Live Cell Imaging of T Cell Pyroptosis

Pyroptosis is a lytic, pro-inflammatory cell death program that is tightly regulated by inflammasomes in most cases. Inflammasome-dependent pyroptosis is best characterized in myeloid cells, but there is growing evidence that this cell death program also functions in human T cells. Several studies have suggested a role for inflammasome components in T-cell biology but often do not unambiguously clarify whether this means that T cells progress to pyroptosis. Pyroptosis has distinct morphological features, such as early loss of membrane integrity and ballooning, that allow it to be distinguished from apoptosis in a microscopic experiment. However, the most stringent definition of inflammasome-dependent pyroptosis is that it is genetically dependent on a pro-inflammatory caspase (caspase-1 or caspase-4) and the pore-forming protein gasdermin D (GSDMD). Therefore, using live cell imaging of T cells in combination with a genetic loss-of-function setup is the most reliable tool for us to unequivocally demonstrate that a T cell undergoes pyroptosis. Parallel live cell imaging of T cells and macrophages is limited due to the fact that T cells do not adhere while macrophages do. This can be overcome by using so-called micro-inserts that hold the cells in a limited area that can be monitored by microscopic field of view. Here we describe in detail how live cell imaging of human T cells and monocyte-derived macrophages undergoing pyroptosis can be performed.