Sensitive detection of antigen-specific T-cells using bead-bound antigen for in vitro re-stimulation

Abstract

Reliable and sensitive detection of antigen specific cells is essential in several fields of research, whether it concerns monitoring responses to infectious agents or exploring the auto-antigen repertoire in autoimmune diseases. Identification of these cells is however difficult, especially when the cells often are rare and methods not sensitive, specific or practical enough. We propose a novel method of processing antigens before stimulation of cells which consists of covalently binding protein antigen to superparamagnetic micro-beads and using denaturing washes to remove contaminants. Peripheral blood mononuclear cells (PBMCs) from healthy donors were stimulated using both cytomegalovirus and tetanus-diphtheria antigen-beads as well as non-antigenic protein-beads as negative control in an IFNγ FluoroSpot assay in order to detect Th1 and CD8+ responses. The responses toward the antigen beads were both antigen specific and sensitive, with a detection threshold of 1 IFNγ producing T-cell per 18,000 PBMCs. • Covalently binding antigen to paramagnetic beads allows for harsh denaturing washes without loss of antigen. • Microbeads are phagocytosed by antigen presenting cells, resulting in efficient uptake, processing and presentation of the antigens. • The method allows the usage of relatively impure starting antigen material and whole PBMC samples without high background levels in follow up cellular assays.