Novel HLA-Cw8-restricted T cell epitopes derived from tyrosinase-related protein-2 and gp100 melanoma antigens.

The identification of T cell epitopes presented by alternative HLA-B and -C alleles may provide a means to counteract the tumor escape mechanism based on the selection of tumor cells no longer susceptible to HLA-A-restricted T cell recognition. Several T cell clones and lines were obtained from T lymphocytes purified from melanoma-infiltrated or noninfiltrated lymph nodes of a patient who remained disease free 8 yr after surgery. Selected T cells recognized the autologous melanoma as evaluated by direct cytolysis and production of cytokines. These effectors were directed against the tyrosinase-related protein-2 (TRP-2) and gp100 melanoma epitopes restricted by HLA-Cw8. The nonamer and decamer peptides containing the sequence ANDPIFVVL (residues 387-395) of TRP-2 and the octamer, nonamer, and decamer peptides containing the sequence SNDGPTLI (residues 71-78) of gp100 reconstituted the epitope for TRP-2- and gp100-specific T cell lines and clones, respectively. However, only the nonameric form of TRP-2 and the nonameric and octameric forms of gp100 were able to induce peptide-specific T cells recognizing the autologous tumor in an HLA-class I-restricted fashion from PBMC of the melanoma patient studied. Together these data indicate that HLA-Cw8 can restrict the recognition of gp100 and TRP-2 epitopes by CTL, and that such peptides could stimulate a patient's PBL, suggesting that these Ags could have contributed to a systemic immunity against melanoma.

A superagonist variant of peptide MART1/Melan A27-35 elicits anti-melanoma CD8+ T cells with enhanced functional characteristics: implication for more effective immunotherapy.

In the present study, we show that a singly substituted peptide derived from the epitope MART1(27-35) and containing a Leu in position 1 (LAGIGILTV; 1L) behaves as a superagonist by in vitro inducing specific T cells with enhanced immunological functions. 1L-specific CTLs can be raised from peripheral blood of HLA-A2+ melanoma patients more efficiently than T cells specific for the cognate peptide. These T cells show a greater sensitivity to native MART1(27-35) when compared with CTL variable raised to parental peptide from the same patients. More importantly, anti-1L but not anti-native T cells display high levels of interferon gamma production at early time points, and readily secreted interleukin-2 in response to native epitope endogenously presented by melanoma cells. Additionally, anti-1L T cells are insensitive to the inhibitory effects of MART1(27-35) natural analogues that antagonize the lytic response of CTLs raised to the cognate peptide. Analysis of T-cell receptor variable beta usage suggests that the native and 1L peptides stimulate different components of the MART1(27-35)-reactive T cell population. These data provide rationale to the use of superagonist analogues of tumor antigens for inducing in vivo immunization potentially able to overcome tumor immune escape and mediate a more significant control of tumor growth.

Human melanoma-reactive CD4+ and CD8+ CTL clones resist Fas ligand-induced apoptosis and use Fas/Fas ligand-independent mechanisms for tumor killing.

Tumor cells have been shown recently to escape immune recognition by developing resistance to Fas-mediated apoptosis and acquiring expression of Fas ligand (FasL) molecule that they may use for eliminating activated Fas+ lymphocytes. In this study, we report that tumor-specific T lymphocytes isolated from tumor lesions by repeated in vitro TCR stimulation with relevant Ags (mostly represented by normal self proteins, such as MART-1/Melan A and gp100) can develop strategies for overcoming these escape mechanisms. Melanoma cells (and normal melanocytes) express heterogeneous levels of Fas molecule, but they result homogeneously resistant to Fas-induced apoptosis. However, CD4+ and CD8+ CTL clones kill melanoma cells through Fas/FasL-independent, granule-dependent lytic pathway. In these lymphocytes, Ag/MHC complex interaction with TCR does not lead to functional involvement of FasL, triggered, on the contrary, by T cell activation with nonspecific stimuli such as PMA/ionomycin. Additionally, melanoma cells express significant levels of FasL (detectable on the cell surface only after treatment with metalloprotease inhibitors), although to a lesser extent than professional immune cells such as Thl clones. Nevertheless, antimelanoma CTL clones resist apoptosis mediated by FasL either in soluble form or expressed by Thl lymphocytes or FasL+ melanoma cells. These results demonstrate that CD4+ and CD8+ antimelanoma T cell clones can be protected against Fas-dependent apoptosis, and thus be useful reagents of immunotherapeutic strategies aimed to potentiate tumor-specific T cell responses.

Immunogenicity of the ALLAVGATK (gp100[17-25]) peptide in HLA-A3.1 melanoma patients.

A T cell line recognizing autologous and allogeneic HLA-A3.1 melanomas was obtained from a disease-free melanoma patient (patient 15392). By transfection of a tumor cDNA library and in vitro sensitization experiments, the ALLAVGATK gp100/Mel17-derived peptide was found to be the epitope recognized by this melanoma-specific T cell line. The role of the ALLAVGATK peptide in the systemic immune response to melanoma of this patient was evaluated. When pulsed on the autologous peripheral blood mononuclear cells, the ALLAVGATK peptide generated tumor-specific HLA-A3-restricted T lymphocytes and a single restimulation in vitro was sufficient to raise gp100-specific T lymphocytes, indicating a high frequency of epitope-specific T cells. gp100-specific T cells were also induced from T lymphocytes purified from tumor-invaded lymph nodes (tumor-associated lymphocytes, TAL). TAL-derived effectors displayed lower peptide affinity and lower tumor recognition than effectors elicited from peripheral blood lymphocytes (PBL). To further evaluate its immunogenicity, ALLAVGATK was used to stimulate PBL derived from six additional HLA-A3.1 melanoma patients and seven healthy donors. After 7 weeks of peptide stimulation in vitro the generation of anti-gp100 and tumor-specific T cell lines was achieved in one out of the six patients analyzed. Taken together these data indicate that an in vivo priming leading to a systemic immunity against gp100 in HLA-A3 melanoma patients may occasionally occur and that the immunogenicity of ALLAVGATK peptide in melanoma patients is comparable to that of other HLA-A2-restricted epitopes derived from gp100/Mel 17 protein.

Multiple melanoma-associated epitopes recognized by HLA-A3-restricted CTLs and shared by melanomas but not melanocytes.

The molecular characterization of melanoma-associated Ags allowed the definition of several HLA class I-presented peptides recognized by T cells. However, no HLA-A3.1-restricted melanoma epitopes have been identified to date. To gain insight into the HLA-A3.1-restricted T cell epitope repertoire of human melanoma, we analyzed the immunologic reactivity of CTLs isolated from tumor-involved or tumor-free lymph nodes in two HLA-A3.1+ melanoma patients. Three CTL lines, clonal or highly oligoclonal in their TCR composition, and two CTL clones were selected for HLA class I-restricted lysis of the autologous tumor and then tested for the recognition of HLA-A3+ and HLA-A3- normal or neoplastic cells of the melanocyte lineage. One CTL recognized a unique HLA-A3.1-restricted Ag expressed only by the autologous tumors, while all the other CTLs defined three HLA-A3.1 epitopes shared by melanomas, but not by melanocytes. Moreover, the epitopes of two CTL lines with different specificity were reconstituted by nonoverlapping fractions of HLA-A3+ melanoma-derived peptides resolved by reverse phase-HPLC, indicating that distinct naturally processed peptides were specifically recognized on melanoma cells in association with HLA-A3.1 molecules. These novel lineage-unrelated HLA-A3.1-restricted melanoma epitopes do not derive from MAGE, BAGE, or GAGE gene families, as evaluated by the COS-7 transfection assay. Our data show that CTLs may recognize HLA-A3.1-class 1 complexes presenting melanoma (but not melanocyte)-associated epitopes that are either unique to a given patient's tumor or that are shared between multiple melanomas.