RESUMO
The 94-kD large tumor (T) antigen specified by simian virus 40 (SV40) is sufficient to induce cell transformation. T antigen contains four H-2Db-restricted cytotoxic T lymphocyte (CTL) recognition epitopes that are targets for CTL clones Y-1, Y-2, Y-3, and Y-5. These epitopes have been mapped to T antigen amino acids 207-215 (site I), 223-231 (sites II and III), and 489-497 (site V), respectively. Antigenic site loss variant cells that had lost one or more CTL recognition epitopes were previously selected by coculturing SV40-transformed H-2Db cells with the site-specific Db-restricted CTL clones. The genetic bases for T antigen CTL recognition epitope loss from the variant cells were identified by DNA amplification and direct sequencing of epitope-coding regions from variant cell DNAs. Cells selected for resistance to CTL clone Y-1 (K-1; K-1,4,5; K-3,1) carry deleted SV40 genomes lacking site I, II, and III coding sequences. Point mutations present within the site II/III coding region of Y-2-/Y-3-resistant cell lines specify the substitution of asparagine for lysine as T antigen amino acid 228 (K-2) or phenylalanine for tyrosine at position 230 (K-3). Point mutations identified within independently selected Y-5 resistant populations (K-5 and K-1,4,5) direct the substitution of isoleucine for asparagine at position 496 (K-5) or the substitution of phenylalanine for isoleucine at position 491 (K-1,4,5) of T antigen. Each substitution causes loss of the relevant CTL recognition epitope, apparently by compromising CTL T cell receptor recognition. These experiments identify specific amino acid changes within a transforming protein that facilitate transformed cell escape from site-specific CTL clones while allowing maintenance of cellular transformation. This experimental model system provides unique opportunities for studying mechanisms of transformed cell escape from active immunosurveillance in vivo, and for analysis of differential host immune responses to wild-type and mutant cell-transforming proteins.
Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , Epitopos/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Células Clonais , DNA , Epitopos/genética , Antígenos H-2/imunologia , Antígeno de Histocompatibilidade H-2D , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Transformação GenéticaRESUMO
Isozyme patterns of lactate and malate dehydrogenases were studied in tumors induced by SV40 and 7,12-dimethylbenz(a)anthracene and in established cultures of cells from these tumors. The expression of B polypeptide subunits of lactate dehydrogenase is suppressed similarly by both agents. This may be due to inactivation of the gene at the locus determining the B polypeptide subunit. Malate dehydrogenase isozyme patterns are not changed significantly by the virus or the carcinogen.
Assuntos
Benzo(a)Antracenos , L-Lactato Desidrogenase/análise , Malato Desidrogenase/análise , Neoplasias Experimentais/enzimologia , Vírus 40 dos Símios , Animais , Transformação Celular Neoplásica , Cricetinae , Técnicas de Cultura , Eletroforese em Gel de Amido , Isoenzimas , Músculos/enzimologia , Neoplasias Experimentais/induzido quimicamenteRESUMO
Recent reports have documented the presence of SV40 large T antigen (T ag) sequences in a number of human tumors and raised the question of whether cellular immunity to T ag is elicited in such individuals. We used HLA-A2.1 transgenic C57BL/6 mice to identify an epitope from T ag recognized by CD8+ CTLs when presented by this human MHC class I molecule. Immunization of HLA-A2.1 transgenic mice with syngeneic T ag-transformed cells resulted in the induction of HLA-A2.1-restricted, T ag-specific CTLs. The target epitope, residues 281-289 (KCDDVLLLL) of T ag, was identified using both cell lines expressing T ag variants and synthetic T ag peptides. Peptide 281-289 bound stably to HLA-A2.1 molecules, effectively sensitized target cells for CTL lysis, and was efficiently processed from endogenous T ag in cells of both mouse and human origin. CTLs were not cross-reactive on the human BK or JC virus T ags. Thus, SV40 T ag 281-289 represents a potential specific CTL recognition epitope for humans.
Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , Antígenos Virais de Tumores/imunologia , Epitopos de Linfócito T/imunologia , Antígeno HLA-A2/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Mapeamento de Epitopos , Humanos , Ativação Linfocitária/imunologia , Camundongos , Fragmentos de Peptídeos/imunologia , Vírus 40 dos Símios/imunologiaRESUMO
The role of CTL tolerance in tumor immunity to SV40 large T antigen (T ag)-induced tumors was studied using T ag transgenic mice of the line 501 (H2b). 501 mice express SV40 T ag under the influence of the alpha-amylase promoter, which leads to the development of osteogenic osteosarcomas late in life and eventual death between 12 and 17 months of age. We determined the ability of 501 mice to respond to the four H2b-restricted T ag CTL epitopes, which include epitope I (T ag 206-215), epitope II/III (T ag 223-231), the immunorecessive epitope V (T ag 489-497), restricted by H2-Db, and epitope IV (T ag 404-411), restricted by H2-Kb. We demonstrate that 501 mice are partially tolerant to the H2b-restricted T ag epitopes. Immunization of 4-month-old 501 mice with T ag-transformed syngeneic cell lines or a recombinant vaccinia virus expressing full-length T ag elicited CTL responses against the H2-Kb-restricted T ag epitope IV only. In contrast, immunization of 4-month-old 501 mice with recombinant vaccinia viruses expressing individual T ag epitopes as minigenes elicited CTLs against epitopes I, IV, and V, but not against epitope II/III. Complete tolerance to epitopes I, IV, and V developed in 501 mice, but the age when tolerance was detected varied for each epitope. Tolerance to epitope I occurred by 6 months of age and was accelerated in the absence of CD4+ T cells. Tolerance to the immunorecessive epitope V was observed in 12-month-old 501 mice but was independent of the presence of osteosarcomas. In contrast, CTLs specific for epitope IV were detected in mice from 3 to 14 months of age but not in mice that had developed osteosarcomas. Analysis of epitope IV-specific CD8+ cells derived from 3-month-old 501 mice with H2-Kb/epitope IV tetramers revealed decreased numbers of epitope IV-specific CD8+ cells in 501 mice relative to C57BL/6 mice, with a further decrease in older 501 mice. Tumor progression resulted in loss of H2-Kb/epitope IV tetramer staining CD8+ cells. Thus, progression to tolerance to individual T ag CTL epitopes in 501 mice is epitope dependent.
Assuntos
Antígenos Virais de Tumores/imunologia , Neoplasias Ósseas/imunologia , Epitopos/imunologia , Osteossarcoma/imunologia , Vírus 40 dos Símios/imunologia , Linfócitos T Citotóxicos/imunologia , Fatores Etários , Animais , Antígenos Virais de Tumores/genética , Neoplasias Ósseas/diagnóstico por imagem , Linhagem Celular , Citocinas/metabolismo , Progressão da Doença , Citometria de Fluxo , Imuno-Histoquímica , Complexo Principal de Histocompatibilidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Osteossarcoma/diagnóstico por imagem , Peptídeos/metabolismo , Regiões Promotoras Genéticas , Radiografia , Vírus 40 dos Símios/genética , Vaccinia virus/metabolismo , alfa-Amilases/genéticaAssuntos
Antígenos de Neoplasias , Transformação Celular Neoplásica , Antígenos de Histocompatibilidade , Imunidade Celular , Neoplasias Experimentais/imunologia , Vírus 40 dos Símios , Animais , Linhagem Celular , Cricetinae , Transfusão de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Quimera por Radiação , Baço/imunologia , Fatores de Tempo , Imunologia de Transplantes , Transplante HomólogoAssuntos
Transformação Celular Neoplásica , Macrófagos/imunologia , Neoplasias Experimentais/imunologia , Vírus 40 dos Símios , Animais , Antígenos de Neoplasias , Células da Medula Óssea , Transplante de Medula Óssea , Feminino , Antígenos de Histocompatibilidade , Transfusão de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Quimera por Radiação , Dióxido de Silício/farmacologia , Baço/imunologia , Imunologia de Transplantes , Transplante HomólogoAssuntos
