Carrier‐mediated uptake and presentation of a major histocompatibility complex class I‐restricted peptide

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Abstract
Antigenic peptides derived from endogenous or viral proteins can associate with class I or class II major histocompatibility complex (MHC) molecules, while exogenous antigens are endocytosed, processed intracellularly and presented on MHC class II molecules. Here we describe a method that allows the presentation of an MHC class I‐restricted antigenic peptide on MHC class I molecules, although it was taken up from the outside. The HLA‐A2‐restricted influenza virus matrix protein‐derived peptide (flu, 57–68) was used either in soluble form or coupled via an S‐S bridge to transferrin (Tf‐flu). Target cells were incubated with flu or Tf‐flu and the effective antigen presentation was detected in a cytotoxicity assay using flu peptide‐specific, HLA‐A2‐restricted CD8+ cytotoxic T lymphocytes. Sensitization of target cells with Tf‐flu required 5 to 10 times higher molar concentrations of peptide compared to sensitization with soluble free peptide. The Tf‐flu construct was taken up by the cells via the Tf receptor (CD71) as the binding of Tf‐flu was blocked by an excess of Tf. In contrast to the flu peptide, cytotoxicity elicited by Tf‐flu was blocked by brefeldin A but not by chloroquine nor inhibitors of intracellular reducing steps, like 1‐buthionine‐(s, r)‐sulfoximine or n‐ethylmaleimide. Presentation of the flu peptide derived from Tf‐flu construct is not hindered in the mutant T2 cell line, which lacks genes coding for transporter proteins for antigenic peptides (TAP1/TAP2) and proteasomes subunits, suggesting that the processing pathway described in this report may involve TAP‐independent steps.