Induction of memory-like CD8(+) T cells and CD4(+) T cells from human naive T cells in culture

Memory T cells are crucial players in vertebrate adaptive immunity but their development is incompletely understood. Here, we describe a method to produce human memory-like T cells from naive human T cells in culture. Using commercially available human T-cell differentiation kits, both purified naive CD8(+) T cells and purified naive CD4(+) T cells were activated via T-cell receptor signaling and appropriate cytokines for several days in culture. All the T-cell activators were then removed from the medium and the cultures were continued in hypoxic condition (1% O-2 atmosphere) for several more days; during this period, most of the cells died, but some survived in a quiescent state for a month. The survivors had small round cell bodies, expressed differentiation markers characteristic of memory T cells and restarted proliferation when the T-cell activators were added back. We could also induce memory-like T cells from naive human T cells without hypoxia, if we froze the activated T cells or prepared the naive T cells from chilled filter buffy coats. Pre-activated human naive CD4(+) or CD8(+) T cells were cultured in activator-free medium to induce cell death for 5 to 10 days. Although no cell survived in normal oxygen condition (20% O2), 2 to 30% of the activated T cells could survive in hypoxia (1% O2) and became memory-like T cells. We named this simple procedure as 'the Death Assay'.