RESUMEN
Cell-mediated immunity directed against human papillomavirus 16 (HPV-16) antigens was studied in 16 patients affected with classic vulvar intra-epithelial neoplasia (VIN), also known as bowenoid papulosis (BP). Ten patients had blood lymphocyte proliferative T cell responses directed against E6/2 (14-34) and/or E6/4 (45-68) peptides, which were identified in the present study as immunodominant among HPV-16 E6 and E7 large peptides. Ex vivo enzyme-linked immunospot-interferon (IFN)-gamma assay was positive in three patients who had proliferative responses. Twelve months later, proliferative T cell responses remained detectable in only six women and the immunodominant antigens remained the E6/2 (14-34) and E6/4 (45-68) peptides. The latter large fragments of peptides contained many epitopes able to bind to at least seven human leucocyte antigen (HLA) class I molecules and were strong binders to seven HLA-DR class II molecules. In order to build a therapeutic anti-HPV-16 vaccine, E6/2 (14-34) and E6/4 (45-68) fragments thus appear to be good candidates to increase HPV-specific effector T lymphocyte responses and clear classic VIN (BP) disease lesions.
Asunto(s)
Epítopos de Linfocito T/inmunología , Papillomavirus Humano 16/inmunología , Proteínas Oncogénicas Virales/inmunología , Infecciones por Papillomavirus/inmunología , Proteínas Represoras/inmunología , Linfocitos T/inmunología , Neoplasias de la Vulva/inmunología , Neoplasias de la Vulva/virología , Adulto , Anciano , Secuencia de Aminoácidos , Proliferación Celular , Epítopos de Linfocito T/metabolismo , Femenino , Antígenos HLA-D/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Interferón gamma/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/inmunología , Persona de Mediana Edad , Proteínas E7 de Papillomavirus , Infecciones por Papillomavirus/virología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Unión Proteica/inmunología , Linfocitos T/citología , Linfocitos T/metabolismo , Factores de Tiempo , Adulto JovenRESUMEN
Little is known about the role of specific hepatitis C virus (HCV) CD8+ T cells in liver damage, especially for the progression of fibrosis, during the highly variable course of chronic C hepatitis. The aim of this study was to investigate the presence of HCV-specific CD8+ T cells in the liver of patients with chronic C hepatitis and to examine their clinical significance by relating the response to liver fibrosis and progression rate, serum viral load, serum aminotransferase levels, inflammatory activity and in situ characteristics of the intrahepatic infiltrate. Fifteen patients were prospectively included in the study. Intrahepatic lymphocytes were tested for interferon gamma (IFNg) production in response to HCV class I-restricted epitopic peptides using enzyme-linked immunospot analysis. Liver biopsy samples were evaluated for fibrosis, fibrosis progression rate, activity, and in situ number of CD8+ cytotoxic lymphocytes and apoptotic cells. An IFNg-specific CD8+ T-cell response was detected in the liver samples of 47% of patients which was significantly related to a lower stage of fibrosis (P = 0.02) and a lower progression rate of fibrosis (P = 0.01). It was neither related to the number of cytotoxic lymphocytes infiltrating the liver nor to hepatocyte apoptosis. In conclusion, our results indicate that the presence of HCV-specific IFNg-secreting T cells in the liver of patients with chronic C hepatitis is associated with low liver fibrosis and fibrosis progression rate, suggesting that these IFNg-secreting T cells might limit the progression of liver damage.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Hepatitis C Crónica/inmunología , Interferón gamma/inmunología , Cirrosis Hepática/inmunología , Adulto , Anciano , Linfocitos T CD8-positivos/metabolismo , Femenino , Hepatitis C Crónica/enzimología , Hepatitis C Crónica/patología , Humanos , Inmunofenotipificación , Interferón gamma/metabolismo , Hígado/enzimología , Hígado/inmunología , Hígado/metabolismo , Cirrosis Hepática/enzimología , Cirrosis Hepática/patología , Cirrosis Hepática/virología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Transaminasas/sangreRESUMEN
The anti-Hu syndrome is the most common paraneoplastic neurologic syndrome but the exact mechanism of immune mediated neuronal injury remains unknown. Anti-Hu antibodies do not appear to play a pivotal role in the pathogenesis of the disease. To assess cell-mediated immunity, we selected 51 peptides from the Hu-D sequence and tested their ability to bind to six common HLA class I molecules. Stable complexes with purified HLA molecules were obtained with 19/51 (37%) selected peptides. Subsequently, the ability of the 19 HLA-binding peptides to stimulate T cells from 10 patients and 10 control subjects was evaluated by detecting IFN-gamma secretion. An anti-peptide T-cell response was observed in 7/10 Hu-positive patients but also in 3/10 control subjects. Overall, a significant T-cell activation occurred in response to 74% (14 out of 19) of the selected peptides in the Hu-positive patients vs. 16% (3 out of 19) in the control group (p < 0.001). In addition, T cells of patients tested within 3 months of the onset of anti-Hu syndrome responded to 82% (14 out of 17) of assessed Hu-D peptides vs. 37% (7 out of 19) in patients tested 1 year or more after developing the syndrome (p < 0.01). Thus, the present study suggests a role of cellular immunity during the course of anti-Hu syndrome.
