Intracellular signaling of gp34, the OX40 ligand: induction of c-jun and c-fos mRNA expression through gp34 upon binding of its receptor, OX40.

We investigated the intracellular signaling events of OX40 ligand (gp34), a member of the TNF family. To elucidate the intracellular signaling via gp34, we prepared a model system in which a human gp34-transfected mouse epithelial cell line was stimulated with a recombinant soluble form of OX40. We demonstrated that OX40 binding resulted in increase in c-jun and c-fos mRNA levels in this transfectant by Northern blot analysis, which was blocked by the pretreatment with anti-gp34 Ab. The studies with various gp34 deletion mutants showed that the cytoplasmic portion including the amino acid sequence 16-21 (RPRFER) was required for the induction of c-jun and c-fos mRNA expression. Furthermore, OX40 binding induced c-jun mRNA expression also in HUVECs, which in our previous study have been shown to express gp34 and interact with activated T cells through the OX40/gp34 pathway. On the other hand, c-fos mRNA was detectable neither in unstimulated HUVECs nor in gp34-stimulated HUVECs. These results indicate that the OX40/gp34 system generates two-way signals and may elicit biological effects on vascular endothelial cells.     

Isolation and characterization of the human adenocarcinoma-associated glycoprotein gp40

The human adenocarcinoma-associated antigen gp40 is a cell surface glycoprotein recognized by murine monoclonal antibody KS1/4. A KS1/4-Sepharose affinity matrix was utilized to purify gp40 from detergent lysates of either tissue culture cells or nude mouse xenograft tumors of the human lung adenocarcinoma cell line P3-UCLA. This single immunoaffinity chromatography step yielded an antigen preparation of approximately 95% purity which was further characterized by immunochemical and enzymatic techniques. The gp40 molecule was shown to have both complex and high-mannose oligosaccharides comprising some 16% of the apparent molecular weight. The antigen preparation was suitable for gas-phase N-terminal amino acid sequencing and the first 16 residues of the N-terminus were determined. Despite considerable molecular heterogeneity, gp40 shows a single N-terminal sequence.     

Evaluation of OX40 ligand as a costimulator of human antiviral memory CD8 T cell responses: comparison with B7.1 and 4-1BBL

CTL are important effectors of antiviral immunity. Designing adjuvants that can induce strong cytotoxic T cell responses in humans would greatly improve the effectiveness of an antiviral vaccination or therapeutic strategy. Recent evidence suggests that, in addition to its well-established role in costimulation of CD4 T cell responses, OX40L (CD134) can directly costimulate mouse CD8 T cells. In this study, we evaluated the role of OX40L in costimulation of human antiviral CD8 T cell responses and compared it with two other important costimulators, B7.1 (CD80) and 4-1BBL (CD137L). Delivery of OX40L to human monocytes using a recombinant replication-defective adenovirus led to greater expansion, up-regulation of perforin, enhanced cytolytic activity, and increased numbers of IFN-gamma- and TNF-alpha-producing antiviral memory CD8 T cells in cultures of total T cells. Synergistic or additive effects were observed when OX40L costimulation was combined with 4-1BBL (CD137L) or B7.1 (CD80) costimulation. In total T cell cultures, at low Ag dose, 4-1BBL provided the most potent costimulus for influenza-specific CD8 T cell expansion, followed by B7.1 (CD80) and then OX40L. For isolated CD8 T cells, 4-1BBL was also the most consistent costimulator, followed by B7.1. In contrast, OX40L showed efficacy in direct activation of memory CD8 T cells in only one of seven donors. Thus, OX40L costimulates human antiviral memory CD8 T cell responses largely through indirect effects and can enhance anti-influenza, anti-EBV, and anti-HIV responses, particularly in combination with 4-1BBL or B7.     

Cutting edge: identification of E-cadherin as a ligand for the murine killer cell lectin-like receptor G1

The killer cell lectin-like receptor G1 (KLRG1) is expressed by NK cells and by T cells. In both humans and mice, KLRG1 identifies Ag-experienced T cells that are impaired in their proliferative capacity but are capable of performing effector functions. In this study, we identified E-cadherin as a ligand for murine KLRG1 by using fluorescently labeled, soluble tetrameric complexes of the extracellular domain of the murine KLRG1 molecule as staining reagents in expression cloning. Ectopic expression of E-cadherin in B16.BL6 target cells did not affect cell-mediated lysis by lymphokine-activated NK cells and by CD8 T cells but inhibited Ag-induced proliferation and induction of cytolytic activity of CD8 T cells. E-cadherin is expressed by normal epithelial cells, Langerhans cells, and keratinocytes and is usually down-regulated on metastatic cancer cells. KLRG1 ligation by E-cadherin in healthy tissue may thus exert an inhibitory effect on primed T cells.     

Molecular characterization of a murine, major A/B type hnRNP protein: mBx

We have previously identified a discrete hnRNP polypeptide of the A/B type, named mBx, as an abundant protein species in murine cells. The molecular characterization of this protein is now accomplished. From all evidence provided, mBx polypeptide represents a new gene product, distinct from the known members of the A/B family A1 and A2/B1. It is, instead, mostly related to a still hypothetical human protein of A/B type, as well as to the Xenopus hnRNPA3 protein species.     

