Molecules of the TNF-R family have been shown to be essential in the regulation of lymphocyte growth and differentiation. The TNF-R family member CD27 binds to a type II transmembrane molecule belonging to the TNF gene family (CD27L) that is identical to the lymphocyte activation Ag CD70. Using transfected mouse fibroblasts expressing human CD70, we demonstrate here that interaction of CD27 with its ligand provides a potent second signal for cytokine production, induction of activation Ags, and proliferation of unprimed CD45RA+, and to a lesser extent, of primed CD45R0+ peripheral blood T cells. In contrast to costimulatory signals delivered via the CD28-ligand B7-1 (CD80), CD70 was found to induce relatively low IL-2, IL-4, and IL-10 but comparable TNF-alpha secretion. Proliferation of CD45RA+, but not of CD45R0+ T cells, was found to be largely resistant to blocking of IL-2/IL-2R interaction. Finally, the finding that CD70 and CD80 cooperate in the induction of T cell proliferation indicates that cooperation of both molecules may be essential for optimal T cell stimulation. The interaction between CD27 and its ligand CD70 might be of particular importance for the recruitment of T cells from the unprimed T cell pool. Moreover, as CD70 expression in vivo is confined to activated B and T lymphocytes, only a limited set of APC are able to generate this specific second signal for T cell expansion.

We have recently described a novel hematopoietic growth factor, referred to as the flt3 ligand, that stimulates the proliferation of sub-populations of hematopoietic cells that are enriched for stem and progenitor cells, This factor is a transmembrane protein that undergoes proteolytic cleavage to generate a soluble form of the protein. We have isolated additional flt3 ligand isoforms by PCR that contain an extra exon and encode what are predicted to be either a soluble form of the ligand or a longer version of the transmembrane protein. We have also isolated cDNAs from murine T cell libraries that encode an isoform of the flt3 ligand that has an unusual C-terminus, This isoform results from a failure to splice out an intron during mRNA processing. The protein encoded by this cDNA is expressed on the cell surface, where it is biologically active. However, this novel isoform does not appear to give rise to a soluble form of the protein, Regulation of mRNA splicing is likely to control the generation of cell bound or soluble forms of this hematopoietic growth factor, Genetic mapping studies localize the gene encoding the flt3 ligand to the proximal portion of mouse chromosome 7 and to human chromosome 19q13.3.

Elk is a member of the eph family of receptor‐like tyrosine kinases. Although its function is unknown, elk is postulated to play a role in nervous system development. Using Northern analysis, we examined the developmental regulation of RNAs encoding elk, and several ligands for the eph family of RTKs, the LERKs. Expression of elk, LERK‐1, and LERK‐2 RNAs is high in all regions examined in the embryonic and postnatal rat brain and decreases to low levels with age. One exception is the adult olfactory bulb which continues to express a moderate level of LERK‐2. In contrast, moderate LERK‐4 expression was limited to the developing hippocampus and cerebral cortex. These data indicate that elk and some of the LERKs may play a role in nervous system development, maintenance, and/or regeneration. © 1995 Wiley‐Liss, Inc.

The human Xq11–Xq21.3 region has been implicated in several inherited disorders including dystonia-parkinsonism (DYT3), sideroblastic anemia and several specific and non-specific forms of mental retardation (MR) syndromes. As part of a positional cloning effort to identify MR genes, we have generated a YAC-based transcript map. We first constructed a YAC/STS framework by extending previously published contigs. This framework map consists of a minimal set of 119 clones, covering approximately 20 Megabases (Mb) and allowing the precise ordering of 71 STSs between DXS136 and DXS472. This YAC contig was then used to define the positions of genes and expressed sequence tags (ESTs) assigned to the Xcen–Xq21.3 region. In addition to the genes previously localized to this part of the X chromosome, 18 transcription units corresponding to additional known genes or gene family members, one pseudogene and 15 novel transcripts were mapped. This transcriptional map incorporates 51 transcription units and provides a useful resource of candidate genes for some of the disorders assigned to this region of the X chromosome.

A family of tyrosine kinase receptors related to the product of the eph gene has been described recently. One of these receptors, elk, has been shown to be expressed only in brain and testes. Using a direct expression cloning technique, a ligand for the elk receptor has been isolated by screening a human placenta cDNA library with a fusion protein containing the extracellular domain of the receptor. This isolated cDNA encodes a transmembrane protein. While the sequence of the ligand cDNA is unique, it is related to a previously described sequence known as B61. Northern blot analysis of human tissue mRNA showed that the elk ligand's mRNA is 3.5 kb long and is found in placenta, heart, lung, liver, skeletal muscle, kidney and pancreas. Southern blot analysis showed that the gene is highly conserved in a wide variety of species. Both elk ligand and B61 mRNAs are inducible by tumour necrosis factor in human umbilical vein endothelial cells. In addition, both proteins show promiscuity in binding to the elk and the related hek receptors. Since these two ligand sequences are similar, and since elk and hek are members of a larger family of eph‐related receptor molecules, we refer to these ligands as LERKs (ligands for eph‐related kinases).

