MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-I PRESENTATION OF EXOGENOUS SOLUBLE TUMOR-ANTIGEN FUSED TO THE B-FRAGMENT OF SHIGA TOXIN

Targeting exogenous antigen into the MHC class I-restricted presentati on pathway is a prerequisite for the induction of cytotoxic T lymphocy tes (CTL) which have been shown to represent an important component of the protective and therapeutic immune response to viral infections an d tumors. In this study, we produced recombinant proteins composed of the receptor-binding non-toxic B-fragment of bacterial Shiga toxin der ived from Shigella dysenteriae associated with an epitope from a model tumor antigen, Mage 1. We show that Shiga B-Mage 1 fusion proteins ca rrying an active or inactive endoplasmic reticulum retrieval signal (t he C-terminal peptides KDEL or KDELGL, respectively) could be presente d by peripheral blood mononuclear cells in an MHC class I-restricted m anner to Mage I-specific CTL. After pulsing B lymphoblastoid cells or dendritic cells with Shiga B-Mage 1 fusion protein, activation of the MHC class I-restricted Mage 1-specific CTL was also demonstrated. In f urther analysis, we showed that treatment with brefeldin A or paraform aldehyde fixation of Epstein-Barr virus-transformed B cells prevented the presentation of the Mage 1 T cell epitope, which excluded extracel lular processing of the antigen. Immunofluorescence analysis also reve aled that the Shiga B-Mage 1 fusion protein was largely excluded from Lamp-2-positive lysosomal structures. Therefore, the ability of Shiga toxin B-fragment to target dendritic cells and B cells and to direct a ntigen into the exogenous class I-restricted pathway makes it an attra ctive non-living and non-toxic vaccine vector.