title: Recognition of breast cancer-associated peptides by tumor-reactive, HLA-class I restricted allogeneic cytotoxic T lymphocytes.
abstract: Strategies to identify tumor-associated antigens rely on the paradigm that tumor-associated peptides presented in the context of HLA-class I are recognized by the cellular immune system. Approaches to isolate tumor-specific cytotoxic T lymphocytes (CTL) from tumor-infiltrating lymphocytes are difficult because long-term growth of the CTL requires autologous tumor cells and lymphocytes (PBL) as feeder cells. In this study, a CTL line (BL.HBL-100 CTL) was generated from PBL from a normal healthy donor by stimulating with irradiated, HLA-class I partially matched breast cancer cell line HBL-100. Activated T lymphocytes generated expressed TCR alpha/beta+ with a predominant CD8+ population after 12 stimulations (98.54% CD8+ vs. 0.18% CD4+). These CTL lysed HLA-A1+, but not HLA-A1-, breast cancer cell lines. Moreover, HLA-A1+, non breast cancer cell lines were not recognized. The lytic activity of BL.HBL-100 CTL against HLA-A1+ breast cancer cell lines was blocked by monoclonal antibodies (MAbs) to HLA-class I and CD8, but not by anti-HLA-class II and CD4. Recognition of HLA-A1+ breast cancer cells by the CTL was dependent on peptides associated with HLA-class I since the lysis was inhibited by acid elution of HLA bound peptides. HBL-100 tumors were grown in severe combined immunodeficient (SCID) mice. Immunohistochemical staining of the HBL-100 tissue harvested from SCID mice demonstrated human breast cancer cells. HLA-class I molecules were affinity purified from the HBL-100 harvested from the SCID mice; class I bound peptides were eluted and separated by RP-HPLC. Pooled HPLC peptide fractions were tested for reconstituting antigenic epitopes recognized by the BL.HBL-100 CTL and found to reside within fraction 40. Our results show that a tumor reactive, HLA-class I restricted CTL was produced by stimulating normal PBL against an HLA-class I matched breast cancer cell line. We also provide evidence for a breast cancer-associated, HLA-class I bound peptide antigen(s) that reconstitutes the antigenic epitope(s) recognized by these CTL.
authors.affiliation: Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA.
authors.forename: T
authors.initials: T
authors.lastname: Nguyen
authors.forename: B
authors.initials: B
authors.lastname: Naziruddin
authors.forename: S
authors.initials: S
authors.lastname: Dintzis
authors.forename: G M
authors.initials: GM
authors.lastname: Doherty
authors.forename: T
authors.initials: T
authors.lastname: Mohanakumar
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D001402:B-Lymphocytes / Q000276:immunology; D001943:Breast Neoplasms / Q000276:immunology* / Q000473:pathology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D003602:Cytotoxicity, Immunologic; D005260:Female; D015395:Histocompatibility Antigens Class I / Q000032:analysis / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D007519:Isoantigens / Q000276:immunology; D051379:Mice; D016513:Mice, SCID; D010455:Peptides / Q000032:analysis / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D015395:Histocompatibility Antigens Class I; D007519:Isoantigens; D010455:Peptides