Failure in expression of structurally altered (CYS164-->TYR) H-2Kb molecules is mitigated with high affinity...

A C164Y somatic mutation in the H-2Kb class I molecule causes a disruption of the alpha 2 domain disulfide bond and results in a loss of H-2Kb cell surface expression by the 69.9.15 cell line. In vitro culture of the somatic cell variant at 30 degrees C induced weak, but reproducible, expression of the H-2Kb mutant molecule on the cell surface, which suggests that a temperature-sensitive mutation was contributing to the H-2Kb null phenotype. Based on the inherent structural instability of the mutant H-2Kb 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-2Kb. Treatment of 69.9.15 cells was performed with acid-eluted cell-derived peptides, as well as synthetic H-2Kb-restricted peptides, ovalbumin (OVA) p257-264 (YSIINFEKL), and vesicular stomatitis virus-nuclear protein p52-59 (RGYVYQGL). Whereas the endogenous and vesicular stomatitis virus peptides were ineffective at inducing H-2Kb expression at either 37 degrees C or 30 degrees C, treatment with the OVA peptide at 30 degrees C gave rise to dose-dependent enhancement in H-2Kb expression, an effect that was independent of exogenous sources of bovine beta 2-microglobulin at the time of peptide treatment. By comparison, expression of H-2Kb remained unaltered when cells were treated with the OVA peptide at 37 degrees C, consistent with the temperature-sensitive expression of the mutant molecules. Decay of H-2Kb from the cell surface was similar for both 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 either a cis-(69.9.15) or trans-acting (RMA-S) defect in heavy chain transport. These data provide novel evidence that transport-defective MHC class I molecules, similar in nature to those encoded by class I genes isolated from human genomic libraries, i.e., the 12.4 pseudogene with a polymorphism at amino acid position 164 (C-->F), are subject to high affinity peptide-induced stabilization which reverses the class I null phenotype.