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

Valmori, Danila; Gileadi, Uzi; Servis, Catherine; Dunbar, P. Rod; Cerottini, Jean-Charles; Romero, Pedro; Cerundolo, Vincenzo; Lévy, Frédéric

doi:10.1084/jem.189.6.895

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Article| March 15 1999

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

Danila Valmori,

Danila Valmori

From the *Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland; the ‡Institute of Molecular Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom; and the §Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland

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Uzi Gileadi,

Uzi Gileadi

From the *Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland; the ‡Institute of Molecular Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom; and the §Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland

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Catherine Servis,

Catherine Servis

From the *Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland; the ‡Institute of Molecular Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom; and the §Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland

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P. Rod Dunbar,

P. Rod Dunbar

From the *Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland; the ‡Institute of Molecular Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom; and the §Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland

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Jean-Charles Cerottini,

Jean-Charles Cerottini

From the *Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland; the ‡Institute of Molecular Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom; and the §Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland

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Pedro Romero,

Pedro Romero

From the *Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland; the ‡Institute of Molecular Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom; and the §Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland

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Vincenzo Cerundolo,

Vincenzo Cerundolo

From the *Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland; the ‡Institute of Molecular Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom; and the §Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland

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Frédéric Lévy

From the *Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland; the ‡Institute of Molecular Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom; and the §Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland

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Author and Article Information

Danila Valmori , Uzi Gileadi , Catherine Servis , P. Rod Dunbar , Jean-Charles Cerottini , Pedro Romero , Vincenzo Cerundolo , Frédéric Lévy

From the *Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, CH-1066 Epalinges, Switzerland; the ‡Institute of Molecular Medicine, Nuffield Department of Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom; and the §Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland

Address correspondence to Frédéric Lévy, Ludwig Institute for Cancer Research, Ch. des Boveresses 155, CH-1066 Epalinges, Switzerland. Phone: 41-21-692-59-98. Fax: 41-21-653-44-74. E-mail: frederic. [email protected]

The expert technical assistance of A.-L. Peitrequin, A. Porret, and N. Montandon is gratefully acknowledged. We thank Dr. I. Miconnet and L. Burri for comments on the manuscript.

U. Gileadi, P.R. Dunbar, and V. Cerundolo were supported by the Medical Research Council of the United Kingdom and the Cancer Research Campaign. F. Lévy was supported in part by a grant from the Swiss Cancer League. This work was partly supported by the Federal Office for Education and Science, Switzerland and the European Community (contract BMH4-CT95-1627).

1

Abbreviations used in this paper: AMC, 7-amido-4-methylcoumarin; BrAAP, branched chain amino acid–preferring; ER, endoplasmic reticulum; E/T, effector-to-target cell ratio; GFP, green fluorescence protein; ha, hemagglutinin epitope; LLnL, N-acetyl-Leu-Leu-norleucinal; MALDI-TOF, matrix assisted laser desorption ionization-time of flight; PGPH, peptidylglutamylpeptide hydrolyzing; rec. v.v., recombinant vaccinia virus; SNAAP, small neutral amino acid–preferring; TAP, transporters associated with antigen presentation; Ub, ubiquitin; UPR, ubiquitin/ protein/reference.

D. Valmori and U. Gileadi contributed equally to this work.

Received: June 19 1998

Revision Received: December 15 1998

Online ISSN: 1540-9538

Print ISSN: 0022-1007

1999

J Exp Med (1999) 189 (6): 895–906.

https://doi.org/10.1084/jem.189.6.895

We have analyzed the presentation of human histocompatability leukocyte antigen-A*0201–associated tumor peptide antigen MAGE-3_271–279 by melanoma cells. We show that specific cytotoxic T lymphocyte (CTL)-recognizing cells transfected with a minigene encoding the preprocessed fragment MAGE-3_271–279 failed to recognize cells expressing the full length MAGE-3 protein. Digestion of synthetic peptides extended at the NH₂ or COOH terminus of MAGE-3_271–279 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-3_271–279–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.

1999

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