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Adjuvant therapy; Breast cancer; Olaparib Terapia adyuvante; Cáncer de mama; Olaparib Teràpia adjuvant; Càncer de mama; Olaparib Background The randomized, double-blind OlympiA trial compared 1 year of the oral poly(adenosine diphosphate-ribose) polymerase inhibitor, olaparib, to matching placebo as adjuvant therapy for patients with pathogenic or likely pathogenic variants in germline BRCA1 or BRCA2 (gBRCA1/2pv) and high-risk, human epidermal growth factor receptor 2-negative, early breast cancer (EBC). The first pre-specified interim analysis (IA) previously demonstrated statistically significant improvement in invasive disease-free survival (IDFS) and distant disease-free survival (DDFS). The olaparib group had fewer deaths than the placebo group, but the difference did not reach statistical significance for overall survival (OS). We now report the pre-specified second IA of OS with updates of IDFS, DDFS, and safety. Patients and methods One thousand eight hundred and thirty-six patients were randomly assigned to olaparib or placebo following (neo)adjuvant chemotherapy, surgery, and radiation therapy if indicated. Endocrine therapy was given concurrently with study medication for hormone receptor-positive cancers. Statistical significance for OS at this IA required P < 0.015. Results With a median follow-up of 3.5 years, the second IA of OS demonstrated significant improvement in the olaparib group relative to the placebo group [hazard ratio 0.68; 98.5% confidence interval (CI) 0.47-0.97; P = 0.009]. Four-year OS was 89.8% in the olaparib group and 86.4% in the placebo group (Δ 3.4%, 95% CI −0.1% to 6.8%). Four-year IDFS for the olaparib group versus placebo group was 82.7% versus 75.4% (Δ 7.3%, 95% CI 3.0% to 11.5%) and 4-year DDFS was 86.5% versus 79.1% (Δ 7.4%, 95% CI 3.6% to 11.3%), respectively. Subset analyses for OS, IDFS, and DDFS demonstrated benefit across major subgroups. No new safety signals were identified including no new cases of acute myeloid leukemia or myelodysplastic syndrome. Conclusion With 3.5 years of median follow-up, OlympiA demonstrates statistically significant improvement in OS with adjuvant olaparib compared with placebo for gBRCA1/2pv-associated EBC and maintained improvements in the previously reported, statistically significant endpoints of IDFS and DDFS with no new safety signals. Funding for this work, which was conducted as a collaborative partnership among the Breast International Group, NRG Oncology, Frontier Science, AstraZeneca, and Merck Sharp & Dohme LLC, a subsidiary of Merck & Co., Inc., Rahway, NJ, U.S.A. (MSD), was provided by the National Institutes of Health (grant numbers: U10CA 180868, UG1CA 189867, and U10CA 180822) and by AstraZeneca as part of an alliance between AstraZeneca and MSD. Provision of olaparib and placebo was from AstraZeneca.

Most patients undergoing autologous hematopoietic stem cell transplantation for malignant diseases suffer recurrences of their neoplasms and die due to the inability of conventional high-dose conditioning regimens to eradicate their malignancies. As a result, intensive efforts to develop more effective conditioning regimens are currently under way at many institutions. Encouraging results have been obtained using targeted radiotherapy with radiolabeled antibodies or bone-seeking isotopes as components of novel conditioning regimens for autologous transplantation of patients with lymphomas, multiple myeloma and bone metastases. Results with radiolabeled antibodies targeting epithelial antigens on solid tumors, however, have been less encouraging. This report reviews the status of clinical studies using myeloablative doses of targeted radiotherapy in patients undergoing autologous stem cell transplantation for hematological malignancies or solid tumors.

