sole pathway activated by LMW-BCGF.

Proliferative responses of a bovine leukemia virus-infected lymphoblastoid B-cell line by its culture supernatant and cytokines.

The growth-promoting activity in the culture supernatant of bovine lymphoblastoid B-cell lines (BL2M3 and BL312) were examined. BL2M3 cells proliferated well in response to conditioned medium (CM) obtained from BL2M3 and BL312 cell cultures. These BL2M3 and BL312 CM were used as sources of BL2M3 cell growth-promoting factor (BL2M3-GPF). BL2M3-GPF was sensitive to acid (pH 2) and alkali (pH 10) and was heat-labile. Proliferative responses of BL2M3 cells were not induced by human recombinant (r)IL 1, rIL 2, rIL 6, granulocyte-colony stimulating factor (rG-CSF) or tumor necrosis factor (rTNF)-alpha. Human low molecular weight B cell-growth factor (LMW-BCGF) was, however, capable of augmenting the proliferation of BL2M3 cells. BL2M3 cells formed clusters in response to LMW-BCGF, whereas they showed single and discete appearance in the presence of BL2M3-GPF. These results suggested that bovine lymphoblastoid B-cell lines might release and respond to the growth-promoting factor for in vitro proliferation of its own cell line, BL2M3.

Induction of proliferation by high molecular weight B cell growth factor or low molecular weight B cell growth factor is associated with increases in intracellular calcium in different subpopulations of human B lymphocytes

The activation of resting B cells with anti-surface Ig is associated with transient increases in intracellular calcium. In the present study, we demonstrate that stimulation of B cells which have already been activated by Staphylococcus aureus Cowan I (Sac), with high molecular weight B cell growth factor (HMW-BCGF) or low molecular weight B cell growth factor (LMW-BCGF), but not IL-2, IL-4, or interferon-γ, is associated with an increase in intracellular calcium, which is modest compared to that seen with anti-Ig (∼100 n M vs ∼400 n M). The increases in intracellular calcium induced by HMW-BCGF or LMW-BCGF occur in distinct but overlapping subpopulations of B cells. Thus, increases in intracellular calcium in human B cells occur not only upon activation but also upon the induction of proliferation by certain (but not all) B cell growth factors. Presumably, the effect of increasing intracellular calcium during the induction of proliferation is to modify a different group of intracellular molecules than those induced during activation.

Effect of a low-molecular-weight B cell growth factor on the proliferation of normal and neoplastic lymphocytes in lymphomas

The usefullness of low-molecular-weight B-cell growth factor (LMW-BCGF) for routine cytogenetic study of B cell non-Hodgkin lymphomas (NHL) was evaluated. Eighteen B cell lymphoma specimens were cultured for 4 days in the presence or absence of LMW-BCGF. After culture, conventional cytogenetic analysis was carried out, and the Morphology Antibody Chromosomes (MAC) method was used to study the frequencies of mitotic cells in different lymphocyte subsets. Our results showed that in 6 of the 14 lymphomas with a chromosomally abnormal clone LMW-BCGF produced an increased frequency of abnormal mitosis. In eight cases, LMW-BCGF did not increase, or even decrease, the frequency of karyotypically abnormal mitoses. In these eight cases, the total number of mitosis was often increased, but the mitotic cells were shown to be T cells. LMW-BCGF seems to have a clearly favorable effect on the cytogenetics of some B cell NHL, but apparently it is not a radical improvement over the routine techniques now being used.

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.

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.

