Addition Of IL-2 Research Articles

Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor.

FLT3 is a member of the type III receptor tyrosine kinase (RTK) family. These receptors all contain an intrinsic tyrosine kinase domain that is critical to signaling. Aberrant expression of the FLT3 gene has been documented in both adult and childhood leukemias including AML, ALL and CML. In addition, 17-27% of pediatric and adult patients with AML have small internal tandem duplication mutations in FLT3. Patients expressing the mutant form of the receptor have been shown to have a decreased chance for cure. Our previous study, using a constitutively activated FLT3, demonstrated transformation of Ba/F3 cells and leukemic development in an animal model. Thus, there is accumulating evidence for a role for FLT3 in human leukemias. This has prompted us to search for inhibitors of FLT3 as a possible therapeutic approach in these patients. AG1296 is a compound of the tyrphostin class that is known to selectively inhibit the tyrosine kinase activity of the PDGF and KIT receptors. Since FLT3 is a close relative of KIT, we wanted to test the possible inhibitory activity of AG1296 on FLT3. In transfected Ba/F3 cells, AG1296 selectively and potently inhibited autophosphorylation of FL-stimulated wild-type and constitutively activated FLT3. Treatment by AG1296 abolished IL-3-independent proliferation of Ba/F3 cells expressing the constitutively activated FLT3 and thus, reversed the transformation mediated by activated FLT3. Inhibition of FLT3 activity by AG1296 in cells transformed by activated FLT3 resulted in apoptotic cell death, with no deleterious effect on their parental counterparts. Addition of IL-3 rescued the growth of cells expressing activated FLT3 in the presence of AG1296. This demonstrates that the inhibition is specific to the FLT3 pathway in that it leaves the kinases of the IL-3 pathway and other kinases further downstream involved in proliferation intact. Several proteins phosphorylated by the activated FLT3 signaling pathway, including STAT 5A, STAT 5B and CBL, were no longer phosphorylated when these cells were treated with AG1296. The activity against FLT3 suggests a potential therapeutic application for AG1296 or similar drugs in the treatment of leukemias involving deregulated FLT3 tyrosine kinase activity and as a tool for studying the biology of FLT3.

Stromal cells provide signals different from cytokines for STAT5 activation in hematopoietic cells.

After detachment from the stromal cells, hematopoietic stem cells are thought to differentiate to the cytokine-dependent stages where their growth and differentiation are promoted by these cytokines. To examine the stromal regulation of hematopoietic stem cells, we previously established a primitive hematopoietic stem-like cell line, THS119, whose growth was dependent on the bone marrow stromal cell line, TBR59, and from which IL-3- (THS119/IL-3) or IL-7- (THS119/IL-7) dependent cell lines were then generated. Using these cell lines, we examined the difference in signals mediated by the stromal cells and cytokines. The cytokine-dependent cell lines (THS119/IL-3 and THS119/IL-7) showed induction of STAT5 phosphorylation and target genes for STAT5 such as CIS, pim-1, p21 and bcl-xL upon addition of IL-3 or IL-7. IL-3 or IL-7 also induced STAT5 phosphorylation and STAT5 target genes of the stromal cell-dependent cell line, THS119, in the absence of stromal cells at levels similar to the cytokine-dependent cell lines. However, quite interestingly, TBR59 stromal cells could not induce STAT5 phosphorylation of THS119 cells, although they did induce STAT5 target genes in THS119 cells. In addition, the mRNAs for STAT5 target genes in THS119 cells on the stromal cells seemed to be more stable than those in the cytokine-dependent cell lines. Expression of the antiapoptotic genes bcl-2 and bcl-xL was higher in the stromal cell-dependent cell line than in the cytokine-dependent cell lines. These results suggested that stromal cells and cytokines may provide different signals for growth and differentiation of the hematopoietic cells.

Ex vivo expansion of human hematopoietic stem cells.

