Marked induction of CD4+CD8+ T cells with multifunctional properties by coculturing CD2+ cells with xenogeneic stromal cells.

A number of T cell subsets have been identified, and the in vitro characterization of these subsets largely depends on an appropriate induction system for each one. In previous studies, we characterized a unique T cell line, HOZOT, which possessed a CD4+CD8+ double positive (DP) phenotype and multifunctional properties including cytotoxic, helper, and regulatory functions. Therefore, this T cell subset has been termed Tchreg cells. In this study, we established a new method of Tchreg cell induction, which consists of three steps and provides more efficient and reproducible results. By using a purified T cell population, the efficiency of Tchreg generation was profoundly increased, and the use of purified CD2+ cells rather than CD3+ cells was shown to be superior. One surprising finding was that at the initial step, stromal cell stimulation induced DP T cells more efficiently than dendritic cells or anti-T cell receptor stimulation, indicating a distinct antigen presenting cell (APC) ability of stromal cells as distinguished from conventional APCs. Even in subsequent steps, the presence of stromal cells was required to maintain the DP phenotype. In the second step, addition of stromal cell-conditioned (but not unconditioned) autologous CD14+ cells instead of interleukin-2 was beneficial for subsequent expansion of Tchreg cells. The improved three-step culture method described here represents a step forward in efforts to achieve clinical application and a good tool for elucidating how Tchreg cells, especially CD4+CD8+ T cells, are generated.

Potent anti-tumor killing activity of the multifunctional Treg cell line HOZOT against human tumors with diverse origins.

The T cell line HOZOT has a unique FOXP3+CD4+ CD8+CD25+ phenotype, exhibits suppressive activity in allogeneic mixed lymphocyte reactions (MLR), and produces IL-10, defining HOZOT as regulatory T cells (Tregs). Interestingly, in addition to possessing a suppressive Treg ability, HOZOT was also found to show cytotoxicity against certain representative human cancer cell types. In order to disclose the range of anti-tumor activity by HOZOT, we screened it by using a panel of twenty human tumor cell lines with different origins. Consequently, HOZOT showed potent cytocidal effects against a wide spectrum of neoplastic cells including carcinomas, sarcomas, mesotheliomas and glioblastomas except for hematopoietic malignancies. Its anti-tumor activity was strong enough with an E:T ratio of 4:1, which is considered to be more effective than that by conventional CTLs. Furthermore, an in vivo representative mouse tumor model by implanting human colon adenocarcinoma cells revealed that adoptive transfer of HOZOT almost completely eradicated disseminated lesions on peritoneum, markedly reduced metastases in lung and liver, and dramatically decreased bloody ascites caused by peritoneal carcinomatosis. Treatment of the tumor model mice by HOZOT with an E:T ratio of 2:1 even indicated the prolongation of their survival, although not reaching obvious statistical significance. In vitro blocking experiments using antibodies and inhibitors suggested that the cytotoxic mechanism of HOZOT against tumors is different from conventional cytotoxic cells such as CTL, NK or NKT cells. Altogether, our studies demonstrated the potent killing activity of HOZOT against a broad range of human malignancies, which indicates that HOZOT is a powerful tool in immunotherapy for advanced stage tumors.

Stromal cells' B7-1 is a key stimulatory molecule for interleukin-10 production by HOZOT, a multifunctional regulatory T-cell line.