Adenoviridae/imunologia , Adenoviridae/isolamento & purificação , Antígenos , Técnicas de Cultura , Soros Imunes , Replicação Viral , Adenoviridae/crescimento & desenvolvimento , Adenoviridae/patogenicidade , Animais , Linhagem Celular , Cricetinae , Efeito Citopatogênico Viral , Vírus Defeituosos , Imunofluorescência , Haplorrinos , Vírus Auxiliares , Humanos , Rim , Transplante de Neoplasias , Neoplasias Experimentais/etiologia , Neoplasias Experimentais/imunologia , Vírus 40 dos Símios , Imunologia de Transplantes , Proteínas Virais , Cultura de VírusAssuntos
Infecções Tumorais por Vírus/imunologia , Animais , Animais Recém-Nascidos , Formação de Anticorpos , Antígenos de Neoplasias/análise , Membrana Celular/imunologia , Inibição de Migração Celular , Isótopos do Cromo , Imunofluorescência , Imunidade Celular , Interferons/análise , Linfócitos/imunologia , Testes de Neutralização , Coelhos , Fatores de Tempo , Infecções Tumorais por Vírus/microbiologia , Infecções Tumorais por Vírus/patologia , Cultura de VírusAssuntos
Antibacterianos/farmacologia , Vírus 40 dos Símios/crescimento & desenvolvimento , Replicação Viral/efeitos dos fármacos , Animais , Bovinos , Técnicas de Cultura , Inibidores Enzimáticos , Imunofluorescência , Herpesviridae/crescimento & desenvolvimento , Soros Imunes , Poliovirus/crescimento & desenvolvimento , Fatores de Tempo , Vaccinia virus/crescimento & desenvolvimentoAssuntos
Transformação Celular Neoplásica , Vírus 40 dos Símios/patogenicidade , Animais , Antígenos de Neoplasias , Antígenos Virais/análise , Vírus do Sarcoma Aviário/patogenicidade , Linhagem Celular/microbiologia , Aberrações Cromossômicas , Transtornos Cromossômicos , Células Clonais/transplante , DNA Viral/metabolismo , Vírus Defeituosos/patogenicidade , Código Genético , Genética Microbiana , Microscopia de Fluorescência , Polyomavirus/patogenicidade , Imunologia de TransplantesAssuntos
Antígenos Virais/análise , Imunofluorescência , Vírus Oncogênicos/imunologia , Animais , Antígenos de Neoplasias , Membrana Celular/imunologia , Transformação Celular Neoplásica , Testes de Fixação de Complemento , Cricetinae , Reações Cruzadas , Testes Imunológicos de Citotoxicidade , DNA de Neoplasias , Epitopos , Hemadsorção , Isoantígenos , Papillomaviridae/imunologia , Polyomaviridae , Vírus 40 dos Símios/imunologiaAssuntos
Antígenos T-Independentes/imunologia , Complexo Principal de Histocompatibilidade , Vírus 40 dos Símios/imunologia , Animais , Antígenos T-Independentes/genética , Transformação Celular Viral , Clonagem Molecular , Citotoxicidade Imunológica , DNA Viral , Genes MHC da Classe II , Genes Virais , Humanos , Células L , Camundongos , Plasmídeos , Vírus 40 dos Símios/genética , Infecções Tumorais por VírusAssuntos
Infecções por Papillomavirus/imunologia , Vírus 40 dos Símios/imunologia , Linfócitos T Citotóxicos/imunologia , Infecções Tumorais por Vírus/imunologia , Sequência de Aminoácidos , Animais , Antígenos Transformantes de Poliomavirus/genética , Linhagem Celular Transformada , Células Clonais/imunologia , Testes Imunológicos de Citotoxicidade , Epitopos/genética , Imunização , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Vírus 40 dos Símios/genética , Virologia/métodosAssuntos
Adenina/análogos & derivados , Adenosina/administração & dosagem , Ciclosporinas/administração & dosagem , Citotoxicidade Imunológica/efeitos dos fármacos , Linfócitos T Citotóxicos/efeitos dos fármacos , Adenina/administração & dosagem , Inibidores de Adenosina Desaminase , Trifosfato de Adenosina/metabolismo , Animais , Sinergismo Farmacológico , Técnicas In Vitro , Masculino , CamundongosRESUMO