There has been great interest in the ex vivo expansion of human long-term repopulating hematopoietic stem cells (LTR-HSCs) for a variety of clinical applications such as umbilical cord blood transplantation. The glucoprotein130 signal, activated by a complex of interleukin 6 (IL-6) and soluble IL-6 receptor (IL-6/sIL-6R), acts dramatically in synergy with the c-Kit or Flk2/Flt3 signal to expand immature human HSCs. We demonstrate a significant ex vivo expansion of human LTR-HSCs capable of repopulating in newly discovered nonobese diabetes/Shi-severe combined immunodeficient (NOD/Shi-SCID) mice. The proportion of human CD45+ cells in recipient marrow was 10 times higher in animals receiving the cultured cells with stem cell factor, Flk2/Flt3 ligand, thrombopoietin, and IL-6/sIL-6R than in those receiving comparable numbers of fresh cord blood CD34+ cells. The expansion rate provided by this combination was estimated to be 4.2-fold by a limiting dilution method. Addition of IL-3 to the culture with the cytokine combination abrogated the repopulating ability of the expanded cells. The culture method with the IL-6/sIL-6R complex and other cytokines may pave the way for ex vivo expansion of human transplantable HSCs suitable for clinical applications.

Stem cells acquire class i recognizing nk cell receptors in vitro

To learn more about human NK cell receptor acquisition during development, cord blood (CB) CD34+Lin−CD38− cells where cultured in direct contact with AFT024 (murine fetal liver), separated from AFT024 by a membrane and in the absence of AFT024 (n = 8). All cultures contained c-kit ligand, flt3 ligand and IL-3 ± IL-7, IL-15 or IL-2. In the absence of one of the lymphoid cytokines, NK cells were KIR and lectin receptor negative. Addition of IL-7, IL-15 or IL-2 resulted in inefficient differentiation of NK cells in the absence of a feeder or when contact with AFT024 was prohibited. In contrast, 300 CB progenitors cultured in direct contact with AFT024 resulted in 22692 ± 10591 NK cells with IL-7, 523110 ± 120220 NK cells with IL-15 and 930310 ± 234250 NK cells with IL-2. The number of NK cells expressing at least one KIR was low under all conditions except for when progenitors were cultured in direct contact with AFT024 and IL-15 (15380 ± 5096) or IL-2 (47108 ± 13852) (n = 9). Ten-fold more cells expressed a lectin receptor as detected by the anti-CD94 mab. However, these studies do not show whether receptor acquisition was determined at the stem cell level or later in development. To address this question, single CD34+Lin−CD38− cells were cultured in direct contact with AFT024. Over 400 single cell progeny were analyzed from cultures containing IL-15 or IL-2. No single cell progeny contained a clonal population of NK cells expressing a single KIR. When single cell NK cell progeny were analyzed for single KIR's, KIR2DL2 (5846 ± 1017, n = 198) was always present at higher numbers than KIR2DL1 (2488 ± 539, n = 197) or KIR3DL1 (1011 ± 152, n = 252) expressing NK cells suggesting a nonrandom sequence of KIR acquisition. Higher numbers of NK cell progeny expressing KIR developed from single fetal liver and CB cells compared to adult BM. Lasting, LIR-1 receptors were acquired during NK cell development (CD94 > LIR-1 > KIR). These results show that NK cell receptor fate occurs at a stage beyond the hematopoietic stem cell.

Growth factor stimulation enhances integrin-mediated migration but not adhesion of cml progenitors

Abnormal progenitor circulation and extramedullary hematopoiesis are characteristic features of chronic myelogenous leukemia (CML). These abnormalities could be related to altered β1 integrin function in CML progenitors. β1 integrins play an important role in growth factor (GF) induced mobilization of normal progenitors from the bone marrow into the circulation. Although expressing β1-integrins at normal levels, CML progenitors demonstrate significantly reduced adhesion to stroma and fibronectin (FN). We evaluated the effect of GF stimulation on CML progenitor adhesion and migration on FN. GF modulation of β1 integrin-mediated adhesion was impaired in CML CFC. Normal CFC incubated with physiological concentrations of GF had significantly enhanced adhesion to FN compared with cells incubated without GF (30.4±4.6% vs. 14±3.6%, p<0.01), whereas CML CFC adhesion to FN was poor regardless of the presence or absence of GF (7.6±1.8% vs. 7.9±2.1%). Addition of IL-3, FL and other GF rapidly enhanced β1 integrin-mediated adhesion of normal CFC to FN, but did not increase CML CFC adhesion. On the other hand, CML CFC demonstrated increase migration across FN-coated transwells (5μm pores) compared with normal CFC (15.7±3.4% vs. 7.3±1.4%, p<0.05) as well as increased spontaneous migration across uncoated transwells (9.9±2.2% vs. 3.2±0.7%, p<0.05). Addition of SCF enhanced migration of normal CFC through uncoated and FN-coated transwells (9.2+1.8% and 19.1+4.1%). In contrast to its lack of effect on adhesion, SCF also enhanced CML CFC migration (FN-: 15.3±4.3% and FN+: 23.9±6.1%). The presence or absence of GF did not affect cell viability or integrin expression. Differential enhancement of migration but not adhesion in response to GF simulation could play a role in abnormal progenitor circulation and localization in CML.