We have previously shown that xenogeneic stromal cell stimulation of naïve T cells resulted in the generation of a new type of regulatory T (Treg) cell termed HOZOT, which has multifunctional properties and a CD4/CD8 double-positive phenotype. Even after the establishment of HOZOT, stromal cells can function as an antigen-presenting cell (APC) by inducing these cells to produce interleukin (IL)-10. When compared with other stimuli, stromal cells showed an IL-10-producing ability comparable to anti-CD3 antibody (Ab) stimulation, and much greater than dendritic cell (DC) stimulation. Distinct from professional APCs, stromal cells express only major histocompatibility complex (MHC) class I and B7-1 costimulatory molecules, and not MHC class II or other costimulatory molecules, such as ICOSL (CD275), PD-L1 (CD274), PD-L2 (CD273), CD40, OX40L (CD252) and 4-1BBL (CD137L) in the absence of stimulation. Blocking experiments revealed that, in addition to anti-H-2K(d) Ab and anti-human CD8 Ab, anti-mouse B7-1 Ab could effectively block IL-10 production, indicating a key role of the B7-1/CD28 pathway. Using stromal cells expressing different levels of B7-1, IL-10 production correlated with the levels of B7-1 expression. Distinct from ICOSL or PD-L1 expressed on DCs (which are regarded as IL-10-inducing costimulatory molecules), this study showed that B7-1 on stromal cells is a key molecule regulating IL-10 production by multifunctional Treg cells, HOZOT.

HOZOTs, novel human regulatory T-cell lines, exhibit helper or suppressor activities depending on dendritic cell or anti-CD3 stimulation.

OBJECTIVE: HOZOT cell lines (HOZOTs) are a new type of regulatory T cells established from human umbilical cord blood without using cytokines. In addition to their unique FOXP3(+)CD4(+)CD8(+)CD25(+) phenotype, HOZOTs are bifunctional and can exert either suppressor or cytotoxic activities. To further characterize HOZOTs, we cocultured HOZOTs with responder T cells under different stimulation conditions and found another function of HOZOTs. MATERIALS AND METHODS: Naïve CD4(+)T cells as responder cells were stimulated with dendritic cells or plate bound anti-CD3 antibody. As effector cells, HOZOTs were added to this culture and proliferation of the responder cells were monitored by (3)H-thymidine incorporation or carboxyfluorescein succinimidyl ester dilution method. To investigate the molecular mechanisms, antibodies specific for interleukin (IL)-2/IL-2R or cell surface molecules were used for blocking experiments. RESULTS: The proliferation of naïve CD4(+)T cells was suppressed by one HOZOT line, HOZOT-4, when the responder cells were stimulated with dendritic cells. However, responder cell proliferation was augmented by HOZOT-4 when these cells were stimulated with anti-CD3 antibody. This opposing function to responder cells was unique to HOZOTs because naturally occurring regulatory T cells suppressed proliferation of both dendritic cell- and anti-CD3-antibody-stimulated cells. IL-2 was not involved in the mechanism of the helper activity of HOZOT-4 as blocking antibodies for IL-2 and IL-2R did not abrogate the helper activity. Moreover, this helper activity could not be reduced by blocking costimulatory pathways such as CD28/B7, CD4/human leukocyte antigen-DR, and intercellular adhesion molecule-1/lymphocyte function-associated antigen-1. CONCLUSION: We demonstrated a new function of HOZOTs as helper T cells in addition to suppressor and cytotoxic activities, characterizing HOZOTs as multifunctional T cells.

Interleukin-8 and RANTES are signature cytokines made by HOZOT, a new type of regulatory T cells.

Distinct cytokine production profiles define the effector functions of both helper and regulatory T cells. Recently, we established novel cytotoxic regulatory T (Treg) cell lines, HOZOT, which have been characterized as IL-10-producing T cells. In this study, we further characterized HOZOT by performing comprehensive analyses of cytokines produced by HOZOTs in order to identify a signature cytokine profile. Using DNA microarrays, we compared the gene expression profiles of HOZOT-4, a representative HOZOT cell line, under three different conditions. Seven genes, including IL-8, IL-10, IL-13, MIP-1alpha, and MIP-1beta, were identified as inducible cytokines when stimulated with stromal cells or anti-CD3/CD28 antibodies. Twelve genes, including IL-2, IL-3, IL-4, IL-22, CCL1, and lymphotactin, were categorized as antibody stimulation-responsive but stromal cell-non-responsive. Three genes, IL-32, RANTES, and CCL23, were constitutively expressed irrespective of stimulation condition. Among these cytokines, we focused on two chemokines, IL-8 and RANTES for further studies, and found that only HOZOT produced both of them at considerable levels whereas other T cell subsets, including Tregs and helper T cells, did not. Kinetic and inhibition experiments revealed contrasting properties for the two chemokines. IL-8 was induced only after stimulation, whereas RANTES mRNA and protein accumulated to high levels even before stimulation. Interestingly, IL-8 mRNA was induced by cycloheximide treatment and RANTES showed reduced mRNA but increased protein expression by antibody stimulation. As a whole, the unique cytokine signature profile consisting of Th1, Th2, and cytolytic T cell cytokines as well as Treg cytokines reflect the multifunctional nature of HOZOT. In particular, the dual production of IL-8 and RANTES by distinct mechanisms is a hallmark of HOZOT.

