Expansion and Determination of Antigen-Reactive T Cells by Flow Cytometry

The detection of antigen-reactive T cells has shown great utility for patient clinical monitoring. However, many of the methods commonly used to detect these cells face major limitations, like the predetermination of the given HLA type. The herein described protocol provides a means of overcoming many of the obstacles associated with the use of multimers and other common approaches in this field. In order to be able to detect rare cells which are below the detection limit of direct ex vivo measurement, in the present protocol, antigen-reactive T cells are first expanded in vitro using libraries of overlapping peptides which span the entire protein of interest and consist of 15 amino acid-long peptides that share a 12-amino-acid overlap. This theoretically allows the detection of T cells to any epitope within a protein of interest and consequently does not require the patient's HLA type to be characterized. Furthermore, this method simultaneously detects CD4+ and CD8+ T cells that produce cytokines upon encounter with antigen and thereby provides a functional insight into the behavior of the responding T cells. In our case, we have investigated the pro- or anti-inflammatory cytokines IL-2, IL-5, IL-10, IL-17, TNF-α, and IFN-γ.