Cytolytic T-cell clones define HLA-A2-restricted human cutaneous melanoma peptide epitopes: correlation with T-cell receptor usage.

PURPOSE

We studied the T-cell receptor alpha chain and beta chain variable region usage in HLA-A2-restricted and melanoma-specific T lymphocytes and correlated such T-cell receptor usage with HLA-A2-presented peptide-specific T-cell recognition.

MATERIALS AND METHODS

Tumor-infiltrating lymphocytes were isolated from a metastatic melanoma lesion and cloned by limiting dilution. Clonal and oligoclonal tumor-infiltrating lymphocytes were analyzed for major histocompatibility complex class I--presented melanoma peptide recognition by using acid-eluted and high-performance liquid chromatography--fractionated melanoma peptides presented by HLA-A2 as targets. The T-cell receptor variable regions of the alpha and beta chains of each individual T-cell clone or oligoclonal T-cell population were analyzed by mRNA extraction and reverse transcribed cDNA by polymerase chain reaction using a panel of T-cell receptor alpha and beta variable region specific primers.

RESULTS

We demonstrated that individual T-cell clones are capable of recognizing peptides within multiple high-performance liquid chromatography fractions containing melanoma epitopes, and that individual high-performance liquid chromatography fractions containing melanoma epitopes can be recognized by T-cell clones exhibiting limited usage of the T-cell receptor alpha and beta variable region chains.

DISCUSSION

These results confirm the heterogeneity of T-cell-defined melanoma antigens in a single individual and suggest the possibility of developing novel antimelanoma therapeutic reagents using either peptides (as immunogens) or the T-cell receptors themselves (as gene therapy when introduced into lymphoid effectors).