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.     

Establishment and characterization of a plasma cell leukaemia cell line dependent for growth on IL-6 and a bi-phenotypic subclone dependent upon both IL-3 and IL-6

Summary A human plasma cell leukaemia cell line (HSM-2) and a subclone (HSM-2.3) have been established from the bone marrow of a patient with bi-phenotypic leukaemia. Proliferation assays using a variety of cytokines demonstrated that HSM-2 proliferated in response to recombinant interleukin-6 (rIL-6), but did not respond to rIL-1, rIL-2, rIL-3, rIL-4, rIL-5, recombinant granulocyte-colony stimulating factor (rG-CSF), or recombinant granulocyte-macrophagecolony stimulating factor (rGM-CSF), and that HSM-2.3 responded to rIL-3 and rIL-6. HSM-2 expressed the CD38 (OKT10), PCA-1, cytoplasmic-IgM, and surface kappa light chain. HSM-2.3 expressed the CD14 (My4), CD33 (My9). CD38 (OKT10), CD19 (B4), CD24 (OKB2), CD10 (J5), PCA-1. HSM-2 and HSM-2.3 are useful tools for analysing the possible role of IL-3 and IL-6 in the oncogenesis of plasma cell leukaemia.     

Mast cells cultured from the peripheral blood of normal donors and patients with mastocytosis originate from a CD34+/Fc epsilon RI- cell population

Mast cells may be cultured from human peripheral blood in the presence of recombinant human stem cell factor (rhSCF). The characteristics of the cells in peripheral blood that give rise to mast cells are unknown. Because mast cell precursors in human marrow are CD34+, human peripheral blood mononuclear cells from patients with mastocytosis and normal controls were sorted on the basis of CD34 expression and the positive and negative cell populations were cultured in rhSCF, recombinant human interleukin-3 (rhIL-3), or both for 6 weeks. Cell cultures were examined every 2 weeks for total and mast cell number and cell differential using Wright Giemsa and acid toluidine blue stains and antibodies to mast cell tryptase and chymase, cell-associated histamine, and expression of CD34, c-kit, Fc epsilon RI, and Fc gamma RII using flow cytometric analysis. The ultrastructural anatomy of mast cells was examined by electron microscopy. Peripheral blood CD34+ cells cultured in rhSCF with or without rhIL-3 gave rise to cell cultures consisting of greater than 80% mast cells by 6 weeks. CD34+ cells cultured in rhIL-3 alone did not give rise to mast cells, whereas rhIL- 3 plus rhSCF increased the final mast cell number eightfold when compared with cells cultured in rhSCF alone. Mast cells increased concomitantly with a decrease in large undifferentiated mononuclear cells. CD34- cells did not give rise to mast cells. Histamine content per cell at 6 weeks was approximately 5 pg. Electron microscopy of 4- week cultures showed immature mast cells containing predominantly tryptase-positive granules that were either homogeneous or contained lattice structures, partial scroll patterns, or central dense cores and mixtures of vesicles, fine granular material, and particles. The CD34+ population at day 0 expressed Kit (65%) and Fc gamma RII (95%), but not Fc epsilon RI, by fluorescence-activated cell sorter analysis. At 6 weeks, CD34+-derived mast cells exhibited Fc epsilon RI in addition to Kit and Fc gamma RII, and were negative for CD34 antigen. Patients with mastocytosis showed a higher number of mast cells per CD34+ cell cultured compared with normal controls. Thus, the mast cell precursor in human peripheral blood is CD34+/Fc epsilon RI- and gives rise to mast cells in the presence of rhSCF with or without rhIL-3, and the number of mast cells arising per CD34+ cell in culture is greater when the CD34+ cells are obtained from patients with mastocytosis compared with normal subjects.     

