Novel p19 protein engages IL-12p40 to form a cytokine, IL-23, with biological activities similar as well as distinct from IL-12.

A novel sequence discovered in a computational screen appears distantly related to the p35 subunit of IL-12. This factor, which we term p19, shows no biological activity by itself; instead, it combines with the p40 subunit of IL-12 to form a novel, biologically active, composite cytokine, which we term IL-23. Activated dendritic cells secrete detectable levels of this complex. IL-23 binds to IL-12R beta 1 but fails to engage IL-12R beta 2; nonetheless, IL-23 activates Stat4 in PHA blast T cells. IL-23 induces strong proliferation of mouse memory (CD4(+)CD45Rb(low)) T cells, a unique activity of IL-23 as IL-12 has no effect on this cell population. Similar to IL-12, human IL-23 stimulates IFN-gamma production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.

Leukocystatin, a new Class II cystatin expressed selectively by hematopoietic cells.

We describe a new cystatin in both mice and humans, which we termed leukocystatin. This protein has all the features of a Class II secreted inhibitory cystatin but contains lysine residues in the normally hydrophobic binding regions. As determined by cDNA library Southern blots, this cystatin is expressed selectively in hematopoietic cells, although fine details of the distribution among these cell types differ between the human and mouse mRNAs. In addition, we have determined the genomic organization of mouse leukocystatin, and we found that in contrast to most cystatins, the leukocystatin gene contains three introns. The recombinant proteins corresponding to these cystatins were expressed in Escherichia coli as N-terminal glutathione S-transferase or FLAGTM fusions, and studies showed that they inhibited papain and cathepsin L but with affinities lower than other cystatins. The unique features of leukocystatin suggests that this cystatin plays a role in immune regulation through inhibition of a unique target in the hematopoietic system.

FLT3 ligand preserves the ability of human CD34+ progenitors to sustain long-term hematopoiesis in immune-deficient mice after ex vivo retroviral-mediated transduction.

Stromal support is required during retroviral-mediated transduction of human bone marrow-derived CD34+ cells to maintain the clonogenicity of the primitive progenitors. We hypothesized that the cytokine FLT3 ligand (FL) might be able to replace the maintenance role provided by the stroma. CD34+ progenitors from human bone marrow were transduced by the retroviral vector LN with the cytokines interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) present in all cultures. Transductions were performed with or without stromal support and with or without the inclusion of 100 U/mL FL. No significant increase in gene transfer into colony-forming cells was obtained by the addition of FL to the cultures. Transduction and survival of more primitive human hematopoietic cells was determined by growth in immune-deficient mice for 7 to 8 months. Human myeloid cells, T lymphocytes, and colony-forming progenitors were recovered from the marrow of mice that had received human cells transduced on stroma or in suspension culture with IL-3, IL-6, SCF, and FL, but not with IL-3, IL-6, and SCF alone. LN provirus was detected by polymerase chain reaction in the marrow recovered from 9 of 10 mice transplanted with human CD34+ cells transduced with stromal support, 5 of 11 mice that received human cells transduced in suspension culture with FL, but none of the 10 mice that received human cells transduced in suspension culture without FL We conclude that FLT3 ligand, in conjunction with IL-3, IL-6, and SCF, preserves the generative capacity of primitive human hematopoietic cells during in vitro transductions in suspension culture.

Biochemical and genetic characterization of multiple splice variants of the Flt3 ligand.

We have performed a comprehensive analysis of cell lines and tissues to compare and contrast the expression patterns of Flt3 ligand (FL), c-Kit ligand (KL), and macrophage colony-stimulating factor as well as their receptors, Flt3, c-Kit, and c-Fms. The message for FL is unusually ubiquitous, whereas that of its receptor is quite restricted, apparently limiting the function of the ligand to fetal development and early hematopoiesis. We have also sequenced a mouse FL genomic clone, revealing how the three splice variant FL mRNAs that we have isolated arise. The chromosomal location of the FL gene has been mapped, by in situ hybridization, to chromosome 7 in mouse and chromosome 19 in human. Natural FL protein has been purified from a stromal cell line and shown to be a 65 kD nondisulfide-linked homodimeric glycoprotein comprised of 30 kD subunits, each containing 12 kD of N- and O-linked sugars. Pulse-chase experiments show that one of the splice variants (T110) is responsible for producing the bulk of soluble FL, but only after it has first been expressed at the cell surface as a membrane-bound form. The other splice-variant forms produce molecules that are either obligatorily soluble (T169) or membrane-bound but released only very slowly (T118). Finally, even though most cell lines express some amount of FL mRNA, we found that very little FL protein is actually made, with T cells and stromal cells being the major producers. The data suggests that FL plays its roles over very short distances, perhaps requiring cell-cell contact.