Assay of lymphokine-activated killer activity generated from bone marrow cells of children with acute lymphoblastic leukemia

We recently reported that low molecular weight B-cell growth factor (LMW-BCGF) plus recombinant interleukin-2 (rIL-2) synergistically induced lymphokine-activated killer (LAK) activity from the bone marrow (BM) cells of children with acute lymphoblastic leukemia (ALL). The kinetics of cell growth, antigenic phenotype, and lytic activity of the generated effector cells were further analyzed in this study. BM cells from ALL patients with active disease and in complete remission (CR) were cultured with a combination of LMW-BCGF and rIL-2. Monoclonal antibodies (anti-CD3 and anti-Leu 19) and immunomagnetic beads were used to separate LAK cells into three subsets: CD3+/Leu 19-, CD3+/Leu 19+, and CD3-/Leu 19+. Cytotoxicity assays with different subsets were performed versus K562, Raji, and autologous leukemic cells, using a 3- hour 51Cr release test. There was a significant cell expansion of 54- fold (mean value) for CD3+ cells and 15-fold for Leu 19+ cells in culture with LMW-BCGF plus rIL-2 for 7 to 14 days, whereas no cell expansion was observed in culture with rIL-2 alone. Although NK activity (K562) was generated from leukemic BM cells in culture with rIL-2 alone, it is only about one third of that generated in culture with rIL-2 plus LMW-BCGF. Analysis of lytic activity of cells generated in the latter cultures demonstrated that CD3-/Leu 19+ cells expressed highest lytic activity against NK-sensitive K562 cells as well as against NK-resistant Raji cells. CD3+/Leu 19+ cells showed median cytotoxicity, and CD3+Leu 19- cells mediated only minimal cytotoxic activity. Also, lytic activity of CD3-/Leu 19+ cells against autologous leukemic blasts was noted in patients with active disease. Our results demonstrate that LAK activity generated from BM cells by LMW-BCGF and r- IL2 is mediated mainly by two types of Leu 19+ cells: CD3-/Leu 19+ NK cells and CD3-/Leu 19+ T cells. Although CD3+ T cells (both Leu 19+ and Leu 19-) mediated less antitumor cytotoxicity than CD3-/Leu 19+ cells, the former cells were the major expanding cell population in culture with LMW-BCGF and rIL-2. The new culture system may be effective in generation of cells with LAK activity for therapeutic use.

Assay of Lymphokine-Activated Killer Activity Generated From Bone Marrow Cells of Children With Acute Lymphoblastic Leukemia

We recently reported that low molecular weight B-cell growth factor (LMW-BCGF) plus recombinant interleukin-2 (rIL-2) synergistically induced lymphokine-activated killer (LAK) activity from the bone marrow (BM) cells of children with acute lymphoblastic leukemia (ALL). The kinetics of cell growth, antigenic phenotype, and lytic activity of the generated effector cells were further analyzed in this study. BM cells from ALL patients with active disease and in complete remission (CR) were cultured with a combination of LMW-BCGF and rIL-2. Monoclonal antibodies (anti-CD3 and anti-Leu 19) and immunomagnetic beads were used to separate LAK cells into three subsets: CD3+/Leu 19-, CD3+/Leu 19+, and CD3-/Leu 19+. Cytotoxicity assays with different subsets were performed versus K562, Raji, and autologous leukemic cells, using a 3-hour 51Cr release test. There was a significant cell expansion of 54-fold (mean value) for CD3+ cells and 15-fold for Leu 19+ cells in culture with LMW-BCGF plus rIL-2 for 7 to 14 days, whereas no cell expansion was observed in culture with rIL-2 alone. Although NK activity (K562) was generated from leukemic BM cells in culture with rIL-2 alone, it is only about one third of that generated in culture with rIL-2 plus LMW-BCGF. Analysis of lytic activity of cells generated in the latter cultures demonstrated that CD3-/Leu 19+ cells expressed highest lytic activity against NK-sensitive K562 cells as well as against NK-resistant Raji cells. CD3+/Leu 19+ cells showed median cytotoxicity, and CD3+Leu 19- cells mediated only minimal cytotoxic activity. Also, lytic activity of CD3-/Leu 19+ cells against autologous leukemic blasts was noted in patients with active disease. Our results demonstrate that LAK activity generated from BM cells by LMW-BCGF and r-IL2 is mediated mainly by two types of Leu 19+ cells: CD3-/Leu 19+ NK cells and CD3-/Leu 19+ T cells. Although CD3+ T cells (both Leu 19+ and Leu 19-) mediated less antitumor cytotoxicity than CD3-/Leu 19+ cells, the former cells were the major expanding cell population in culture with LMW-BCGF and rIL-2. The new culture system may be effective in generation of cells with LAK activity for therapeutic use.

