Sensitive Detection Of Antigen-Specific T-Cells Using Bead-Bound Antigen For In Vitro Re-Stimulation

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 IFN gamma 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 IFN gamma 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. (C) 2019 The Authors. Published by Elsevier B.V.