Asunto(s)
Carcinoma de Células Pequeñas/inmunología , Inmunidad Celular/inmunología , Proteínas del Tejido Nervioso/inmunología , Síndromes Paraneoplásicos del Sistema Nervioso/inmunología , Proteínas de Unión al ARN/inmunología , Linfocitos T/inmunología , Carcinoma de Células Pequeñas/complicaciones , Células Cultivadas , Proteínas ELAV , Antígenos HLA-A/inmunología , Antígenos HLA-A/metabolismo , Humanos , Interferón gamma/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Síndromes Paraneoplásicos del Sistema Nervioso/complicaciones , Fragmentos de Péptidos/inmunología , Proteínas de Unión al ARN/metabolismo , Estadísticas no Paramétricas , Síndrome , Linfocitos T/metabolismoRESUMEN
Cytotoxic T-lymphocyte (CTL) responses directed to different human immunodeficiency virus (HIV) epitopes vary in their protective efficacy. In particular, HIV-infected cells are much more sensitive to lysis by anti-Gag/p17(77-85)/HLA-A2 than to that by anti-polymerase/RT(476-484)/HLA-A2 CTL, because of a higher density of p17(77-85) complexes. This report describes multiple processing steps favoring the generation of p17(77-85) complexes: (i) the exact COOH-terminal cleavage of epitopes by cellular proteases occurred faster and more frequently for p17(77-85) than for RT(476-484), and (ii) the binding efficiency of the transporter associated with antigen processing was greater for p17(77-85) precursors than for the RT(476-484) epitope. Surprisingly, these peptides, which differed markedly in their antigenicity, displayed qualitatively and quantitatively similar immunogenicity, suggesting differences in the mechanisms governing these phenomena. Here, we discuss the mechanisms responsible for such differences.