Adherent lymphokine-activated killer cells suppress autologous human normal bone marrow progenitors

We have generated a homogeneous population of recombinant interleukin-2 (rIL-2)-stimulated effector cells termed adherent lymphokine-activated killer cells (A-LAK) from peripheral blood mononuclear cells (PBMNC) of 14 normal individuals and tested the effect of A-LAK cells on autologous hematopoietic bone marrow (BM) progenitor growth. Enrichment of A-LAK from PBMNC depended on the propensity of A-LAK precursors to adhere to plastic and proliferate in the presence of rIL-2. The resultant population had the morphologic appearance of large granular lymphocytes, and the majority of cells (73% +/- 4%) expressed the CD56+/CD3- phenotype associated with rIL-2-stimulated natural killer (NK) cells. The A-LAK population had potent lytic activity in chromium release assays against both NK-sensitive (K562) and NK-resistant (Raji) targets. When BM mononuclear cells (BMMNC) were coincubated with autologous A-LAK and rIL-2 (1,000 U/mL) added at the start of culture, dose-dependent suppression of burst-forming unit-erythroid (BFU-E) and colony-forming unit mix (CFU-MIX) colony growth was observed at effector to target ratios (E:T) ranging from 0.25:1 to 5:1 (maximal suppression BFU-E = 85% +/- 6%; CFU-MIX = 95% +/- 3%). This suppression was rIL-2 dose-dependent, and no suppression was seen in the absence of rIL-2. Depletion of BM monocytes and T lymphocytes did not alter A-LAK suppression of progenitors coincubated with A-LAK cells. Addition of polyclonal neutralizing antibodies against both interferon-gamma (IFN-gamma) and tumor necrosis facto alpha (TNF-alpha) to the coincubation culture completely abrogated the suppressive effect of A-LAK on BFU-E and CFU-MIX colony growth while each neutralizing antibody used alone had intermediate effects. In contrast to coincubation studies, 36 hours of preincubation of A-LAK cells with autologous BM (E:T 2.2:1) and rIL-2 (1,000 U/mL) followed by plating of preincubated BM cells in hematopoietic progenitor culture produced significant suppression of day 14 BFU-E (47% +/- 5%), but spared the more primitive CFU-MIX (7% +/- 9%), suggesting a divergent effect of A-LAK cells on hematopoietic progenitors at different stages of differentiation. Addition of neutralizing antibodies against IFN-gamma and TNF-alpha in preincubation failed to abrogate the suppressive effect of A-LAK on BFU-E colony growth, suggesting that this suppression occurs by a different mechanism than that seen in coincubation studies. Previous studies have demonstrated that the A-LAK population has cytotoxic and proliferative advantages over other killer cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Impaired in vitro growth of purified (CD34+) hematopoietic progenitors in human immunodeficiency virus-1 seropositive thrombocytopenic individuals.

In this report the role played by human immunodeficiency virus type-1 (HIV-1) in the pathogenesis of HIV-1-related thrombocytopenia was investigated. CD34+ hematopoietic stem/progenitor cells were purified from the bone marrow (BM) of HIV-1(+) thrombocytopenic patients, HIV-1(+) nonthrombocytopenic individuals, HIV-1(-) patients with immune thrombocytopenic purpura, and HIV-1(-) normal donors. CD34+ cells from HIV-1(+) thrombocytopenic individuals alone showed a reduced capacity to give rise to megakaryocytic colonies (CFU-Meg) and also a progressive and significant decline in cell number when placed in liquid culture containing recombinant human interleukin-3 (rIL-3). This decline involved not only megakaryocyte but also erythroid and granulocyte/macrophage progenitors. The defects in megakaryocyte colony formation and CD34+ cell growth did not result from a productive HIV-1 infection of CD34+ cells. Moreover, HIV-1 DNA was absent from CD34+ cells in 10 of 12 thrombocytopenic patients examined. On the other hand, the decreased survival/proliferation of CD34+ cells in liquid culture, within the HIV-1(+) thrombocytopenic patients, was correlated with the presence of HIV-1 p24 antigen in BM plasma. These results demonstrate an impairment of CD34+ cells in HIV-1(+) individuals presenting thrombocytopenia as the only hematologic manifestation. Furthermore, these findings suggest that increased viral replication in the BM microenvironment may cause this impairment and possibly contributes to HIV-induced thrombocytopenia.     

Differentiation of a human eosinophilic leukemia cell line (EoL-1) by a human T-cell leukemia cell line (HIL-3)-derived factor.

