Isolation of naturally processed peptides recognized by cytolytic T lymphocytes (CTL) on human melanoma cells in association with HLA-A2.1

Authors

  • Thomas Wölfel,

    Corresponding author
    1. I. Medizinische Klinik und Poliklinik, Johannes Gutenberg-Universität, D-55131 Mainz, Germany
    • I. Medizinische Klinik und Poliklinik, Johannes Gutenberg-Universität, D-55131 Mainz, Germany
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  • Jörg Schneider,

    1. I. Medizinische Klinik und Poliklinik, Johannes Gutenberg-Universität, D-55131 Mainz, Germany
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  • Karl-Hermann Meyer Zum Büschenfelde,

    1. I. Medizinische Klinik und Poliklinik, Johannes Gutenberg-Universität, D-55131 Mainz, Germany
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  • Hans-Georg Rammensee,

    1. Max-Planck-Institut für Biologie, Abteilung Immungenetik, Universität Tübingen, D-72076 Tübingen, Germany
    Current affiliation:
    1. Deutsches Krebsforschungszentrum, Abteilung Tumorvirus-Immunologies, D-69120 Heidelberg, Germany
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  • Olaf Rötzschke,

    1. Max-Planck-Institut für Biologie, Abteilung Immungenetik, Universität Tübingen, D-72076 Tübingen, Germany
    Current affiliation:
    1. Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138, U.S.A.
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  • Kirsten Falk

    1. Max-Planck-Institut für Biologie, Abteilung Immungenetik, Universität Tübingen, D-72076 Tübingen, Germany
    Current affiliation:
    1. Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138, U.S.A.
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Abstract

Cytolytic T lymphocyte (CTL) clones have previously been derived from peripheral blood of melanoma patient SK29(AV). They lyse autologous melanoma cells but not autologous Epstein-Barr virus (EBV)-transformed B lymphocytes. Immunoselection experiments indicate that these CTL clones recognize 4 different antigens (Aa, Ab, B, C) in association with a single HLA restriction element, HLA-A2.1. While the expression of antigens B and C appears to be confined to SK29-melanoma cells, antigens Aa and Ab are shared by a high proportion of allogeneic HLA-A2-positive melanoma lines. HLA-A2.1 and total HLA class 1 molecules have now been purified from SK29-melanoma cells using affinity chromatography and associated peptides have been eluted. Peptide pools eluted from HLA-A2.1 and total class 1 were separated by reversed phase high performance liquid chromatography (HPLC). Individual HPLC fractions were tested for their ability to sensitize target cells for recognition by SK29-CTL clones. The presence of antigens Aa, Ab, B and C was detected in distinct HPLC fractions that were identical for both peptide pools. As target for detection of peptide antigens in HPLC fractions, the use of the HLA-A2.1-positive antigen processing mutant cell line CEM x 721.174.T2 (T2), pre-incubated with anti-HLA-A2 monoclonal antibody (MAb) MA2.1, was shown to be essential. Single-peak target-sensitizing activity was found for antigens Ab and B, whereas multi-peak sensitizing activity was reproducibly detected for antigens Aa and C. We reason that at least some of these melanoma peptide antigens might occur in biochemically distinct isoforms. © 1994 Wiley-Liss, Inc.

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