Erythropoietin (EPO) and stem cell factor (SCF) are two important factors in human erythropoiesis. We have recently demonstrated that SCF and EPO synergistically activate mitogen-activated protein (MAP) kinase, thereby promoting growth of human erythroid colony-forming cells (ECFCs). In the present study, we have examined the intracellular mechanisms by which SCF and EPO maintain survival of these cells. In the absence of SCF and EPO, human ECFCs underwent rapid apoptosis. The process was significantly inhibited by addition of a single factor and was totally prevented in the presence of both factors. Treatment of ECFCs with wortmannin, a specific inhibitor of phosphoinositide 3-kinase (PI3K), inhibited the antiapoptotic effect of SCF but had no effect on that of EPO, indicating that SCF but not EPO inhibits apoptosis through the PI3K pathway. In contrast, treatment of ECFCs with PD98059, a specific inhibitor of MAP kinase/ERK kinase (MEK), inhibited cell growth but had no effect on the antiapoptotic activity of either SCF or EPO, suggesting that SCF and EPO prevent apoptosis of human ECFCs independent of the extracellular signal-regulated kinase (ERK) pathway. Interestingly, both EPO and SCF induced activation of PI3K. However, through PI3K, SCF caused activation of protein kinase B (PKB), an anti-apoptosis signal, whereas EPO led to activation of ERKs. Furthermore, the SCF- and EPO-maintained expression of antiapoptotic protein Bcl-XL was correlated with the activation of ERKs and was inhibited by PD98059, suggesting that Bcl-XL may not have a major role in preventing apoptosis of human ECFCs. Phosphorylated BAD was not affected by SCF, EPO or wortmannin. Taken together with our previous results, the present study indicates that SCF and EPO support survival and growth of human ECFCs through different signalling pathways and that they transduce distinctly different signals through activation of PI3K.

We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34+ cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34+ cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34+ cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34+ cells were subfractionated into two populations of IL-6R–negative (CD34+ IL-6R−) and IL-6R–positive (CD34+ IL-6R+) cells by fluorescence-activated cell sorting. The CD34+IL-6R− cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34+ IL-6R+cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo.

We recently showed that c-kit signal synergizes with glycoprotein (gp)130 signal mediated by a complex of interleukin (IL)-6 and soluble IL-6 receptor (IL-6/sIL-6R) to stimulate the expansion of human primitive hematopoietic progenitor cells and erythropoietin-independent erythropoiesis. In the present study, we examined the effect of a ligand for Flt3 (FL), whose receptor tyrosine kinase is closely related to c-kit, in combination with IL-6/sIL-6R on human hematopoiesis in vitro. In serum-containing methylcellulose clonal culture of cord blood CD34+ cells, whereas FL alone stimulated only granulocyte-macrophage (GM) colony formation, erythroid bursts and mixed colonies in addition to GM colonies were induced by FL with IL-6/sIL-6R, but not IL-6/sIL-6R alone. In suspension culture, CD34+ cells generated a small number of myeloid cells in the presence of FL or IL-6/sIL-6R alone. However, the addition of IL-6/sIL-6R to the culture with FL induced the generation of a significant number of erythroid cells and megakaryocytes in addition to myeloid cells. The combination of FL and IL-6/sIL-6R also induced a remarkable expansion of GM colony- and erythroid burst-forming cells and multipotential progenitors, although FL or IL-6/sIL-6R alone induced the generation of only a small number of progenitors for GM colonies. The synergistic effects of FL and IL-6/sIL-6R were confirmed in serum-free clonal and suspension cultures. In addition, the addition of anti-human gp130 monoclonal antibodies abrogated the synergistic action. These results indicate that Flt3 signal, as well as c-kit signal, synergizes with gp130 signal to stimulate human myelopoiesis, erythropoiesis and megakaryopoiesis, and the expansion of primitive multipotential hematopoietic progenitor cells.

Polycythemia vera (PV) is a clonal hematologic disease characterized by hyperplasia of the three major bone marrow lineages. PV erythroid progenitor cells display hypersensitivity to several growth factors, which might be caused by an abnormality of tyrosine phosphorylation. In the present study, we have investigated protein tyrosine phosphatase (PTP) activity in highly purified erythroid progenitor cells and found that the total PTP activity in the PV cells was twofold to threefold higher than that in normal cells. Protein separation on anion-exchange and gel-filtration columns showed that the increased activity was due to a major PTP eluted at approximately 170 kD. This enzyme was sensitive to PTP inhibitors and it did not cross-react with antibodies to SHP-1, SHP-2, or CD45. Subcellular fractionation showed that the PTP localized with the membrane fraction, where its activity was increased by threefold in PV erythroid progenitors when compared with normal cells. As the erythroid progenitors progressively matured, activity of the PTP declined rapidly in the normal cells but at a much slower rate in the PV cells. These studies suggest that a potentially novel membrane or membrane-associated PTP, representing a major PTP activity, may have an important role in proliferation and/or survival of human erythroid progenitors and that its hyperactivation in PV erythroid progenitors might be responsible for the increased erythropoiesis in PV patients.