Asunto(s)
Presentación de Antígeno/inmunología , Cisteína Endopeptidasas , Epítopos de Linfocito T/inmunología , Productos del Gen gag/inmunología , Antígenos VIH/inmunología , Infecciones por VIH/inmunología , Transcriptasa Inversa del VIH/inmunología , VIH-1/inmunología , Antígeno HLA-A2/inmunología , Epítopos Inmunodominantes/inmunología , Complejos Multienzimáticos , Linfocitos T Citotóxicos/inmunología , Proteínas Virales , Secuencia de Aminoácidos , Transporte Biológico , Endopeptidasas/metabolismo , Epítopos de Linfocito T/metabolismo , Productos del Gen gag/metabolismo , Antígenos VIH/metabolismo , Transcriptasa Inversa del VIH/metabolismo , Humanos , Epítopos Inmunodominantes/metabolismo , Complejo Mayor de Histocompatibilidad , Datos de Secuencia Molecular , Complejo de la Endopetidasa Proteasomal , Precursores de Proteínas/metabolismo , Proteínas/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia HumanaRESUMEN
In many types of cancer, p53 frequently accumulates in tumor cells and anti-p53 antibodies can be detected. However, only four CD8(+) T-cell epitopes from p53 have been identified in humans so far. To further analyze the development of a T-cell response against p53, peptides having binding motifs specific for HLA-A1, -A2, -A3, -A24, -B7, -B35, -B44, and -B51 molecules have been defined. The HLA-binding capacity of those peptides was tested, and the stability of formed complexes was defined. Thirteen peptides that bound to HLA-A24 and -B44 molecules are presented. The positive peptides were then used to detect the anti-p53 response of CD8(+) T lymphocytes from patients with bladder cancer. Six peptides, presented by HLA-A2, -B51, or -A24, were able to stimulate T cells from two patients (among 16) with tumor cells that strongly accumulated p53. On the contrary, p53 peptides systematically failed to stimulate T cells from healthy donors or patients with low or undetectable levels of p53 in their tumor cells. These results have led to the identification of four new potential T CD8(+) epitopes from p53: 194-203 associating with HLA-B51 and 204-212, 211-218, and 235-243 associating with HLA-A24.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Péptidos/inmunología , Proteína p53 Supresora de Tumor/inmunología , Neoplasias de la Vejiga Urinaria/inmunología , Células Cultivadas , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Antígenos HLA-A/inmunología , Antígenos HLA-A/metabolismo , Antígeno HLA-A2/inmunología , Antígeno HLA-A2/metabolismo , Antígeno HLA-A24 , Antígenos HLA-B/inmunología , Antígenos HLA-B/metabolismo , Antígeno HLA-B51 , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Activación de Linfocitos , Péptidos/metabolismo , Unión Proteica , Proteína p53 Supresora de Tumor/química , Neoplasias de la Vejiga Urinaria/terapiaRESUMEN
First and foremost among the many factors that influence epitope presentation are the degradation of Ag, which results in peptide liberation, and the presence of HLA class I molecules able to present the peptides to T lymphocytes. To define the regions of HIV-1 Nef that can provide multiple T cell epitopes, we analyzed the Nef sequence and determined that there are 73 peptides containing 81 HLA-binding motifs. We tested the binding of these peptides to six common HLA molecules (HLA-A2, -A3, -A24, -B7, -B8, and -B35), and we showed that most of them were efficient binders (54% of motifs), especially peptides associating with HLA-A3, -B7/35, and -B8 molecules. Nef peptides most frequently recognized by T cells of HIV-1-infected individuals were 90-97, 135-143, 71-81, 77-85, 90-100, 73-82, and 128-137. The frequency of T cell recognition was not directly related to the strength of peptide-HLA binding. The generation of Nef epitopes is crucial; therefore, we investigated the digestion by the 20S proteasome of a large peptide, Nef(66-100). This fragment was efficiently cleaved, and NH(2)-terminally extended precursors of epitope 71-81 were recognized by T cells of an HIV-1-infected individual. These results suggest that a high frequency of T cell recognition may depend on proteasome cleavage.
Asunto(s)
Linfocitos T CD8-positivos/metabolismo , Cisteína Endopeptidasas/metabolismo , Epítopos de Linfocito T/metabolismo , Productos del Gen nef/inmunología , Productos del Gen nef/metabolismo , VIH-1/inmunología , Antígenos HLA/metabolismo , Complejos Multienzimáticos/metabolismo , Fragmentos de Péptidos/inmunología , Secuencias de Aminoácidos/inmunología , Secuencia de Aminoácidos , Presentación de Antígeno , Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Línea Celular Transformada , Seropositividad para VIH/enzimología , Seropositividad para VIH/inmunología , VIH-1/enzimología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Hidrólisis , Epítopos Inmunodominantes/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal , Unión Proteica/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaAsunto(s)
Citotoxicidad Inmunológica/fisiología , Antígenos HLA/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/inmunología , Proteínas Recombinantes de Fusión/farmacología , Células Cultivadas , Células Clonales , Clonación Molecular , Citotoxicidad Inmunológica/efectos de los fármacos , Escherichia coli , Antígenos HLA/farmacología , Antígenos HLA-G , Antígenos de Histocompatibilidad Clase I/farmacología , Humanos , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
Backbone modifications have been introduced into the melanoma derived peptide MART-1(27-35) to increase its binding to class I major histocompatibility complex HLA-A2 molecule, and ultimately to enhance its immunogenicity. Each analogue was obtained by replacing one peptide bond at a time in the natural epitope by the aminomethylene (CH2-NH) surrogate. All analogues displayed an increased resistance to proteolysis. Interestingly, the comparative results showed that five analogues bound more efficiently to HLA-A2 than the parent peptide. On the other hand, two pseudopeptide/HLA-A2 complexes were recognized by one melanoma-specific T cell clone. Close examination of the impact of such modifications at the molecular level provides useful supports for the rational design of stable compounds with applications in anti-tumour specific immunotherapy and in vaccine development.
