Generation of human tumor‐reactive cytotoxic T cells against peptides presented by non‐self HLA class I molecules

The cyclin‐D1 protein, which was found to be overexpressed in various human tumors, promotes cell cycle progression from the G1 into the S phase. It is normally expressed at low levels in several tissues and is likely to induce immunological tolerance. We have recently shown in a murine system that T cell tolerance to a widely expressed protein was circumvented by raising cytotoxic T lymphocytes (CTL) from major histocompatability complex mismatched donors. In this study, we tested whether it is possible to raise human allo‐restricted CTL against the cyclin‐D1 protein. The human cell line T2 is deficient in the genes encoding the transporter associated with antigen processing (TAP), resulting in inefficient loading of HLA‐A2 class I molecules with endogenous peptides. Thus, a large number of A2 molecules can bind exogenously supplied synthetic peptides. Peripheral blood mononu clear cells from HLA‐A2‐negative donors were stimulated with T2 cells presenting cyclin‐D1‐derived synthetic peptides. Cloning of bulk cultures revealed that a large proportion of CTL clones were peptide specific. One peptide induced CTL which lysed cyclin‐D1‐expressing breast cancer cells, but not control Epstein‐Barr virus‐transformed B lymphoid cells. The results show that HLA‐A2‐negative donors can be used to isolate tumor‐reactive CTL spe cific for cyclin‐D1 peptides presented by HLA‐A2 class I molecules.