Erythropoietin (EPO) is the primary humoral regulator of erythropoiesis and no other factor has previously been reported to support proliferation and terminal maturation of erythroid cells from hemopoietic stem cells. Here we show that stimulation of glycoprotein (gp130) by a combination of recombinant human soluble interleukin 6 receptor (sIL-6R) and IL-6 but not sIL-6R or IL-6 alone can support proliferation, differentiation, and terminal maturation of erythroid cells in the absence of EPO from purified human CD34+ cells in suspension culture containing stem cell factor (SCF). A number of erythroid bursts and mixed erythroid colonies also developed in methylcellulose culture under the same combination. The addition of anti-gp130 monoclonal antibodies but not anti-EPO antibody to the same culture completely abrogated the generation of erythroid cells. These results clearly demonstrate that mature erythroid cells can be emerged from hemopoietic progenitors without EPO in vitro. Together with the previous reports that human sera contain detectable levels of sIL-6R, IL-6, and SCF, current data suggest that gp130 signaling in association with c-kit activation may play a role in human erythropoiesis in vivo.

gp130, a signal-transducing receptor component of interleukin 6 (IL-6), associates with an IL-6 and IL-6 receptor (IL-6) complex and transduces signals. To examine the role of gp130 signaling in the expansion of human hemopoietic progenitor cells, we tested the effects of a recombinant soluble human IL-6 receptor (sIL-6R) and/or IL-6 in combination with other cytokines on purified human umbilical cord blood CD34+ cells, using methylcellulose clonal assay and suspension culture in the presence or absence of serum. A combination of sIL-6R and IL-6 (sIL-6R/IL-6), but not sIL-6R or IL-6 alone, was found to dramatically stimulate expansion of hemopoietic progenitor cells as well as CD34+ cells in the presence of stem cell factor. Significant generation of multipotential hemopoietic progenitors over a period of 3 weeks in suspension culture and efficient formation of colonies, especially multilineage and blast cell colonies, in methylcellulose assay supplemented with a combination of sIL-6R/IL-6 together with stem cell factor were observed in serum-containing and serum-free culture. Addition of anti-gp130 monoclonal antibodies or anti-IL-6R monoclonal antibodies to the above cultures dose-dependently inhibited the expansion of progenitor cells in suspension culture and also completely blocked the formation of multilineage colonies in methylcellulose culture. These findings demonstrated that the significant expansion of human primitive hemopoietic progenitors could be achieved with the gp130 and c-Kit signalings initiated by the sIL-6R/IL-6 complex in the presence of stem cell factor and suggested the possible application of this method for ex vivo expansion of CD34+ cells for bone marrow transplantation.

We previously demonstrated the feasibility of generating therapeutic numbers of cytotoxic T lymphocyte (CTL) clones expressing a CD20-specific scFvFc:CD3zeta chimeric T cell receptor (cTCR), making them specifically cytotoxic for CD20+ B lymphoma cells. However, the process of generating and expanding he CTL clones was laborious, the CTL clones expressed the cTCR at low surface density, and they exhibited suboptimal proliferation and cytotoxicity. To improve the performance of the CTLs in vitro and in vivo, we engineered "second-generation'' plasmid constructs containing a translational enhancer (SP163) and CD28 and CD137 costimulatory domains in cis with the CD3zeta intracellular signaling domain of the cTCR gene. Furthermore, we verified the superiority of generating genetically modified polyclonal T cells expressing the second-generation cTCR rather than T cell clones. Our results demonstrate that SP163 enhances the surface expression of the cTCR; that the second-generation cTCR improves CTL activation, proliferation, and cytotoxicity; and that polyclonal T cells proliferate rapidly in vitro and mediate potent CD20-specific cytotoxicity. This study provides the preclinical basis for a clinical trial of adoptive T cell immunotherapy for patients with relapsed CD20+ mantle cell lymphoma and indolent lymphomas.