Asunto(s)
Epítopos/química , Proteínas de Neoplasias/química , Proteínas de Neoplasias/síntesis química , Secuencia de Aminoácidos , Aminoácidos/química , Línea Celular , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Epítopos/inmunología , Fluorenos/química , Antígeno HLA-A2/metabolismo , Humanos , Melanoma/inmunología , Melanoma/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/inmunología , Péptidos/química , Unión Proteica , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
CD8(+) T lymphocytes, which are major immune effectors, require primary stimulation by dendritic cells (DC) presenting MHC class I molecule-bound epitopes. Sensitization to exogenous protein epitopes that are not synthesized in DC, such as cross-priming, is obtained through pathways leading to their association with MHC class I. To follow class I-restricted pathways in human DC, we have tracked a lipopeptide derived from the conserved HLA-A*0201-restricted HIV-1 reverse transcriptase 476-484 epitope, by N-terminal addition of an Nepsilon-palmytoyl-lysine. Indeed, lipopeptides elicit cytotoxic responses from CD8(+) T lymphocytes, whereas peptides without a lipid moiety do not. The lipopeptide and its parent peptide were labeled unequivocally by rhodamine to study their entry into immature monocyte-derived human DC by confocal microscopy. The lipid moiety induced endocytosis of the lipopeptide, assessed by rapid entry into vesicles, colocalization with Dextran-FITC and dependence on energy. Internalization occurred even when actin filaments were depolymerized by Cytochalasin B. This internalization induced functional stimulation of specific CD8(+) T lymphocytes in IFN-gamma ELISPOT assays. The peptide alone was not visualized inside the DC and was presented through direct surface association to HLA-A*0201. Therefore, lipopeptides are a unique opportunity to define precisely the pathways that lead exogenous proteins to associate with MHC class I molecules in DC. The results will also be useful to design lipopeptide vaccines.
Asunto(s)
Presentación de Antígeno , Linfocitos T CD8-positivos/inmunología , Comunicación Celular/inmunología , Células Dendríticas/inmunología , Antígenos VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Linfocitos T CD8-positivos/citología , Línea Celular , Células Dendríticas/citología , Endocitosis/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Activación de LinfocitosRESUMEN
The design of heteroclytic antigens with high MHC binding capacity is of particular interest to overcome the weak immunogenicity of peptide epitopes derived from tissue antigens expressed by tumors. In the present study, double-substituted peptide analogues of the tumor-associated antigen MART-1(27-35) incorporating a substitution at a primary anchor residue and a beta-amino acid residue at different positions in the sequence were synthesized and evaluated for binding to the human histocompatibility class I molecule HLA-A2 and for recognition by tumor-infiltrating lymphocytes. Interestingly, by combining a Leu for Ala substitution at P2 (which alone is deleterious for antigenic activity) with a beta-amino acid substitution at a putative TCR contact residue, recognition by tumor-infiltrating lymphocytes was partially restored. The analogue [Leu(28),beta-HIle(30)]MART-1(27-35) displays both a higher affinity to HLA-A2 and a more prolonged complex stability compared to [Leu(28)]MART-1(27-35). Overall, these results suggest that double-substitution strategies and beta-amino acid replacements at putative TCR contact residues might prove useful for the design of epitope mimics with high MHC binding capacity.