The production of IL-10 by human regulatory T cells is enhanced by IL-2 through a STAT5-responsive intronic enhancer in the IL-10 locus.

STAT5 molecules are key components of the IL-2 signaling pathway, the deficiency of which often results in autoimmune pathology due to a reduced number of CD4(+)CD25(+) naturally occurring regulatory T (Treg) cells. One of the consequences of the IL-2-STAT5 signaling axis is up-regulation of FOXP3, a master control gene for naturally occurring Treg cells. However, the roles of STAT5 in other Treg subsets have not yet been elucidated. We recently demonstrated that IL-2 enhanced IL-10 production through STAT5 activation. This occurred in two types of human Treg cells: a novel type of umbilical cord blood-derived Treg cell, termed HOZOT, and Tr1-like Treg cells, IL-10-Treg. In this study, we examined the regulatory mechanisms of IL-10 production in these Treg cells, focusing specifically on the roles of STAT5. By performing bioinformatic analysis on the IL-10 locus, we identified one STAT-responsive element within intron 4, designated I-SRE-4, as an interspecies-conserved sequence. We found that I-SRE-4 acted as an enhancer element, and clustered CpGs around the I-SRE-4 were hypomethylated in IL-10-producing Treg cells, but not in other T cells. A gel-shift analysis using a nuclear extract from IL-2-stimulated HOZOT confirmed that CpG DNA methylation around I-SRE-4 reduced STAT5 binding to the element. Chromatin immunoprecipitation analysis revealed the in situ binding of IL-2-activated STAT5 to I-SRE-4. Thus, we provide molecular evidence for the involvement of an IL-2-STAT5 signaling axis in the expression of IL-10 by human Treg cells, an axis that is regulated by the intronic enhancer, I-SRE-4, and epigenetic modification of this element.

IL-2 activation of STAT5 enhances production of IL-10 from human cytotoxic regulatory T cells, HOZOT.

OBJECTIVE: Interleukin (IL)-10 is an immunosuppressive cytokine produced by many cell types, including T cells. We previously reported that a novel type of regulatory T (Treg) cells, termed HOZOT, which possesses a FOXP3+CD4+CD8+CD25+ phenotype and dual suppressor/cytotoxic activities, produced high levels of IL-10. In this study, we examined the mechanisms of high IL-10 production by HOZOT, focusing on Janus activating kinase (JAK)/signal transducers and activators of transcription (STAT) signaling pathway. MATERIALS AND METHODS: We prepared five different types of T cells, including HOZOT from human umbilical cord blood. Cytokine productions of IL-10, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) were compared among these T cells after anti-CD3/CD28 antibody stimulation in the presence or absence of IL-2. Specific inhibitors for JAK/STAT, nuclear factor-kappaB (NF-kappaB), and nuclear factor for activated T cell (NFAT) were used to analyze signal transduction mechanisms. RESULTS: IL-10 production by HOZOTs was greatly enhanced by the addition of IL-2. Little or no enhancement of IFN-gamma and TNF-alpha production was observed under the same conditions. The enhancing effect of IL-2 was specific for both HOZOT and IL-10-secreting Treg cells. T helper type 2 cells, whose IL-10 production mechanisms involve GATA-3, failed to show IL-2-mediated enhancement of IL-10. Similar enhancing effects of IL-15 and IFN-alpha suggested a major role of JAK/STAT activation pathway for high IL-10 production. Further inhibitor experiments demonstrated that STAT5 rather than STAT3 was critically involved in this mechanism. CONCLUSION: Our results demonstrated that IL-2 selectively enhanced production of IL-10 in HOZOT primarily through activation of STAT5, which synergistically acts with NF-kappaB/NFAT activation, implying a novel regulatory mechanism of IL-10 production in Treg cells.

