RESUMEN
Melanoma-reactive HLA-A x 0201-restricted cytotoxic T lymphocyte (CTL) lines generated in vitro lyse autologous and HLA-matched allogeneic melanoma cells and recognize multiple shared peptide antigens from tyrosinase, MART-1, and Pme117/gp100. However, a subset of melanomas fail to be lysed by these T cells. In the present report, four different HLA-A x 0201+ melanoma cell lines not lysed by melanoma-reactive allogeneic CTL have been evaluated in detail. All four are deficient in expression of the melanocytic differentiation proteins (MDP) tyrosinase, Pme117/gp100, gp75/ trp-1, and MART-1/Melan-A. This concordant loss of multiple MDP explains their resistance to lysis by melanoma-reactive allogeneic CTL and confirms that a subset of melanomas may be resistant to tumor vaccines directed against multiple MDP-derived epitopes. All four melanoma lines expressed normal levels of HLAA x 0201, and all were susceptible to lysis by xenoreactive-peptide-dependent HLA-A x 0201-specific CTL clones, indicating that none had identifiable defects in antigen-processing pathways. Despite the lack of shared MDP-derived antigens, one of these MDP-negative melanomas, DM331, stimulated an effective autologous CTL response in vitro, which was restricted to autologous tumor reactivity. MHC-associated peptides isolated by immunoaffinity chromatography from HLA-A1 and HLA-A2 molecules of DM331 tumor cells included at least three peptide epitopes recognized by DM331 CTL and restricted by HLA-A1 or by HLA-A x 0201. Recognition of these CTL epitopes cannot be explained by defined, shared melanoma antigens; instead, unique or undefined antigens must be responsible for the autologous-cell-specific anti-melanoma response. These findings suggest that immunotherapy directed against shared melanoma antigens should be supplemented with immunotherapy directed against unique antigens or other undefined antigens, especially in patients whose tumors do not express MDP.
Asunto(s)
Antígenos de Diferenciación/inmunología , Antígenos de Neoplasias/inmunología , Melanoma/inmunología , Glicoproteínas de Membrana , Monofenol Monooxigenasa/deficiencia , Proteínas de Neoplasias/deficiencia , Oxidorreductasas , Proteínas/inmunología , Linfocitos T Citotóxicos/inmunología , Presentación de Antígeno , Antígenos de Diferenciación/genética , Antígenos de Neoplasias/genética , Diferenciación Celular , Cromatografía de Afinidad , Citotoxicidad Inmunológica , Epítopos/inmunología , Antígeno HLA-A1/inmunología , Humanos , Antígeno MART-1 , Masculino , Melanoma/genética , Melanoma/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/inmunología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Pigmentación , Proteínas/genética , Células Tumorales Cultivadas , Antígeno gp100 del MelanomaRESUMEN
The immunosuppressive activity of tumor cells may be mediated by tumor-derived cytokines such as transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10). A human breast cancer cell line derived from malignant ascites (BRC 173) secreted TGF-beta, but not IL-10, into tissue culture supernatant (TCS). BRC 173 TCS suppressed natural killer (NK) and lymphokine-activated killer (LAK) cell activity and also blocked the generation of HLA-A*0201-restricted tumor-reactive cytotoxic T-lymphocyte (CTL) lines in vitro. Human alpha 2-macroglobulin (alpha 2M), a plasma protein and cytokine carrier that binds isoforms in the TGF-beta family, was tested for its ability to neutralize the immunosuppressive activity in BRC 173 TCS. alpha 2M was converted to its activated conformation by reaction with methylamine (alpha 2M-MA) and then incubated with normal human peripheral blood lymphocytes (PBL) in the presence of IL-2 and BRC 173 TCS. Lysis of NK targets (K562) and LAK cell targets (DM6 melanoma) by the PBL was examined after 6 days of culture. PBL cultured in IL-2, without TCS or alpha 2M-MA, were lytic for both target cells. BRC 173 TCS substantially suppressed the lytic activity of the PBL in the presence of IL-2. When TGF-beta-neutralizing antibody was added to the PBL culture medium with IL-2 and TCS, a majority of the lytic activity was restored. alpha 2M-MA (280 nM) neutralized almost all of the immunosuppressive activity in the TCS, restoring 80-100% of the lytic activity without any apparent effect on the activity of IL-2. The ability of alpha 2M-MA to counteract immunosuppressive cytokines in breast cancer TCS was evident in serum-containing and serum-free medium. These studies demonstrate the activated alpha 2M can function as a selective cytokine neutralizer to thereby promote the activation of NK, LAK, and tumor-specific CTL responses.
Asunto(s)
Interleucina-2/inmunología , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/farmacología , alfa-Macroglobulinas/farmacología , Neoplasias de la Mama/inmunología , Células Cultivadas/efectos de los fármacos , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Medio de Cultivo Libre de Suero , Citocinas/farmacología , Femenino , Humanos , Tolerancia Inmunológica/efectos de los fármacos , Interleucina-10/análisis , Células Asesinas Activadas por Linfocinas/efectos de los fármacos , Células Asesinas Naturales/efectos de los fármacos , Pruebas de Neutralización , Linfocitos T Citotóxicos/efectos de los fármacos , Factor de Crecimiento Transformador beta/análisis , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/inmunología , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Human CD8+ CTL recognize peptides bound to class I MHC molecules on the surface of melanoma cells. Several peptides derived from melanocyte lineage-specific proteins have been identified as epitopes for HLA-A2 restricted melanoma-reactive CTL. Because less than half of melanoma patients express HLA-A2, it is important to identify CTL epitopes restricted by other common MHC molecules including HLA-A1 and -A3. We have generated HLA-A3-restricted human CTL that recognize one or more shared melanoma Ags. All of the melanomas recognized by one of these CTL lines express Pmel-17/gp100, and those that fail to express this Ag are not lysed. This CTL line also specifically recognizes the lymphoblastoid line C1R-A3 following infection with a recombinant vaccinia encoding the melanocyte lineage-specific protein Pmel-17/gp100. Thus, at least one Pmel-17/ gp100 peptide is an epitope for this CTL line. We have identified ALLAVGATK (Pmel-17/gp100 residues 17-25) as an epitope for this CTL line and have shown that it is naturally processed and presented by HLA-A3 on melanoma cells. A second HLA-A3-restricted melanoma-reactive CTL line recognizes at least one additional shared epitope. These findings suggest that cellular immune responses directed against multiple shared melanoma epitopes exist in the 20 to 25% of melanoma patients who express HLA-A3. In addition, immunotherapy directed against Pmel-17/gp100 and other shared melanoma Ags may be useful in a large subset of these patients.