CD27 is a lymphocyte-specific member of the TNF/NGF-R family that is highly induced on T cells after TCR stimulation, primarily on lymphocytes belonging to the unprimed CD45RA+ subset. However, after prolonged activation both in vivo and in vitro, CD27 expression is gradually switched off. In search of physiologic signals that influence CD27 expression, we found that B cell lines can mediate down-regulation of CD27 surface expression on activated T cells via direct cell-to-cell contact and that preincubation of activated T cells with CD27 mAbs prevented this modulation. Based on these observations, we hypothesized that a ligand for CD27L is expressed on B cell lines and that ligand interaction would result in down-modulation of the molecule from the T cell surface. To identify this putative CD27L, mAbs were raised against the Burkitt cell line Ramos and screened for their ability to block down-modulation of CD27 on activated T cells. Using this approach, we isolated a mAb, designated 2F2, that inhibits CD27 down-regulation in a dose-dependent manner. We demonstrate that this mAb recognizes CD27L as it 1) reacted to mouse fibroblasts expressing the recently cloned CD27L, a novel surface-expressed member of the TNF gene family, and 2) prevented binding of a soluble CD27-Fc construct to a CD27L expressing cell line. Down-regulation of CD27 from the T cell surface by recombinant CD27L was shown to be at least partially caused by release of soluble CD27. The 2F2 mAb enabled us to begin to analyze the biochemical properties, tissue distribution, and function of the CD27L on human cells. From cell lysates of 125I surface-labeled Burkitt cells a complex immunoprecipitation pattern with dominant bands of 29, 55, and 122 to 127 kDa was obtained using the 2F2 mAb. Phenotypical analyses showed that freshly isolated lymphocytes lacked CD27L expression, but that expression of the molecule could be induced after in vitro stimulation of T and B cells. No expression was found on cells of the myeloid lineage or on endothelial cells. Finally, blocking of naturally expressed CD27L by the 2F2 mAb exerted a potent inhibitory effect on the proliferation of T cells in response to allogeneic B cells and PHA. The data presented in this paper identify CD27 and its ligand as potentially important structures involved in cellular interactions between T and B lymphocytes.

Using a fragment of the murine flt3 ligand as a probe, we have succeeded in cloning a human flt3 ligand from a human T-cell lambda gt10 cDNA library. The human and murine ligands are 72% identical at the amino acid level. Analysis of multiple cDNA clones shows that alternative splicing of the human flt3 mRNA can occur at a number of positions. A recombinant soluble form of the human flt3 ligand stimulates the proliferation and colony formation of a subpopulation of human bone marrow cells that are CD34+ and are enriched for primitive hematopoietic cells. In addition, the human flt3 ligand also stimulates the proliferation of cells expressing murine flt3 receptors. Northern blot analysis shows widespread expression of flt3 ligand mRNA transcripts in human tissues.

Cloning of a ligand for the murine flt3/flk-2 tyrosine kinase receptor was undertaken using a soluble form of the receptor to identify a source of ligand. A murine T cell line, P7B-0.3A4, was identified that appeared to express a cell surface ligand for this receptor. A cDNA clone was isolated from an expression library prepared from these cells that was capable, when transfected into cells, of conferring binding to a soluble form of the flt3/flk-2 receptor. The cDNA for this ligand encodes a type I transmembrane protein that stimulates the proliferation of cells transfected with the flt3/flk-2 receptor. A soluble form of the ligand stimulates the proliferation of defined subpopulations of murine bone marrow and fetal liver cells as well as human bone marrow cells that are highly enriched for hematopoietic stem cells and primitive uncommitted progenitor cells.

Macrophage inflammatory protein (MIP)-1 alpha, part of a family termed chemokines, has been implicated in suppression of hemopoietic stem and progenitor cell proliferation. The chemokine family has been organized into two subgroups with MIP-1 alpha, MIP-1 beta, macrophage chemotactic and activating factor (MCAF) and RANTES belonging to one subgroup, and GRO-alpha, MIP-2 alpha (GRO-beta), MIP-2 beta (GRO-gamma), platelet factor 4 (PF4), IL-8, and neutrophil activating peptide (NAP)-2 belonging to the other. These molecules were evaluated for effects on colony formation by human bone marrow multipotential (CFU-GEMM), erythroid (BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells. None of the chemokines stimulated colony formation in the absence of CSF, or influenced colony formation stimulated by a single growth factor such as granulocyte-macrophage-CSF or erythropoietin. However, MIP-1 alpha, MIP-2 alpha, PF4, IL-8, and MCAF suppressed in dose-response fashion colony formation of immature subsets of myeloid progenitor cells stimulated by GM-CSF plus steel factor. Effects were apparent on low density and CD34 HLA-DR(+)-sorted marrow cells in which up to 88.4% of the cells were composed of progenitor cells, suggesting direct effects on the progenitors themselves. Up to 2500-fold less of each chemokine could be used to demonstrate synergistic suppression when any two of these five chemokines were used together at low concentrations, effects also apparently directly on the progenitors. In contrast, MIP-1 beta, MIP-2 beta, GRO-alpha, NAP-2, and RANTES were not suppressive nor did they synergize with MIP-1 alpha, MIP-2 alpha, PF4, IL-8, or MCAF to suppress. However, a fivefold excess of MIP-1 beta blocked the suppressive effects of MIP-1 alpha. Similarly, a fivefold excess of either MIP-2 beta or GRO-alpha blocked the suppressive effects of IL-8 and PF4. These suppressing, synergizing and blocking effects may be of relevance to blood cell regulation.