Cytokines in the tumor necrosis factor (TNF) family regulate development and function of the immune system. We have isolated a new member of this family, designated Apo-2 ligand (Apo-2L), via an expressed sequence tag. Apo-2L is a 281-amino acid protein, related most closely to Fas/Apo-1 ligand. Transfected Apo-2L is expressed at the cell surface with its C terminus exposed, indicating a type II transmembrane protein topology. Like Fas/Apo-1 ligand and TNF, the C-terminal extracellular region of Apo-2L (amino acids 114 -281) exhibits a homotrimeric subunit structure. Soluble Apo-2L induces extensive apoptosis in lymphoid as well as non-lymphoid tumor cell lines. The effect of Apo-2L is not inhibited by soluble Fas/Apo-1 and TNF receptors; moreover, expression of human Fas/Apo-1 in mouse fibroblasts, which confers sensitivity to induction of apoptosis by agonistic anti-Fas/Apo-1 antibody, does not confer sensitivity to Apo-2L. Hence, Apo-2L acts via a receptor which is distinct from Fas/Apo-1 and TNF receptors. These results suggest that, along with other family members such as Fas/Apo-1 ligand and TNF, Apo-2L may serve as an extracellular signal that triggers programmed cell death. TNF1 family cytokines modulate host defense mechanisms via a corresponding family of receptors (1-3). Most TNF family cytokines are expressed as type II transmembrane proteins, whose C-terminal extracellular domain is processed proteolytically to form a soluble homotrimeric molecule. In contrast, the members of the TNF receptor (TNFR) family are type I transmembrane proteins. In both families, sequence homology is found mainly in the extracellular regions, which mediate ligand-receptor binding.Members of the TNF family have diverse biological actions, including T cell co-stimulation, induction of B cell proliferation and differentiation, and macrophage activation (1-3). In addition, certain TNF family members regulate the elimination of unwanted immune cells by inducing programmed cell death (apoptosis). For example, Fas/Apo-1 ligand (Fas/Apo-1L) plays a key role in peripheral deletion of self-reactive lymphocytes, as suggested by the autoimmune phenotype of the mouse mutations lpr and gld, which occur, respectively, in the Fas/Apo-1 receptor or ligand genes (4, 5). In addition, Fas/Apo-1L is involved in apoptosis of B cells and CD4 ϩ T cells subsequent to stimulation by antigen, while TNF mediates a similar function in CD8ϩ T cells (4 -7). In this article, we describe a new member of the TNF cytokine family. We have designated this protein Apo-2L, because it resembles the Fas/Apo-1L in its amino acid sequence, as well as in its ability to induce apoptosis. Apo-2L appears to act via a receptor which is distinct from Fas/Apo-1 and TNF receptors, suggesting that its biological role is unique. EXPERIMENTAL PROCEDURESCloning of Apo-2L cDNA-To isolate a full-length cDNA that contains the sequence of expressed sequenced tag (EST) HHEA47M, a gt11 bacteriophage library of human placental cDNA (ϳ1 ϫ 10 6 clones) (HL1075b, Clontech) was screene...

Fas ligand (FasL) is produced by activated T cells and natural killer cells and it induces apoptosis (programmed cell death) in target cells through the death receptor Fas/Apol/CD95. One important role of FasL and Fas is to mediate immune-cytotoxic killing of cells that are potentially harmful to the organism, such as virus-infected or tumour cells. Here we report the discovery of a soluble decoy receptor, termed decoy receptor 3 (DcR3), that binds to FasL and inhibits FasL-induced apoptosis. The DcR3 gene was amplified in about half of 35 primary lung and colon tumours studied, and DcR3 messenger RNA was expressed in malignant tissue. Thus, certain tumours may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL.

Natural killer cell stimulatory factor (NKSF) is a 70-kD heterodimeric cytokine that was initially isolated from conditioned medium of human B lymphoblastoid cell lines. The effects of recombinant NKSF on the function of human peripheral blood NK cells were examined. NKSF directly augmented the cytolytic activity of freshly isolated NK cells. Both CD56dim and CD56bright NK cells demonstrated enhanced cytotoxicity after brief exposure to NKSF. In contrast, highly purified T lymphocytes did not exhibit major histocompatibility complex- unrestricted cytotoxicity after short-term culture with NKSF. Like interleukin 2 (IL-2), NKSF augmented the lysis of NK-sensitive, NK- resistant, and antibody-coated targets. Both NKSF and IL-2 induced marked upregulation of several NK cell adhesion molecules known to participate in cytolysis, including CD2, CD11a, and CD54. However, NKSF activates NK cells through a pathway distinct from that of IL-2, since the presence of anti-IL-2 receptor (anti-IL-2R) antibodies or IL-4 did not inhibit the effects of NKSF. NKSF by itself induced very little proliferation of resting NK cells. NK cells preactivated in vitro with IL-2 demonstrated enhanced proliferation to NKSF, but the degree of proliferation was always inferior to that induced by IL-2 alone. Moreover, NKSF strongly inhibited IL-2-induced proliferation of either resting or preactivated NK cells. This inhibition was not the result of decreased IL-2R expression, because NKSF-activated NK cells expressed higher levels of both IL-2Rs p75 and p55. Furthermore, NKSF did not inhibit the proliferation of mitogen-activated T cells, indicating a selective effect on NK cell proliferation. Human NK cells expanded in vivo by prolonged continuous infusions of IL-2 remained fully responsive to NKSF. Picomolar concentrations of NKSF were as effective as nanomolar concentrations of IL-2 in augmenting the cytolytic activity of NK cells expanded in vivo by IL-2. NKSF may play an important role in the regulation of human NK cell function, and its possible use as a therapeutic cytokine deserves further investigation.

Recently, several lines of evidence have indicated an expanded role for thrombopoietin (TPO) and its receptor, c-mpl, in hematopoiesis. In addition to being the primary physiological regulator of platelet production, it is now apparent that TPO also acts during early hematopoiesis. To futher define the role of TPO in early hematopoiesis we have identified discrete murine and human stem cell populations with respect to c-mpl expression and evaluated their potential for hematopoietic engraftment. Fluorescence-activated cell sorter analysis of enriched stem cell populations showed the presence of c-mpl expressing subpopulations. Approximately 50% of the murine fetal liver stem cell–enriched population, AA4+Sca+c-kit+, expressed c-mpl. Analysis of the murine marrow stem cell population LinloSca+c-kit+ showed that 70% of this population expressed c-mpl. Expression of c-mpl was also detected within the human bone marrow CD34+CD38− stem cell progenitor pool and approximately 70% of that population expressed c-mpl. To rigorously evaluate the role of TPO/c-mpl in early hematopoiesis we compared the repopulation capacity of murine stem cell populations with respect to c-mpl expression in a competitive repopulation assay. When comparing the fetal liver progenitor populations, AA4+Sca+c-kit+c-mpl+and AA4+Sca+c-kit+c-mpl−, we found that stem cell activity segregates with c-mpl expression. This result is complemented by the observation that the LinloSca+ population of c-mplgene-deficient mice was sevenfold less potent than LinloSca+ cells from wild-type mice in repopulating activity. The engraftment potential of the human CD34+CD38−c-mpl+ population was evaluated in a severe combined immunodeficient-human bone model. In comparison to the CD34+CD38−c-mpl− population, the CD34+CD38−c-mpl+ cells showed significantly better engraftment. These results demonstrate a physiological role for TPO and its receptor, c-mpl, in regulating early hematopoiesis.