Akira Harashima

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.

miR-155, a Modulator of FOXO3a Protein Expression, Is Underexpressed and Cannot Be Upregulated by Stimulation of HOZOT, a Line of Multifunctional Treg.

MicroRNAs (miRNAs) play important roles in regulating post-transcriptional gene repression in a variety of immunological processes. In particular, much attention has been focused on their roles in regulatory T (Treg) cells which are crucial for maintaining peripheral tolerance and controlling T cell responses. Recently, we established a novel type of human Treg cell line, termed HOZOT, multifunctional cells exhibiting a CD4+CD8+ phenotype. In this study, we performed miRNA profiling to identify signature miRNAs of HOZOT, and therein identified miR-155. Although miR-155 has also been characterized as a signature miRNA for FOXP3+ natural Treg (nTreg) cells, it was expressed quite differently in HOZOT cells. Under both stimulatory and non-stimulatory conditions, miR-155 expression remained at low levels in HOZOT, while its expression in nTreg and conventional T cells remarkably increased after stimulation. We next searched candidate target genes of miR-155 through bioinformatics, and identified FOXO3a, a negative regulator of Akt signaling, as a miR-155 target gene. Further studies by gain- and loss-of-function experiments supported a role for miR-155 in the regulation of FOXO3a protein expression in conventional T and HOZOT cells.

NF-ATc2 induces apoptosis in Burkitt's lymphoma cells through signaling via the B cell antigen receptor

Cross‐linking of the B cell antigen receptor (BCR) with an anti‐IgM antibody has been shown to induce dramatic apoptosis in type I Burkitts lymphoma (BL) cells. However, the apoptotic mechanism triggered via BCR remains unknown. Here we reports a mechanism of BCR ligation‐induced apoptosis involving protein phosphatase calcineurin and its specific substrate, transcriptional factor NF‐AT. In response to BCR cross‐linking, endogenous calcineurin was rapidly activated, and this facilitated nuclear translocation of NF‐ATc2, a subtype of NF‐AT members. Interestingly, nuclear‐imported NF‐ATc2 functioned pro‐apoptotically in BL cells. The effect of NF‐ATc2 was efficiently blocked with FK506, which prevented its nuclear translocation through inactivation of calcineurin. In addtion, TR3 induction during BCR cross‐linking was reduced by FK506 and the VIVIT peptide, which is a highly selective inhibitor for NF‐AT. This strongly suggests that activation of NF‐ATc2 by calcineurin is essential for TR3 recruitment, and that TR3 can be considered as a candidate for death effector in BCR‐mediated apoptosis. Therefore, NF‐ATc2 plays a crucial role in BCR‐mediated apoptosis in type IBL, providing greater insight into unique BL characteristics through BCR signaling.

Human bone marrow stroma‐dependent cell line MOLP‐5 derived from a patient in leukaemic phase of multiple myeloma

The novel multiple myeloma (MM) cell line MOLP‐5 and its homologous sister cell line B407, a lymphoblastoid cell line (LCL), were established from the peripheral blood of a 71‐year‐old Japanese patient with Bence‐Jones κ‐type multiple myeloma (stage IIIB with hyperammonaemia and hypercalcaemia). The growth of MOLP‐5 cells is constitutively dependent on bone marrow stroma (BST) cells; none of the cytokines tested nor the culture supernatant of the bone marrow stroma cells could support the growth of MOLP‐5. Wright–Giemsa‐stained MOLP‐5 cells showed typical plasma cell morphology with abundant cytoplasm and one to three nuclei. The immunoprofile of MOLP‐5 corresponds to that seen typically in primary MM cells: positive for cytoplasmic immunoglobulin (Ig) κ light chain, CD28, CD29, CD38, CD40, CD44, CD49d, CD54, CD56, CD58, CD71, CD138 and PCA‐1; the cells were negative for surface Ig and various other B‐cell, T‐cell and myelomonocyte‐associated immunomarkers. Interleukin 6 (IL‐6) receptor mRNA was found in the reverse transcriptase polymerase chain reaction (RT‐PCR) analysis. IL‐6 and IL‐10 could induce cellular proliferation in short‐term induction experiments. IL‐6 or IL‐10 production was not detected by specific enzyme‐linked immunoabsorbent assay (ELISA). MOLP‐5 cells expressed parathyroid hormone‐related protein (PTHrP) at the mRNA level. Cytogenetic analysis showed the typical t(11; 14) chromosome abnormality. The novel MOLP‐5 cell line together with the B407 B‐LCL sister line will be useful model systems in the investigation of the biology of MM.

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 iniluence development and lineage commibnent 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. ’Ihe 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 γ /δ T-cell receptor (TCR) type and 2 α/β 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. TCFl 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 leukemiaderived cell lines.