Differentiation of a human eosinophilic leukemia cell line, EoL-1, induced by the culture supernatant of a human ATL cell line, HIL-3 (HIL-3 sup) was compared with differentiation induced by defined cytokines. HIL-3 sup induced EoL-1 cells to express eosinophilic granules and segmented nuclei after 6 to 9 days of incubation. HIL-3 sup also induced the expression of Fc epsilon receptor II (Fc epsilon RII/CD23) and an eosinophil differentiation antigen EO-1 mainly on eosinophilic granule (+) cells. Furthermore, HIL-3 sup induced EoL-1 cells to respond to an eosinophil chemotactic factor, platelet activating factor. HIL-3 cells express messenger RNA (mRNA) of interleukin-5 (IL-5), macrophage colony-stimulating factor (M-CSF), and IL-3 but not granulocyte CSF (G-CSF). Granulocyte-macrophage CSF (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) were detected in the HIL-3 sup. Recombinant IL-2 (rIL-2), rIL-3, rIL-4, rIL-5, rM-CSF, and rGM-CSF did not induce eosinophilic granules. rG-CSF induced a few eosinophilic granule (+) cells, and TNF-alpha, which did not induce eosinophilic granules by itself, enhanced the ability of G-CSF to induce them. However, G-CSF and TNF-alpha did not induce the expression of Fc epsilon RII and EO-1 antigen. Moreover, anti-G-CSF, anti-TNF-alpha, anti-GM-CSF, anti-IL-3, and anti-IL-5 antibodies did not suppress the effect of HIL-3 sup on the differentiation of EoL-1 cells. All the data suggest that HIL-3 sup contains an unidentified factor that induces differentiation of EoL-1 cells, and that EoL-1 cells and HIL-3 sup provide an important model for the examination of differentiation mechanisms and functions of eosinophils.     

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.     

Kinetics of the appearance of Fc epsilon RI-bearing cells in interleukin-3-dependent mouse bone marrow cultures: correlation with histamine content and mast cell maturation.

While it is known that mast cells arise from pluripotential hematopoietic cells and express their mature phenotypes in tissues, the sequence of events in maturation is incompletely understood. To study early mast cells, we sorted cells from interleukin-3 (IL-3)-dependent mouse bone marrow cultures on the basis of Fc epsilon RI and examined their morphology, histamine content, and growth characteristics. Flow cytometric analysis and sort showed that the Fc epsilon RI-bearing (Fc epsilon RI+) cells increased from 0% on day 0 to 90% by day 21 and that the total number of Fc epsilon RI+ cells increased from 0 at the start of culture to 3.75 x 10(5) cells by day 21 from an initial population of 1 x 10(5) cells. The dissociation rate of 125I-labeled IgE from early cultured cells resembled the dissociation rate of mouse IgE from mature murine mast cells. Mean fluorescence intensity increased over time, reflecting an increase in IgE receptor density. Fc epsilon RI+ cells were also positive for Fc gamma RII/III. Morphologic studies showed gradual acquisition of metachromatic granules in the Fc epsilon RI+ cells, which was paralleled by an increase in histamine content. Sorted Fc epsilon RI+ cells, when placed in liquid suspension culture, gave rise to pure mast cell populations. Fc epsilon RI+ cells sorted at day 3 and cultured in agarose with IL-3 gave rise to 4,800 small and 150 medium-size mast cell colony-forming units per 10(6) cells, while Fc epsilon RI- cells gave rise to 23 medium-size and 49 large mast cell colony-forming units per 10(6) cells. Fc epsilon RI+ cells grown in granulocyte-macrophage colony-stimulating factor (CSF) or macrophage-CSF did not give rise to colony-forming units. These results show that Fc epsilon RI+ cells have proliferative potential, but that there also is a population of mast cell progenitor cells that have not yet expressed Fc epsilon RI, and such individual progenitor cells have greater potential for proliferation than cells that express Fc epsilon RI.     

Soluble human CD4 elicits an antibody response in rhesus monkeys that inhibits simian immunodeficiency virus replication.

Rhesus monkeys infected with the simian immunodeficiency virus of macaques (SIVmac) demonstrate significant virologic and clinical improvement as a result of treatment with human recombinant soluble CD4 (rsCD4). We show that human rsCD4 does not efficiently inhibit SIVmac replication in bone marrow macrophages of rhesus monkeys and does not significantly augment bone marrow hematopoietic colony formation in vitro. However, plasma of human rsCD4-treated rhesus monkeys does exhibit significant anti-SIVmac activity in vitro. Plasma of these animals efficiently blocks SIVmac replication in peripheral blood lymphocytes and bone marrow macrophages. It also increases granulocyte/macrophage colony formation in vitro by bone marrow cells of SIVmac-infected monkeys. This plasma and the IgG fraction of plasma from a rhesus monkey immunized with human rsCD4 in adjuvant demonstrate reactivity with a soluble form of the rhesus monkey CD4 molecule, exhibit binding to CD4+ but not CD8+ concanavalin A-activated rhesus monkey peripheral blood lymphocytes, and precipitate the CD4 molecule from surface-labeled activated rhesus monkey peripheral blood lymphocytes. Moreover, anti-viral activity is demonstrable in the IgG fraction of plasma from a human rsCD4-immunized monkey. These studies raise the possibility that a modified human CD4 molecule serving as an immunogen might elicit an antibody response that could potentially induce a beneficial therapeutic response in human immunodeficiency virus-infected individuals.     

