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.

ABSTRACTgpl3O, a signal-transducing receptor component of interleukin 6 (IL-6), associates with an IL-6 and IL-6 receptor (IL-6R) complex and transduces signals. To examine the role of gpl3O signaling in the expansion of human hemopoietic progenitor cells, we tested the effects of a recombinant soluble human and/or 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 antigpl3O 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 gpl3O 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.

SummaryWe recently demonstrated that stimulation of gp 130 by a combination of soluble interleukin 6 receptor (slL-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| 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, slL-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-6P, -cells contained a larger number of LTC-IC than did the CD34+IL-6R + cells. In a serum-free suspension ofCD34+IL-61:( -cells, the addition of slL-6P, to the combination oflL-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-61:(-populations, and the activation of gp130 on these progenitors can be achieved by a complex of IL-6-slL-6R, but not by IL-6 alone. The present culture system using IL-6, slL-6R., and SCF may provide a novel approach for ex vivo expansion of human primitive hematopoietic progenitors.

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.

Summary. 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-X L was correlated with the activation of ERKs and was inhibited by PD98059, suggesting that Bcl-X L 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.

SummaryErythropoietin (EPO) is the primary humoral regulator of erythropoiesis and no other factor has previously been reported to support proliferation and terminal maturation oferythroid cells from hemopoietic stem cells. Here we show that stimulation ofglycoprotein (gp130) by a combination of recombinant human soluble intedeukin 6 receptor (slL-6R) and IL-6 but not slL-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 ofanti-gpl30 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 slL-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.

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.

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.