A novel, rapid and sensitive heterotypic cell adhesion assay for CD2-CD58 interaction, and its application for testing inhibitory peptides

Abstract

The immunoglobulin CD2 is a cell adhesion molecule that mediates T-cell activation by binding to its receptor CD58 on antigen-presenting cells (APCs). Modulation or inhibition of this interaction has been shown to be therapeutically useful. E-rosetting assay is usually applied in the study of the modulation of CD2-CD58 interaction. In this study, we demonstrated a novel, rapid and sensitive heterotypic cell adhesion assay for CD2-CD58 interaction. The CD2 expression on the surface of Jurkat cells and the CD58 expression on the Caco-2 cells were confirmed by flow cytometry and ELISA studies, respectively. Then Jurkat cells were fluorescent-labeled with 2 μM of BCECF-AM for 45 min at 37°C before adding to confluent Caco-2 monolayers cultured in 96-well culture dishes. After 30 min, non-adherent Jurkat cells were removed by washing with PBS, while the monolayer-associated Jurkat cells were lysed with 0.5 ml of 2% Triton X-100 in 0.1 M NaOH. Fluorescence (FL) was quantitated using a microplate fluorescence analyzer with BCECF's excitation maximum of 485 nm and emission maximum of 535 nm. This method was successfully applied for testing inhibitory peptides to CD2-CD58 interaction. © 2004 Elsevier B.V. All rights reserved.