Polycythemia vera. II. Hypersensitivity of bone marrow erythroid, granulocyte-macrophage, and megakaryocyte progenitor cells to interleukin-3 and granulocyte-macrophage colony-stimulating factor.

Polycythemia vera (PV) is a clonal disease of the hematopoietic stem cell characterized by a hyperplasia of marrow erythropoiesis, granulocytopoiesis, and megakaryocytopoiesis. We previously reported that highly purified PV blood burst-forming units-erythroid (BFU-E) are hypersensitive to recombinant human interleukin-3 (rIL-3). Because these cells may be only a subset, and not representative of marrow progenitors, we have now studied partially purified marrow hematopoietic progenitor cells. Dose-response experiments with PV marrow BFU-E showed a 38-fold increase in sensitivity to rIL-3 and a 4.3-fold increase in sensitivity to recombinant human erythropoietin (rEpo) compared with normal marrow BFU-E. In addition, PV marrow colony-forming units-granulocyte-macrophage (CFU-GM) and CFU-megakaryocyte (CFU-MK) also showed a marked hypersensitivity to rIL-3 and to human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Dose-response curves with rGM-CSF and blood BFU-E showed a 48-fold increase in sensitivity. No effect of rIL-4, rIL-6, human recombinant granulocyte-CSF (rG-CSF), or macrophage-CSF (rM-CSF) was evident, nor was there any effect of PV cell-conditioned medium on normal BFU-E, when compared with normal cell-conditioned medium. Autoradiography with 125I-rEpo showed an increase in Epo receptors after maturation of PV BFU-E to CFU-E similar to that shown with normal BFU-E, but no increase of specific binding of 125I-rIL-3 by PV CD34+ cells was seen compared with normal CD34+ cells. These studies show that PV marrow hematopoietic progenitor cells are hypersensitive to rIL-3 and rGM-CSF, similar to PV blood BFU-E. While the mechanism does not appear to be due to enhanced binding of rIL-3, the hypersensitivity of PV progenitor cells to IL-3 and GM-CSF may be a key factor in the pathogenesis of PV.     

Reduced expression of vascular cell adhesion molecule-1 on bone marrow stromal cells isolated from marrow transplant recipients correlates with a reduced capacity to support human B lymphopoiesis in vitro

A common sequela to allogeneic or autologous bone marrow transplantation (BMT) is a delay in the reconstitution of a functional B-cell immune response. Therefore, we examined whether the posttransplant BM microenvironment is deficient in supporting the proliferation and/or differentiation of B-cell precursors. BM stromal cell cultures were established from patients who received allogeneic or autologous BMT for acute lymphoblastic leukemia, Hodgkin's disease, or non-Hodgkin's lymphoma. These cultures were then compared with normal donor BM stromal cell cultures for expression of adhesion molecules and the capacity to support the adhesion and interleukin-7 (IL-7)-dependent growth of normal B-cell precursors. Analysis of BM stromal cell cultures established from 28 BMT recipients showed a significantly reduced expression of cell surface vascular cell adhesion molecule-1 (VCAM-1/CD106), compared with normal donor BM stromal cells. Transplant BM stromal cell CD106 expression was responsive to regulatory cytokines in a manner qualitatively comparable with normal donor BM stromal cells. The level of B-cell precursor adhesion to transplant BM stromal cells correlated with the level of CD106 expression. Of 19 evaluable transplant BM stromal cell cultures, eight exhibited a reduced capacity to support the growth of CD19+/light chain- normal B-cell precursors. The capacity of transplant BM stromal cells to support B-cell precursor growth correlated with the level of CD106 expression, and the level of B-cell precursor adhesion. Our collective results may provide new mechanistic insight into why B-cell recovery is delayed post-BMT and underscore the importance of VCAM-1/CD106 in regulating B lymphopoiesis.     

