HPV 16-Derived Synthetic Peptides with Ability to Upregulate MHC Class I Expression on RMA-S or T2 Cells, as Detected by Enzyme Immunoassay

Abstract

The measurement of the ability of peptides to interact with MHC class I molecules has been greatly facilitated in recent years, by the introduction of mutant cell lines with surface expression of unstable MHC class I molecules lacking endogenously bound peptides^1,2. The murine lymphoma cell line RMA-S, a mutant of the T cell lymphoma line RMA, was generated by selection for low surface class I expression¹. RMA-S expresses only about 5% of class I K^b and D^b molecules compared to the wild type cell line RMA¹. These MHC class I molecules are unstable at physiological. temperature, due to lack of normal. peptides in the antigen binding groove¹. However, they can be stabilized by supplementing the culture medium with exogenous peptide with ability to bind to the allele in question^1,2. The binding ability of a given peptide can thereafter be quantified by measuring the amount of class I molecules by immunoprecipitation or by quantitative immunofluorescence^1,2,3,4,5. Although the use of immunofluorescence represents a significantly more rapid assay than immunoprecipitation, it still requires a considerable amount of time on a Fluorescence Activated Cell Sorter (FACS), which was not available. In the past 5 years, our laboratory has synthesized a large amount of peptides (>2000), derived from a variety of viruses including HPV. All these peptides are approximately 20 residues long, which is much longer than the optimal. size of a MHC class I binding peptide (8–10 residues long)⁶. In the present study, we wished to 1) develop a rapid assay that does not require a FACS for measurement of the ability of synthetic peptides to interact with MHC class I and 2) investigate whether 20-mer peptides could be used for screening for agretopes, in spite of their suboptimal. length.