A fast live-cell assay to detect antigen-specific CD8+ T cells based on upregulation of integrin-mediated adhesion

Antigen stimulation of T-cell receptors on T cells induces an immediate increase in ligand-binding affinity and clustering in the plasma membrane of beta2-integrins, resulting in a strong binding to intercellular adhesion molecules (ICAMs) on target cells. This firm adhesion, e.g. of cytotoxic T cells to virus-infected or tumor cells with formation of cytotoxic synapses is required for target cell lysis. Therefore, upregulation of integrin-mediated adhesion after stimulation identifies functional effector T cells. Here we present a novel flow cytometry assay to enumerate antigen-specific CD8+ T cells using soluble multimeric ICAM1-Fc-F(ab’)2 fluorescent complexes to identify integrin activation induced after short-term stimulation in vitro. By optimizing and validating this assay we were able to identify pathogen-, tumor- or vaccine-induced CD8+T cells, and show its feasibility for assessing and isolating live, functional antigen-specific CD8+T cells. Performing coexpression analyses, we found that antigen-stimulated cells that expressed cytokines and mobilized CD107a were predominantly highly adherent CD8+T cells. Finally, CD8+ T cells that upregulated integrins were shown to mediate cytolytic activity in an antigen-specific manner. The most striking advantage of this method is the short-time incubation (several minutes) that is necessary to identify and isolate viable antigen-specific cytotoxic T cells by assessing upregulation of T cell adhesiveness, a critical step for elaborating multiple effector functions, including cytotoxicity and cytokine production.