PTP-MEG2 is an intracellular protein tyrosine phosphatase with a putative lipid-binding domain at the N-terminus. The present study reports expression, purification, and characterization of the full-length form of the enzyme plus a truncated form containing the catalytic domain alone. Full-length PTP-MEG2 was expressed with an adenovirus system and purified from cytosolic extracts of human 293 cells infected with the recombinant adenovirus. The purification scheme included chromatographic separation of cytosolic extracts on fast flow Q-Sepharose, heparin-agarose, l-histidyldiazobenzylphosphonic acid agarose, and hydroxylapatite. The enrichment of PTP-MEG2 from the cytosol was about 120-fold. The truncated form of PTP-MEG2 was expressed in E. coli cells as a non-fusion protein and purified by using a chromatographic procedure similar to that used for the full-length enzyme. The purified full-length and truncated enzymes showed single polypeptide bands on SDS-polyacrylamide gel electrophoresis under reducing conditions and behaved as monomers on gel exclusion chromatography. With para-nitrophenylphosphate and phosphotyrosine as substrates, both forms of the enzyme exhibited classical Michaelis-Menten kinetics. Their responses to pH, ionic strength, metal ions, and protein phosphatase inhibitors are similar to those observed with other characterized tyrosine phosphatases. Compared with full-length PTP-MEG2, the truncated DeltaPTP-MEG2 displayed significantly higher V(max) and lower K(m) values, suggesting that the N-terminal putative lipid-binding domain may have an inhibitory role. The full-length and truncated forms of PTP-MEG2 were also expressed as GST fusion proteins in E. coli cells and purified to near homogeneity through affinity columns. However, the specific phosphatase activities of the GST fusion proteins were 10-25-fold below those obtained with the correspondent non-fusion proteins.

P(0), a major structural protein of peripheral myelin, belongs to the immunoglobulin superfamily. Sequence comparison of P(0) with PZR, a tyrosine phosphatase SHP-2 binding protein we recently cloned, revealed the presence of an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracellular portion of the P(0) molecule. To study the role of this putative ITIM in signal transduction, we have expressed P(0) in HT-1080 and 293 cells. Stimulation of the transfected cells with pervanadate, a powerful inhibitor of tyrosine phosphatases, resulted in tyrosine phosphorylation of P(0) and its association with several tyrosine-phosphorylated proteins. Mutation of Y(220) embedded in the ITIM to phenylalanine abolished the tyrosine phosphorylation and the association. Tyrosine phosphorylation of P(0) and its association with other signaling proteins were also observed in pervanadate-treated RN22 Schwannoma cells, which express endogenous P(0). Furthermore, injection of pervanadate induced tyrosine phosphorylation of P(0) in peripheral nerves of newborn but not adult mice. The physiological importance of the ITIM in P(0) is implied by the fact that a naturally occurred P(0) mutant with a disrupted ITIM has a dominant role in causing Dejerine-Scotts syndrome. Taken together, P(0) is phosphorylated on Try(220). The presence of an ITIM in P(0) and its ability to mediate protein-protein interaction through tyrosine phosphorylation indicate that P(0) is not merely a structural protein but may also be a crucial player in cell signaling.

