Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues: implications for tumor vaccine development.
To identify shared epitopes for melanoma-reactive CTL restricted by MHC molecules other than HLA-A*0201, six human melanoma patient CTL lines expressing HLA-A1 were screened for reactivity against the melanocyte differentiation proteins Pmel-17/gp100, MART-1/Melan-A, and tyrosinase, expressed via recombinant vaccinia virus vectors. CTL from five of the six patients recognized epitopes from tyrosinase, and recognition of HLA-A1+ target cells was strongly correlated with tyrosinase expression. Restriction by HLA-A1 was further demonstrated for two of those tyrosinase-reactive CTL lines. Screening of 119 synthetic tyrosinase peptides with the HLA-A1 binding motif demonstrated that nonamer, decamer, and dodecamer peptides containing the sequence KCDICTDEY (residues 243-251) all reconstituted the CTL epitope in vitro. Epitope reconstitution in vitro required high concentrations of these peptides, which was hypothesized to be a result of spontaneous modification of cysteine residues, interfering with MHC binding. Substitution of serine or alanine for the more N-terminal cysteine prevented modification at that residue and permitted target cell sensitization at peptide concentrations 2 to 3 orders of magnitude lower than that required for the wild-type peptide. Because spontaneous modification of sulfhydryl groups may also occur in vivo, tumor vaccines using this or other cysteine-containing peptides may be improved by amino acid substitutions at cysteine residues.
Kittlesen
D J
DJ
Department of Surgery, The Beirne Carter Center for Immunology, University of Virginia, Charlottesville 22908, USA.
Thompson
L W
LW
Gulden
P H
PH
Skipper
J C
JC
Colella
T A
TA
Shabanowitz
J
J
Hunt
D F
DF
Engelhard
V H
VH
Slingluff
C L
CL
Shabanowitz
J A
JA
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019496:Cancer Vaccines / Q000737:chemistry / Q000276:immunology*; D003545:Cysteine; D003602:Cytotoxicity, Immunologic; D015788:HLA-A1 Antigen / Q000235:genetics / Q000276:immunology*; D006801:Humans; D016056:Immunodominant Epitopes / Q000737:chemistry / Q000276:immunology*; D008545:Melanoma / Q000201:enzymology / Q000276:immunology*; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000276:immunology*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D012694:Serine; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology / Q000276:immunology*; D014407:Tumor Cells, Cultured
D019496:Cancer Vaccines; D015788:HLA-A1 Antigen; D016056:Immunodominant Epitopes; D010446:Peptide Fragments; D012694:Serine; D014442:Monophenol Monooxygenase; D003545:Cysteine; D000409:Alanine