BIOENGINEERED SOLUBLE HLA-B7 - GENESIS, CHARACTERIZATION, AND OCCURRENCE OF DIMERIZATION

A soluble, secreted form of HLA-B7 was engineered by replacing the exo ns encoding the transmembrane and cytoplasmic domains of the B7 gene w ith a CI. The modified gene, gsB7, transfected into J27.2 or C1R cell lines, produced a secreted protein, sB7, serologically recognized as B 7. Size fractionation showed one species of sB7 at the approximate to 55 kD expected for an sB7 alpha-chain-beta(2)m heteroduplex, and anoth er at approximate to 120 kD which had the same constituent chains and was a dimer of the 55-kD species. Dimer formation appeared to be relat ed to protein concentration but not to disulfide bridging. The sB7 hea vy chain on SDS-PAGE showed a doublet at approximate to 39 and approxi mate to 42 kD; enzyme analysis indicated that the two bands differed o nly by a carboxyl terminal polypeptide. Analysis of gsB7 transfectants ' mRNA by Northern blots and PCR revealed message fully spliced or wit h retained CI, accounting for the 13- and 42-kD bands, respectively, a nd apparently untranslated message with 13 retained. sB7 was not detec table on the surface of gsB7 transfectants by CTLs, nor did it inhibit those CTLs. Production of the sB7 protein provides a ready, consisten t source of soluble class I antigen for-further study, including test materials for tolerogenicity studies in animal models.