Stem cell factor and interleukin-4 induce murine bone marrow cells to develop into mast cells with connective tissue type characteristics in vitro

In this study, we have developed a method to obtain mast cells with connective tissue type mast cell (CTMC) characteristics directly from mouse bone marrow (BM) cells. BM cells were grown for 3 weeks in presence of interleukin-4 (IL-4) plus stem cell factor (SCF). SCF alone poorly supported growth and development of mast cells. IL-4 dose-dependently enhanced the expression of c-kit and high-affinity receptor for IgE (Fc(epsilon)RI) on the cell surface of SCF-cultured BM cells. Furthermore, cytoplasmic granulation and histamine synthesis of BM-derived mast cells were increased in presence of IL-4 and SCF. Histochemical staining demonstrated that granules were safranin positive. BM-derived mast cells could be activated for granule exocytosis (beta-hexosaminidase release) and lipid mediator generation (LTC4 production) via Fc(epsilon)RI after sensitization with IgE and subsequent crosslinking with multivalent antigen. In addition, mast cells derived from BM cells cultured with SCF plus IL-4 could be activated by substance P, a nonimmunologic stimulus, to release beta-hexosaminidase. The results presented indicate that IL-4 and SCF both have a prominent role in the development of mast cells from murine BM cells in vitro. Mast cells can directly be derived from BM cells in presence of SCF and IL-4 and the cultured cells show typical hallmarks of CTMC, indicating that precursor cells for CTMC may be present in BM. The described culture procedure may be useful to investigate the molecular aspects of the development of committed mast cell lineages.     

A recombinant single-chain IL-7/HGFbeta hybrid cytokine induces juxtacrine interactions of the IL-7 and HGF (c-Met) receptors and stimulates the proliferation of CFU-S12, CLPs, and pre-pro-B cells

A novel recombinant interleukin-7/hepatocyte growth factor beta-chain (IL-7/HGFbeta) hybrid cytokine was constructed as a single chain (sc) composed of IL-7 and HGFbeta connected by a flexible linker. Unlike recombinant (r) IL-7, which stimulated pro-B cells and pre-B cells only, scIL-7/HGFbeta stimulated the proliferation of pre-pro-B cells, common lymphoid progenitors (CLPs), and colony-forming unit (CFU)-S12 in cultures of IL-7-/- mouse BM cells. When injected in vivo, 3- to 4-fold more splenic B-lineage cells appeared in recipients of bone marrow (BM) cells from the scIL-7/HGFbeta-stimulated cultures than from rIL-7-stimulated cultures. Moreover, on a per-cell basis, scIL-7/HGFbeta culture-generated cells produced 16- to 20-fold more BM and splenic B-lineage cells than did normal BM cells. Antibody blocking, receptor phosphorylation, and confocal microscopy demonstrated that scIL-7/HGFbeta signals though both the IL-7 and HGF (c-Met) receptors, which form IL-7R/c-Met complexes on the surface of CLPs and pre-pro-B cells. In addition, the IL-7Ralpha chain, gammac chain, and c-Met were coisolated from purified CLPs and pre-pro-B cells on scIL-7/HGFbeta affinity gels, indicating that they are major components of the IL-7/HGFbeta receptor. Hence, the present results demonstrate that the IL-7/HGFbeta hybrid cytokine efficiently and selectively stimulates the most primitive B-lineage precursors in BM by inducing juxtacrine interactions between the IL-7 and c-Met receptors.     

Interleukin-7 induces the proliferation of normal human B-cell precursors.

In the present study, we investigated the effects of human recombinant interleukin-7 (IL-7) on the proliferation of enriched hematopoietic cells isolated from human adult and fetal bone marrow (BM). In cultures of CD34+ cells, IL-7 was found to induce dose-dependent incorporation of 3H-thymidine (3H-TdR), but had no demonstrable effect on the development of myeloid colony-forming cells. Numbers of B-cell precursors (BCP), initially present within CD34+ populations and which included a CD34+CD20+ subset, were significantly increased when CD34+ BM cells were cultured in the presence of IL-7. This effect was most striking on CD20+ BCP, and resulted at least partly from higher numbers of cycling cells as indicated by Hoechst 33342 fluorescence (Calbiochem, Behring Diagnostics, La Jolla, CA). These results indicate that IL-7 promotes the growth of BCP within the CD34+ compartment. In line with the B-lineage affiliation of CD34+ target cells, committed BCP (CD10+ CD19+ surface IgM-) isolated from BM were also found to proliferate in response to IL-7. Interestingly, this effect of IL-7 was strongly potentiated by the addition of IL-3. Taken together, and in accordance with previous observations on murine cells, our data indicate that IL-7 acts as a growth factor during the ontogeny of human B lymphocytes.     