Asunto(s)
Epítopos/química , Antígeno HLA-A2/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Melanoma/química , Proteínas de Neoplasias/química , Fragmentos de Péptidos/síntesis química , Cromatografía Líquida de Alta Presión , Electroforesis Capilar , Humanos , Isoantígenos/química , Isoantígenos/metabolismo , Espectroscopía de Resonancia Magnética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales CultivadasRESUMEN
Human papilloma virus type 16 (HPV-16) is the HPV most frequently associated with cervical carcinoma in humans. For the prevention or treatment of cervical carcinoma, the E6 and E7 oncoproteins appear to be good targets for vaccine-induced cytotoxic T lymphocytes (CTL). Lipopeptide vaccination is an efficient way of stimulating cellular responses. However, to synthesize effective lipopeptides, it is necessary to define which epitopes are immunogenic. In this study we first determined that peptide 80 - 88 of the E6 protein was recognized by CTL from a healthy donor in association with the HLA-B18 molecule. We then defined the HLA-B18 anchoring peptide motif by testing the binding of various short peptides with the HLA-B18 molecule and showed that it was related to the HLA-A1-specific peptide motif. Furthermore, in analyzing the potential E7 epitopes susceptible to associating with HLA-B18, we demonstrated that peptide E7 44 - 52 gave the strongest binding. It could also be recognized by CTL from peripheral blood mononuclear cells (PBMC) of the same healthy donor. Finally, with PBMC from a patient with a cervical intraepithelial neoplasia grade 3, we found CTL which recognized the E6 80 - 88 epitope. We have hence identified two peptides encoded by the E6 and E7 proteins which are presented by the HLA-B18 molecule and could be included in a vaccine against HPV-16.
Asunto(s)
Antígenos HLA-B/metabolismo , Proteínas Oncogénicas Virales/inmunología , Proteínas Represoras , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Sitios de Unión , Antígeno HLA-B18 , Humanos , Datos de Secuencia Molecular , Proteínas E7 de Papillomavirus , Fragmentos de Péptidos/inmunologíaRESUMEN
There is considerable evidence that peptides corresponding to MHC class I-restricted epitopes can be used as immunogens or immunomodulators. Pseudopeptides containing isosteric replacements of the amide bond provide more stable analogues, which may even have enhanced biologic activity. But there have been very few studies on the use of pseudopeptides to initiate or modulate the cellular immune response. This study describes the immunogenicity of a partially modified retro-inverso pseudopeptide of an influenza virus epitope and shows that this pseudopeptide modulates the cytokine profile expressed by CD8+ CTL generated from primed precursors. Moreover, the pseudopeptide is much more efficient at low concentration than the wild-type epitope to stimulate IFN-gamma secretion by CD8+ T effector cells. These results are analyzed with reference to changes in the conformation of the MHC molecule/peptide complex deduced from molecular modeling. The findings support the idea that partially modified retro-inverso analogues can be used as altered peptide ligands to enhance the stimulation of natural epitope-specific CTL and to modify their functional properties. Hence, pseudopeptide ligands might be promising tools for use in immunotherapy.
Asunto(s)
Citocinas/biosíntesis , Epítopos de Linfocito T/inmunología , Virus de la Influenza A/inmunología , Fragmentos de Péptidos/farmacología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Proteínas de la Matriz Viral/farmacología , Línea Celular , Citocinas/genética , Pruebas Inmunológicas de Citotoxicidad , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito T/farmacología , Antígeno HLA-A2/química , Antígeno HLA-A2/metabolismo , Humanos , Interferón gamma/biosíntesis , Interferón gamma/genética , Activación de Linfocitos/efectos de los fármacos , Modelos Moleculares , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , ARN Mensajero/análisis , Linfocitos T Citotóxicos/química , Proteínas de la Matriz Viral/síntesis química , Proteínas de la Matriz Viral/metabolismoRESUMEN
Recent studies have shown that transfusions of HLA-compatible donor lymphocytes may induce complete remission in marrow-grafted patients with relapses of acute myeloblastic leukemia (AML). We investigated the in vitro generation of antileukemia T-cell clones obtained from the peripheral blood mononuclear cells of a partially HLA-compatible donor (HLA-A2 and B7 molecules in common with the leukemic blasts) after stimulation with a pool of naturally processed peptides extracted from leukemic blast cells collected at diagnosis from a patient with hyperleucocytosis AML. We recovered a significant quantity of peptides that bound to the HLA-A2 or HLA-B7 molecules that were able to induce cytolytic T-lymphocyte (CTL) lines and clones specific for the eluted AML peptides and restricted to the HLA-A2 or B7 molecules. Such CTL line did not recognize the patient's nonleukemic cells, and one clone was able to interact with the leukemic blasts from which the naturally processed peptides had been eluted. Such T-cell clones might provide a rationale for the development of adoptive immunotherapy and could be used to improve the efficiency of HLA-compatible T-lymphocyte transfusions and the graft-versus-leukemia response in patients with AML.
