Characterization of antigen association with accessory cells: specific removal of processed antigens from the cell surface by phospholipases.
To characterize the basis for the cell surface association of processed antigen with the antigen-presenting cell (APC) we analyzed its sensitivity to enzymatic digestion. Antigen-exposed APC that are treated with phospholipase and then immediately fixed lose their ability to stimulate antigen-plus-Ia-specific T-T hybridomas. This effect is seen with highly purified phospholipase A2 and phospholipase C. In addition it is observed with three distinct antigens--ovalbumin, bovine insulin, and poly(LGlu56LLys35LPhe9) [(GluLysPhe)n]. The effect of phospholipases is highly specific. Identically treated APC are equivalent to controls in their ability to stimulate alloreactive hybridomas specific for precisely the same Ia molecule that is corecognized by antigen-plus-Ia-specific hybrids. Furthermore, the antigen-presenting function of enzyme-treated, fixed APC can be reconstituted by the addition of exogenous in vitro processed or "processing independent" antigens. In parallel studies 125I-labeled avidin was shown to specifically bind to APC that were previously exposed and allowed to process biotin-insulin. Biotin-insulin-exposed APC that are pretreated with phospholipase bind significantly less 125I-labeled avidin than do untreated, exposed APC. Identical enzyme treatment does not reduce the binding of avidin to a biotinylated antibody already bound to class II major histocompatibility complex molecules of APC. At least some of the biotin-insulin surface sites are immunologically relevant, because the presentation of processed biotin-insulin by fixed APC is blocked by avidin. This effect is specific. Avidin binding to biotin-insulin-exposed APC does not inhibit allospecific stimulation nor the presentation of unconjugated insulin. These studies demonstrate that phospholipase effectively removes processed cell surface antigen.
Falo
L D
LD
Haber
S I
SI
Herrmann
S
S
Benacerraf
B
B
Rock
K L
KL
D000818:Animals; D000911:Antibodies, Monoclonal; D000938:Antigen-Presenting Cells / Q000276:immunology*; D000954:Antigens, Surface / Q000032:analysis*; D002460:Cell Line; D002462:Cell Membrane / Q000276:immunology; D006825:Hybridomas / Q000276:immunology; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D010740:Phospholipases / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010738:Type C Phospholipases / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases