Abstract
Previous work from this laboratory has indicated that murine memory T cells differ from virgin T cells in that the former are more resistant to agents that alter intracellular [Ca]i. We have used this difference to devise a method for separating virgin from memory T cells by centrifugation over an ionomycin-containing Percoll step gradient after brief exposure to 2 microM ionomycin. Under these conditions, those T cells that are most sensitive to ionomycin-induced changes in [Ca]i become more dense and therefore travel further into the Percoll/ionomycin gradient than cells that are more resistant to ionomycin. We show that the ionomycin-resistant cell population is enriched for cells that express high levels of Pgp-1 (CD44), and low levels of CD45RB, and thus appears to consist largely of memory T cells. Both CD4 and CD8 cells can be divided into Pgp-1hi and Pgp-1lo subsets in this way. Cells recovered from such a gradient and washed to remove the ionomycin appear normally functional, i.e., neither more nor less responsive to mitogens and costimuli than untreated cells. Limiting dilution methods show that the ionomycin-sensitive (virgin) subset contains most of the Con A-responsive precursors for cytotoxicity, and most of the cells able to produce IL-2 in responses to Con A or staphylococcal enterotoxin B. Ag-specific helper memory cells are, however, found predominantly in the ionomycin-resistant fraction of the spleen and draining lymph nodes of mice infected with Schistosoma mansoni. Changes in resistance to calcium signal development may represent a fundamental distinction between virgin and memory T cells, and could contribute to differences in activation requirements between these two cell subsets.
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