Identification of a thymic epithelial cell subset sharing expression of the class Ib HLA-G molecule with fetal trophoblasts.

HLA-G is the only class I determinant of the major histocompatibility complex (MHC) expressed by the trophoblasts, the fetal cells invading the maternal decidua during pregnancy. A unique feature of this nonclassical HLA molecule is its low polymorphism, a property that has been postulated to play an important role in preventing local activation of maternal alloreactive T and natural killer cells against the fetus. Yet, the mechanisms by which fetal HLA-G can be recognized as a self-MHC molecule by the maternal immune system remain unclear. Here we report the novel observation that HLA-G is expressed in the human thymus. Expression is targeted to the cell surface of thymic medullary and subcapsular epithelium. Thymic epithelial cell lines were generated and shown to express three alternatively spliced HLA-G transcripts, previously identified in human trophoblasts. Sequencing of HLA-G1 transcripts revealed a few nucleotide changes resulting in amino acid substitutions, all clustered within exon 3 of HLA-G, encoding for the alpha2 domain of the molecule. Our findings raise the possibility that maternal unresponsiveness to HLA-G-expressing fetal tissues may be shaped in the thymus by a previously unrecognized central presentation of this MHC molecule on the medullary epithelium.

Cell adhesion and migration are regulated at distinct stages of thymic T cell development: the roles of fibronectin, VLA4, and VLA5.

T cell development in the thymus requires the establishment of stable interactions with cell-selecting elements such as the cortical epithelium followed by a regulated movement of selected progenitors to the medulla. Cell adhesion and migration are mediated by integrins in a number of biological systems though little is known regarding their function in the thymus. We demonstrated previously that immature CD3loCD69lo double positive human thymocytes adhere avidly to FN via the integrin, VLA4. We now demonstrate that the interaction of mature CD3hiCD69hi thymic subsets with FN triggers migration rather than firm adhesion. Migration requires the engagement of VLA4 in cooperation with VLA5 and both receptors regulate the persistence and directionality of movement. While migration capability is linked to maturation state, ligand concentration determines the efficiency of migration. In fact, FN and the alternatively spliced CS1 site are predominant in the thymic medulla, suggesting an instructive role of this ECM protein in vivo. Our studies identify a novel VLA4 and VLA5/FN-mediated pathway likely to be involved in regulating cell traffic between the cortex and medulla of the thymus. Moreover, the data provides evidence that VLA4 exists in at least two functional states at distinct stages of T cell development. While different states of VLA4 activation have been described on cell lines, this represents the first evidence supporting a biological significance for this integrin property.

Expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins in the developing pancreas: roles in the adhesion and migration of putative endocrine progenitor cells.

Cell-cell and cell-matrix interactions play a critical role in tissue morphogenesis and in homeostasis of adult tissues. The integrin family of adhesion receptors regulates cellular interactions with the extracellular matrix, which provides three-dimensional information for tissue organization. It is currently thought that pancreatic islet cells develop from undifferentiated progenitors residing within the ductal epithelium of the fetal pancreas. This process involves cell budding from the duct, migration into the surrounding mesenchyme, differentiation, and clustering into the highly organized islet of Langerhans. Here we report that alpha(v)beta(3) and alpha(v)beta(5), two integrins known to coordinate epithelial cell adhesion and movement, are expressed in pancreatic ductal cells and clusters of undifferentiated cells emerging from the ductal epithelium. We show that expression and function of alpha(v)beta(3) and alpha(v)beta(5) integrins are developmentally regulated during pancreatic islet ontogeny, and mediate adhesion and migration of putative endocrine progenitor cells both in vitro and in vivo in a model of pancreatic islet development. Moreover, we demonstrate the expression of fibronectin and collagen IV in the basal membrane of pancreatic ducts and of cell clusters budding from the ductal epithelium. Conversely, expression of vitronectin marks a population of epithelial cells adjacent to, or emerging from, pancreatic ducts. Thus, these data provide the first evidence for the contribution of integrins alpha(v)beta(3) and alpha(v)beta(5) and their ligands to morphogenetic events in the human endocrine pancreas.

Bead-based sandwich hybridization characteristics of oligonucleotide-alkaline phosphatase conjugates and their potential for quantitating target RNA sequences.

The hybridization characteristics of oligonucleotide-alkaline phosphatase conjugate probes were examined in bead-based sandwich hybridization reactions using single-stranded nucleic acid targets and oligonucleotide-polystyrene capture beads. Enzymatic activity was monitored using a chemiluminescent substrate and calibration plots of chemiluminescent signal versus conjugate concentration were used to estimate the sandwich hybridization efficiencies. Improved hybridization behavior was noted using glycerol as an additive and by increasing the length of the probe and alkyl spacer of the conjugates. The chemiluminescent assay is at least as sensitive as those employing 32P-labeled probes and can detect as little as 10-20 amol of target RNA. The linear relationship of chemiluminescent signal versus target assayed provides a method for quantitating unknown target samples. A single human immunodeficiency virus type 1 infected cell in a background of 10(6) uninfected cells is facilely detected when this enzyme-based detection assay is prefaced with a self-sustained sequence-replication amplification reaction.

Vascular cell adhesion molecule-1 is expressed by cortical thymic epithelial cells and mediates thymocyte adhesion. Implications for the function of alpha4beta1 (VLA4) integrin in T-cell development.

T-cell development requires a series of discrete selection and activation signals delivered to maturing progenitors in the thymic cortex and medulla. We have previously shown the constitutive activity of the integrin, alpha4beta1 (VLA4), on a unique subpopulation of immature cortical thymocytes and proposed a role for integrin-mediated adhesion in positive selection by cortical epithelium. In the present report we show that thymic epithelial cell lines express vascular cell adhesion molecule-1 (VCAM-1) a high-affinity ligand for apha4beta1, and that VCAM-1 mediates thymocyte binding to these lines. Immunohistochemistry and confocal microscopy show that VCAM-1 is selectively expressed in situ by thymic epithelium in the cortex and corticomedullary junction, two locations at which VCAM-1 could determine the interaction between immature thymocytes and selecting elements on epithelial cells. In parallel, we confirmed that fibronectin (FN), the alternative ligand for alpha4beta1, is expressed predominantly in the medulla. These results suggest that VCAM-1 is an adhesive ligand in the thymic cortex for the activated form of alpha4beta1 constitutively expressed during development by immature double positive thymocytes. The structural segregation of the alternative ligand, FN, to the medulla suggests that medullary FN may regulate the migration, development, and export of more mature thymocytes.