FAILURE IN EXPRESSION OF STRUCTURALLY ALTERED (CYS164-]TYR) H-2K(B) MOLECULES IS MITIGATED WITH HIGH-AFFINITY PEPTIDE-LIGAND

A C164Y somatic mutation in the H-2K(b) class I molecule causes a disr uption of the alpha 2 domain disulfide bond and results in a loss of H -2K(b) cell surface expression by the 69.9.15 cell line, In vitro cult ure of the somatic cell variant at 30 degrees C induced weak, but repr oducible, expression of the H-2K(b) mutant molecule on the cell surfac e, which suggests that a temperature-sensitive mutation was contributi ng to the H-2K(b) null phenotype. Based on the inherent structural ins tability of the mutant H-2K(b) molecules synthesized by 69.9.15 cells, we sought to determine the ability of high affinity peptide-ligand to counteract the null expression of H-2K(b), Treatment of 69.9.15 cells was performed with acid-eluted cell-derived peptides, as well as synt hetic H-2K(b)-restricted peptides, ovalbumin (OVA) p257-264 (YSIINFEKL ), and vesicular stomatitis virus-nuclear protein p52-59 (RGYVYQGL), W hereas the endogenous and vesicular stomatitis virus peptides were ine ffective at inducing H-2K(b) expression at either 37 degrees C or 30 d egrees C, treatment with the OVA peptide at 30 degrees C gave rise to dose-dependent enhancement in H-2K(b) expression, an effect that was i ndependent of exogenous sources of bovine beta 2-microglobulin at the time of peptide treatment, By comparison, expression of H-2K(b) remain ed unaltered when cells were treated with the OVA peptide at 37 degree s C, consistent with the temperature-sensitive expression of the mutan t molecules, Decay of H-2K(b) from the cell surface was similar for bo th 69.9.15 and RMA-S cells, an indication that binding of OVA p257-264 provided the same level of stability for class I molecules with eithe r a cis(69.9.15) or trans-acting (RMA-S) defect in heavy chain transpo rt, These data provide novel evidence that transport-defective MHC cla ss I molecules, similar in nature to those encoded by class I genes is olated from human genomic libraries, i,e,, the 12.4 pseudogene with a polymorphism at amino acid position 164 (C --> Fl, are subject to high affinity peptide-induced stabilization which reverses the class I nul l phenotype.