The simian virus 40 (SV40) tumor or T antigen synthesized in transformed or infected cells is highly immunogenic, inducing both antibody and cytotoxic T lymphocyte (CTL) responses. In the C57BL/6 (H-2b) strain of mice the CTL response is directed to discrete sites on T antigen. To date, five CTL recognition sites have been identified using CTL clones, deletion mutants and overlapping synthetic peptides. The CTL sites I, II and III are clustered in the amino-terminal one-third of T antigen, whereas sites IV and V are located in the carboxyl one-third. Using synthetic peptides, the site I has been tentatively assigned to residues 205 to 215 of T antigen and sites II and III map to residues 220 to 233. Site V maps to amino acids 489 to 503. The location of site IV remains undefined but probably falls between amino acids 368 and 511. The CTL sites I, II, III and V are H-2Db-restricted, whereas site IV is H-2Kb-restricted. CTL sites II and III can be distinguished using H-2Db class I mutants which present the same peptide differentially to CTL clones specific for sites II and III. The multiplicity of CTL sites on SV40 T antigen contributes to the overall immunosurveillance in the host against SV40 carcinogenesis. In the event of a loss of a particular site due to mutation or deletion, the remaining CTL sites continue to provide an effective target for CTL-mediated surveillance. Similar events may also contribute toward controlling papovavirus infections in humans.
Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , Vírus 40 dos Símios/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Mutação , Neoplasias/genética , Neoplasias/imunologia , Vírus 40 dos Símios/genética , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/imunologiaRESUMO
Specific cell-mediated immunity to SV40 tumor-specific transplantation antigen (TSTA) in BALB/c mice undergoing progressive tumorigenesis by syngeneic SV40-transformed cells (VLM) was investigated in vivo using a tumor-cell neutralization test. Specific cellular reactivity to SV40 TSTA was not detected in BALB/c mice bearing large tumors (10-15 mm mean diameter) but was demonstrable after tumor excision. Specific cytotoxic reactivity against syngeneic SV40-transformed cells in vivo could be restored to lymphoid cells from VLM tumor-bearing mice either by culturing the lymphoid cells in vitro or by treating them with papain or trypsin. Enzyme-treated lymphoid cells from MCA tumor-bearing BALB/c mice had no cytotoxic reactivity against VLM cells. These studies suggest that tumor-bearing hosts possess lymphocytes which are sensitized to the TSTA of the tumor but that the reactivity of these lymphocytes is blocked.
Assuntos
Antígenos de Neoplasias , Antígenos de Histocompatibilidade , Imunidade Celular , Linfócitos/imunologia , Neoplasias Experimentais/imunologia , Vírus 40 dos Símios/imunologia , Animais , Linhagem Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Epitopos , Imunização , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Peptídeo Hidrolases/farmacologia , Estimulação Química , Transplante IsogênicoRESUMO
SV40-transformed cells of C57BL/6 (B6) mouse origin (H-2b) express four distinct predominant antigenic sites, I, II, III, and IV, on SV40 large tumor (T) Ag that are recognized by SV40 T Ag-specific CTL clones. In this study, we selected SV40 T Ag-positive cell lines which had lost one or more of the antigenic sites, by in vitro cocultivation of a SV40-transformed B6 mouse kidney cell line (K-0) with SV40 T Ag site-specific CTL clones, Y-1 (site I specific), Y-2 (site II specific), Y-3 (site III specific), and Y-4 (site IV specific). All of the CTL-resistant cell lines expressed large quantities of cell surface H-2 class I Ag. K-1 cells selected by CTL clone Y-1 lost the expression of antigenic sites I, II, and III, but not site IV. K-2 and K-3 cells