IDENTIFICATION OF CONSERVED AMINO-ACIDS IN MURINE B7-1 IGV DOMAIN CRITICAL FOR CTLA4 CD28-B7 INTERACTION BY SITE-DIRECTED MUTAGENESIS - A NOVEL STRUCTURAL MODEL OF THE BINDING-SITE/

The B7:CD28/CTLA4 interaction plays a major role in T eel responses. I mmune intervention targeted at this interaction has demonstrated a vas t potential in enhancing tumor immunity and blocking autoimmunity and transplant rejection. However, the structural basis for this interacti on is unclear. While we and others have performed site-directed mutage nesis to define amino acids involved in binding CD28 and CTLA4, these residues are localized in different regions, and it is unlikely for al l of them to be directly involved. In addition, the effect of the muta tions on the overall conformation of B7 has not been systematically ev aluated. In this study, we have carried out site-directed mutagenesis to define the amino acids within B7-1 IgV-like domain which participat e B7:CD28/CTLA4 interaction. Four anti-B7-1 mAbs that recognize three independent antigenic epitopes on B7-1 were used to monitor the effect of mutations on the overall conformation of B7-1. Of the five mutatio ns in the IgV domain that we have produced, D113 > A appears to interf ere with cell surface expression and/or overall conformation of B7-1, while four others do not significantly affect the overall conformation and cell surface expression of B7-1. Among them, G115 > A and Y91 > A eliminated B7-1 binding to both CD28Ig and CTLA4Ig; our previously re ported mutants L109 > A and W88 > A selectively affect the B7-1 bindin g to either CD28Ig or CTLA4Ig. Structural modeling of B7-1 based on th e structure of immunoglobulin revealed that these four and other previ ously identified critical amino acids in both IgV- and IgC-like domain s can form a localized structure. (C) 1998 Elsevier Science Ltd. All r ights reserved.