Asunto(s)
Presentación de Antígeno , Antígenos de Neoplasias/inmunología , Leucemia Mieloide Aguda/inmunología , Linfocitos T Citotóxicos/inmunología , Antígeno HLA-A2/inmunología , Antígeno HLA-B7/inmunología , Humanos , Leucemia Mieloide Aguda/patología , Activación de Linfocitos , Péptidos/inmunología , Células Tumorales CultivadasRESUMEN
Tumor cells can have different morphologic or metabolic phenotypes and display genetic instability. Thus they could also vary in their ability to present epitopes to the immune system. We have analyzed the presentation of H-2 Kb- and Db-restricted cytotoxic T lymphocyte (CTL) epitopes of a tumor-associated antigen by three cell lines derived from hepatocarcinomas developed in vivo by mice transgenic for SV40 T targeted to the liver. SV40 T is the obvious tumor-specific antigen and epitopes derived from this antigen were therefore studied. The study included four already known epitopes that can be presented by SV40-transformed kidney cells and two new CTL epitopes that were identified in the present work. CTL lines specific for each epitope were obtained from C57BL/6 mice and were used to map the presentation of SV40 T peptides by the hepatocarcinoma cells. These tumor cells were derived from the same tissue, induced by the same agent and all naturally presented peptide p232-240 from p53. Despite these common features, they all had different patterns of spontaneous presentation of SV40 T CTL epitopes. The mechanisms underlying this disparity are discussed, together with the possible consequences for establishing immunotherapeutic strategies.
Asunto(s)
Antígenos Transformadores de Poliomavirus/inmunología , Epítopos de Linfocito T/inmunología , Neoplasias Hepáticas Experimentales/inmunología , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Transformadores de Poliomavirus/metabolismo , Mapeo Epitopo , Antígenos H-2/inmunología , Antígenos H-2/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Péptidos/inmunología , Péptidos/metabolismo , Células Tumorales CultivadasRESUMEN
The p53 protein is accumulated in tumor cells of many human cancers and can elicit in vivo humoral and proliferative responses. Rare reports about p53-mediated tumor recognition by CTLs have remained questioned. We therefore studied a panel of breast tumor and melanoma cell lines that we assayed for the presence of accumulated p53 and surface HLA-A2 and for the presentation of p53 epitopes. From PBMC of a healthy donor, we have generated a CTL line, D5/L9V, directed against HLA-A2-restricted peptide 264-272 from wild-type p53. It efficiently lysed breast adenocarcinomas MCF-7, MCF7/RA1, and MDA-MB-231, and melanoma M8, which all accumulate the p53 protein. Using competition assays, we made sure that tumor lysis by D5/L9V was due to recognition of endogenously produced p53 peptide 264-272 associated with the HLA-A2.1 molecule on the surface of these tumor cells. Cells with undetectable levels of wild-type p53, such as lymphoblastoid cells and melanoma M74, were not recognized by D5/L9V. Neither were breast tumor cell line MCF7/ADR nor melanoma line M44 because of HLA loss. This study therefore shows that it is possible to obtain in vitro CTL lines that specifically recognize a p53 epitope spontaneously presented by a variety of HLA-A2+ transformed cell lines provided they display abnormal patterns of p53 expression. This work points out that breast tumors and melanomas share a p53 epitope, and raises hopes for future immunotherapeutic approaches.