Isolation and characterization of a human T lymphocyte-associated glycoprotein (gp40)

The biosynthetic and structural characteristics of the human thymocyte/T cell antigen defined by the monoclonal antibody WT1 have been studied. WT1 identified a monomeric cell surface glycoprotein of Mr = 40,000 ( gp40 ). Cross-absorption experiments and two-dimensional gel analyses indicate that WT1 and another monoclonal antibody, 3A1, react with the same structure. This glycoprotein was asymmetrically inserted into the rough endoplasmic reticulum as a transmembrane structure. At this stage, the polypeptide chain possessed two N-linked, "high-mannose" type glycans; these were subsequently processed into endo-H-insensitive, complex oligosaccharides during intracellular transport to the cell surface. Inhibition of N-linked glycosylation with tunicamycin failed to block the processing of the nonglycosylated Mr = 29,000 polypeptide to a glycoprotein of Mr = 33,000. Cleavage of the mature Mr = 40,000 form with endo-F yielded a similar Mr = 33,000 product. The kinetics of synthesis of the Mr = 33,000 intermediate in conjunction with gal-NAc oligosaccharidase digestion indicated the presence of O-linked glycans in the mature cell surface WT1 antigen. The fully processed cell surface form of the polypeptide also contains covalently associated fatty acid, and was labeled by 32P phosphate, the predominantly labeled phosphoamino acid being phosphoserine. We also demonstrate biochemically that the reactivity of WT1 with cells from a few patients with acute myeloid leukemia reflects genuine expression of the gp40 structure on myeloid cells.     

IL-1 enhances T cell-dependent antibody production through induction of CD40 ligand and OX40 on T cells.

IL-1 is a proinflammatory cytokine that plays pleiotropic roles in host defense mechanisms. We investigated the role of IL-1 in the humoral immune response using gene-targeted mice. Ab production against SRBC was significantly reduced in IL-1alpha/beta-deficient (IL-1(-/-)) mice and enhanced in IL-1R antagonist(-/-) mice. The intrinsic functions of T, B, and APCs were normal in IL-1(-/-) mice. However, we showed that IL-1(-/-) APCs did not fully activate DO11.10 T cells, while IL-1R antagonist (-/-) APCs enhanced the reaction, indicating that IL-1 promotes T cell priming through T-APC interaction. The function of IL-1 was CD28-CD80/CD86 independent. We found that CD40 ligand and OX40 expression on T cells was affected by the mutation, and the reduced Ag-specific B cell response in IL-1(-/-) mice was recovered by the treatment with agonistic anti-CD40 mAb both in vitro and in vivo. These observations indicate that IL-1 enhances T cell-dependent Ab production by augmenting CD40 ligand and OX40 expression on T cells.     

Molecular epidemiology of 58 new African human T-cell leukemia virus type 1 (HTLV-1) strains: identification of a new and distinct HTLV-1 molecular subtype in Central Africa and in Pygmies.

To gain new insights on the origin, evolution, and modes of dissemination of human T-cell leukemia virus type I (HTLV-1), we performed a molecular analysis of 58 new African HTLV-1 strains (18 from West Africa, 36 from Central Africa, and 4 from South Africa) originating from 13 countries. Of particular interest were eight strains from Pygmies of remote areas of Cameroon and the Central African Republic (CAR), considered to be the oldest inhabitants of these regions. Eight long-term activated T-cell lines producing HTLV-1 gag and env antigens were established from peripheral blood mononuclear cell cultures of HTLV-1 seropositive individuals, including three from Pygmies. A fragment of the env gene encompassing most of the gp21 transmembrane region was sequenced for the 58 new strains, while the complete long terminal repeat (LTR) region was sequenced for 9 strains, including 4 from Pygmies. Comparative sequence analyses and phylogenetic studies performed on both the env and LTR regions by the neighbor-joining and DNA parsimony methods demonstrated that all 22 strains from West and South Africa belong to the widespread cosmopolitan subtype (also called HTLV-1 subtype A). Within or alongside the previously described Zairian cluster (HTLV-1 subtype B), we discovered a number of new HTLV-1 variants forming different subgroups corresponding mainly to the geographical origins of the infected persons, Cameroon, Gabon, and Zaire. Six of the eight Pygmy strains clustered together within this Central African subtype, suggesting a common origin. Furthermore, three new strains (two originating from Pygmies from Cameroon and the CAR, respectively, and one from a Gabonese individual) were particularly divergent and formed a distinct new phylogenetic cluster, characterized by specific mutations and occupying in most analyses a unique phylogenetic position between the large Central African genotype (HTLV-1 subtype B) and the Melanesian subtype (HTLV-1 subtype C). We have tentatively named this new HTLV-1 genotype HTLV-1 subtype D. While the HTLV-1 subtype D strains were not closely related to any known African strain of simian T-cell leukemia virus type 1 (STLV-1), other Pygmy strains and some of the new Cameroonian and Gabonese HTLV-1 strains were very similar (>98% nucleotide identity) to chimpanzee STLV-1 strains, reinforcing the hypothesis of interspecies transmission between humans and monkeys in Central Africa.