IL-2-independent generation of FOXP3(+)CD4(+)CD8(+)CD25(+) cytotoxic regulatory T cell lines from human umbilical cord blood.

OBJECTIVE: Since the existence of mouse naturally occurring CD4(+)CD25(+) T regulatory (Treg) cells was demonstrated, a variety of human Treg subsets have been identified as distinct T cell populations. Here we show the establishment of novel Treg cell lines possessing unique characteristics. METHODS: Novel Treg cell lines, designated HOZOT, were generated by coculturing human umbilical cord blood cells with mouse stromal cell lines in the absence of exogenous IL-2 or other cytokines. HOZOT were characterized and compared with CD4(+)CD25(+) Treg cells in terms of the CD phenotype, FOXP3 expression, suppressor activity against allogeneic MLR, anergy property, and IL-10 production. RESULTS: HOZOT were generated and expanded as normal lymphoblastoid cells with cytotoxic activity against the cocultured stromal cells. HOZOT consisted of three subpopulations as defined by phenotype: CD4(+)CD8(+), CD4(+)CD8(dim), and CD4(-)CD8(+). All three subpopulations showed both suppressor and cytotoxic activities. While HOZOT's expression of FOXP3, CD25, GITR, and cytoplasmic CTLA-4 implied a similarity to naturally occurring CD4(+)CD25(+) Treg cells, these two Treg cells differed in IL-2 responsiveness and IL-10 production. CONCLUSIONS: Our studies introduce a new method of generating Treg cells in an IL-2-independent manner and highlight a unique Treg cell type with cytotoxic activity and a phenotype of FOXP3(+)CD4(+)CD8(+)CD25(+).

Transcription factor expression in B-cell precursor-leukemia cell lines: preferential expression of T-bet.

A number of transcription factors (TFs) have been reported that play crucial roles in hematopoiesis. However, only little is known about how these factors are involved in the mechanisms of hematopoietic development and lineage commitment. To investigate the roles of TFs in human B-cell precursors (BCPs), the present study analyzed the expression of the following 16 hematopoietic TFs: AML1, C/EBPalpha, C/EBPbeta, C/EBPgamma, C/EBPepsilon, E2A, Ets-1, GATA-1, GATA-2, GATA-3, Ikaros, IRF-1, Pax5, PU.1, T-bet and TCF-1 in 30 human BCP-leukemia cell lines. All BCP-leukemia cell lines were found to be positive for the expression of AML1, C/EBPgamma, E2A, Ets-1, IRF-1, Pax5 and PU.1 at the mRNA level. The mRNA expression of C/EBPalpha, C/EBPbeta, C/EBPepsilon, GATA-2, Ikaros, T-bet and TCF-1 was detected in 2 to 29 of the cell lines. Eight BCP-cell lines showed positivity for the dominant negative Ikaros isoform Ik6, while others were positive for expression of Ik1, 2, 3 and 4. GATA-1 and GATA-3 were universally negative. The expression of C/EBPalpha, PU.1 and T-bet was positive at the protein level in five, 29 and four out of 30 BCP-cell lines, respectively. Cell lines were stimulated with interleukin (IL)-7 and/or interferon (IFN)-gamma to investigate the regulation of TF expression. T-bet was clearly induced in the two cell lines NALM-19 and NALM-29 after stimulation. C/EBPbeta and IRF-1 were up-regulated in both cell lines and TCF-1 was down-regulated in NALM-19. No significant changes were observed for the other 12 TFs. The present report could provide useful information in the study of the role of TFs on normal and malignant human BCPs.

Acute myeloid leukemia cell lines MOLM-17 and MOLM-18 derived from patient with advanced myelodysplastic syndromes.

