Anti-tumor CTLs recognize peptides derived from cellular proteins and presented on MHC class I. molecules, presentation of these peptides is generally considered as A-867744 strictly tumor-specific [1]. CTLs that recognize tumor antigens have often been isolated from the blood or tumors of cancer patients [2-4]. The antigenic peptides recognized by these CTLs are typically produced in the cytosol through degradation of cellular proteins by a multicatalytic protease called the proteasome [5]. Peptides resulting from proteasomal degradation are then transported into the lumen of the endoplasmic reticulum (ER) by A-867744 the transporter associated with antigen processing (TAP). In the ER, peptide loading onto MHC class I-2m dimers is facilitated by the peptide-loading NFBD1 complex, which is composed of TAP, tapasin (TPN), ERp57, calreticulin (CRT), MHC class I heavy chain (HC) and 2m (reviewed in [6]). Association of MHC class I molecules with a peptide is essential for their stability and transport to the cell surface, and proteins such as tapasin play a crucial role for optimal assembly of peptide-MHC class I complexes [6]. We report here the identification of an antigenic peptide derived from tumor protein MAGE-A1 (160-169) and presented to CTLs by HLA-B*44:02. Although the peptide is well presented when processed intracellularly, it almost completely fails to associate with HLA-B*44:02 when pulsed onto target cells from outside. Using a variety of cellular and biochemical approaches, we investigated the conditions required for the exogenous loading of this tapasin-dependent peptide. Results Isolation of a CTL recognizing a MAGE-A1 peptide In order to identify new antigenic peptides derived from the MAGE-A1 protein, CD8+ T cells from a hemochromatosis patient were subjected to 4 weekly stimulations with autologous presenting cells transduced with a recombinant canarypoxvirus, ALVAC, containing the coding sequence of MAGE-A1. CTL clone LB1801-461/G4.2 (hereinafter called CTL 4) was isolated after A-867744 cloning of the responsive population by limiting dilution. CTL 4 specifically lysed autologous EBV-B cells infected with a vaccinia virus construct driving expression of the MAGE-A1 protein (Fig. 1A, left panel). The MAGE-A1 positive melanoma cell line LB373-MEL4.1 was also recognized by CTL 4, demonstrating that the MAGE-A1 peptide was presented on melanoma cells after processing of the endogenous MAGE-A1 protein (Fig. 1A, right panel). Figure 1 CTL 4 recognizes a peptide derived from MAGE-A1 and restricted by HLA-B*44:02 Identification of the MAGE-A1 antigenic peptide recognized by CTL 4 Melanoma cell line LB373-MEL4.1, which is recognized by CTL 4, and patient LB1801, from which CTL 4 was derived, share HLA-A*02:01, HLA-B*44:02 and HLA-Cw5, suggesting that the peptide recognized by CTL 4 is presented by one of these HLA molecules. We therefore transfected COS-7 cells with cDNA constructs encoding MAGE-A1 and either HLA-A*02:01, HLA-B*44:02 or HLA-Cw5 and assessed their recognition in T cell assays. CTL 4 only recognized cells transfected with MAGE-A1 and HLA-B*44:02 (Fig. 1B), demonstrating that its cognate antigenic peptide is presented by A-867744 HLA-B*44:02. To further define the peptide-coding sequence, we transfected COS-7 cells with plasmids encoding HLA-B*44:02 and a series of truncated MAGE-A1 plasmid constructs. These cells were then tested for recognition by CTL 4 (Fig. 1C). A minigene construct encoding a 9-amino acid fragment (EADPTGHSY) located between positions 161 and 169 of the MAGE-A1 protein and preceded by a start-methionine sensitized transfected cells to CTL recognition. Because the peptide binding motif to HLA-B44 is characterized by a glutamic acid in position 2 and an aromatic amino acid in position 9 or 10 [7], it was likely that the endogenous peptide recognized by CTL 4 contained the lysine residue present at position 160 of the MAGE-A1 protein instead of the start-methionine of the minigene construct (Fig. 1C). To address this possibility, various synthetic peptides overlapping the MAGE-A1160-169 sequence were loaded exogenously on autologous HLA-B*44:02+ target cells (LB1801-EBV) and tested for their ability to activate CTL 4 (Fig. 1D). However, surprisingly none of the peptides tested was recognized by CTL 4 even when applied at 1M (Fig. 1D, left panel). In contrast, HLA-A*01:01+ A-867744 target cells loaded with the exact same set of peptides were efficiently recognized by CTL MZ2-82/30, which is specific for peptide EADPTGHSY presented by HLA-A*01:01 [8] (Fig. 1D, right panel). Peptide candidate KEADPTGHSY was only weakly recognized by CTL 4, at doses of 10 M and higher (Fig. 2A, higher panel). Strikingly, we observed that CTL recognition of peptide KEADPTGHSY was drastically improved when the HLA-B*44:02+ target cells were fixed with paraformaldehyde (PFA) prior to exogenous loading (Fig. 2A). This was the case when using the autologous EBV-B cells LB1810-EBV or the HLA-B*44:02+ 721.220 cells back-transfected with tapasin (so that they express sufficient amounts of surface HLA-B*44:02). Because fixed APC are devoid of any metabolic and processing capability, this.