Expression of a Single-Chain HLA class I Molecule in a Human Cell Line: Presentation of Exogenous Peptide and Processed Antigen to Cytotoxic T Lymphocytes

We have synthesized a recombinant gene encoding a single-chain HLA-A2 / β2-microglobulin (β2m) molecule by linking β2m through its carboxyl terminus via a short peptide spacer to HLA-A2 (A*0201). This gene has been expressed in the β2m-deficient colorectal tumor cell line DLD-1. Transfection of this cell with the single-chain construct was associated with conformationally correct cell surface expression of a class I molecule of appropriate molecular mass. The single-chain HLA class I molecule presented either exogenously added peptide or (after interferon-γ treatment) endogenously processed antigen to an influenza A matrix-specific, HLA-A2-restricted cytotoxic T-lymphocyte line. The need for interferon γ for the processing and presentation of endogenous antigen suggests that DLD-1 has an antigen-processing defect that can be up-regulated, a feature that may be found in other carcinomas. Our data indicate that single-chain HLA class I constructs can form functional class I molecules capable of presenting endogenously processed antigens. Such molecules should be of use for functional studies, as well as providing potential anticancer immunotherapeutic agents or vaccines.