The two acute myelomonocytic leukemia sister cell lines MOLM-17 and MOLM-18 and the Epstein-Barr-virus positive non-malignant B-lymphoblastoid cell lines (B-LCLs) B422 and B423 were established from the bone marrow sample of a 60-year-old Japanese male in the advanced leukemic phase of refractory anemia with excess of blasts, a subtype of myelodysplastic syndromes (MDS). MOLM-17/-18 are proliferatively responsive to the growth factors present in the culture supernatant of the 5637 cell line. The B-LCLs are constitutively growth factor-independent. MOLM-17 and B422 were established at eight months after the initial diagnosis, while MOLM-18 and B423 were derived from a sample one month later. Immunophenotyping of the first leukemia sample revealed a mixed lineage leukemia immunophenotype with positivity for terminal deoxynucleotidyl transferase (TdT), CD13 and CD19; the second sample revealed solely myeloid characteristics with positivity for CD13, CD41 and CD61, whereas TdT was negative. MOLM-17/-18 showed immunomarker profiles typical of the myelomonocytic lineage. The karyotype analysis of MOLM-17/-18 revealed various non-random numerical and structural abnormalities including del(5)(q?), -7, der(11)add(11)(p11.2)add(11)(q23), add(17)(p11.2), add(18)(p11.2), -20, -22 as common aberrations. Treatment with tumor necrosis factor-alpha induced pronounced cellular differentiation of both cell lines into macrophage-like cells. The overall profile of MOLM-17/-18 based on their extensive immunological, cytogenetic and functional characterization suggests that these cell lines together with the paired B-LCLs B422 and B423 may represent scientifically significant in vitro models which could facilitate investigations into the pathobiology of MDS.

5-Azacytidine supports the long-term repopulating activity of cord blood CD34(+) cells.

DNA methylation plays important roles in a wide range of biological phenomena, especially in the embryonic development and tumorigenesis. However, correlations between differentiation and DNA methylation have not been clarified well in each differentiation system. In this study, we focused our attention on regulatory roles of DNA methylation in normal hematopoietic differentiation using a demethylating reagent, 5-azacytidine (5-AzaC). As a source of hematopoietic progenitor cells, we used CD34(+) cells prepared from human umbilical cord blood and examined the effects of 5-AzaC on the colony-forming activity and the long-term culture-initiating (LTC-IC) activity of these cells. 5-AzaC treatment increased LTC-IC frequency 1.57- to 2.50-fold as compared to the nontreated control. In parallel to this, immunoblotting analysis showed that the intensity of overall DNA methylation decreased after 5-AzaC treatment. These results indicated the involvement of DNA methylation and demethylation in controlling immaturity of hematopoietic progenitor cells and the usefulness of 5-AzaC for regulating this immaturity.

Transcription factor expression in cell lines derived from natural killer-cell and natural killer-like T-cell leukemia-lymphoma.

Although a number of transcription factors (TFs) have been identified that play a pivotal role in the development of hematopoietic lineages, only little is known about factors that may influence development and lineage commitment of natural killer (NK) or NK-like T (NKT)-cells. Obviously to fully appreciate the NK- and NKT-cell differentiation process, it is important to identify and characterize the TFs effecting the NK- and NKT-cell lineage. Furthermore, these TFs may play a role in NK- or NKT-cell leukemias, in which the normal differentiation program is presumably disturbed. The present study analyzed the expression of the following 13 TFs: AML1, CEBPA, E2A, ETS1, GATA1, GATA2, GATA3, IKAROS, IRF1, PAX5, PU1, TBET and TCF1 in 7 malignant NK-cell lines together with 5 malignant NKT-cell lines, 5 T-cell acute lymphoblastic leukemia (ALL) cell lines including 3 gamma/delta T-cell receptor (TCR) type and 2 alpha/beta TCR type, and 3 B-cell precursor (BCP) leukemia cell lines. AML1, E2A, ETS1, IKAROS and IRF1 were found to be positive for all cell lines tested whereas GATA1 turned out to be universally negative. CEBPA, PAX5 and PU1 were negative for all cell lines tested except in the three positive BCP-cell lines. GATA2 was positive for 3/5 T-cell lines but negative for the other cell lines. GATA3 was positive for 7/7 NK-, 4/5 NKT-, 5/5 T- and 2/3 BCP-cell lines. TBET was positive for all NK- and NKT-cell lines and negative for all T- and BCP-cell lines except one BCP-cell line. In contrast to the expression of TBET, TCF1 was negative for all NK- and NKT-cell lines, being positive for 4/5 T- and 1/3 BCP-cell lines. Expression analysis of TFs revealed that NK- and NKT-cell lines showed identical profiles, clearly distinct from those of the other T-ALL or BCP-ALL leukemia-derived cell lines..

