Flow cytometric detection of perforin upregulation in human CD8 T cells

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Abstract
Perforin and granzymes work synergistically to induce apoptosis in target cells recognized by cytotoxic T lymphocytes. While perforin is readily detectable by flow cytometry in resting CD8 T cells, upregulation of perforin in activated cells is thought to require proliferation. However, perforin undergoes numerous conformational changes during its maturation, which may affect the ability of conventional antibodies to recognize newly synthesized perforin. Polychromatic flow cytometry was used to detect perforin and cytokine production following stimulation of ex vivo human CD8 T cells. Two different anti-perforin antibodies, clones B-D48 and δG9, were used to discriminate various forms of perforin after cellular activation. We provide evidence for the rapid upregulation of perforin protein, which may contribute to the ability of CD8 T cells to kill multiple targets over time. The δG9 clone recognizes the granule-associated conformation of perforin, while the B-D48 clone is able to detect perforin in multiple forms. Finally, we show there is variability in the ability of CD8 T cells to upregulate perforin. Human CD8 T cells are capable of new perforin production immediately following activation. This work defines a novel flow cytometric procedure that can be used to more completely assess the cytotoxic capacity of human CD8 T cells. © 2008 International Society for Advancement of Cytometry