ANALYSIS OF CORECEPTOR VERSUS ACCESSORY MOLECULE FUNCTION OF CD8 AS ACORRELATE OF EXOGENOUS PEPTIDE CONCENTRATION

Substitution in the alpha 3 domain of class I molecules can ablate the recognition of target cells by CD8 dependent cytotoxic T lymphocytes. This effect has been attributed to a destruction of the CD8 alpha bin ding site on the class I molecule, a hypothesis which is consistent wi th results obtained in conjugate binding assays. To assess the relativ e contribution to CTL activation of CD8 functioning as either a corece ptor or an accessory molecule, we have compared the ability of H-2K(b) ovalbumin reactive CTL to lyse M12.C3 or T2 cells transfected with an H-2K(b) gene encoding a wild type or mutant (CD8 nonbinding) alpha 3 domain. To establish that the substitution in the alpha 3 domain does not alter the ability of the H-2K(b) molecule to bind the antigenic pe ptide, we have compared the binding of the ovalbumin derived H-2K(b) r estricted peptide (SIINFEKL) to T2 cells expressing either the CD8 bin ding or the CD8 nonbinding form of H-2K(b). This peptide conjugated wi th FITC bound equally well to T2 cells expressing either form of H-2K( b). Upon binding of this peptide, both forms of the H-2K(b) molecule u nderwent the same conformational change as revealed by increases in th e expression of particular serological epitopes. Furthermore, inhibiti on of the binding of the SIINFEKL peptide to both the wild type and mu tant H-2K(b) was observed following pretreatment of the cells with sim ilar amounts of other H-2K(b) restricted peptides derived from Sendai and Vesicular Stomatitis viruses. When the transfected M12 cells were tested for their ability to serve as targets for an anti-H-2K(b) ovalb umin CTL clone, cells expressing the mutant H-2K(b) molecule required the addition of 100-fold more exogenous peptide than did cells express ing the wild type molecule in order to obtain significant lysis. These data strengthen the previous hypothesis that CD8 functions much more efficiently as a coreceptor than as an accessory molecule for T cell e ffector function.