Naturally processed and concealed HLA‐A2.1‐restricted epitopes from tumor‐associated antigen tyrosinase‐related protein‐2

Abstract

In this study, a computer-assisted reverse immunology approach was utilized in order to identify potentially antigenic peptides derived from the differentiation antigen TRP-2, a melanosomal protein frequently expressed in melanoma. Among the seven peptides complying with HLA-A2.1-binding motifs, two induced specific CD8(+) cytotoxic T lymphocytes. HLA-A2.1(+) melanoma cells expressing TRP-2 were lysed by clones specific for TRP-2(360-368) (TLDSQVMSL) peptide, thus identifying it as a naturally processed epitope. Other T-cell clones directed against TRP-2(476-484) (VMGTLVALV) were unable to lyse HLA-matched TRP-2(+) cell lines. The role of intracellular proteolytic processing in the generation of this epitope was investigated by transfecting mini-genes encoding the TRP-2(476-484) peptide alone or carrying N- or C-terminal extensions. Specific T-cell clones recognized target cells expressing the cytotoxic T-lymphocyte (CTL)-defined epitope or its C-terminally extended precursor, but failed to recognize cells expressing the N-terminally extended TRP-2(476-484) peptide, suggesting the presence of a negative processing signal (NPS). Regarding C-terminus-flanking regions, mutational analysis indicates that the GLY485 residue plays a key role in the processing of the TRP-2(476-484) epitope. Interestingly, proteasome inhibitors preventing the generation of the MART-1/Melan-A(27-35) immunodominant melanoma tumor-associated antigen (TAA) promoted detectable presentation of TRP-2(476-484) epitope in HLA-A2.1(+) and TRP-2(+) tumor lines, as witnessed by cytokine release by specific T-cell clones.

Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules

“…They also explain the reported paradoxically increased presentation of the MAGE-A3 peptide upon treatment with proteasome inhibitors (34), which efficiently block the activity of subunit β5, responsible for destruction of the antigenic peptide, but not that of β1, responsible for production of the C terminus of the antigenic peptide. The presentation of other antigenic peptides is increased by lactacystin (36)(37)(38) and, in two cases, by transfection of β5i (36,38), suggesting that the processing of these peptides also depends on the intermediate proteasome β5i.…”

High density oligonucleotide array analysis of interferon-α2a sensitivity and transcriptional response in melanoma cells

“…These results were consistent with our previously published data, showing that D10, HLA-A2.1-positive, melanoma cells were effectively killed by HLA-A2. 1-restricted CTL lines recognizing epitopes derived from MART-1/Melan-A, pmel-17/gp 100, tyrosinase or TRP-2 proteins (Spagnoli et al, 1995;Noppen et al, 2000). In contrast, ME59 HLA-A2.1 positive cells, that did not express the genes under investigation failed to be killed by the specific CTL (Spagnoli et al, 1995).…”

Selective responsiveness to common gamma chain cytokines in peripheral blood‐derived cytotoxic T lymphocytes induced by Melan‐A/MART‐127–35targeted active specific immunotherapy

“…13,14 Briefly, cells were cultured in 60-well Terasaki plates (Nunc, Glostrup, Denmark) at 0.3 cells per well in 20 ml volumes, in the presence of 10,000 irradiated allogenic PBMC/well, in RPMI 1640 medium, supplemented with 1 mM sodium pyruvate, 2 mM nonessential amino-acids, 2 mM L-glutamine, 10 mM HEPES buffer and kanamycin (all from Gibco BRL, Paisley, UK) and 5% pooled human serum (RPMI-HS), to which rIL-2 (200 units/ml) and purified phytohemagglutinin (PHA, 0.5 mg/ml, Remel, Dartford, UK) were added. After 10-14 days wells where cell growth was microscopically detectable were expanded in RPMI-HS supplemented with 100 units/ml rIL-2 and screened for antigen-specific cytotoxic activity (see below).…”