Mouse splenic and bone marrow cell populations that express high-affinity Fc epsilon receptors and produce interleukin 4 are highly enriched in basophils.

Splenic and bone marrow cells from normal mice, and from mice that have been polyclonally activated by injection of anti-IgD antibody, contain cells that produce interleukin 4 (IL-4) in response to crosslinkage of Fc epsilon receptors (Fc epsilon R) or Fc gamma R or to ionomycin. Isolated Fc epsilon R+ cells have recently been shown to contain all of the IL-4-producing capacity of the nonlymphoid compartment of spleen and bone marrow. Here, purified Fc epsilon R+ cells are shown to be enriched in cells that contain histamine and express alcian blue-positive cytoplasmic granules. By electron microscopy, the vast majority of cytoplasmic granule-containing cells are basophils; they constitute approximately 25% and approximately 50%, respectively, of Fc epsilon R+ spleen and bone marrow cells from anti-IgD-injected mice. The Fc epsilon R- populations contain cells that form colonies typical of mast cells. The Fc epsilon R+ populations also contain cells that, upon culture with IL-3, form colonies of alcian blue-positive cells, but (in contrast to colonies derived from Fc epsilon R- populations) the colonies are small, and all the cells die within 2-3 weeks. The Fc epsilon R+ cells synthesize histamine during a 60-hr culture with IL-3, while the Fc epsilon R- cells do not. These results indicate that IL-4-producing Fc epsilon R+ cells are highly enriched in basophils.     

Regulation of human B-cell precursor adhesion to bone marrow stromal cells by cytokines that exert opposing effects on the expression of vascular cell adhesion molecule-1 (VCAM-1)

Self-renewal and differentiation of B-cell precursors is dependent on interactions with bone marrow (BM) stromal cells and associated extracellular matrix. We have recently developed an interleukin (IL)-7- dependent, BM-derived stromal cell culture that supports the growth of normal human B-cell precursors. In the current study, we have characterized the constitutive expression, cytokine-regulated expression, and function of adhesion molecules on BM stromal cells that are critical for adhesion of B-cell precursors. Flow cytometric analysis showed that cultured adult BM stromal cells expressed higher constitutive levels of vascular cell adhesion molecule (VCAM)-1 than intercellular adhesion molecule (ICAM)-1 (CD54). IL-1 beta upregulated VCAM-1 and CD54 in a dose-dependent manner, whereas IL-4 upregulated VCAM-1, but had no effect on CD54. In contrast, transforming growth factor (TGF)-beta decreased the level of BM stromal cell VCAM-1. Using an assay to measure the adhesion of 51Cr-labeled B-cell precursors to BM stromal cells, we observed a direct correlation between cytokine- regulated levels of VCAM-1 and the capacity of stromal cells to support the adhesion of B-cell precursors. Blocking studies using a panel of monoclonal antibodies (MoAb) showed that adhesion of B-cell precursors to untreated and cytokine-treated (IL-1 beta, IL-4) BM stromal cells was mediated by very late antigen (VLA)-4 (CD49d/CD29) and VCAM-1. Adhesion of B-cell precursors could also be enhanced by direct stimulation with MoAb to the CD29 subunit. Our collective results indicate that B-cell precursor/BM stromal cell adhesion is mediated by a VLA-4-VCAM-1 interaction, which in turn can be regulated at the level of the BM stromal cell by cytokines that specifically increase or decrease cell surface VCAM-1.     