Expansion of human NOD/SCID-repopulating cells by stem cell factor, Flk2/Flt3 ligand, thrombopoietin, IL-6, and soluble IL-6 receptor.

Here, we demonstrate a significant ex vivo expansion of human hematopoietic stem cells capable of repopulating in NOD/SCID mice. Using a combination of stem cell factor (SCF), Flk2/Flt3 ligand (FL), thrombopoietin (TPO), and a complex of IL-6 and soluble IL-6 receptor (IL-6/sIL-6R), we cultured cord blood CD34(+) cells for 7 days and transplanted these cells into NOD/SCID mice. Bone marrow engraftment was judged successful when recipient animals contained measurable numbers of human CD45(+) cells 10-12 weeks after transplantation. When cells were cultured with SCF+FL+TPO+IL-6/sIL-6R, 13 of 16 recipients were successfully engrafted, and CD45(+) cells represented 11.5% of bone marrow cells in engrafted recipients. Cells cultured with a subset of these factors were less efficiently engrafted, both as measured by frequency of successful transplantations and prevalence of CD45(+) cells. In animals receiving cells cultured with all 4 factors, human CD45(+) cells represented various lineages, including a large number of CD34(+) cells. The proportion of CD45(+) cells in recipient marrow was 10 times higher in animals receiving these cultured cells than in those receiving comparable numbers of fresh CD34(+) cells, and the expansion rate was estimated at 4.2-fold by a limiting dilution method. Addition of IL-3 to the cytokine combination abrogated the repopulating ability of the expanded cells. The present study may provide a novel culture method for the expansion of human transplantable hematopoietic stem cells suitable for clinical applications.

Thrombopoietin, Interleukin-11, and Early-Acting Megakaryocyte Growth Factors in Human Myeloid Leukemia Cells

In this study we report our data on effects of early-acting megakaryocyte growth factors, particularly the c-mpl ligand also known as thrombopoietin (TPO) and interleukin-11 (IL-11), on cell proliferation and apoptosis (Apo) of primary acute myeloid leukemia (AML) cells. A proliferative response to TPO was noticed in the majority of AML samples (17/19) with an average increase of S-phase cells from 7.8% ± 1.5 to 14.5% ± 2.1 (p=0.0006). Resulting cell cycle activation did not always correlate with expression of the c-mpl receptor, although it was coupled, in the majority of samples, by an average decrease of apoptotic cells from 13% ± 0.7 to 8.8% ± 1.8 (p=0.05). Clonogenic cell growth (CFU-L) was confirmed in 5/17 of the samples with a mean colony number of 21.4 ± 9.6 × 105 cells plated. Conversely, effects of IL-11 on AML cells demonstrated that cell cycle changes (recruitment from G0 to S phase) were promoted only in a minority of samples (2/14) and there was little, if any, effect on CFU-L growth (mean colony number=17.5 ± 9.5) or Apo (from 13% ± 0.7 to 13.3±1.9). Combination of TPO with IL-11 induced a slight increase of clonogenic cell growth, while the addition of IL-3 or SCF to the c-mpl ligand significantly raised the mean colony numbers up to 119.2 ± 68.3 and 52.9±22.1 × 105 cells plated, respectively. In summary, TPO shows activity on AML cells by stimulating their proliferation in a significant proportion of cases and generally protecting the majority of AML blast cells from induction of Apo. Conversely, IL-11 exerts little effect on the cell cycle activation and Apo. These data help to understand regulation of myeloid leukemia cell growth and should be considered in the clinical use of early-acting megakaryocyte growth factors in acute leukemia.