Effect of interferon-alpha on the expression and release of the CD23 molecule in hairy cell leukemia

Hairy cells are stimulated to DNA synthesis by low molecular weight B cell growth factor (LMW-BCGF) and this proliferative response is suppressed by interferon (IFN)-alpha, both in vitro and in vivo. The suggestion that the CD23 molecule (Fc epsilon II receptor) might be involved in the signalling pathway of LMW-BCGF prompted us to study the expression of this molecule on hairy cells and its modulation by IFN- alpha. By flow cytometry and direct binding experiments with anti CD23 monoclonal antibodies, the presence of the CD23 antigen was detected in 7 of 12 cases tested, on variable percentages of cells, ranging from low to medium expression. In vitro incubation of hairy cells with IFN- alpha, which elicits a suppression of the proliferative response of these cells to LMW-BCGF, induced a parallel significant reduction of CD23 expression in only three cases. Similarly, a transient in vivo decrease of CD23 expression, concommitant with an inhibition of the LMW- BCGF response, could be detected in only one of three patients injected with IFN-alpha. Soluble sCD23/IgE-binding factor (BF) was quantitated in the serum from six other patients with hyperleukocytic hairy cell leukemia (HCL) undergoing a clinical trial of IFN-alpha therapy. Before treatment, these patients presented higher concentrations of the cleaved soluble form of the CD23 molecule than normal controls. Within a few weeks of IFN-alpha administration, these levels markedly decreased, paralleling a diminution of blood leukemic cells. Of interest, no such diminution was noticed for another patient resistant to IFN-alpha therapy. These results show that the proliferative response of hairy cells to LMW-BCGF is not linked to the expression of the CD23 marker. Besides, when the latter molecule was present, its decrease following IFN-alpha treatment, which could be detected in some cases, was not necessarily required for the suppression of the LMW-BCGF response and is thus not mandatory for the therapeutic efficacy of IFN- alpha. Our results point out that quantitation of serum sCD23/IgE-BF, whether related to a process of autocrine proliferation or not, is a parameter of potential importance for therapy monitoring.

Induction of human IgE synthesis by CD4+ T cell clones. Requirement for interleukin 4 and low molecular weight B cell growth factor.

We have analyzed in detail the precise requirements for the induction of human IgE synthesis using several experimental approaches with purified B cells and well-characterized alloantigen-specific CD4+ T cell clones expressing different profiles of lymphokine secretion. Using these clones under cognate conditions in which the B cells expressed alloantigens recognized by the cloned T cells, we have confirmed that IL-4 is required for the induction of IgE synthesis, but we have clearly demonstrated that IL-4 by itself is not sufficient. With several cloned CD4+ T cell lines, including an IL-4-producing clone that could not induce IgE synthesis, and cloned T cells pretreated with cyclosporin A to inhibit lymphokine synthesis, we showed that Th cell-B cell interactions are necessary for IgE synthesis, and that low molecular weight B cell growth factor (LMW-BCGF) and IL-4, in combination, are lymphokines of major importance in the induction of IgE synthesis. Together our results indicate that optimal induction of an IgE-specific response requires the exposure of B cells to a particular complex of signals that include (a) a signal(s) involving Th-B cell interaction that primes B cells to receive additional signals from soluble lymphokines, (b) a specific B cell proliferative signal provided by LMW-BCGF, and (c) a specific B cell differentiation signal provided by IL-4.

