Cytotoxic T-Cell Adherence Assay (CAA)

Abstract

Cytotoxic T lymphocytes (CTL) play an important role in the control of intracellular pathogens through their capacity to lyse infected target cells or secrete immunostimulatory cytokines, such as IFN-γ or TNF-α (1–3). These features of cytotoxic T cells are termed “effector” mechanisms and have been measured extensively in the past using assays, such as cytokine ELISAs, or radioactive chromium release from target cells (4,5). However, several other important changes occur within CTL in conjunction with these effector mechanisms. Triggering of the T-cell receptor (TCR) by cognate peptide/major histocompatability complex (MHC) class I complexes leads to a cascade of intracellular signaling events which, among other outcomes, results in increased cell surface adhesion to extracellular matrix and surrounding cells (6,7). Increased surface adhesion may be caused by the provision of new cell surface adhesion receptors from the intracellular pool or conformational changes in existing cell surface receptors following antigen-specific TCR triggering (8). Several studies have now demonstrated the specific binding of activated CTL to plate-bound MHC class I molecules and fibronectin using an assay system that involves wash steps to remove unbound cells (9–11). In the current chapter, the increased adhesiveness of CTL, following specific peptide/MHC class I recognition, forms the basis of a simple, peptide-specific assay, the cytotoxic T-cell adherence assay (CAA) (12), which correlates well with more traditional effector assays, such as ⁵¹Cr release, and does not involve washing steps.