Chemotaxis of primitive hematopoietic cells in response to stromal cell-derived factor-1.

Stromal cell-derived factor-1 (SDF-1) provides a potent chemotactic stimulus for CD34(+) hematopoietic cells. We cultured mobilized peripheral blood (PB) and umbilical cord blood (CB) for up to 5 weeks and examined the migratory activity of cobblestone area-forming cells (CAFCs) and long-term culture-initiating cells (LTC-ICs) in a transwell assay. In this system, SDF-1 or MS-5 marrow stromal cells placed in the lower chamber induced transmembrane and transendothelial migration by 2- and 5-week-old CAFCs and LTC-ICs in 3 hours. Transmigration was blocked by preincubation of input CD34(+) cells with antibody to CXCR4. Transendothelial migration of CB CAFCs and LTC-ICs was higher than that of PB. We expanded CD34(+) cells from CB in serum-free medium with thrombopoietin, flk-2 ligand, and c-kit ligand, with or without IL-3 and found that CAFCs cultured in the absence of IL-3 had a chemotactic response equivalent to noncultured cells, even after 5 weeks. However, addition of IL-3 to the culture reduced this response by 20-50%. These data indicate that SDF-1 induces chemotaxis of primitive hematopoietic cells signaling through CXCR4 and that the chemoattraction could be downmodulated by culture ex vivo.

A combination of megakaryocyte growth and development factor and interleukin-1 is sufficient to culture large numbers of megakaryocytic progenitors and megakaryocytes for transfusion purposes.

Chemotherapy-induced thrombocytopenia is a major risk factor in cancer treatment. The transfusion of autologous ex vivo expanded megakaryocytes could be a new therapy to shorten the period of thrombocytopenia. Therefore we investigated, in a liquid culture system, the effect of various cytokine combinations composed of pegylated megakaryocyte growth and development factor (PEG-rHuMGDF), interleukin-1 (IL-1), IL-3, IL-6, IL-11 and stem cell factor (SCF) on the proliferation and differentiation of CD34+ cells, in order to define the most optimal and minimum levels of cytokine combinations for megakaryocyte expansion. Besides PEG-rHuMGDF, IL-1 was found to be important for optimal megakaryocyte expansion. Depletion of either SCF, IL-6 or IL-11 did not exert a large effect, but the absence of IL-1 strongly diminished the number of megakaryocytic cells. Addition of IL-3 to the combination PEG-rHuMGDF, IL-1, IL-6, IL-11 and SCF significantly reduced the number of megakaryocyte progenitors (CD34+CD41+ cells) and the number of CFU-Meg. Furthermore, we found a strong correlation between the number of CD34+CD41+ cells and the number of CFU-Meg obtained after 8 d culture. Our study shows that optimal ex vivo expansion of megakaryocytes is achieved by the combination of PEG-rHuMGDF and IL-1. The numbers of megakaryocytes and megakaryocyte progenitors (CD34+CD41+) obtained in our liquid culture system with the growth factor combination PEG-rHuMGDF and IL-1 are suitable for transfusion purposes.

Mouse granulated metrial gland cell cytotoxicity of Wehi 164 cells: is there a role for interleukin-3 and tumour necrosis factor-alpha?

Mouse granulated metrial gland (GMG) cells are maternal, cytotoxic cells found in the uterine wall in pregnancy. In vivo, they are cytotoxic to layer 1 labyrinthine placental cells. In an in vitro system, granulated metrial gland cells are highly cytotoxic to Wehi 164 fibrosarcoma cells. In this study the role of interleukin (IL)-3 and tumour necrosis factor (TNF)-alpha in granulated metrial gland cell cytotoxic activity was investigated. Chromium-release cytotoxicity assays using mouse metrial gland effector cells and Wehi 164 target cells with the addition of IL-3 or an antibody to TNF-alpha. An antibody to TNF-alpha reduced the level of cytotoxic activity. IL-3 had no effect on the level of cytotoxicity. Mouse granulated metrial gland cells kill using a TNF-alpha mediated mechanism which is independent of IL-3 stimulation.