Potentiation of the proliferative response of human B lymphocytes to low molecular weight B cell growth factor (LMW-BCGF) by fibroblast growth factors (FGFs)

Both acidic and basic fibroblast growth factor (FGF), although devoid alone of growth-promoting ability on resting or activated human lymphoid B cells, were found to markedly increase the proliferative response of anti-μ-chain or SAC preactivated B cell blasts to the low molecular weight B cell growth factor (LMW-BCGF) and to enhance the costimulatory response of resting B cells to anti-μ-chain and LMW-BCGF. This potentiating effect was also observed for a LMW-BCGF-dependent B cell tumor derived from a lymphocytic nodular lymphoma. Other growth factors acting on fibroblasts, such as epidermal growth factor, α-thrombin, platelet-derived growth factor, and insulin-like growth factor-I did not display such enhancing effect on LMW-BCGF-driven proliferation. Activated, but not resting B cells were found to bear receptor sites for FGFs and from kinetics experiments, it is suggested that LMW-BCGF induces competence expression for FGFs in those cells. Moreover, the LMW-BCGF-elicited generation of inositol-trisphosphate resulting from polyphosphoinositides hydrolysis was increased in the presence of FGF.

Biphenotypic leukemic lymphocyte precursors in CD2+CD19+ acute lymphoblastic leukemia and their putative normal counterparts in human fetal hematopoietic tissues

During detailed immunophenotypic analyses of marrow blasts from 336 acute lymphoblastic leukemia (ALL) patients, a very small percentage of cases reactive with B-cell-directed as well as T-cell-directed monoclonal antibodies (MoAbs) were identified. Five ALL cases were biphenotypic since they coexpressed CD2 (Tp50) and CD19 (Bp95) antigens at the single-cell level. The composite immunophenotype of these biphenotypic ALL cases was [TdT+HLA-ABC+CD2+CD3-CD10+CD13-CD14-CD16- CD19+CD20+ ++-CD21-CD33-CD34+Bgp95-C mu- slg-]. Low-molecular-weight B- cell growth factor (LMW-BCGF), recombinant interleukin-2 (rIL-2), and rIL-3 stimulated the proliferative activity of biphenotypic leukemic lymphocyte precursors without inducing differentiation. In the presence of the phorbol ester TPA, leukemic blasts from two cases differentiated along the B precursor pathway to the [CD2-CD10+CD19+CD20+C mu+slg-] pre- B cell stage. Biphenotypic ALL cases did not share a common configuration and gene rearrangement pattern of the immunoglobulin heavy chain genes or T-cell receptor (TCR) genes. Three cases had rearranged C mu genes but germline TCR genes, one case showed rearrangement of both C mu and TCR genes, and the remaining case had rearranged TCR genes but germline C mu genes. All five patients attained prompt remission after standard induction chemotherapy. Three to four years after initial diagnosis, four patients are now off chemotherapy and remain alive in their first remission. One patient relapsed at 3 years, 7 months, but promptly achieved complete remission after reinduction chemotherapy and remains in second remission off chemotherapy greater than 3 years after her reinduction therapy. With two-color immunofluorescence staining techniques and multiparameter flow cytometric analyses, we identified a small population of CD2+CD19+ lymphoid cells in fetal livers (FLs) and fetal bone marrows (FBMs), which may represent the putative normal counterparts of biphenotypic ALL blasts. A CD2+CD19+ normal biphenotypic lymphoid precursor cell line, designated FL 8.2 CD2+, was established from an FL of 8-weeks of gestational age by Epstein-Barr virus (EBV)-induced blastoid transformation. The composite immunophenotype of FL 8.2 CD2+ cell line was [TdT+HLA-ABC+HLA-DR+ CD2+CD5-CD7-CD10+/-CD13-CD19+CD20-CD21+ CD22+CD33-CD34+/-Bgp95-CDw40+C mu-slgD-slgM-]. FL 8.2 CD2+ cells showed germline patterns of immunoglobulin heavy-chain joining region, heavy- chain constant region, kappa light-chain constant region genes, and TCR beta-chain genes. Cross-linking of CD2 as well as CD19 antigens on FL 8.2 CD2+ cells caused an increase of intracellular ionized calcium.(ABSTRACT TRUNCATED AT 400 WORDS)

Biphenotypic Leukemic Lymphocyte Precursors in CD2+CD19+ Acute Lymphoblastic Leukemia and Their Putative Normal Counterparts in Human Fetal Hematopoietic Tissues