We have recently shown that stimulation of glycoprotein (gp) 130, the membrane-anchored signal transducing receptor component of IL-6, by a complex of human soluble interleukin-6 receptor (sIL-6R) and IL-6 (sIL-6R/IL-6), potently stimulates the ex vivo expansion as well as erythropoiesis of human stem/progenitor cells in the presence of stem cell factor (SCF). Here we show that sIL-6R dose-dependently enhanced the generation of megakaryocytes (Mks) (IIbIIIa-positive cells) from human CD34(+) cells in serum-free suspension culture supplemented with IL-6 and SCF. The sIL-6R/IL-6 complex also synergistically acted with IL-3 and thrombopoietin (TPO) on the generation of Mks from CD34(+) cells, whereas the synergy of IL-6 alone with TPO was barely detectable. Accordingly, the addition of sIL-6R to the combination of SCF + IL-6 also supported a substantial number of Mk colonies from CD34(+) cells in serum-free methylcellulose culture, whereas SCF + IL-6 in the absence of sIL-6R rarely induced Mk colonies. The addition of monoclonal antibodies against gp130 to the suspension and clonal cultures completely abrogated the megakaryopoiesis induced by sIL-6R/IL-6 in the presence of SCF, whereas an anti-TPO antibody did not, indicating that the observed megakaryopoiesis by sIL-6R/IL-6 is a response to gp130 signaling and independent of TPO. Furthermore, human CD34(+) cells were subfractionated into two populations of IL-6R-negative (CD34(+) IL-6R-) and IL-6R-positive (CD34(+) IL-6R+) cells by fluorescence-activated cell sorting. The CD34(+) IL-6R- cells produced a number of Mks as well as Mk colonies in cultures supplemented with sIL-6R/IL-6 or TPO in the presence of SCF. In contrast, CD34(+) IL-6R+ cells generated much less Mks and lacked Mk colony forming activity under the same conditions. Collectively, the present results indicate that most of the human Mk progenitors do not express IL-6R, and that sIL-6R confers the responsiveness of human Mk progenitors to IL-6. Together with the presence of functional sIL-6R in human serum and relative unresponsiveness of human Mk progenitors to IL-6 in vitro, current results suggest that the role of IL-6 may be mainly mediated by sIL-6R, and that the gp130 signaling initiated by the sIL-6R/ IL-6 complex is involved in human megakaryopoiesis in vivo.

Stem cell factor (SCF) and erythropoietin (EPO) work synergistically to support erythropoiesis, but the mechanism for this synergism is unknown. By using purified human erythroid colony-forming cells (ECFC), we have found that SCF and EPO synergistically activate MAP kinase (MAPK, ERK1/2), which correlates with the cell growth and thus may be responsible for the synergistic effects. Treatment of the cells with PD98059 and wortmannin, inhibitors of MEK and PI-3 kinase, respectively, inhibited the synergistic activation of MAPK and also the cell growth, further supporting this conclusion. Wortmannin only inhibits MAPK activation induced by EPO but not that by SCF, suggesting that SCF and EPO may activate MAPK through different pathways, which would facilitate synergy. Furthermore, EPO, but not SCF, led to activation of STAT5, whereas SCF and wortmannin had no effect on the EPO-induced STAT5 activation, suggesting that STAT5 is not involved in the synergistic action of SCF and EPO. Together, the data suggest that synergistic activation of MAPK by SCF and EPO is essential for expanded erythropoiesis.© 1998 by The American Society of Hematology.

Flow cytometric study revealed that almost all CD34+ cells in human umbilical cord blood expressed interferon-gamma receptor (IFN-gammaR). To clarify the precise functional roles of IFN-gammaR in human CD34+ cells, we examined the effect of IFN-gamma alone and in combination with various cytokines on the growth of haemopoietic progenitor cells in CD34+ cells using a serum-free clonal culture. Surprisingly, IFN-gamma alone supported only megakaryocyte (MK) colonies in a dose-dependent manner with a plateau level at 1000 U/ml of IFN-gamma. IFN-gamma at 1000 U/ml induced 10 +/- 1.2 MK colonies from 1 x 10(3) CD34+ cells, whereas thrombopoietin (TPO), interleukin (IL)-3, stem cell factor (SCF) or IL-6 alone induced 22 +/- 4.0, 22 +/- 4.2, 4 +/- 0.6 and 0 MK colonies, respectively. The addition of anti-IFN-gamma monoclonal antibody (mAb) to the IFN-gamma culture completely abrogated MK colony formation, whereas the mAb had no effect on TPO-dependent production of MK colonies. In contrast, although anti-TPO polyclonal Ab almost completely blocked TPO-dependent MK colony formation, it failed to inhibit the generation of MK colonies induced by IFN-gamma, suggesting that the observed effect of IFN-gamma on the proliferation of human MK progenitor cells is independent of TPO. The addition of IFN-gamma to culture with TPO or SCF significantly augmented the development of MK colonies, whereas it did not affect IL-3-dependent MK colony formation. Additionally, IFN-gamma induced the increase of DNA content of cultured glycoprotein IIb/IIIa-positive megakaryocytes. These results suggest that IFN-gamma may have regulatory roles in human megakaryocytopoiesis.