Survival of human leukaemic B-cell precursors is supported by stromal cells and cytokines: association with the expression of bcl-2 protein.

We searched for cytokines with the potential to support the survival of human B-cell precursor acute lymphoblastic leukaemia (pre-B ALL) cells. 47 patients with pre-B ALL were classified into four stages: stage I, CD19+CD10-CD20-; stage II, CD19+CD10+CD20-; stage III, CD19+CD10+CD20+cytoplasmic mu-heavy chain (cmu)-; stage IV, CD19+CD10+CD20+cmu. Interleukin (IL)-3 receptor alpha chain (IL-3Ralpha) was expressed in all stages, whereas the expressions of IL-7Ralpha and IL-2Ralpha were pronounced in stages IV and II, respectively. Neither IL-3, IL-7 nor IL-2 supported the survival of pre-B ALL cells. When pre-B ALL cells were layered on stromal, MS-10, cells, viability of the pre-B ALL cells increased. Addition of IL-3 to culture containing MS-10 cells enhanced the survival of pre-B ALL cells in all cases, whereas addition of IL-7 augmented the survival of pre-B ALL cells of some cases of stage III and all cases of stage IV. The survival of pre-B ALL cells was also supported by the conditioned media of MS-10 cells. Stromal-cell-derived factor 1 (SDF-1) supported the survival of pre-B ALL cells. Effects of the conditioned media of MS-10 cells were abrogated by an anti-SDF-1 neutralizing antibody. The extent of survival of pre-B ALL cells supported by stromal cells and IL-3 and IL-7, correlated with the expression level of bcl-2 protein. The effects of stromal cells may be in part related to SDF-1.

Single Adult Human CD34+/Lin−/CD38− Progenitors Give Rise to Natural Killer Cells, B-Lineage Cells, Dendritic Cells, and Myeloid Cells

AbstractMarrow stromal cultures support adult CD34+/Lin−/HLA-DR− or CD34+/Lin−/CD38− cell differentiation into natural killer (NK) or myeloid cells, but unlike committed lymphoid progenitors (CD34+/Lin−/CD45RA+/CD10+), no B cells are generated. We tested whether different microenvironments could establish a developmental link between the NK and B-cell lineages. Progenitors were cultured in limiting dilutions with interleukin-7 (IL-7), flt3 ligand (FL), c-kit ligand (KL), IL-3, IL-2, and AFT024, a murine fetal liver line, which supports culture of transplantable murine stem cells. NK cells, CD10+/CD19+ B-lineage cells and dendritic cells (DC) developed from the same starting population and IL-7, FL, and KL were required in this process. Single cell deposition of 3,872 CD34+/Lin−/CD38− cells onto AFT024 with IL-7, FL, KL, IL-2, and IL-3 showed that a one time addition of IL-3 at culture initiation was essential for multilineage differentiation from single cells. Single and double lineage progeny were frequently detected, but more importantly, 2% of single cells could give rise to at least three lineages (NK cells, B-lineage cells, and DC or myeloid cells) providing direct evidence that NK and B-lineage differentiation derive from a common lymphomyeloid hematopoietic progenitor under the same conditions. This study provides new insights into the role of the microenvironment niche, which governs the earliest events in lymphoid development.

Single Adult Human CD34+/Lin−/CD38− Progenitors Give Rise to Natural Killer Cells, B-Lineage Cells, Dendritic Cells, and Myeloid Cells