During detailed immunophenotypic analyses of marrow blasts from 336 acute lymphoblastic leukemia (ALL) patients, a very small percentage of cases reactive with B-cell-directed as well as T-cell-directed monoclonal antibodies (MoAbs) were identified. Five ALL cases were biphenotypic since they coexpressed CD2 (Tp50) and CD19 (Bp95) antigens at the single-cell level. The composite immunophenotype of these biphenotypic ALL cases was [TdT+HLA-ABC+CD2+CD3-CD10+CD13-CD14-CD16-CD19+CD20+ ++-CD21-CD33-CD34+Bgp95-C mu- slg-]. Low-molecular-weight B-cell growth factor (LMW-BCGF), recombinant interleukin-2 (rIL-2), and rIL-3 stimulated the proliferative activity of biphenotypic leukemic lymphocyte precursors without inducing differentiation. In the presence of the phorbol ester TPA, leukemic blasts from two cases differentiated along the B precursor pathway to the [CD2-CD10+CD19+CD20+C mu+slg-] pre-B cell stage. Biphenotypic ALL cases did not share a common configuration and gene rearrangement pattern of the immunoglobulin heavy chain genes or T-cell receptor (TCR) genes. Three cases had rearranged C mu genes but germline TCR genes, one case showed rearrangement of both C mu and TCR genes, and the remaining case had rearranged TCR genes but germline C mu genes. All five patients attained prompt remission after standard induction chemotherapy. Three to four years after initial diagnosis, four patients are now off chemotherapy and remain alive in their first remission. One patient relapsed at 3 years, 7 months, but promptly achieved complete remission after reinduction chemotherapy and remains in second remission off chemotherapy greater than 3 years after her reinduction therapy. With two-color immunofluorescence staining techniques and multiparameter flow cytometric analyses, we identified a small population of CD2+CD19+ lymphoid cells in fetal livers (FLs) and fetal bone marrows (FBMs), which may represent the putative normal counterparts of biphenotypic ALL blasts. A CD2+CD19+ normal biphenotypic lymphoid precursor cell line, designated FL 8.2 CD2+, was established from an FL of 8-weeks of gestational age by Epstein-Barr virus (EBV)-induced blastoid transformation. The composite immunophenotype of FL 8.2 CD2+ cell line was [TdT+HLA-ABC+HLA-DR+ CD2+CD5-CD7-CD10+/-CD13-CD19+CD20-CD21+ CD22+CD33-CD34+/-Bgp95-CDw40+C mu-slgD-slgM-]. FL 8.2 CD2+ cells showed germline patterns of immunoglobulin heavy-chain joining region, heavy-chain constant region, kappa light-chain constant region genes, and TCR beta-chain genes. Cross-linking of CD2 as well as CD19 antigens on FL 8.2 CD2+ cells caused an increase of intracellular ionized calcium.(ABSTRACT TRUNCATED AT 400 WORDS)

The p24 leucocyte membrane antigen: modulation associated with lymphocyte activation and differentiation.

Monoclonal antibodies of the CD9 cluster recognize a 24 kD protein (p24) found on platelets and endothelium, and expressed by lymphocytes at restricted stages of maturation and activation. In this study, we explore the possibility that p24 is involved in the response of lymphocytes to signals delivered by lymphokines. p24 is expressed only very weakly by resting B lymphocytes. We found no increase in expression when cells were activated with anti-immunoglobulin together with interleukin-4, or induced to proliferate by low-molecular weight B cell growth factor (LMW-BCGF). Culture of activated B cells with B cell differentiation factor was associated with an increased mean expression of p24. In cells from a patient with chronic lymphocytic leukaemia (CLL), culture with LMW-BCGF up-regulated p24 expression. Resting T cells (p24-negative) were induced to express p24 strongly when activated with antibody against CD3. CD9 antibody did not modulate B or T cell responses to activation stimuli. The results suggest that the p24 molecule is not involved in the primary interaction of cells with lymphokine, but rather may be involved in a secondary reaction, such as ion flux, which follows as a consequence of the action of lymphokines on cells.

Activation of the phosphatidylinositol metabolic pathway by low molecular weight B cell growth factor.

