Anti-idiotypic antibodies derived against C8, C9 and perforin bind homologous restriction factor.
A pore-forming protein (PFP/perforin/cytolysin), stored in the cytoplasmic granules of cytolytic lymphocytes, lyses a variety of target cells but not the cytolytic lymphocytes. In the complement (C) system, a C8-binding protein (C8bp) or homologous restriction factor (HRF) has been described that protects cells against lysis mediated by homologous C. C8bp/HRF is known to bind to C8 and C9 and has also been suggested to protect lymphocytes against perforin-mediated lysis. Here, using an anti-idiotypic antibody approach, several polyclonal antisera were raised against IgGs that are specific for mouse perforin, and human C8 and C9. These anti-idiotypic antisera were shown to react against an overlapping epitope(s) on C8bp/HRF as indicated by the following evidence: (i) all three types of antisera reacted against partially purified C8bp/HRF and against a 65 kDa protein band in cell lysates; reactivity was only observed against disulfide-reduced antigens; (ii) the three antibodies react with a protein band in normal erythrocytes (E) but not with type III E of patients with paroxysmal nocturnal hemoglobinuria or with a mutant B lymphoblastoid cell line, both of which cell types are known to be deficient in C8bp/HRF; and (iii) the three antibodies compete with each other for binding to C8bp/HRF. Type III E and the C8bp/HRF-deficient mutant lymphoblastoid cell line, however, are as susceptible to perforin-mediated lysis as type I E and wild-type lymphoblastoid cell line, respectively, indicating that C8bp/HRF does not play a role in protecting cells against perforin-mediated lysis. These paradoxical findings suggest that perforin may share with C8 and C9 the same domain(s) that bind to C8bp/HRF and yet, unlike C8 and C9, perforin is not inactivated by this type of putative interaction. Since C8 and C9 are now readily available, the anti-idiotypic approach described here provides a convenient protocol for production of antisera specific for C8bp/HRF.
Laboratory of Cellular Physiology and Immunology, Rockefeller University, New York, NY 10021.
J D
JD
Young
S
S
Jiang
C C
CC
Liu
C S
CS
Hasselkus-Light
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology*; D001798:Blood Proteins / Q000276:immunology* / Q000502:physiology; D018957:CD59 Antigens; D002352:Carrier Proteins / Q000276:immunology* / Q000502:physiology; D003185:Complement C8 / Q000276:immunology*; D003186:Complement C9 / Q000276:immunology*; D000939:Epitopes / Q000032:analysis; D006801:Humans; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000276:immunology*; D051379:Mice; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011817:Rabbits
D000888:Antibodies, Anti-Idiotypic; D001798:Blood Proteins; D018957:CD59 Antigens; D002352:Carrier Proteins; D003185:Complement C8; D003186:Complement C9; D000939:Epitopes; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; C051590:CD59 protein, human; D054353:Perforin