Immunogenicity of the ALLAVGATK (gp10017 – 25) peptide in HLA-A3.1 melanoma patients

Abstract

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 ALLAV-GATK peptide generated tumor-specific HLA-A3-restricted T lymphocytes and a single rest-imulation 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 lympho-cytes 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.

Human Tumor-Derived Heat Shock Protein 96 Mediates In Vitro Activation and In Vivo Expansion of Melanoma- and Colon Carcinoma-Specific T Cells

“…As Ag-specific T cells, we used: the HLA-A*0201-restricted MART-1-specific CD8 ϩ T cell clone A42, autologous to 501mel (20) and recognizing the immunodominant epitope MART-1 [27][28][29][30][31][32][33][34][35] , and the CD8 ϩ tumorinfiltrating lymphocyte (TIL) line (GDN-TIL), an oligoclonal T cell line recognizing MART-1 27-35 epitope in a HLA-A*0201-restricted fashion (20). PBMCs from HLA-A*0201 ϩ melanoma or colon carcinoma patients were purified from peripheral blood by Ficoll gradient, aliquoted, and stored in liquid nitrogen.…”

“…Monocytes, purified by adherence from PBMCs of HLA-A*0201 ϩ donors, were incubated overnight at 37°C with hsp96 from colon carcinoma or melanoma cells (at 10 g/ml), or with MART-1 [27][28][29][30][31][32][33][34][35] or CEA 571-579 peptides (10 M). T cells specific for MART-1 (A42 T cell clone) or CEAs (oligoclonal T cell lines, composed by Ͼ90% CEA-specific CD8 ϩ T cells, as assessed by HLA/tetramer staining) were then added.…”

“…1C). A significant inhibition of the lysis of monocytes loaded with 501mel-derived hsp96 was achieved when T2 ϩ MART-1 [27][28][29][30][31][32][33][34][35] , but not T2 ϩ CEA 571-579 , was added to the assay at 20-fold excess. The killing by the GDN-TIL of HLA-A*0201 ϩ monocytes pulsed with 501mel-derived hsp96 was reduced by the addition of irrelevant hsp96 (i.e., derived from MART-1-negative colon carcinoma cells), in a dose-dependent manner (Fig.…”

“…In contrast, no competition was observed when a 2-fold excess of BSA was used as control (data not shown). These latter data suggest that colon carcinoma-and melanomaderived hsp96 competed for the binding to monocytes through the same receptor, but only hsp96 from MART-1 ϩ melanoma cells contain the relevant T cell epitope (i.e., MART-1 [27][28][29][30][31][32][33][34][35] ). Chaperone activity of hsp96 was then analyzed in human colon carcinoma cells.…”

Identification of a Mutated Receptor-Like Protein Tyrosine Phosphatase κ as a Novel, Class II HLA-Restricted Melanoma Antigen

“…PBMCs of patient 15392 obtained at 70 mo after surgery, during the disease-free period of follow-up, were stimulated in vitro with PYYFAAELP PRNLPEP peptide as previously described (17). Briefly, PBMCs (2 ϫ 10 6 /well) were stimulated weekly with autologous peptide-pulsed monocytes.…”