The possible role of phosphatidylinositol breakdown in the induction of proliferation of human activated B cells by low molecular weight B cell growth factor (LMW-BCGF) was examined. LMW-BCGF was found to induce a rapid rise in the concentration of inositol trisphosphate (InsP3) in [3H]inositol-loaded B cell blasts, obtained by prior anti-mu antibody activation. A concomitant decrease in the concentration of phosphatidylinositol 4,5-bisphosphate could be detected at the same time. Maximum generation of InsP3 occurred within 15-30 s after the addition of the LMW-BCGF ligand to the activated B cells, then was followed by a slow decrease and return to control values. The amount of InsP3 generated by phosphatidylinositol hydrolysis was dependent on the concentration of LMW-BCGF. This effect was only detected in B cells already preactivated by a first signal such as anti-mu antibody and not in resting unstimulated B cells. In contrast, under similar conditions, interleukin 2, another B cell growth-promoting lymphokine, did not alter the rate of formation of the various phosphatidylinositol breakdown products. An augmentation of the [Ca2+]i concentration was also detected in activated B cells upon addition of LMW-BCGF and this increase could be blocked by TMB-8, a specific inhibitor of endoplasmic reticulum calcium release. Hydrolysis of phosphoinositides thus represents an essential component in the mechanism of transduction of the signal provided by LMW-BCGF.

Immunobiologic differences between normal and leukemic human B-cell precursors.

The early stages of normal human B-cell differentiation were studied by flow cytometry and cell sorting based on expression of CD10 (CALLA) and CD19 antigens in fetal liver. Both CD10+ CD19+ and CD10+ CD19- precursor populations proliferated in vitro to form B-cell precursor colonies under stimulation from low molecular weight B-cell growth factor (L-BCGF) or recombinant interleukin 3 but did not respond to high molecular weight B-cell growth factor (H-BCGF). The colonies derived from the CD10+ CD19- fraction showed induction of CD19 expression in 10-50% of growing cells, suggesting that CD10 expression precedes CD19 expression in B-cell ontogeny. This hypothesis was corroborated by less-differentiated marker profiles of the progeny of CD10+ CD19- B-cell precursors as compared to CD10+ CD19+ B-cell precursors in BCGF-stimulated cultures and by higher percentages of CD10+CD19- versus CD10-CD19+ B-cell precursors. CD19 crosslinking on normal fetal liver or bone marrow B-cell precursors was associated with an increase in cytoplasmic calcium concentration, but was inhibitory for colony formation. Leukemic B-cell precursors from acute lymphoblastic leukemias (ALLs) differed from normal B-cell precursors in their in vitro proliferative responses, since (i) they responded not only to L-BCGF and rIL-3 but also to H-BCGF and (ii) their proliferation was stimulated rather than inhibited by CD19 crosslinking. A clonogenic leukemic counterpart for the CD10+CD19- normal B-cell precursor population does not exist among malignant cells from B-cell precursor ALL patients, suggesting that the CD19 receptor may be involved in leukemogenesis of human B-cell precursor ALL.

B lymphocyte/carcinoma antigen (BLCa): functional study in B cells.

BLCa is an antigen expressed on the surface of B lymphocytes and certain carcinomas. In this study we have demonstrated that BLCa is distinct from known B cell antigens classified by 'clusters of differentiation' (CD). In particular, the monoclonal antibody MA6, which identifies BLCa, can be distinguished from antibodies of the CDw40 group, which detect an antigen of similar molecular weight expressed also on B cells and certain carcinomas. The expression of BLCa on B cells was measured as tonsil B cells were activated (by anti-Ig and interleukin-4 (IL-4)), induced to proliferate (by low molecular weight B Cell Growth Factor (LMW-BCGF), and induced to differentiate (by B Cell Differentiation Factor, BCDF). Expression of BLCa increased in response to LMW-BCGF. The effect of inclusion of MA6 antibody in cultures with the B cell stimuli was also investigated. MA6 showed an anti-proliferative effect which was antibody-dose dependent, but did not otherwise inhibit or co-operate with anti-Ig. IL-4, LMW-BCGF or BCDF.