Optimization of a Multi-Parametric Activation-Induced Marker (AIM) Assay for Identifying Antigen-Specific CD4+ and CD8+ T-cells

Interest in methods to identify and isolate reactive, antigen-specific CD4+ and CD8+T-cells has increased due to their potential for use as immunotherapeutics in cancer and infectious disease. Current methods use one or two markers of T-cell activation to identify the cells of interest after stimulating and culturing PBMCs for up to two weeks in vitro. Here we describe an activation-induced marker (AIM) assay that uses five surface markers to reliably identify all subsets of activated CD4+ and CD8+T-cell populations after overnight stimulation. Conditions have been optimized to either capture activation marker expression during the stimulation or surface stain for expression post-stimulation in the absence of protein transport inhibitors. We successfully used this approach to identify subsets of T cells expressing CD134(OX40), CD137 (4-1BB), CD154 (CD40L), CD278 (ICOS) and CD69, capturing the total antigen-specific population and enabling characterization of typically hard-to-identify T helper subsets including Th2 and Tfh cells. As there is nointracellular staining component, this method allows for sorting of the populations of interest. This assay is also compatible with downstream applications such as single-cell transcriptional profiling and in vitro expansion – both of which require intact and viable antigen-specific T cells.