Induction of CD28 on the new myeloma cell line MOLP-8 with t(11;14)(q13;q32) expressing delta/lambda type immunoglobulin.

The novel multiple myeloma (MM) cell line MOLP-8 carrying the t(11;14) (q13;q32) was established from the peripheral blood of a 52-year-old Japanese male patient with Bence-Jones delta/lambda type MM (stage IIIA with hyperammonemia). The growth of MOLP-8 cells is constitutively independent of exogenous growth factors or feeder cells. MOLP-8 cells grow mainly as free floating single cells and slightly adherent on the bottom of the plastic culture flask. Wright-Giemsa-stained MOLP-8 cells show the typical plasma cell morphology with abundant cytoplasm, heterogeneous cell size and one to three nuclei. The immunoprofile of MOLP-8 corresponds to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) delta/lambda chains, CD10, CD29, CD38, CD40, CD44, CD49b, CD49d, CD54, CD56, CD58, CD71, CD138 and PCA-1; the cells were negative for surface Igs and various other B-cell, T-cell and myelomonocyte-associated immunomarkers. CD28 became positive after co-culture of MOLP-8 cells with bone marrow adherent stromal (BST) feeder cells for a week. About 30% of MOLP-8 cells adhered strongly to the BST cells, but the cellular adhesion was clearly inhibited by addition of either anti-CD29 or anti-CD106 monoclonal antibody, suggesting a specific cellular adhesion through alpha4beta1-integrin-VCAM-1 interaction. The novel MOLP-8 cell line together with the present myeloma cell lines will present useful model systems in the investigation of the biology of MM.

CD45 tyrosine phosphatase inhibits erythroid differentiation of umbilical cord blood CD34+ cells associated with selective inactivation of Lyn.

CD45 is a membrane-associated tyrosine phosphatase that dephosphorylates Src family kinases and Janus kinases (JAKs). To clarify the role of CD45 in hematopoietic differentiation, we examined the effects of anti-CD45 monoclonal antibody NU-L(PAN) on the proliferation and differentiation of umbilical cord blood CD34(+) cells. NU-L(PAN) showed a prominent inhibition of the proliferation of CD34(+) cells induced by the mouse bone marrow stromal cell line MS-5 or erythropoietin (EPO). However, NU-L(PAN) did not affect the proliferation induced by interleukin 3. NU-L(PAN) also inhibited MS-5-induced or EPO-induced erythroid differentiation of CD34(+) cells. The cells stimulated with EPO in the presence of NU-L(PAN) morphologically showed differentiation arrest at the stage of basophilic erythroblasts after 11 days of culture, whereas the cells treated with EPO without NU-L(PAN) differentiated into mature red blood cells. The Src family kinase Lyn and JAK2 were phosphorylated when erythroblasts obtained after 4 days of culture of CD34(+) cells in the presence of EPO were restimulated with EPO. Overnight NU-L(PAN) treatment before addition of EPO reduced the phosphorylation of Lyn but not that of JAK2. Simultaneously, the enhancement of Lyn kinase activity after restimulation with EPO was reduced by NU-L(PAN) treatment. These results indicate selective inactivation of Lyn by CD45 activated with NU-L(PAN) and could partly explain the inhibitory mechanism on erythropoiesis exhibited by EPO. These findings suggest that CD45 may play a pivotal role in erythropoiesis.

