CD5 expression by B lymphocytes and its regulation upon Epstein–Barr virus transformation

Abstract

Dim expression of CD5 on human B lymphocytes has been used to delineate B1 and B2 subsets. Nevertheless, others have suggested that the molecule is an activation marker and does not predicate a subset distinction. We have used enzymatic amplification staining, a technology that enhances the resolution of flow cytometric analysis of cell surface molecules by as much as 100-fold, to determine that essentially all human B cells express CD5. Furthermore, we show that this expression is regulated during EpsteinBarr virus transformation. C D5, a 67-kDa surface glycoprotein of the scavenger receptor cysteine-rich family, appears on T lymphocytes early in their development and is abundantly expressed on all mature T cells. The expression of the molecule on B lymphocytes and its role in defining separate lineages of these cells is controversial (1, 2). It has been postulated that the low-level expression of CD5 by a subset of B lymphocytes defines a separate lineage that is associated with autoreactive antigenic specificity (2, 3). Alternatively, others have shown that CD5 expression on B cells can be enhanced by various activating agents, prompting the suggestion that CD5 is a B cell activation antigen and that B cells comprise a single lineage (4, 5).We have developed a powerful technology, enzymatic amplification staining (EAS), that significantly enhances the resolution of flow cytometric analysis of cell surface molecules (6). By using EAS, we have achieved a 100-fold enhancement in the fluorescent signal. The enhanced signal allows for the detection of molecules that could not be observed previously. Thus, we have been able to define a subpopulation of peripheral blood cells stimulated in vitro that express Fas ligand (CD178), and we have demonstrated the validity of EAS in this case by correlating the staining with functional activity (6). EAS has also been used to provide a high-resolution immunophenotype of leukemic cells from patient samples. It was shown that enzymatic amplification gives a more powerful assessment of the clonality of the leukemic cells (7).Some investigators have proposed that human B lymphocytes can be divided into B1 and B2 subsets based on the expression of CD5 (2, 3); however, others have shown that CD5 expression on B cells can be up-regulated by various activating agents, which suggests that CD5 is a B cell activation antigen (4, 5). To address these alternative models, we used EAS to assess the expression of CD5 on human B lymphocytes. We found that essentially all B cells express CD5 constitutively at low levels, and we also found that CD5 expression is regulated by Epstien-Barr virus (EBV) transformation. Flow Cytometric Analysis. For standard amplification staining (indirect staining), we incubated cells with biotinylated primary antibodies followed by an incubation with streptavidin conjugated to fluorescein isothiocyanate. For EAS, we obtained kits from Flow-Amp Systems (Cleveland), and followed the manufacturer's instructions. The incubations were all performed for 10 min at room...

Selection of the Alternative Exon 1 from thecd5Gene Down-Regulates Membrane Level of the Protein in B Lymphocytes

“…Thus, the density of CD5 on the surface of T cells is 30-fold higher than in B1a cells (25). In T lymphocytes themselves, there is less CD5 on immature than on mature cells (26). Furthermore, there is evidence that the proportion of CD5-expressing B cells declines with age (27), and that the density of CD5 molecules on B cells is reduced by IL-4 which is important for their differentiation (28), but increased by PMA stimulation (29).…”

Human CD5 promotes B-cell survival through stimulation of autocrine IL-10 production

“…Indeed, other inducers than BCR, such as EBV, are able to modulate CD5 expression. 10 It could be argued that the distinction between CD5 ϩ and CD5 Ϫ subsets is an artifact because all B cells seem to express little CD5 if analyzed by using an amplification technique. 10 This, however, does not raise questions about our results because we showed that normal human peripheral B cells that up-regulate CD5 also up-regulate IL-10 in parallel, making our results applicable to all B cells.…”

“…10 It could be argued that the distinction between CD5 ϩ and CD5 Ϫ subsets is an artifact because all B cells seem to express little CD5 if analyzed by using an amplification technique. 10 This, however, does not raise questions about our results because we showed that normal human peripheral B cells that up-regulate CD5 also up-regulate IL-10 in parallel, making our results applicable to all B cells. The respective roles of BCR and CD5 in IL-10 production could not be directly addressed in de novo CD5 ϩ peripheral B cells, mainly because we were unable to dissociate CD5 expression from BCR stimulation using several inhibitors of BCR-signaling pathways (H.G.-G., unpublished data, March 2001).…”

“…6,7 In contrast, the differentiation hypothesis holds that every B cell is able to acquire B-1 cell characteristics. In brief, the notion of B-cell lineage based on differential CD5 expression is controversial because of the lack of clear identification of a fetal progenitor destined to become a B-1 B cell and the demonstration that CD5 Ϫ cells can acquire CD5 expression in vivo 8 or in vitro 9 after B-cell receptor (BCR) stimulation This up-regulation supports the initial view that CD5 is an activation marker 10 ; however, a definitive function for this antigen remains to be established.…”

Correlation between ZAP‐70, phospho‐ZAP‐70, and phospho‐Syk expression in leukemic cells from patients with CLL

“…The samples were analyzed for the expression of ZAP-70, phospho-ZAP-70, phospho-Syk, and p21cip1 by Pathfinder Biotech using enzymatic amplification staining (EAS) as previously described (10,(12)(13)(14). EAS is a validated, catalyzed reporter deposition technology based on the enzymatic activity of peroxidase.…”