Assessing Antigen-Specific T Cell Responses Through IFN-γ Enzyme-Linked Immune Absorbent Spot (ELISpot).

T cells are instrumental in protecting the host against invading pathogens and the development of cancer. To do so, they produce effector molecules such as granzymes, interleukins, interferons, and perforin. For the development and immunomonitoring of therapeutic applications such as cell-based therapies and vaccines, assessing T cell effector function is paramount. This can be achieved through various methods, such as 51 Cr release assays, flow cytometry, and enzyme-linked immune absorbent spot (ELISpot) assays. For T cell ELISpots, plates are coated with antibodies directed against the effector molecule of interest (e.g., IFN-g). Subsequently, peripheral blood mononuclear cells (PBMCs) or isolated T cells are cultured on the plate together with stimuli of choice, and the production of effector molecules is visualized via labeled detection antibodies. For clinical studies, ELISpot is currently the gold standard to determine antigen-specific T cell frequencies. In contrast to 51 Cr release assays, ELISpot allows for the exact enumeration of responding T cells, and compared to flow cytometry, ELISpot is more cost-effective and high throughput. Here, we optimize and describe, in a step-by-step fashion, how to perform a controlled IFN-γ ELISpot experiment to determine the frequency of responding or antigen-specific T cells in healthy human volunteers. Of note, this protocol can also be employed to assess the frequency of antigen-specific T cells induced in, e.g., vaccination studies or present in cellular products.