ISOLATION OF NATURALLY PROCESSED PEPTIDES RECOGNIZED BY CYTOLYTIC T-LYMPHOCYTES (CTL) ON HUMAN-MELANOMA CELLS IN ASSOCIATION WITH HLA-A2.1

Cytolytic T lymphocyte (CTL) clones have previously been derived from peripheral blood of melanoma patient SK29(AV). They lyse autologous me lanoma cells but not autologous Epstein-Barr virus (EBV)-transformed B lymphocytes. Immunoselection experiments indicate that these CTL clon es recognize 4 different antigens (Aa, Ab, B, C) in association with a single HLA restriction element, HLA-A2.1. While the expression of ati gens B and C appears to be confined to SK29-melanoma cells, antigen Aa and Ab are shared by a high proportion of allogeneic HLA-A2-positive melanoma lines. HLA-A2.1 and total HLA class I molecules have now been purified from SK29-melanoma cells using affinity chromatography and a ssociated peptides have been eluted. Peptide pools eluted from HLA-A2. 1 and total class I were separated by reversed phase high performance liquid chromatography (HPLC). Individual HPLC fractions were tested fo r their ability to sensitize target cells for recognition by SK29-CTL clones. The presence of antigens Aa, Ab, B and C was detected in disti nct HPLC fractions that were identical for both peptide pools. As targ et 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.T 2 (T2), pre-incubated with anti-HLA-A2 monoclonal antibody (MAb) MA2.1 , was shown to be essential. Single-peak target-sensitizing activity w as found for antigens Ab and B, whereas multi-peak sensitizing activit y was reproducibly detected for antigens Aa and C. We reason that at l east some of these melanoma peptide antigens might occur in biochemica lly distinct isoforms. (C) 1994 Wiley-Liss, Inc.