Ex Vivo Enumeration of Flu-Reactive CD4+ T Cells

Accurate assessments of the frequencies of antigen-specific T cells ex vivo from human tissue samples have been difficult to achieve, especially for autoimmune diseases such as multiple sclerosis or type 1 diabetes. Conventional approaches involve the expansion of T cells in vitro prior to assessing the frequencies of antigen-specific cells with fluorescently labeled tetramers of peptide-loaded major histocompatibility complex (MHC) receptors. This talk will describe an alternative approach that uses functionalized arrays of subnanoliter wells to isolate and stimulate T cells ex vivo. Activation of the cells is monitored by the release of cytokines (IL-4, IL-10, IL-17, IFNG) from single cells using microengraving. To validate this technique, arrays of microwells were coated a combination of anti-CD3/CD28 antibodies or major histocompatibility complex (MHC) monomers, and loaded with peripheral blood mononuclear cells (PBMCs). Retrieving and proliferating T cells activated ‘on-chip' by peptide-loaded MHC monomers confirmed the specificity of the responses measured by microengraving. The application of this technique to assess directly the frequencies of subsets of flu-reactive T cells ex vivo will be presented.