Regulation of cytotoxic T cells by ecto-nicotinamide adenine dinucleotide (NAD) correlates with cell surface GPI-anchored/arginine ADP-ribosyltransferase.
This report demonstrates that incubation of cytotoxic T cells with NAD causes suppression of their ability to proliferate in response to stimulator cells or to lyse targets. Effects are evident after incubation for 3 h with concentrations of NAD as low as 1 microM and are sustained for many hours after removal of NAD from culture media. Suppression is a result of the failure of CTL to form specific conjugates with targets as well as a lower level of activation in response to TCR-mediated stimulation, although TCR-mediated transmembrane signaling is demonstrable. Metabolites of NAD such as nicotinamide, ADP-ribose, and cyclic-ADP-ribose have no detectable effect, indicating that NAD-glycohydrolase or ADP-ribose cyclase do not mediate suppression. Incubation of intact CTL with [32P]NAD leads to incorporation of 32P into a particulate, subcellular fraction, a reaction that is not inhibitable by ADP-ribose. Hydroxylamine, but not mercuric ion releases [32P]ADP-ribose, whereas phosphodiesterase releases [32P]AMP from the particulate subcellular fraction, suggesting that labeling is a result of enzymatic mono-ADP-ribosylation of arginines. In support of this, treatment of intact CTL with phosphatidylinositol-specific phospholipase C releases an arginine-specific ADP-ribosyltransferase and causes insensitivity to ecto-NAD suppression. These results suggest that a GPI-anchored ADP-ribosyltransferase uses ecto-NAD to ADP-ribosylate proteins that regulate CTL function.
Wang
J
J
Department of Microbiology, Norris Comprehensive Cancer Center, University of Southern California, School of Medicine, Los Angeles 90033.
Nemoto
E
E
Kots
A Y
AY
Kaslow
H R
HR
Dennert
G
G
D036002:ADP Ribose Transferases / Q000097:blood*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003601:Cytotoxicity Tests, Immunologic; D005434:Flow Cytometry; D017261:Glycosylphosphatidylinositols / Q000097:blood*; D008213:Lymphocyte Activation; D007959:Lymphocyte Culture Test, Mixed; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009243:NAD / Q000502:physiology*; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases; D011948:Receptors, Antigen, T-Cell / Q000502:physiology; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism / Q000502:physiology*
D017261:Glycosylphosphatidylinositols; D011948:Receptors, Antigen, T-Cell; D009243:NAD; D036002:ADP Ribose Transferases; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium