Immortalization of equine trophoblast cell lines of chorionic girdle cell lineage by simian virus-40 large T antigen.

Immortalized cell lines have many potential experimental applications including the analysis of molecular mechanisms underlying cell-specific gene expression. We have utilized a recombinant retrovirus encoding the simian virus-40 (SV-40) large T antigen to construct several immortalized cell lines of equine chorionic girdle cell lineage - the progenitor cells that differentiate into the equine chorionic gonadotropin (eCG) producing endometrial cups. Morphologically, the immortalized cell lines appear similar to normal chorionic girdle cells. Derivation of the immortalized cell lines from a chorionic girdle cell lineage was verified by immunological detection of cell-surface antigens specific to equine invasive trophoblasts. The cell lines differed, however, from mature chorionic girdle cells or endometrial cup cells in that they did not produce eCG and did express MHC class I molecules. Thus, these cell lines appear to have been arrested at a stage of development prior to final differentiation into endometrial cup cells. It was also determined that some of these cell lines as well as endometrial cups express the estrogen receptor-related receptor beta gene, but not the glial cell missing gene (GCMa) both of which are expressed in the murine and human placenta. Among these cell lines, three (eCG 50.5, 100.6 and 500.1) express eCG alpha mRNA. Since regulation of eCG alpha subunit gene is largely unknown, we investigated the signal transduction pathways regulating the eCG alpha subunit gene. Both activators of protein kinase A (PKA) and protein kinase C (PKC) induced the expression of eCG alpha subunit expression 3.2 (P<0.05)- and 1.9 (P<0.05)-fold respectively, in the eCG 500.1 cell line. However, activation of these pathways failed to induce eCG beta subunit expression. In conclusion, lines of equine trophoblast cells have been immortalized that display markers characteristic of those with the equine chorionic girdle and endometrial cup cell lineage. A subset of these cells expresses the eCG alpha subunit gene which is responsive to activators of the PKA and PKC signal transduction pathways.

Binding and antigen presentation of ceramide-containing glycolipids by soluble mouse and human CD1d molecules.

We have purified soluble mouse and human CD1d molecules to assess the structural requirements for lipid antigen presentation by CD1. Plate-bound CD1d molecules from either species can present the glycolipid alpha-galactosyl ceramide (alpha-GalCer) to mouse natural killer T cells, formally demonstrating both the in vitro formation of antigenic complexes, and the presentation of alpha-GalCer by these two CD1d molecules. Using surface plasmon resonance, we show that at neutral pH, mouse CD1 and human CD1d bind to immobilized alpha-GalCer, unlike human CD1b, which requires acidic pH for lipid antigen binding. The CD1d molecules can also bind both to the nonantigenic beta-GalCer and to phosphatidylethanolamine, indicating that diverse lipids can bind to CD1d. These studies provide the first quantitative analysis of monomeric lipid antigen-CD1 interactions, and they demonstrate that the orientation of the galactose, or even the nature of the polar head group, are likely to be more important for T cell receptor contact than CD1d binding.

The role of CD1 molecules in immune responses to infection.

Recent work on CD1 molecules has demonstrated that human CD1b and a lipoglycan from mycobacteria that CD1b presents colocalize to late endosomes. Presentation of this lipoglycan by CD1b requires antigen uptake via the mannose receptor. CD8(+) CD1-restricted T cells can decrease the load of intracellular mycobacteria by granule release. TCR-transgenic and CD1-deficient mice have provided insights into the role of CD1 in the T helper responses required for the clearance of some microorganisms.

Analysis of MHC class I expression in equine trophoblast cells using in situ hybridization.