Asunto(s)
Antígenos de Neoplasias/inmunología , Neoplasias de la Mama/inmunología , Melanoma/inmunología , Linfocitos T Citotóxicos/inmunología , Proteína p53 Supresora de Tumor/inmunología , Células Presentadoras de Antígenos/inmunología , Células Cultivadas , Citotoxicidad Inmunológica , Relación Dosis-Respuesta Inmunológica , Humanos , Péptidos/química , Péptidos/inmunología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
This study investigates the generation of primary melanoma cell-specific cytotoxic T lymphocytes (CTLs) in vitro. Induction of peptide-specific CTLs from unfractionated naive peripheral blood mononuclear cells from HLA-A2 healthy donors was assessed using 2 recently described 9-mer epitopes from the melanoma tumor antigen Melan-A/MART-1. The need for help from CD4+ T lymphocytes for the long-lasting induction of CTLs and the capacity of the peptide-induced CTL lines to recognize many melanoma cells were evaluated. CTL lines were obtained reproducibly when CD4+ T-lymphocyte help was provided during the primary stimulation either in an autologous way, in the case of tetanus toxoid antigen (TT) responder donors, or with allogeneic TT-activated T-helper cells, separated by an insert well, in the case of tetanus toxoid non-responder donors. We also investigated helper T-cell-derived factors that are produced by TT-activated lymphocytes. Our results strongly suggest that a complex network of cytokines like interleukin-2 (IL-2), interferon-gamma, IL-6 and IL-1 exerts stimulatory effects for the initiation process of CTLs. In contrast, cytokine-like IL-4 might inhibit generation of cytolytic activity if provided by TT-activated T cells at early stages of induction. Our approach can be used to generate CTLs of a desired specificity for clinical use in adoptive immunotherapy protocols.
Asunto(s)
Antígenos de Neoplasias/inmunología , Citocinas/biosíntesis , Leucocitos Mononucleares/inmunología , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Células Cultivadas , Citotoxicidad Inmunológica , Antígeno HLA-A2/biosíntesis , Humanos , Interferón gamma/biosíntesis , Leucocitos Mononucleares/citología , Activación de Linfocitos , Antígeno MART-1 , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Oligopéptidos/química , Oligopéptidos/farmacología , Reacción en Cadena de la Polimerasa , Complejo Receptor-CD3 del Antígeno de Linfocito T/análisis , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Linfocitos T Citotóxicos/citología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Chronic myeloid leukemia (CML) is characterized cytogenetically by a t(9;22) translocation which generates a hybrid bcr-abl gene, encoding a p210(bcr-abl) fusion protein. The induction in vitro of leukemia-specific T cells reactive with p210(bcr-abl) is a strategy developed for an immunological therapeutic approach in CML. Peptides from the junction region of this chimeric protein have been considered as potential targets for a cytotoxic response against leukemic cells. However, only a few peptides encompassing the two p210(bcr-abl) breakpoints have been shown to bind to the most common HLA class I molecules, which limits the number of patients who could benefit from this approach. We assume that the presence of chimeric BCR-ABL protein in leukemic cells may affect processing and delivery of peptides, possibly giving rise to new epitopes at the cell surface. We selected 162 peptides from the whole sequence of this protein, including 14 peptides of the b2a2 and b3a2 junctions, which had an anchor motif for a common HLA class I molecule. We tested their ability to bind to eight HLA class I molecules (HLA-A1, -A2, -A3, -A11, -B7, -B8, -B27, -B44). We identified 48 peptides from outside the junction region, with intermediate or strong binding capacities to these HLA class I molecules contrasting with only six junction peptides with a moderate binding capacity to HLA-A3/A11, -B8, or -B44 molecules. Moreover, cytotoxic T lymphocyte lines specific for various peptides outside the junction were generated from peripheral blood mononuclear cells of HLA-A2 or -B7 healthy donors and from one CML patient. These results contribute to evaluation of immunity to the BCR-ABL chimeric protein. Further studies are required to investigate whether such epitopes are correctly processed and presented by leukemic cells.
