The Fas antigen (CD95) on human lymphoid cells: epitope analysis with ten antibodies.

The expression of CD95 antigen was examined on adult and cord blood lymphocytes using a highly sensitive immunofluorescence/flow cytometric procedure. CD95 was expressed by the majority of circulating blood T cells in adults, and by a smaller proportion of CD4+ and CD8+ T cells in cord blood. The majority of circulating B cells did not react with seven CD95 antibodies, but three antibodies did stain B cells. In tonsil sections, CD95 was expressed throughout the tissue but germinal centres showed generally stronger staining than the surrounding follicular mantle and interfollicular areas. This was confirmed by flow cytometry, which showed expression preferentially on B cells with a germinal centre phenotype. Because different antibodies stained different proportions of B cells, CD95 epitopes were examined by inhibition, additive binding and protease susceptibility studies using a panel of ten CD95 antibodies. B cells apparently reacting selectively with CD95 antibodies were sorted and CD95 mRNA was reverse transcribed to cDNA and analyzed, in order to confirm the presence of CD95 in cells which reacted selectively and to explore the possible existence of CD95 isoforms. The major cDNA band was identical in the two populations. Inhibition of N-glycosylation suggested that the epitopes detected differentially could not be accounted for by differential N-glycosylation.

IgE+ cells in the peripheral blood of atopic, nonatopic, and bee venom-hypersensitive individuals exhibit the phenotype of highly differentiated B cells.

We have analyzed IgE+ cells in peripheral blood of atopic donors, donors hypersensitive to bee venom, and nonatopic control donors with two- and three-color flow cytometry. Although the percentage of IgE+ cells varied among these groups, the overall phenotypic patterns were similar. Most IgE+ cells do not display typical B-cell markers, such as CD19, CD20, and CD21. A significant proportion of these cells stain for CD38, indicating that they are more differentiated. IgE+ cells express Fc gamma RII and CD45RO, an isoform associated with an advanced level of differentiation. The majority of IgE+ cells do not coexpress other surface immunoglobulin isotypes. In the case of bee venom-hypersensitive donors, we have been able to identify a small population of IgE+ cells with a specificity for phospholipase A2, a major immunogenic component of bee venom. The phospholipase A2+ cells display a phenotype similar to that of the IgE+ cells.

An organ fragment culture model to study lymphocyte activation in human lymphoid tissue.

We have established and evaluated an organ fragment culture model for the study of human lymphocyte activation and differentiation. Small fragments of tonsillar tissue were cultured on Gelfoam for periods of up to 7 days. Monoclonal antibody in the medium was able to diffuse into the tissue, as demonstrated by subsequent detection of antibody-coated cells. Phytohaemagglutinin added to the culture medium caused activation of T and B cells, as indicated by changes in expression of a number of markers. Antibody against human IgM (added as a F(ab')2 fragment) together with IL-4 caused B cell activation, detectable by an increased expression of CD23 and other markers. Cell viability fell gradually in culture, but useful data could nevertheless be obtained from culture periods up to 7 days. The organ fragment culture provides a model for the study of T and B cell activation which maintains, at least in part, the intercellular interactions and the native microenvironment of lymphoid tissue.