Mouse IL-17: a cytokine preferentially expressed by alpha beta TCR + CD4-CD8-T cells.

A novel cytokine originally designated murine CTLA-8 was described as a cDNA isolated from an activated T cell hybridoma produced by fusing a mouse cytotoxic T cell clone and a rat T lymphoma. This cDNA, which contains mRNA instability sequences characteristic of many cytokines, encoded a putative secreted protein that was homologous to the ORF13 gene of Herpesvirus saimiri. The human homolog to this molecule has recently been identified as the proinflammatory cytokine IL-17. We describe the isolation of a cDNA encoding mouse IL-17 from a cDNA library generated from alpha beta TCR + CD4-CD8- thymocytes using a subtraction technique that enriched for activation specific genes. This cDNA shares 87.3% amino acid identity to the previously described murine CTLA-8. Comparison of murine CTLA-8 to a cDNA we isolated from activated rat splenocytes revealed that murine CTLA-8 is, in fact, the rat homolog of IL-17. Mouse IL-17 mRNA is specifically expressed by activated alpha beta TCR + CD4-CD8- T cells, a small subset with a potentially important role in immune regulation. Mouse, rat, and human IL-17 can induce IL-6 secretion in mouse stromal cells, indicating that all homologs can recognize the mouse receptor.

DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes.

Intercellular adhesion molecules play an important role in the generation of T lymphocyte-mediated immune responses. Here, we describe a novel accessory molecule, DNAX accessory molecule-1 (DNAM-1), that is constitutively expressed on the majority of peripheral blood T lymphocytes. DNAM-1 is a 65 kDa transmembrane glycoprotein consisting of 318 aa including two immunoglobulin-like domains. Anti-DNAM-1 monoclonal antibody (MAb) inhibits T and NK cell-mediated cytotoxicity against a variety of tumor cell targets and blocks cytokine production by alloantigen-specific T cells. In addition, DNAM-1 is a tyrosine-phosphorylated signal-transducing molecule that participates in primary adhesion during cytotoxic T lymphocyte (CTL)-mediated cytotoxicity.

Flt3 ligand induces proliferation of quiescent human bone marrow CD34+CD38- cells and maintains progenitor cells in vitro.

Flt3 is a class III tyrosine kinase receptor expressed on primitive human and murine hematopoietic progenitor cells (HPC). In previous studies using stroma-free short term assays, Flt3 ligand (FL) has been shown to induce proliferation of HPC at proportions similar to or less than c-kit ligand (steel factor, SF). Using long term stromal cocultivation assays, we studied the effects of FL on proliferation and differentiation of a highly primitive and cytokine nonresponsive subpopulation of human HPC, CD34+cd38- cells. Cell Proliferation was significantly greater with FL than with SF, when used individually or in combinations with interleukin-3 (IL-3) and/or IL-6. The effect of FL was greater on bone marrow (BM) CD34+CD38- cells than the more cytokine responsive cord blood CD34+CD38- cells. Little or no effect was seen with FL on more mature CD34+CD38+ cells from either BM or cord blood. The frequency of colony-forming units (CFU) and high proliferative potential-colony forming cells (HPP-CFC) during early culture ( < or = 30 days) was increased by both SF and FL to similar levels. However, in the LTC-IC period (35 to 60 days) and extended long-term culture initiating cell (ELTC-IC) period ( > 60 days), the frequency of CFU and HPP-CFC was significantly greater in cultures containing FL than those without FL (P < .0025). Fluorescence-activated cell sorter analysis of cultures after 21 days showed a significantly higher percentage of cells remained CD34+ in the combination of FL, IL-3, IL-6, and SF (F/3/6/S) than in 3/6/S (0.78% +/- 0.52% v 0.21% +/- 0.29% respectively, mean +/- SD). Cloning efficiency of BM CD34+CD38- cells was significantly increased by the addition of FL to the combination of 3/6/S (mean 11.7% v 0.5%, P < .0001). These data show that FL is able to induce proliferation of CD34+CD38-cells that are nonresponsive to other early acting cytokines and to improve the maintenance of progenitors in vitro.

