On-chip activation and subsequent detection of individual antigen-specific T cells

The frequencies of antigen-specific CD4+ T cells in samples of human tissue have been difficult to determine accurately ex vivo, particularly for autoimmune diseases such as multiple sclerosis or type 1 diabetes. Conventional approaches involve the expansion ofprimaryTcells in vitro to increase the numbers of cells, and a subsequent assessment of the frequencies of antigen-specific T cells in the expanded population by limiting dilution or by using fluorescently labeled tetramers of peptide-loaded major histocompatibility complex (MHC) receptors. Here we describe an alternative approach that uses arrays of subnanoliter wells coated with recombinant peptide- loaded MHC class II monomers to isolate and stimulate individual CD4+ T cells in an antigen-specific manner. In these experiments, activation was monitored using microengraving to capture two cytokines (IFNυ and IL 17) released from single cells. This new method should enable direct enumeration of antigen-specific CD4+ T cells ex vivo from clinical samples.