1. Amino acid identity and/or position determines the proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE-3271-279
I Miconnet, C Servis, J C Cerottini, P Romero, F Lévy J Biol Chem. 2000 Sep 1;275(35):26892-7. doi: 10.1074/jbc.M000701200.
The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of major histocompatibility complex class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. We have previously shown that a cytolytic T lymphocyte-defined antigenic peptide derived from the MAGE-3 tumor-associated antigen (MAGE-3(271-279), FLWGPRALV in one-letter code) is not presented at the surface of melanoma cell lines expressing the MAGE-3 protein. By using purified proteasome and MAGE-3(271-279) peptides extended at the C terminus by 6 amino acids, we identified predominant cleavages after residues 278 and 280 but no detectable cleavage after residue Val(279), the C terminus of the antigenic peptide. In the present study, we have investigated the influence of Pro(275), Leu(278), and Glu(280) on the proteasomal digestion of MAGE-3(271-285) substituted at these positions. We show that positions 278 and 280 are major proteasomal cleavage sites because they tolerate most amino acid substitutions. In contrast, the peptide bond after Val(279) is a minor cleavage site, influenced by both distal and proximal amino acid residues.
2. Analysis of MAGE-3-specific cytolytic T lymphocytes in human leukocyte antigen-A2 melanoma patients
D Valmori, D Liénard, G Waanders, D Rimoldi, J C Cerottini, P Romero Cancer Res. 1997 Feb 15;57(4):735-41.
The MAGE-3 gene is a member of a multigene family that is selectively expressed by subsets of different human tumor types, including malignant melanoma, but not by normal tissues except for testis and placenta. A cytolytic T lymphocyte (CTL)-defined MAGE-3 antigen, corresponding to the MAGE-3 peptide 271-279 associated with the human leukocyte antigen (HLA)-A2 molecule, has been recently identified using T lymphocytes from a normal individual stimulated in vitro with peptide-pulsed autologous antigen-presenting cells. Because MAGE-3 is expressed in 76% of metastatic melanomas, the HLA-A2-restricted MAGE-3 antigen should be expressed by approximately 37% of Caucasians bearing a metastatic melanoma tumor, thus representing an attractive candidate for the elicitation of specific CTL immune responses in vivo. In this study, we determined the proportion of HLA-A2+ melanoma patients displaying detectable MAGE-3 peptide 271-279-specific CTL precursors in peripheral blood. Peptide-specific CTL populations were obtained from at least 4 of 11 HLA-A2+ patients. Peptide-specific CTL lines derived from these populations readily lysed HLA-A2-positive target cells that were pulsed with MAGE-3 peptide 271-279 at nanomolar concentrations yet were unable to recognize (as assessed by cytolysis and cytokine production) MAGE-3-expressing autologous or allogeneic HLA-A2-positive melanoma lines. Similarly, the CTL lines failed to recognize MAGE-3-negative HLA-A2-positive tumor lines after transfection with the MAGE-3 gene, although they were able to recognize COS-7 cells transfected with MAGE-3. In contrast, HLA-A1-positive melanoma lines transfected with MAGE-3 were efficiently recognized by CTL lines directed against the MAGE-3 peptide 168-176, a known HLA-A1-restricted CTL epitope. These results suggest that the expression level of the MAGE-3 peptide 271-279, unlike that of MAGE-3 peptide 168-176, in melanomas may be too low to allow efficient recognition by specific CTLs. Thus, it appears that despite the presence of CTL precursors against MAGE-3 peptide 271-279 in some HLA-A2+ melanoma patients, the usefulness of this peptide for specific immunotherapy of melanoma may be limited.
3. Induction of human leukocyte antigen (HLA)-A2-restricted and MAGE-3-gene-derived peptide-specific cytolytic T lymphocytes using cultured dendritic cells from an HLA-A2 esophageal cancer patient
S Kanaoka, S Yamasaki, T Okino, N Inoue, Y Shimada, M Kaneko, A Otaka, N Fujii, M Imamura J Surg Oncol. 1999 May;71(1):16-21. doi: 10.1002/(sici)1096-9098(199905)71:13.0.co;2-a.
Background and objectives: Using peripheral blood mononuclear cells (PBMCs) from a 10-year survivor with established human leukocyte antigen (HLA)-A2(+) and MAGE-3(+) esophageal cancer cell line (KYSE-170), we examined the induction of HLA-A2-restricted and MAGE-3-gene-derived peptide (FLWGPRALV, amino acids 271-279)-specific cytolytic T lymphocytes (CTLs). Methods: Autologous dendritic cells (DCs) cultured with granulocyte-macrophage colony stimulating factor and interleukin-4 were used as antigen presenting cells. PBMCs were stimulated by peptide-pulsed DCs in vitro. Results: PBMC cocultured with FLWGPRALV-pulsed DCs could induce the relevant peptide-specific CTLs, which had tumor necrosis factor production and specific cytotoxicity against relevant peptide-pulsed autologous DCs (34%, effector:target ratio = 40:1). Moreover, they showed specific cytotoxicity against the autologous esophageal cancer cell line KYSE-170 (17%, effector:target ratio = 40:1). Conclusions: These results suggest that FLWGPRALV-pulsed cultured DCs would be a potent candidate for peptide vaccine against HLA-A2(+) and MAGE-3(+) esophageal cancer.