Novel mechanisms of suppressor activity exhibited by cytotoxic regulatory T cell lines, HOZOT

OBJECTIVE Regulatory T (Treg) cells, which play a central role in maintaining immune tolerance, can be grouped into different subtypes, such as naturally occurring Treg, type-1 T regulatory, and Th3. The suppressor activities of Treg cells are mediated through several molecular mechanisms, including immunosuppressive cytokines, cell surface molecules, and cytolytic molecules. In a previous report, we described a novel regulatory human T-cell line (termed HOZOT). The line was established by cocultivating human umbilical cord blood cells with mouse stromal cells in the absence of exogenous cytokines. In this study, we investigated the mechanism of HOZOTs suppressor activity. MATERIALS AND METHODS Suppressor activity of HOZOT was evaluated in vitro by assessing their inhibition of allogeneic mixed lymphocyte reaction, in which CD4+CD25(-) responder T cells were stimulated by dendritic cells (DCs). Responder T cells as well as DCs were prepared from umbilical cord blood using magnetic-activated cell sorting separation system. DNA microarray analysis was performed to search for specific molecules involved in HOZOTs suppressor mechanisms. RESULTS We confirmed that suppressing effects were observed in all three subpopulations of CD4/CD8 phenotype. We ruled out possible involvement of HOZOTs cytotoxic activity as well as participation of surface molecules, including cytotoxic T-lymphocyte-associated protein-4, programmed death-1, and glucocorticoid-inducible tumor necrosis factor receptor in suppressor. The supernatant obtained from HOZOT and DC coculture revealed mixed lymphocyte reaction inhibitory activity, indicating the presence of a soluble factor, which mediates suppressor function. Blocking experiments demonstrated that interleukin-10 and transforming growth factor-beta were not responsible factors. CONCLUSIONS HOZOT exerted suppressor activity in the absence of cell contact mechanisms, which are distinct from those of naturally occurring Treg, type-1 T regulatory, and Th3.

Transforming growth factor-β1 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.

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.

Establishment and characterization of a novel ALL-L3 cell line (BALM-18): induction of apoptosis by anti-IgM and inhibition of apoptosis by bone marrow stroma cells.

A human acute lymphoblastic leukemia (ALL) cell line, BALM-18, was established from the peripheral blood specimen of a patient with B cell ALL L3 type (ALL-L3) at diagnosis using bone marrow stroma cells (BST) as feeder cells. The primary leukemia cells did not grow without feeder cells. As with the primary leukemia cells, BALM-18 showed an immunophenotype of Burkitts lymphoma group I [CD10+, CD20+, CD23-, CD39-, CD77+] and carried the t(8;14)(q24;q32) chromosomal abnormality which is highly associated with ALL-L3 and Burkitts lymphoma. It also revealed a significantly low level of bcl-2 protein. Strikingly, anti-human IgM antibody did induce apoptosis in induction experiments. However, it was reversed by the addition of anti-CD40 antibody or BST cells, whereas the culture supernatant of the stroma cells did not show any effect on the inhibition of apoptosis. BALM-18 may be useful for analyzing both the mechanisms of anti-IgM induced apoptosis and signaling during the inhibition of apoptosis by CD40 or BST cells.

A novel ALL-L3 cell line, BALM-25, expressing both immunoglobulin light chains.

A human acute lymphoblastic leukemia (ALL)-derived cell line, BALM-25, was established from the bone marrow specimen of a 59-year-old male patient with B-cell ALL L3 type (ALL-L3) at diagnosis. Immunophenotyping indicated mature B-cell characteristics including expression of cell surface and cytoplasmic immunoglobulin (Ig) chains, CD10, CD19, CD20, CD38, CD39, CD40, CD71, NU-B1 and HLA class II. T-cell and myeloid associated antigens tested were negative except CD5. BALM-25 cells have a morphological appearance typical for L3-type lymphoblasts. Regarding the expression of Ig chains, while the original leukemia cells expressed Ig λδμ and hence a single light (L) chain isotype, the established line revealed double L chain expression both at the cell surface and the cytoplasmic level. Definitive double L chain expression was confirmed by flow cytometry and Western blot analysis. Southern blot analysis demonstrated rearrangement of the IgJH, the Cκ and the Cλ genes. Cytogenetic analysis of BALM-25 revealed the following numerical and structural abnormalities: 55, X, add(X)(q12), + 2, add(3)(p21), + 5, add(7)(p13), add(11)(p11.2), add(11)(q?23), add(12)(p11.2), add(14)(q22), − 15, + 16, + 16,add(18)(11.2), + 20, + mar1, + mar2, + mar3, + mar,inc. The established cell line, BALM-25, provides an unlimited supply of cell material for analyzing the unique (patho)physiology of Ig expression in general and for clarifying the pathogenesis of this type of B-cell malignancy in particular.