GENERATION OF FUNCTIONAL HLA-A2 MOLECULES COVALENTLY ATTACHED TO ANTIGENIC PEPTIDES

Abstract

Major Histocompatibility Complex (MHC) class I molecules expressed at the cell surface are associated with a large number of different peptides so that the density of a given MHC/peptide complex is relatively low. These experiments describe the properties of an MHC class I molecule covalently attached to a single antigenic peptide. This single chain fusion protein consisting of HLA-A2 (Human leucocyte antigen class I) and the HTLV-1 Tax (amino acid residues 11-19) peptide derived from the human T cell leukemia virus which causes neurological diseases has been used as a test system for induction of an HLA-A2 restricted cytotoxic T lymphocyte (CTL) response against the Tax peptide. The results indicate that this fusion protein can induce such CTL responses. Prokaryotic and eukaryotic expression systems were employed to generate the fusion protein. In the case of the prokaryotic system, the fusion protein corresponding to the DNA construct named "pA2Taxprok" has been tested in an in vitro binding assay for complex formation with radiolabelled beta-2-microglobulin (B₂m). The results of this assay showed that exogenous Tax peptide was required for correct folding of the molecule, suggesting that the covalently attached peptide was not situated correctly or at all in the binding groove of HLA-A2. In the case of the eukaryotic expression system, the DNA construct corresponding to the Tax peptide covalently linked to the HLA-A2 molecule has been inserted into the plasmid named "A2rsvneo" expression vector and transformed into competent prokaryotic cells. Positive clones were identified and sequenced. One of these positive clones was transfected into the HLA-A2 negative cell line HMY.CIR [a human plasma cell line that does not express endogenous HLA-A or HLA-B antigen] and the resulting transfectant has been used successfully to generate an anti-Tax 11-19 T cell line. Results of ⁵¹ Cr-release assays have shown that the transfectants bearing the single chain construct appear to be equally sensitive targets for lysis by anti-Taxi 11-19 CTL as are HLA-A2 positive cells that are loaded exogenously with the Tax peptide.