Transfection of IL-2 augments CTL response to human melanoma cells in vitro: immunological characterization of a melanoma vaccine.

We have transfected human melanoma cell line 518A2 with the cDNA encoding interleukin-2 (IL-2) or granulocyte-macrophage colony-stimulating factor (GM-CSF), and compared cytokine-producing clones for their ability to induce melanoma-specific cytotoxic T lymphocytes (CTL) from autologous peripheral blood mononuclear cells (PBMC) in vitro. The parental cell line expressed HLA-A1, HLA-A2, ICAM-1, LFA-3, in addition to the common CTL antigens MAGE-1, MAGE-3, tyrosinase, gp100, and Melan-A/MART-1. Stimulation of autologous PBMC responders with the IL-2-transfected clone 518/IL2.14 specifically induced CTL lines reactive with all cell lines derived from the autologous patient. Strikingly, GM-CSF-transfected 518A2 cells did not induce anti-tumor CTL reactivity. CTL induction against 518/IL2.14 was independent of HLA class II expression or CD4 help. The parental cell line 518A2 gained immunogenic properties when high concentrations of IL-2 were supplied exogenously, indicating that IL-2 produced and present at high levels locally by itself enhanced immunogenicity. From the autologous CTL line reactive with 518/IL2.14, clones were generated against an as yet unknown antigen, which was present in all autologous melanoma cell lines as well as in 7 of 15 HLA-A2+ melanoma cell lines tested, but not in melanocytes. These results will be discussed with respect to the possibility of using IL-2-transfected melanoma cells as a vaccine for treatment of patients with melanoma.

Peptide-pulsed dendritic cells induce tumoricidal cytotoxic T lymphocytes from healthy donors against stably HLA-A*0201-binding peptides from the Melan-A/MART-1 self antigen.

The melanoma antigen Melan-A/MART-1 was screened for the presence of potential HLA-A*0201-binding cytotoxic T lymphocytes (CTL) epitopes. The immunodominant nonamer epitope AAGIGILTV demonstrated weak binding to T2 but a significant half-life of binding to HLA-A*0201 in contrast to the decamer EAAGIGILTV. In addition to the immunodominant CTL epitope, we describe two peptides, GILTVILGV and ALMDKSLHV, that display stable binding to HLA-A*0201. Using cultured autologous dendritic cells pulsed with these peptides, CTL lines were induced from peripheral blood lymphocytes that displayed reactivity with HLA-A2+, Melan-A/MART-1+ melanoma cells. CTL reactivity against the immunodominant epitope could be induced with the nonamer epitope alone, but not with the decamer variant. CTL clones generated from an (EAAGIGILTV + AAGIGILTV)-induced CTL line recognize the appropriate melanoma cells and normal melanocytes. Upon further characterization of one of these CTL clones, it was found to be of surprisingly high affinity considering that it is directed against a self antigen. This study demonstrates that immunogenic peptides can be selected based on stability (half-life) of peptide/HLA binding. In addition, cultured DC were found to efficiently induce CTL responses in vitro against such selected peptides, and some of these CTL were capable of recognizing endogenously processed antigen.

Induction and characterization of cytotoxic T-lymphocytes recognizing a mutated p21ras peptide presented by HLA-A*0201.

The ras oncogene is frequently found to be activated in human cancer through point mutations at codons 12, 13 or 61. We explored whether these altered p21ras protein sequences contain peptide sequences that can activate naive CD8+ cytotoxic T lymphocytes (CTL). Several wild-type and mutated p21ras peptides were identified that carry a binding motif for human leukocyte antigen (HLA)-A*0201. Two peptides were found to bind strongly to this allele. CD8+ CTL bulk cultures specifically reacting with one of these peptides could be induced, using processing-defective T2 cells loaded with peptide CLLDILDTAGL as stimulators. The peptide is derived from p21ras, position 51-61, and carries a 61 Gln-->Leu mutation. In contrast, a 9-mer peptide CLLDILDTA corresponding to amino acid sequence 51-59 of wild-type p21ras did not yield reactive CTL cultures. T-cell clones with low affinity for the 11-mer peptide were isolated from CLLDILDTAGL-reactive bulk cultures. These T cells did not lyse melanoma cells transfected with 61-Leu N-ras, although lysis was found when these transfectants were pulsed with the 11-mer peptide. Possibly, T cells of higher affinity may be required to demonstrate processed peptide on the cell surface. The combined experiments suggest that a peptide derived from mutated p21ras can be recognized by HLA class I-restricted CTL, whereas an analogous wild-type p21ras peptide may not be immunogenic.