Resistance of cytolytic lymphocytes to perforin-mediated killing. Inhibition of perforin binding activity by surface membrane proteins.
The mechanism whereby cytolytic lymphocytes protect themselves from killing mediated by their own cytotoxic protein, perforin, was studied. By using a competition assay, we demonstrated that the resistance of cells to perforin-mediated cytolysis is inversely correlated with their ability to absorb perforin, with tumor cells and noncytotoxic lymphocytes that are susceptible to perforin-mediated lysis being able to absorb perforin from the supernatant much better than CTL. The evidence implies that there is molecule on cytolytic lymphocytes that interferes with perforin-binding activity, resulting in the inability of perforin to lyse these cells. The molecule is most likely a surface protein or complex of proteins because its activity decreases after CTL treatment with the proteolytic enzymes trypsin and papain, and the activity can be recovered by incubation of the treated CTL cells at 37 degrees C for 6 h. The recovery can be blocked by emetine, cycloheximide, and actinomycin D, inhibitors of protein and RNA/DNA synthesis. The protein contains carbohydrate groups that play an important role in the function of the protein, as indicated by the fact that inhibition of glycosylation by tunicamycin and cleavage of sialic acid from the protein with neuraminidase result in a significant increase of perforin binding to CTL. Cross-linkage of CTL membrane proteins with glutaraldehyde and formaldehyde and blockage of the functional domains of the protein with an antiserum against CTL also inhibit the activity of this protein. Temperature-dependence studies that allow for a dissociation of the binding and pore-forming stages of perforin-mediated hemolysis suggest that the protective protein interferes at the perforin-binding stage.
Jiang
S B
SB
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021.
Ojcius
D M
DM
Persechini
P M
PM
Young
J D
JD
D000818:Animals; D002470:Cell Survival; D005557:Formaldehyde / Q000494:pharmacology; D005976:Glutaral / Q000494:pharmacology; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000633:toxicity*; D051379:Mice; D009439:Neuraminidase / Q000494:pharmacology; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011485:Protein Binding; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects*; D014357:Trypsin / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology
D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; D014415:Tunicamycin; D054353:Perforin; D005557:Formaldehyde; D009439:Neuraminidase; D014357:Trypsin; D005976:Glutaral