The B7h-ICOS costimulatory pair is a member of the B7-CD28 superfamily that activates and controls T cell- and B cell-mediated immune responses. Previous work has identified a novel mode of regulation wherein cell-surface B7h on B cells is rapidly downregulated upon B cell receptor (BCR) signaling or ICOS binding. Although BCR-mediated B7h downregulation occurs through ectodomain shedding by a metalloprotease, ICOS-mediated B7h downregulation does not depend on metalloprotease activity. In order to study the mechanism of ICOS-mediated B7h downregulation, we developed a rapid non-cloning mutagenesis approach, based on the cyclical packaging rescue (CPR) technique, to generate a cellular library expressing a saturating number of point mutants in the B7h gene. Using this mutant library, we mapped the ICOS-binding surface of murine B7h, and identified specific B7h residues that are critical for binding the ICOS receptor.

To investigate the type of protease involved in ICOS-mediated B7h downregulation, we used a panel of inhibitors for different classes of proteases, and show for the first time that ICOS-mediated downregulation occurs through B7h shedding by a serine protease. Additionally, to identify B7h mutants that are resistant to ICOS-mediated downregulation, we used the B7h mutant library that we generated by CPR to select for loss of B7h downregulation by ICOS. Although we screened a saturating number of B7h mutants, we did not identify any point mutations within B7h that abrogated ICOS-mediated B7h downregulation, in accordance with previous data where chimeric mutants containing large domain swaps between B7h and the downregulation-insensitive B7.2 protein were necessary for resistance to ICOS-mediated B7h shedding (Logue et al., 2006). Therefore, using a chemical mutagen that induces frameshift mutations, we identified a somatic cell mutant resistant to ICOS-mediated B7h downregulation. Characterization of this genetic mutant indicated that it is specifically defective in ICOS-mediated soluble B7h release, and that the defect abrogates the activity of a component of the serine protease pathway that mediates ICOS-induced B7h down regulation.