(464a) Multidimensional Analysis of Cytokine Secretion From Individual T Cells Using Quantitative Microengraving

The activation of T cells is an essential process in the coordination of both cellular and humoral immune responses. The release of cytokines is one of the important functions carried out by T cells during either a protective immune response to pathogens or autoimmune responses. The types and duration of cytokine secretion highly depend on the nature of disease and the immune system of the host. Dynamic characterization of the T cell response following activation would provide new insight into both fundamental T cell biology and how to monitor clinical treatments of disease. Conventional analytical methods, such as intracellular staining and enzyme-linked immunospot, are not able to monitor cytokine secretion at the single-cell level with temporal resolution. To provide dynamic and systematic analyses of the cytokine responses of single T cells, we developed quantitative microengraving that enables serial, kinetically-resolved snapshots of the cytokine profiles from ~10⁴ individual cells in parallel. Each snapshot (~1 hour) quantitatively measures the rates of secretion of up to four cytokines simultaneously from single cells. Different combinations of these cytokines define the functional status of individual cells at a particular time period. Consecutive measurements taken over time yield the dynamic profile of cytokine secretion for each cell following stimulation. In this talk, we will describe the kinetic profiles of cytokine secretion by mitogen-stimulated primary human T cells. Specifically, we report on the release of IFNγ, IL-2, and TNFα from individual cells over 17 h of stimulation. Analysis of these measured profiles shows the cellular responses are highly dynamic and diverse. Combining information on the lineages of the cells from image-based cytometry, we identified distinct patterns of cytokine profiles for subclasses of T cells. Initial work to use these data to construct models describing the intracellular cytokine network and the implied regulatory mechanism of cytokine production in T cells will also be discussed.