Modulation of Proteasomal Activity Required for the Generation of a Cytotoxic T Lymphocyte–defined Peptide Derived from the Tumor Antigen MAGE-3

Abstract

We have analyzed the presentation of human histocompatability leukocyte antigen-A*0201–associated tumor peptide antigen MAGE-3271–279 by melanoma cells. We show that specific cytotoxic T lymphocyte (CTL)-recognizing cells transfected with a minigene encoding the preprocessed fragment MAGE-3271–279 failed to recognize cells expressing the full length MAGE-3 protein. Digestion of synthetic peptides extended at the NH2 or COOH terminus of MAGE-3271–279 with purified human proteasome revealed that the generation of the COOH terminus of the antigenic peptide was impaired. Surprisingly, addition of lactacystin to purified proteasome, though partially inhibitory, resulted in the generation of the antigenic peptide. Furthermore, treatment of melanoma cells expressing the MAGE-3 protein with lactacystin resulted in efficient lysis by MAGE-3271–279–specific CTL. We therefore postulate that the generation of antigenic peptides by the proteasome in cells can be modulated by the selective inhibition of certain of its enzymatic activities.

Proteasome-Assisted Identification of a SSX-2-Derived Epitope Recognized by Tumor-Reactive CTL Infiltrating Metastatic Melanoma

“…The peptides that were predicted to bind to a given HLA class I molecule with the highest affinity were selected, and their immunogenicity was tested. Although this method allowed the identification of a number of promising peptides, several of the latter were not produced by tumor cells as demonstrated by the poor recognition of cells expressing the corresponding protein of interest endogenously (15)(16)(17). Hence, a crucial step for a correct application of this latter method is the demonstration that the generated CTL can recognize target cells expressing the antigenic protein.…”

“…Proteasomes were purified from outdated human blood as previously described (17). Synthetic peptide (2 nmol) was digested with 16 g purified proteasome for 10, 20, 40, and 80 min at 37°C.…”

“…The reactions were stopped by adding 2% TFA. After lyophilization, the samples were analyzed by MALDI-TOF as described (17). The identification of proteolytic fragments was based on digestion times that were variable for each peptide.…”

“…This approach, named reverse immunology, has been applied to a number of tumor-associated proteins but has led, in several instances, to the generation of peptide-specific CTLs that are unable to lyse Ag-presenting tumor cells (14,15). This was shown to be mainly due to the fact that these peptides are normally not efficiently processed by the cellular machinery (16,17). Recently, an improved reverse immunology strategy has been described (18).…”

Testing mouse mammary tumor virus superantigen as adjuvant in cytotoxic T‐lymphocyte responses against a melanoma tumor antigen

“…v-ELA and v-ELA/SAg use the ubiquitin/protein/reference system to generate the Melan-A epitope. 10,32,33 Both encode the same linear fusion product, in which ubiquitin is located between the reference GFP and the Melan- analogue was not only expressed but also correctly presented by HLA-A*0201 and recognized by CD8 ϩ T cells, we determined the presentation of the Melan-A 26 -35 A27L peptide analogue by rVVinfected HLA-A*0201 ϩ NA8-MEL cells to a Melan-A-specific CD8 ϩ CTL line. NA8 cells infected with v-ELA or v-ELA/SAg efficiently stimulated CD8 ϩ CTLs for IFN-␥ secretion and the response observed with v-ELA/SAg was even superior to that of v-ELA (Fig.…”