Marrow stromal cultures support adult CD34+/Lin−/HLA-DR− or CD34+/Lin−/CD38− cell differentiation into natural killer (NK) or myeloid cells, but unlike committed lymphoid progenitors (CD34+/Lin−/CD45RA+/CD10+), no B cells are generated. We tested whether different microenvironments could establish a developmental link between the NK and B-cell lineages. Progenitors were cultured in limiting dilutions with interleukin-7 (IL-7), flt3 ligand (FL), c-kit ligand (KL), IL-3, IL-2, and AFT024, a murine fetal liver line, which supports culture of transplantable murine stem cells. NK cells, CD10+/CD19+ B-lineage cells and dendritic cells (DC) developed from the same starting population and IL-7, FL, and KL were required in this process. Single cell deposition of 3,872 CD34+/Lin−/CD38− cells onto AFT024 with IL-7, FL, KL, IL-2, and IL-3 showed that a one time addition of IL-3 at culture initiation was essential for multilineage differentiation from single cells. Single and double lineage progeny were frequently detected, but more importantly, 2% of single cells could give rise to at least three lineages (NK cells, B-lineage cells, and DC or myeloid cells) providing direct evidence that NK and B-lineage differentiation derive from a common lymphomyeloid hematopoietic progenitor under the same conditions. This study provides new insights into the role of the microenvironment niche, which governs the earliest events in lymphoid development.

Differential regulation of CD95 (Fas/APO-1) expression in human blood eosinophils

CD95 (Fas, APO-1) is a cell surface receptor expressed on many cells including eosinophils which mediates apoptosis when ligated by agonistic antibodies or its natural ligand FasL. Since inhibition of apoptosis may play an important role in controlling tissue eosinophilia, we investigated the expression of CD95 on purified peripheral blood eosinophils from normal donors. Freshly isolated eosinophils expressed CD95 on the cell surface as well as CD95-specific mRNA at low levels which did not change during 24-h culture. Incubation of eosinophils with IL-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) did not modulate the basal expression of CD95. IFN-gamma as well as TNF-alpha, however, induced a significant, dose- and time-dependent increase in CD95 mRNA and cell surface expression as measured by reverse transcription-PCR and flow cytometry. Co-stimulation with IFN-gamma and TNF-alpha had synergistic effects on the CD95 surface expression on eosinophils. Addition of IL-3, IL-5 or GM-CSF to IFN-gamma- and TNF-alpha-stimulated eosinophils caused in a reduction of CD95 expression. Functional activity for CD95 following incubation with IFN-gamma and TNF-alpha was demonstrated by increased apoptosis in response to cross-linking with FasL. From these data, we conclude that IFN-gamma and TNF-alpha can up-regulate cell surface expression of CD95 on eosinophils, which leads to an increased susceptibility of eosinophils to Fas-mediated apoptosis. Thus, our results suggest that receptors involved in eosinophil apoptosis can be regulated by antagonistic cytokines.

Complementary and antagonistic effects of IL‐3 in the early development of human megakaryocytes in culture

The effect of IL-3 on the early steps in the growth and development of megakaryocytes (MK) in culture has been studied. Although thrombopoietin (TPO) by itself could support the development of mature CD41+ MK from pre-MK, the number of cells produced was greatly augmented by the addition of IL-3 and SCF. IL-3 was also able to support the growth of MK colonies in semi-solid media (CFU-MK). The CD41+ cells that developed in suspension cultures containing IL-3 differed phenotypically from those that developed without this agent. Cells grown in the presence of IL-3 lost CD34 expression more rapidly, expressed lower levels of the platelet glycoproteins gpIIb-IIIa and Ib and achieved lower degrees of polyploidy than in the absence of IL-3. The inhibitory effects of IL-3 were not a consequence of the dilution of the mature cells by increased numbers of immature cells since it was observed under conditions in which IL-3 did not stimulate MK growth. The results obtained in these cultures suggest that IL-3 plays an important role in early MK development, but inhibits further maturation after endoreduplication begins. Thus, prolonged contact with IL-3 results in the appearance of cells that do not mature normally.

Fetal liver organ cultures allow the proliferative expansion of pre-B receptor-expressing pre-B-II cells and the differentiation of immature and mature B cells in vitro.