Human FLT3/FLK2 receptor tyrosine kinase is expressed at the surface of normal and malignant hematopoietic cells.

FLT3/FLK2 is a receptor tyrosine kinase (RTK) which is thought to play an important role in early stages of hematopoiesis. Monoclonal antibodies (mAbs) against the extracellular domain of human FLT3 were generated to study the cell surface expression of this class III RTK on normal bone marrow cells and on leukemic blasts from patients with acute leukemias. Functional analysis of five mAbs (SF1 series) revealed that all of them can mimic to variable extents the activity of the FLT3 ligand (FL) upon receptor activation and modulation, while only one mAb weakly inhibited ligand binding. Using flow cytometry, we detected surface expression of FLT3 on cell lines of the myeloid (4/8) and B lymphoid (7/10) lineages. On normal human bone marrow cells, the expression of FLT3 is restricted, in agreement with a presumed function of this receptor at the level of the stem cells and early committed progenitors. Expression of FLT3 was found on a fraction of CD34-positive and CD34-negative cells. Three-color analysis further revealed that most of the CD34 FLT3+ cells coexpress CD117 (KIT) at a high level. Finally, FLT3 is expressed on leukemic blasts of 18/22 acute myeloid leukemias (AML) and 3/5 acute lymphoid leukemias (ALL) of the B lineage, providing a possible application in diagnosis and therapy of these diseases.

Expression and signal transduction of the FLT3 tyrosine kinase receptor.

FLT3 is a receptor tyrosine kinase of 130-55 kDa expressed on normal bone marrow stem and early progenitor cells and on leukemic blasts from patients with acute leukemias. The FLT3 ligand, FL, is a new cytokine which acts on hematopoietic progenitors in synergy with other cytokines. FLT3 transduces FL-mediated signal through interaction with a number of cytoplasmic substrates.

FLT3/FLK2 ligand promotes the growth of murine stem cells and the expansion of colony-forming cells and spleen colony-forming units.

The effect of FLT3/FLK2 ligand (FL) on the growth of primitive hematopoietic cells was investigated using ThyloSca1+ stem cells. FL was observed to interact with a variety of factors to initiate colony formation by stem cells. When stem cells were stimulated in liquid culture with FL plus interleukin (IL)-3, IL-6, granulocyte colony-stimulating factor (G-CSF), or stem cell factor (SCF), cells capable of forming colonies in secondary methylcellulose cultures (CFU-c) were produced in high numbers. However, only FL plus IL-6 supported an increase in the number of cells capable of forming colonies in the spleens of irradiated mice (CFU-s). Experiments with accessory cell-depleted bone marrow (Lin- BM) showed that FL alone lacks significant colony-stimulating activity for progenitor cells. Nevertheless, FL enhanced the growth of granulocyte-macrophage progenitors (CFU-GM) in cultures containing SCF, G-CSF, IL-6, or IL-11. In these assays, FL increased the number of CFU-GM initiating colony formation (recruitment), as well as the number of cells per colony (synergy). Many of the colonies were macroscopic and contained greater than 2 x 10(4) granulocytes and macrophages. Therefore, FL appears to function as a potent costimulus for primitive cells of high proliferative potential (HPP). FL was also observed to costimulate the expansion of CFU-GM in liquid cultures of Lin- BM. In contrast, FL had no growth-promoting affects on progenitors committed to the erythrocyte, megakaryocyte, eosinophil, or mast cell lineages.

NFATx, a novel member of the nuclear factor of activated T cells family that is expressed predominantly in the thymus.

The nuclear factor of activated T cells (NFAT) regulates cytokine gene expression in T cells through cis-acting elements located in the promoters of cytokine genes. Here, we report the cDNA cloning, chromosomal localization, and initial characterization of a transcription factor related to NFATp and NFATc. The novel molecule, designated NFATx, exhibits in its middle a region very similar to the Rel homology domain in NFATc and NFATp. The amino-terminal region of NFATx also shows significant similarities to corresponding sequences in NFATc and NFATp and contains three copies of a conspicuous 17-residue motif of unknown function. We provide evidence showing that NFATx can reconstitute binding to the NFAT-binding site from the interleukin 2 promoter when combined with AP1 (c-Fos/c-Jun) polypeptides and that NFATx is capable of activating transcription of the interleukin 2 promoter in COS-7 cells when stimulated with phorbol ester and calcium ionophore. NFATx mRNA is preferentially and remarkably found in the thymus and at lower levels in peripheral blood leukocytes. The expression pattern of NFATx, together with its functional activity, strongly suggests that NFATx plays a role in the regulation of gene expression in T cells and immature thymocytes.

