Does co‐aggregation of the CD45 and CD3 antigens inhibit T cell antigen receptor complex‐mediated activation of phospholipase C and protein kinase C?

Abstract

The binding of agonistic monoclonal antibodies (mAb) to the CD3 antigen in T cells induces a rapid increase in tyrosine phosphorylation, inositive phosphate (IP) production, a rise in intracellular calcium and protein kinase C (PKC) activation. These intracellular signals have been implicated in the control of interleukin-2 and interleukin-2R receptor gene expression, thereby regulating T cell proliferation. Previous studies have shown that co-ligation of the CD45 and CD3 antigens inhibits CD3-induced tyrosine phosphorylation, IP production, calcium signals and T cell proliferation. It has therefore been suggested that the CD45 antigen uncouples the T cell receptor (TcR) from mitogenic signal pathways. In this study co-ligation of the CD3 and CD45 antigens with precisely constructed bispecific mAb did not inhibit CD3-induced T cell proliferation, IP production, calcium signals, diacylglycerol production or PKC activation. Furthermore, co-ligation of CD3 and CD45 antigens already cross-linked with IgM mAb did not lead to inhibition of CD3-induced calcium signals. Inhibitions of CD3-induced intracellular signals were observed following co-ligation of IgG CD45 and CD3 mAb with anti-IgG (F(ab')2 fragments. However, comparable inhibitions were also noted following co-ligation of CD3 with other abundant cell-surface antigens such as CD5 and LFA-1, and inhibitions were only observed when the CD3 mAb used required cross-linking to induce signals. These results suggested that the inhibitory effects of CD45 IgG mAb were not specific and were caused by the prevention of CD3-CD3 cross-linking following CD3 antigen co-ligation with other cell surface molecules. These findings are inconsistent with a specific inhibitory role for the CD45 phosphotyrosine phosphatase in uncoupling the TcR from mitogenic signal pathways.

CD3 antigen‐mediated calcium signals and protein kinase C activation are higher in CD45R0+ than in CD45RA+ human T lymphocyte subsets

“…Furthermore, TcR-induced increases in tyrosine phosphorylation, phosphoinositide metabolism and intracellular calcium ([Ca2+]i) no longer occur in CD4S-deficient mutant T leukemia cell lines, but are restored by CD45 cDNA transfection [21]. These findings point to a positive role for CD45 in maintaining theTcR in a primed state in which it is able to transduce signals [22].The reported physical association between CD45 molecules and the TcR also indicates an important role for CD45 in regulating TcR signal transduction mechanisms [23].…”

The role of phosphotyrosine phosphatases in haematopoietic cell signal transduction

“…What has become clear, however, is that the earlier results, obtained by co-ligating CD45 with various receptors and then observing generalised inhibitory effects, were not due to the inhibitory actions of CD45 per se, but to the nonspecific prevention of the receptor aggregation necessary for effective signal transduction (18). Nevertheless, such artifactual results should not detract from the highly selective effects on lymphocyte signalling of certain CD45 mAbs added to cells without cross-linking to other receptors.…”

Long wavelength fluorophores and cell‐by‐cell correction for autofluorescence significantly improves the accuracy of flow cytometric energy transfer measurements on a dual‐laser benchtop flow cytometer

“…Antibodies were labeled with fluorescent dyes as described previously (1), with slight modifications. Briefly, covalent binding of the bifunctional succimidyl ester derivatives of sulfoindocyanine dyes (Cy3 and Cy5; Amersham, Braunschweig, Germany) to the lysyl‐ϵ amino groups of antibodies was carried out in bicarbonate buffer at pH 8.3.…”

“…Although generally applied biochemical and immunological approaches (i.e., cocapping, coprecipitation, chemical cross‐linking) have provided valuable information about the topography of cell surface protein interactions, such techniques have several disadvantages. For example, the necessary application of extraction or isolation procedures prevent the investigation of proteins in their natural environment and may, on the one hand, disrupt protein–protein interactions or, on the other hand, induce the formation of artificial protein aggregates, the latter being a main concern in case of high expression of cell surface glycoproteins (1). In contrast, fluorescence resonance energy transfer (FRET) offers a suitable and convenient alternative because it can be performed on live cells without major interference with the physiologic condition of the cells.…”