We describe the phenotypic and functional properties of B lineage cells developing in fetal liver organ cultures (FLOC) of mouse embryos at day 14 or 15 of gestation which contain pro/pre-B-I cells. FLOC B cell development proceeds to mature IgM+, IgD+ and CD23+ lipopolysaccharide-reactive B cells within a culture period of 5-6 days. The phenotypes and relative proportions of pro/pre-B-I, pre-B-II, immature and mature B cells from FLOC were similar to that seen in livers freshly isolated from age-matched, i.e. newborn, mice. More importantly, the numbers of cells recovered in the different B lineage subpopulations from FLOC were close to those developed in vivo. Hence, in contrast to single-cell suspension cultures of fetal liver, FLOC allow the proliferative expansion of pre-B cell receptor-expressing pre-B-II cells. FLOC from embryos of mice with targeted mutations in the RAG-2 and lambda5 genes, which cannot expand by proliferative expansion of their pre-B-II compartment in vivo because they cannot express a pre-B cell receptor on their surface, show this same defect in vitro. FLOC are accessible to the action of mAb and cytokines. Thus, addition of anti-IL-7 receptor mAb to FLOC of normal mice inhibits B cell development at the transition of pro/pre-B-I to pre-B-II cells. This inhibition is reversed by addition of excess rIL-7. Addition of IL-7 alone stimulates the proliferation of pro/pre-B-I cells and inhibits their differentiation to pre-B-II and immature B cells, as it does in single-cell suspension cultures. FLOC should be useful to study the effects of other mAb, cytokines, ligands and other molecules on early B cell development.

INTERLEUKIN 3 (IL-3), BUT NOT STEM CELL FACTOR (SCF) INCREASES SELF-RENEWAL BY HUMAN ERYTHROID BURST-FORMING UNITS (BFU-E) IN VITRO

Interleukin 3 (IL-3) and stem cell factor (SCF) are both important regulators of early haemopoietic cell development. Here, we have compared their effects on the kinetics of erythroid burst formation by BFU-E in normal adult bone marrow. We grew the BFU-E in the presence of erythropoietin (Epo) alone, Epo+IL-3 or Epo+SCF and scored the numbers of subcolonies in individual bursts after 14 days. The data were plotted as the cumulative distribution of the numbers of subcolonies per erythroid burst then linearised by logarithmic transformation. Analysis of the data revealed that IL-3 increases the numbers of subcolonies in BFU-E whilst SCF increases the size of the subcolonies themselves. Experiments involving combinations of Epo+IL-3+SCF and the delayed addition of Il-3 or SCF indicated that the actions of IL-3 and SCF are largely independent of one another. We conclude that: (1) Il-3 acts at an earlier stage of erythroid differentiation than SCF, and (2) it may be possible to classify haemopoietic growth factors according to their effects on cell kinetics in vitro.

Replication-Defective Canarypox (ALVAC) Vectors Effectively Activate Anti–Human Immunodeficiency Virus-1 Cytotoxic T Lymphocytes Present in Infected Patients: Implications for Antigen-Specific Immunotherapy

In the attempt to develop immunotherapeutic strategies for acquired immunodeficiency syndrome capable of activating effector cells in an antigen-specific manner while maintaining the broadest possible T-cell repertoire, we evaluated two canarypox (ALVAC)-based vectors for their capacity to induce ex vivo activation/expansion of human immunodeficiency virus (HIV)-specific CD8+ cytotoxic lymphocyte precursors (CTLp) obtained from HIV-1–infected donors. These two vectors, vCP205 encoding HIV-1 gp120 + TM (28 amino acid transmembrane anchor sequence) in addition to Gag/protease and vCP300 encoding gp120 + Gag/protease as well as Nef and Pol CTL determinants, are pancytotropic but replication incompetent in mammalian cells. Bulk peripheral blood mononuclear cells (PBMCs) or enriched CD8+ T cells were stimulated for 10 days with autologous ALVAC-infected PBMCs in the presence of different cytokine combinations (interleukin-2 [IL-2], IL-4, IL-7, and IL-12). Activation by ALVAC constructs was highly antigen-specific, because vCP205 elicited only Env and Gag CTL, whereas vCP300 elicited broader reactivities against Env, Gag, Pol, and Nef determinants. The ALVAC activation of CTLp was IL-2 dependent and enhanced by the addition of IL-7, whereas IL-4 and IL-12 failed to augment cytotoxic reactivities elicited by these constructs. The expansion of enriched CD8+ T cells after activation with vCP300 was higher in patients with CD4 counts greater than 400 cells/μL. Two rounds of in vitro stimulation (IVS) with vCP300 resulted in nearly an eightfold expansion of CD8+ lymphocytes over a 25-day period. After the second IVS, an average 3.2-fold increase among the different antigen-specific CTL frequencies was achieved. These studies clearly show that HIV-recombinant ALVAC vectors represent powerful polyvalent antigenic stimuli for activation and expansion of the CD8 lymphocyte response that occurs as a result of HIV infection.