Transforming growth factor-beta(1) augments granulocyte-macrophage colony-stimulating factor-induced proliferation of umbilical cord blood CD34(+) cells with an associated tyrosine phosphorylation of STAT5.

OBJECTIVE: Several investigators have reported that transforming growth factor (TGF)-beta(1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) synergistically support cell proliferation. However, the mechanisms involved have not been elucidated. To clarify the mechanisms of the synergistic action of TGF-beta(1) and GM-CSF, we compared the activation states of STAT5 and mitogen-activated protein kinase in CD34(+) cells and in GM-CSF-dependent hematopoietic cell lines. MATERIALS AND METHODS: Human CD34(+) cells and GM-CSF-dependent cell lines (FKH-1, YNH-1, and M-07e) were stimulated with 1.25 ng/mL GM-CSF and/or 0.25 ng/mL TGF-beta(1), and 1.25 ng/mL GM-CSF and/or 0.25 ng/mL, 0.025 ng/mL TGF-beta(1), respectively, and cell proliferation was analyzed by [3H]thymidine uptake. Expression of signal transduction proteins and their phosphorylation states were determined by Western blotting. RESULTS: TGF-beta(1) synergistically enhanced the GM-CSF-augmented growth of CD34(+) cells and FKH-1 cells, but inhibited the growth of YNH-1 and M-07e cells. Tyrosine phosphorylation of STAT5 induced by GM-CSF was enhanced by stimulation with the combination of TGF-beta(1) and GM-CSF (TGF-beta(1)/GM-CSF) compared with that induced by GM-CSF alone in CD34(+) cells and FKH-1 cells. However, combinations of TGF-beta(1)/GM-CSF caused inhibition of GM-CSF-induced tyrosine phosphorylation in M-07e cells. No significant difference was observed in mitogen-activated protein kinase activation between CD34(+) cells and FKH-1 cells stimulated with GM-CSF/TGF-beta(1) or GM-CSF alone. CONCLUSIONS: Results suggest that TGF-beta(1) may augment GM-CSF-induced proliferation of CD34(+) cells in association with enhanced tyrosine phosphorylation of STAT5. Our data suggest a novel mechanism for the synergistic enhancement of cellular growth induced by the combination of TGF-beta(1) and GM-CSF.

Novel B-cell acute lymphoblastic leukemia sister cell lines BALM 19-23 and BALM-26 with interclonal proliferative and phenotypic heterogeneity from a patient with hypercalcemia.

A series of human acute lymphoblastic leukemia (ALL) cell lines, BALM-19, -20, -21, -22, -23 (BALM 19-23) and BALM-26 were established from a patient with B-cell characteristics of ALL L2 type. All cell lines were derived from bone marrow specimens, BALM 19-23 from a sample taken at diagnosisand BALM-26 from one at relapse. Like the original leukemia cells, the established lines present various B-cell characteristics, being positive for cell surface immunoglobulin (Ig) chains but also for nuclear terminal deoxynucleotidyl transferase; hence the cell lines should be assigned to B-cell category B-IV. As a unique feature, the cell lines expressed the CD33 myeloid antigen in addition to the common B-cell markers. Heterogeneous antigen expression among the different cell lines was found regarding CD35, CD39, CD45RA, CD78 and CD95. The malignant nature of the cell lines was documented by negativity for the Epstein-Barr virus and by the occurrence of clonal non-random structural chromosome abnormalities. The patient's serum showed hypercalcemia, prompting further investigation of the established cell lines which expressed parathyroid hormone related peptide (PTHrP) mRNA as examined by reverse transcriptase polymerase chain reaction. The established B-cell ALL sister cell lines, BALM 19-23 and BALM-26, could provide useful material for clarifying the pathogenesis of this type of B-cell malignancy. The scientific significance of this panel of cell lines lies in the availability of a series of clonally derived but phenotypically different sister cell lines established at different phases of the disease.