FLK-2/FLT-3 ligand regulates the growth of early myeloid progenitors isolated from human fetal liver.

The effects of the recently identified FLK-2/FLT-3 ligand (FL) on the growth of purified human fetal liver progenitors were investigated under serum-deprived culture conditions. FL alone was found to stimulate modest proliferation in short-term cultures of CD34++ CD38+ lineage (Lin)- light-density fetal liver (LDFL) cells and the more primitive CD34++ CD38- Lin- LDFL cells. However, the low levels of growth induced by FL were insufficient for colony formation in clonal cultures. Synergism between FL and either granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) or KIT ligand (KL) was observed in promoting the growth of high-proliferative potential (HPP) colony-forming cells (CF) and/or low-proliferative potential (LPP)-CFC in cultures of CD34++ CD38+ Lin- and CD34++ CD38- Lin- LDFL-cells. FL, alone or in combination with other cytokines, was not found to affect the growth of CD34+ Lin- LDFL cells, the most mature subpopulation of fetal liver progenitors investigated. The growth of the most primitive subset of progenitors studied, CD34++ CD38- Lin- LDFL cells, required the interactions of at least two cytokines, because only very low levels of growth were observed in response to either FL, GM-CSF, IL-3 or KL alone. However, the results of delayed cytokine-addition experiments suggested that individually these cytokines did promote the survival of this early population of progenitors. Although two-factor combinations of FL, KL, and GM-CSF were observed to promote the growth of early progenitors in a synergistic manner, neither of these factors was found to make fetal liver progenitors more responsive to suboptimal concentrations of a second cytokine. Only myeloid cells were recovered from liquid cultures of CD34++ CD38- Lin- LDFL cells grown in the presence of combinations of FL, KL, and GM-CSF. These results indicate that FL is part of a network of growth factors that regulate the growth and survival of early hematopoietic progenitors.

Ligand for FLT3/FLK2 receptor tyrosine kinase regulates growth of haematopoietic stem cells and is encoded by variant RNAs.

The FLT3/FLK2 receptor tyrosine kinase is closely related to two receptors, c-Kit and c-Fms, which function with their respective ligands, Kit ligand and macrophage colony-stimulating factor to control differentiation of haematopoietic and non-haematopoietic cells. FLT3/FLK2 is thought to be present on haematopoietic stem cells and found in brain, placenta and testis. We have purified to homogeneity and partially sequenced a soluble form of the FLT3/FLK2 ligand produced by mouse thymic stromal cells. We isolated several mouse and human complementary DNAs that encode polypeptides with identical N termini and different C termini. Some variants contain hydrophobic transmembrane segments, suggesting that processing may be required to release soluble ligand. The purified ligand enhances the response of mouse stem cells and a primitive human progenitor cell population to other growth factors such as interleukins IL-3 and IL-6 and to granulocyte-macrophage colony-stimulating factor, and also stimulates fetal thymocytes.

Antibodies to murine CD40 stimulate normal B lymphocytes but inhibit proliferation of B lymphoma cells.

A rat anti-mouse CD40 antiserum has been prepared by hyperimmunisation of Lewis rats with a highly purified preparation of the recombinant extracellular domain of murine CD40. This antiserum specifically binds CD40-expressing L cell transfectants, but not untransfected L cells, and induces vigorous proliferation of highly purified small dense B cells obtained from the spleens of unstimulated mice. Anti-CD40-induced B cell proliferation can be augmented by the addition of IL-4 and is inhibited by purified recombinant soluble mouse CD40. Interestingly the same anti-CD40 antiserum specifically inhibits the in vitro growth of A.20 murine B lymphoma cells. The specificity of this inhibition can be demonstrated by reversing the effect with purified recombinant soluble mouse CD40. These data implicate CD40 as a possible target for therapeutic intervention in the treatment of B lymphomas.

Interleukin-1 receptor antagonist activity of a human interleukin-1 inhibitor.

Three interleukin-1 inhibitors have been purified to homogeneity from medium conditioned by human monocytes. Partial sequence analysis and digestion with N-glycanase indicate that these are glycosylation forms of a single protein. The protein binds to the interleukin-1 receptor but has no interleukin-1-like activity, even at very high concentrations, and is therefore a pure receptor antagonist.

Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist.

Human monocytes induced with adherent IgG secrete an interleukin-1 receptor antagonist which could be important for the in vivo regulation of IL-1 activity. A complementary DNA for this molecule has been isolated from a human monocyte library. Analysis of monocyte RNA indicates that the gene is transcriptionally regulated. The sequence of the receptor antagonist indicates that it is structurally similar to IL-1 beta. Expression of the cDNA in Escherichia coli yields IL-1 receptor antagonist activity.

An IL-1 inhibitor from human monocytes. Production and characterization of biologic properties.

Previous studies have described a 22 kD IL-1 inhibitor in the supernatant of human monocytes cultured on adherent immune complexes (J. Immunol. 134:3868, 1985). The studies reported herein further detail the conditions of production and biological properties of this IL-1 inhibitor. The inhibitor was produced by human monocytes cultured on adherent human IgG with maximal production between 8 and 24 hr. The IL-1 inhibitor was not performed in the cells but required transcription and new protein synthesis. The inhibitor blocked IL-1 augmentation of PHA-induced murine thymocyte proliferation but not IL-2-induced stimulation of CTLL or HT-2 cell lines. In addition, the inhibitor blocked IL-1-stimulated collagenase production from rabbit articular chondrocytes and IL-1-induced PGE2 production from human fibroblasts and synovial cells. The IL-1 inhibitor was not transforming growth factor beta (TGF beta) as determined by: the failure of anti-TGF beta antibodies to reduce IL-1 inhibitory activity, the separation of TGF beta from the IL-1 inhibitor by ion exchange chromatography, and the failure of TGF beta to inhibit IL-1-induced PGE2 production from synovial cells. IL-1 and the inhibitor showed no immunological cross-reactivity by Western blot analysis. The inhibitor specifically blocked binding of IL-1 to its receptor on the murine thymoma cell line EL4-6.1. These results indicate that a specific inhibitor of IL-1-induced immune and inflammatory cell responses is produced by monocytes cultured on adherent immune complexes or adherent IgG. This IL-1 inhibitor may be of importance in modulating the effects of IL-1 in the monocyte microenvironment.

Thymocytes with the predicted properties of pre-T cells.

T cell receptor synthesis in thymocytes was examined by the differential immunoprecipitation of receptors from the surfaces and interiors of metabolically labeled newborn and adult thymocytes. Precipitated molecules were then analyzed for size, charge, and state of glycosylation. Our experiments identified cells within the thymic cortex that contained a large pool of cytoplasmic-free receptor beta chain. The beta chain in this pool was synthesized and degraded rapidly and bore only high-mannose N-linked oligosaccharides. This pool was found predominantly in cells that lacked surface alpha/beta receptors and appeared in ontogeny before cells expressing surface alpha/beta. These results are consistent with a model in which the progenitor of cells with surface alpha/beta expression is the T cell equivalent of the pre-B cell, which has rearranged and expressed beta chain, but not alpha chain.

Assembled topographic antigenic determinants of pigeon cytochrome c.

The specificity of the IgG fraction of the sera of several rabbits hyperimmunized with glutaraldehyde-polymerized pigeon cytochrome c was examined by fluorescence-quenching titration and a sensitive competitive plate-binding radioimmunoassay developed for the analysis of small amounts of antiserum. Four pigeon cytochrome c-specific Fab fragments were found to bind simultaneously to the immunogen. Competition assays, using an extensive set of naturally occurring, chemically prepared hybrid and enzymically modified cytochromes c, implicated in antibody binding all seven amino acid residues at which the immunogen differs from the homologous rabbit cytochrome c. Thus, rabbits produce small amounts of three antibody populations directed against the regions of serine 15, alanine 44, and glycine 89, respectively, on pigeon cytochrome c, and a large amount of the population which binds to an assembled topographic determinant composed of isoleucine 3, glutamine 100, alanine 103, and lysine 104. The latter four residues are from the amino-terminal and carboxyl-terminal alpha-helices, and cluster where the two helices cross each other on the back surface of the molecule. Antibodies against native pigeon cytochrome c reacted very poorly with the several cyanogen bromide-cleaved fragments of the molecule, consisting of residues 1 to 65, 1 to 80, 66 to 104, and 81 to 104.