We recently demonstrated that stimulation of gp130 by a combination of soluble interleukin 6 receptor (sIL-6R) and IL-6 but not IL-6 alone significantly stimulates the ex vivo expansion of primitive hematopoietic progenitors and the generation of erythroid cells from human CD34+ cells in the presence of stem cell factor (SCF). Here, we show that gp130 is found low positively on most CD34+ cells, whereas IL-6R is expressed on only 30-50% of these cells. Although most of the colonies generated from FACS-sorted CD34+IL-6R+ cells were granulocyte/macrophage (GM) colonies, CD34+IL-6R- cells gave rise to various types of colonies, including erythroid bursts, GM, megakaryocytes, and mixed colonies in methylcellulose culture with a combination of IL-6, sIL-6R, and SCF. Similar results were obtained in culture supplemented with a combination of IL-3, IL-6, SCF, granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. A limiting dilution analysis of long-term culture-initiating cells (LTC-IC) showed that the CD34+IL-6R- cells contained a larger number of LTC-IC than did the CD34+IL-6R+ cells. In a serum-free suspension of CD34+IL-6R- cells, the addition of sIL-6R to the combination of IL-6 and SCF dramatically increased the total and multipotential progenitors, whereas CD34+IL-6R+ cells failed to do so under the same conditions. These results indicate that most of the erythroid, megakaryocytic, and primitive human hematopoietic progenitors are included in the IL-6R- populations, and the activation of gp130 on these progenitors can be achieved by a complex of IL-6-sIL-6R, but not by IL-6 alone. The present culture system using IL-6, sIL-6R, and SCF may provide a novel approach for ex vivo expansion of human primitive hematopoietic progenitors.

The hemopoietic system is characterized by continuous cell turnover supported by a small populations of cells termed hemopoietic stem cells. It is generally held that multipotential hemopoietic stem cells possess extensive capabilities to self-renew and generate various committed progenitor cells(1). Intercellular communication in the hemopoietic system is mediated by soluble factors called cytokines, whose functional characteristics are now partly explained by their receptor system (2–5). Most cytokine receptor systems consist of a multi-chain complex, a ligand-specific receptor chain (α-chain) and a signal transducing chain (β-chain), the latter of which is often utilized in common by several receptor complexes(2,6).

Glycoprotein (gp) 130, a receptor component for interleukin 6 (IL-6), can associate with a soluble IL-6 receptor (sIL-6R) – IL-6 complex. To examine the role of gp130 signaling in human hematopoietic progenitor-cell proliferation and differentiation, we studied the effects of the sIL-6R – IL-6 complex in combination with other cytokines on human CD34+ cells in clonal and suspension cultures. The sIL-6R – IL-6 complex, but not sIL-6R or IL-6 alone, in the presence of stem-cell factor (SCF) produced dramatic increases in the populations of various cell lineages, including erythroid cells and various hematopoietic progenitors, in suspension culture. Significant numbers of colonies of (particularly) multilineage and blast cells were generated in methylcellulose culture supplemented with a combination of sIL-6R – IL-6 complex and SCF. Addition of anti-gp130 monoclonal antibodies (MAbs) and anti-IL-6R MAbs to the above-mentioned cultures dose-dependently inhibited the generation of cells of various lineages and of progenitor cells in suspension culture and completely blocked multilineage colony production in methylcellulose culture; an anti-erythropoietin antibody did not cause inhibition. These findings demonstrate that both proliferation and differentiation of hematopoietic progenitor cells can be induced through gp130 and c-Kit signaling, indicating that progenitor cells are responsive to the sIL-6R – IL-6 complex, even though they do not express IL-6R. Together with previous studies showing that detectable levels of sIL-6R, IL-6, and SCF are present in human serum, these results suggest that gp130 signaling may play an important role in human hematopoiesis in vivo.