Receptor-specific inhibition of bone marrow erythropoiesis by recombinant DNA-derived interleukin-2

Interleukin-2 (IL-2) induces differential secretion of lymphokines by IL-2 receptor (IL-2R)-positive and IL-2R-negative T cells. We studied T cell IL-2R-specific modulation of adult bone marrow erythropoiesis by recombinant IL-2 (rIL-2). I3-2R were induced by CD3 T cell surface determinant-triggering and analyzed by cytofluorography. Bone marrow monocyte and T cell-depleted (NAB-T) target cells were assessed for early erythroid progenitor expression (BFU-E) in the presence of 0 to 10(3) U/mL of rIL-2, rIL-2 had no significant effect on BFU-E expression in the absence of T cells or in the presence of IL-2R- negative T cells. rIL-2 caused a dose-dependent inhibition (75% to 90%) of BFU-E in the presence of autologous IL-2R-positive T cells. The addition of anti-IL2-receptor antibody to cultures containing rIL-2 plus IL-2R-positive T cells entirely abrogated rIL-2-mediated inhibition of BFU-E. In the presence of rIL-2 (10(2) U/mL) production of interferon gamma (IF-gamma) by adult marrow CD3-triggered IL-2R- positive T cells was increased 37- to 125-fold compared to IL-2R- negative T cells. rIF-gamma caused a dose-dependent (88% +/- 17% at 10(3) U/mL) inhibition of adult BFU-E in the presence of CD3-triggered autologous T cells. rIL2-mediated inhibition of adult BFU-E in the presence of IL-2R-positive T cells was partially abrogated (52% +/- 16%) following addition of monospecific IF-gamma antibody. These results demonstrate (a) rIL-2 modulation of adult marrow erythropoiesis is selectively dependent upon both the presence or absence of autologous T cells and the IL-2R status of these T cells; and (b) rIL-2- induced inhibition of adult marrow erythropoiesis is mediated in part by release of IF-gamma from IL-2R-positive T cells.     

Resistance of primary isolates of human immunodeficiency virus type 1 to neutralization by soluble CD4 is not due to lower affinity with the viral envelope glycoprotein gp120.

Recombinant soluble CD4 (rsCD4) has potent antiviral activity against cell line-adapted isolates of the human immunodeficiency virus type 1 (HIV-1) but low activity toward HIV-1 primary isolates from patients. A simple hypothesis proposed to explain this discrepancy, which questions the therapeutic utility of soluble CD4-based approaches, is that the major envelope glycoprotein, gp120, of patient virus has lower affinity for CD4 than does gp120 from laboratory viruses. To test this hypothesis, we have produced pairs of low- and high-passage HIV-1 isolates which, depending on culture passage history, display dramatically different sensitivities to neutralization by rsCD4. Here, we present evidence that the HIV-1 major envelope glycoprotein cDNAs cloned from one such isolate pair show only minor differences in their deduced gp120 primary structures, and these occur outside regions previously shown to be involved in CD4 interactions. In addition, recombinant gp120 from a low-passage rsCD4-resistant patient virus binds rsCD4 with high affinity, equal to that previously measured for recombinant gp120 from high-passage cell line-adapted virus isolates. These data indicate that differences in CD4-gp120 affinity do not account for rsCD4 resistance in HIV-1 recently isolated from patients.     

Signal requirements for the generation of T-lymphocytes from T-depleted bone marrow cells: a sequential study

We have investigated the sequence of signals provided by a B- and null-cell-derived prothymocyte-differentiating activity (PTDA), phytohemagglutinin (PHA), and interleukin 2 (IL2) to the generation of mature T-lymphocytes by T-depleted bone marrow (BM) cells. Sequential studies show that preincubation of CD2-, CD3-, CD4-, CD6-, and CD8- BM cells with PTDA, but not with recombinant (r) IL2 or PHA increased their capacity to proliferate in liquid culture and to form agar T-cell colonies provided both PHA and rIL2 were added to the cultures. In contrast, the growth of T-cell-containing BM was significantly enhanced in both liquid and agar culture following its preincubation with rIL2 as well as with PTDA. The selective effect of PTDA on CD2-, CD3-, CD4-, CD6-, CD8- BM cells was abolished by adding a CD7 monoclonal antibody to the T-cell-purging coctail. Cell marker studies performed on T-cell-depleted BM-derived liquid or agar cultures have shown that they contain up to 70%-85% CD2+, CD3+, CD4+, CD8- cells. No IL1 or IL2 could substitute for PTDA, nor have these activities, as well as interferon (IFN), IL3, IL4, or granulocyte-macrophage colony-stimulating activity (GM-CSA) been detected so far in PTDA-containing preparations. These results indicate that PTDA can trigger marrow T-cell precursors into PHA-responsive T cells, which, following activation by PHA, require IL2 for growth. It is suggested that this may represent a thymus-independent alternative pathway for T-cell differentiation and activation.