Down-regulation of major histocompatibility complex (MHC) genes by trophoblast cells is considered to be a primary mechanism preventing maternal immune rejection of the fetal-placental unit in mammalian pregnancy by rendering these cells, which form the primary barrier between mother and fetus, relatively non-antigenic. In situ hybridization with probes encoding human and horse MHC class I genes was used to characterize the pattern of MHC class I mRNA expression in the various forms of horse trophoblast. Strong hybridization signals were observed in the invasive trophoblast cells of chorionic girdle tissue. In contrast, no hybridization signal specific for MHC class I mRNA transcripts was observed in the descendent endometrial cup trophoblast cells. In the non-invasive trophoblast cells of the allantochorion, no hybridization signals specific for horse MHC class I mRNA transcripts were consistently detected. In parallel to the in vivo results, strong hybridization signals were observed in the small, mononuclear cells present in chorionic girdle cell explant cultures, but not in the population of large binucleate cells corresponding to endometrial cup cells. The results obtained using in situ hybridization are consistent with the hypothesis that expression of MHC class I genes may be controlled at the transcriptional level in horse invasive and non-invasive trophoblast cells, and suggest that down-regulation of MHC class I antigen expression in endometrial cup cells may be accomplished by the same mechanisms in vivo and in vitro.

Nonclassical behavior of the thymus leukemia antigen: peptide transporter-independent expression of a nonclassical class I molecule.

The thymus leukemia (TL) antigen is a major histocompatibility complex-encoded nonclassical class I molecule. Here we present data demonstrating that expression of the TL antigen, unlike other class I molecules, is completely independent of the function of the transporter associated with antigen processing (TAP). The TL antigen is expressed by transfected TAP-2-deficient RMA-S cells when these cells are grown at 37 degrees C. In transfected RMA cells, the kinetics of arrival of TL antigen on the cell surface are similar to those of a classical class I molecule. The kinetics are not altered in TAP-deficient RMA-S cells, demonstrating that surface TL expression in TAP-deficient cells is not due to the stable expression of a few molecules that leak out by a TAP-independent pathway. Soluble TL molecules produced by Drosophila melanogaster cells are highly resistant to thermal denaturation, unlike peptide-free classical class I molecules synthesized by these insect cells. In addition, these soluble TL molecules are devoid of detectable bound peptides. The results demonstrate that the TL antigen is capable of reaching the surface without bound peptide, although acquisition of peptide or some other ligand through a TAP-independent pathway cannot be formally excluded. We speculate that the ability of the TL antigen to reach the cell surface, under conditions in which other class I molecules do not, may be related to a specialized function of the TL molecule in the mucosal immune system, and possibly in the stimulation of intestinal gamma delta T cells.

Analysis of horse and donkey gonadotrophin genes using Southern blotting and DNA hybridization techniques.

The number of genes encoding the common alpha-subunit and hormone-specific beta-subunits of the equine gonadotrophins (FSH, LH and CG) were investigated in the horse (Equus caballus), donkey (E. asinus) and 2 horse x donkey hybrids (the mule and hinny). The Southern technique, involving restriction enzyme digestion, blotting and DNA hybridization to 32P-labelled DNA probes was used to estimate the copy number for each gene and to assess the extent to which equids resemble primates, the only other animals that secrete a CG during pregnancy. These methods indicated that, in common with mammals, there was a single copy of the equine gonadotrophin alpha-and FSH beta-subunit genes. Also in common with other mammals, our results are consistent with there being a single LH beta-subunit gene but, unlike primates, no evidence of multiple LH/CG beta-subunit genes was obtained. There was no restriction fragment length polymorphism (RFLP) within species using the 4 enzymes chosen for this study, but the RFLP pattern for the beta-subunit genes differed between species, giving rise to species-specific 'fingerprints'. The mule and hinny Southern blots showed a combination of the horse and donkey fingerprints, consistent with the presence of both genomes in these hybrids and consistent with the expression of both horse and donkey CG by hybrid conceptuses. In man and baboons, multiple genes code for the beta-subunit as a consequence of LH beta-subunit gene duplication.(ABSTRACT TRUNCATED AT 250 WORDS)