Asunto(s)
Proteínas de Fusión bcr-abl/inmunología , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Sitios de Unión/genética , Línea Celular , Línea Celular Transformada , Proteínas de Fusión bcr-abl/genética , Antígeno HLA-B7/metabolismo , Herpesvirus Humano 4 , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Unión Proteica , Células Tumorales CultivadasRESUMEN
RAS oncogenic proteins are frequently found mutated in human cancers, where they are known to be implicated in the tumoral process. Mutations occur preferentially at positions 12, 13 or 61. Identification of potential T cell epitopes is the first step to determine it RAS mutated proteins can generate tumor specific antigens which could be further used as targets for cancer immunotherapy protocols. We have investigated the capacity of synthetic wild-type and mutant RAS derived peptides encompassing positions 12 and 13 to bind to three frequent HLA-A alleles: HLA-A*0201, HLA-A*0301 and HLA-A*1101. Binding was evaluated by two methods using TAP-defective cell lines: a cytometric assay based on HLA molecules stabilization at the cell surface, and an assembly assay detecting interactions between solubilized HLA molecules and peptides. Positive HLA binding was observed for two sets of synthetic peptides, one specific for HLA-A*0201 allele (RAS 5-14), and the other one specific for HLA-A*0301 and HLA-A*1101 alleles (RAS 8-16). Interestingly, the different substitutions at positions 12 and 13 were not equivalent for HLA binding. These observations will be useful for the in vitro generation of restricted CD8+ T lymphocytes specific for mutated RAS proteins and recognizing tumoral cells expressing such RAS mutations.
Asunto(s)
Alelos , Antígenos HLA-A/genética , Fragmentos de Péptidos/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Línea Celular , Humanos , Mutación , Relación Estructura-Actividad , Linfocitos T Citotóxicos/inmunologíaRESUMEN
The cytotoxic T lymphocyte (CTL) response directed against the immunodominant peptide M.58-66 from the matrix of influenza A virus presented by the HLA-A2.1 molecule is characterized by a restricted T cell repertoire. This limitation may be due to selective pressure induced by endogenous homologous ligands responsible for both positive and negative selection in the thymus and partial activation in peripheral T cell responses. We have used three self-protein-derived peptides homologous to M.58-66 to study their HLA-A2.1 binding capacity and recognition by M.58-66-specific HLA-A2.1-restricted CTLs. We show that they antagonize M.58-66-reactive T cells, presumably by the formation of altered HLA-A2.1 complex conformations. The results are discussed with reference to the role of endogenous ligands homologous to antigenic peptides in T cell repertoire selection, tolerance, and overall regulation of the immune response.
Asunto(s)
Autoantígenos/fisiología , Antígeno HLA-A2/metabolismo , Virus de la Influenza A/inmunología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Péptidos/inmunología , Péptidos/farmacología , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/inmunología , Proteínas de la Matriz Viral/metabolismo , Citotoxicidad Inmunológica/efectos de los fármacos , Antígeno HLA-A2/genética , Humanos , Ligandos , Péptidos/metabolismo , Conformación Proteica , Homología de Secuencia de Aminoácido , Linfocitos T Citotóxicos/efectos de los fármacosRESUMEN
The possibility to identify epitopes presented by tumor cells to cytotoxic T lymphocytes (CTL) has given rise to new fields in tumor immunology. The tumor suppressor gene product p53 is a good candidate antigen because it is involved in the tumorigenesis of many cancers. It accumulates in an inactivated form due to mutation or formation of heterodimers with an oncogene product. Epitopes from the mutant or wild-type p53 proteins are thought to be presented by tumor cells and to induce a tumor-specific CTL response. To identify such epitopes, mouse wild-type p53 peptides encompassing the H-2 Db anchoring motif were tested for their association with the Db molecule. Positive peptides were assayed for their ability to induce CTL in C57BL/6 mice. CTL specific for one wild-type p53 peptide, p232-240, were isolated and found to lyse hepatocarcinoma cell lines established from mice transgenic for simian virus 40 large T antigen which overexpress p53. These results show that the p232-240 epitope from wild-type p53 is naturally processed and presented in H-2b tumor cells.