Replication-Defective Canarypox (ALVAC) Vectors Effectively Activate Anti–Human Immunodeficiency Virus-1 Cytotoxic T Lymphocytes Present in Infected Patients: Implications for Antigen-Specific Immunotherapy

AbstractIn the attempt to develop immunotherapeutic strategies for acquired immunodeficiency syndrome capable of activating effector cells in an antigen-specific manner while maintaining the broadest possible T-cell repertoire, we evaluated two canarypox (ALVAC)-based vectors for their capacity to induce ex vivo activation/expansion of human immunodeficiency virus (HIV)-specific CD8+ cytotoxic lymphocyte precursors (CTLp) obtained from HIV-1–infected donors. These two vectors, vCP205 encoding HIV-1 gp120 + TM (28 amino acid transmembrane anchor sequence) in addition to Gag/protease and vCP300 encoding gp120 + Gag/protease as well as Nef and Pol CTL determinants, are pancytotropic but replication incompetent in mammalian cells. Bulk peripheral blood mononuclear cells (PBMCs) or enriched CD8+ T cells were stimulated for 10 days with autologous ALVAC-infected PBMCs in the presence of different cytokine combinations (interleukin-2 [IL-2], IL-4, IL-7, and IL-12). Activation by ALVAC constructs was highly antigen-specific, because vCP205 elicited only Env and Gag CTL, whereas vCP300 elicited broader reactivities against Env, Gag, Pol, and Nef determinants. The ALVAC activation of CTLp was IL-2 dependent and enhanced by the addition of IL-7, whereas IL-4 and IL-12 failed to augment cytotoxic reactivities elicited by these constructs. The expansion of enriched CD8+ T cells after activation with vCP300 was higher in patients with CD4 counts greater than 400 cells/μL. Two rounds of in vitro stimulation (IVS) with vCP300 resulted in nearly an eightfold expansion of CD8+ lymphocytes over a 25-day period. After the second IVS, an average 3.2-fold increase among the different antigen-specific CTL frequencies was achieved. These studies clearly show that HIV-recombinant ALVAC vectors represent powerful polyvalent antigenic stimuli for activation and expansion of the CD8 lymphocyte response that occurs as a result of HIV infection.

Direct evidence for the commitment of hematopoietic stem cells to T, B and myeloid lineages in murine fetal liver.

We established an experimental system in vitro to examine the developmental capacity of individual hematopoietic progenitors to generate T, B and myeloid (M) cells. By using this system we analyzed the process of lineage commitment of hematopoietic progenitors in murine fetal liver (FL). It is known that small numbers of B and M cells, in addition to T cells, are generated in a co-culture of hematopoietic progenitors and a deoxyguanosine-treated fetal thymus (FT) lobe. We tried to enhance the growth of B and M cells by the addition of IL-7, IL-3 and stem cell factor into the co-culture. This cytokine-supplemented FT organ culture was used to examine the developmental capacity of individual hematopoietic progenitors in FL. Single cells of lineage marker (Lin)-c-kit+Sca-1+ (Sca-1+) and Lin-c-kit+Sca-1-(Sca-1-) populations from the FL harvested at day 12 of gestation were cultured for 10 days, and the phenotypes of cells generated in each lobe were analyzed with a flow cytometer. All progenitors in the Sca-1- population were shown to be committed to generate only T, B or M cells. On the other hand, multipotent progenitors, which are capable of generating T, B and M cells, as well as unipotent progenitors committed to the T, B or M lineage were found in the Sca-1+ population. Bipotent progenitors generating M and T cells and those generating M and B cells were also found in the Sca-1+ population, which probably represent progenitors in the process of commitment. However, no bipotent progenitors generating T and B cells were detected.