Differential sensitivity of virgin and memory T lymphocytes to calcium ionophores suggests a buoyant density separation method and a model for memory cell hyporesponsiveness to Con A.
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.
Miller
R A
RA
Department of Pathology, Boston University School of Medicine, MA.
Flurkey
K
K
Molloy
M
M
Luby
T
T
Stadecker
M J
MJ
D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D002118:Calcium / Q000502:physiology; D002469:Cell Separation; D002499:Centrifugation, Density Gradient; D003208:Concanavalin A / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D006649:Histocompatibility Antigens / Q000032:analysis; D007111:Immunity, Cellular; D007156:Immunologic Memory; D015759:Ionomycin / Q000494:pharmacology*; D017493:Leukocyte Common Antigens; D008213:Lymphocyte Activation; D051379:Mice; D016201:Receptors, Lymphocyte Homing / Q000032:analysis; D012555:Schistosomiasis mansoni / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology
D015703:Antigens, CD; D015704:CD4 Antigens; D016827:CD8 Antigens; D006649:Histocompatibility Antigens; D016201:Receptors, Lymphocyte Homing; D003208:Concanavalin A; D015759:Ionomycin; D017493:Leukocyte Common Antigens; D002118:Calcium