Use of anti-CD3/CD28 mAb coupled magnetic beads permitting subsequent phenotypic analysis of activated human T cells by indirect immunofluorescence

Abstract

Functional analysis of T lymphocytes requires in vitro stimulation of these cells under experimental conditions that mimic as closely as possible physiological in vivo stimulation and that involve antigen/T cell receptor (TCR)-mediated activation. Because of the low frequency of antigen-specific T cells in human clinical samples, stimulation with a combination of anti-CD3 and anti-CD28 monoclonal antibodies (mAbs) is a preferred method. Interaction of these mAbs with their ligand results in modulation of the mAb–ligand complex from the cell surface. However, as a result of incomplete modulation, CD3/CD28 mAb complexes often remain at the cell surface, thereby precluding subsequent indirect immunofluorescence and flow cytometry analysis using mouse immunoglobulin (Ig)-specific antibodies. This is of importance in situations in which no specific fluorochrome-conjugated mAbs are available, such as in screening procedures of Ig-containing hybridoma culture supernatants. We propose here the use of CD3/CD28 mAbs, linked to magnetic beads allowing standardization of the activation conditions, optimal activation of T cells and complete modulation of antigen–antibody complexes from the cell surface.