selected by CTL clones Y-2 and Y-3, respectively, were found to be negative for sites II and III but expressed sites I and IV. K-4 cells selected by CTL clone Y-4 lost the expression of only site IV. K-1,4 cells (sites I-, II-, III-, IV-) were selected from K-1 cells by cocultivation with CTL clone Y-4, K-2,4 cells (sites I+, II-, III-, IV-) were selected from K-2 cells by CTL clone Y-4. K-3,1 cells (sites I-, II-, III-, IV+) were selected from K-3 cells by CTL clone Y-1, and K-3,1,4 cells (sites I-, II-, III-, IV-) were selected from K-3,1 cells by CTL clone Y-4. From K-4 cells, K-4,1 cells (sites I-, II-, III-, IV-) and K-4,3 cells (sites I+, II-, III-, IV-) were selected by CTL clone Y-1 and Y-3, respectively. The antigenic site loss variant cell lines K-1, K-1,4, K-3,1 K-3,1,4, K-4,1, and K-4,3 synthesized SV40 T Ag molecules of 75, 75, 78, 78, 81, and 88 kDa, respectively. Expression of wild-type SV40 T Ag in the antigenic site loss variants by infection with SV40 or transfection with cloned SV40 DNA restored the CTL recognition sites on the variant cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Variação Antigênica , Antígenos Transformantes de Poliomavirus/imunologia , Epitopos/imunologia , Vírus 40 dos Símios/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos Transformantes de Poliomavirus/genética , Antígenos Transformantes de Poliomavirus/isolamento & purificação , Linhagem Celular Transformada , Células Clonais/imunologia , Epitopos/genética , Imunidade Inata , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Vírus 40 dos Símios/genética , Transfecção , Infecções Tumorais por Vírus/imunologiaRESUMO
The ability of hamster lymphoid cells to become cytotoxic in vitro to xenogeneic human cells after stimulation with concanavalin A (ConA) was investigated by using the cell-mediated (51)Cr release test. Cytotoxicity developed by 12 to 16 hr with peak values acquired within 24 hr of stimulation with ConA. The cytotoxicity was found to be mediated by the lymphoid cells themselves rather than by soluble substances. Spleen cells were more efficient than lymph node cells in releasing radioactivity from human cells. The concentration of mitogen was found to be critical in the development of cytotoxicity. ConA concentrations of 12.5 mug or less per culture conferred upon the lymphoid cells the capacity to release (51)Cr from target cells whereas higher concentrations were ineffective and even inhibitory. The cell-mediated cytotoxicity could be reversed by methyl alpha-d-glucopyranoside, but only during the first 8 to 12 hr of stimulation. The lytic reaction did not require the presence of ConA.
Assuntos
Testes Imunológicos de Citotoxicidade , Lectinas , Linfócitos/imunologia , Animais , Medula Óssea/imunologia , Células da Medula Óssea , Linhagem Celular , Isótopos do Cromo , Concanavalina A , Cricetinae , Humanos , Linfonodos/citologia , Baço/citologiaRESUMO
The E3/19-kDa glycoprotein (E3/19K) coded by adenovirus type 2 (Ad2) is known to inhibit the cell-surface expression of major histocompatibility complex (MHC) class I antigens by binding to the MHC antigens intracellularly, and thus reduces recognition of antigens by MHC-restricted cytotoxic T lymphocytes (CTL). We have studied the effect of the E3/19K expression in SV40-infected monkey cells, TC-7/H-2Kb and TC-7/H-2Db expressing transfected H-2Kb and H-2Db antigens, respectively, on the cell-surface H-2 class I antigens and on lysis of the cells by SV40 large tumor (T)-antigen-specific H-2Kb- and H-2Db-restricted CTL clones. H-2Db antigen expression on TC-7/H-2Db cells was drastically reduced by infection with Ad2 but not with an E3/19K-negative SV40-Ad2 hybrid virus, Ad2+ND1, as early as 12 hr postinfection. However, H-2Kb antigen expression on Ad2-infected TC7/H-2Kb cells remained unaltered, even at 24 hr postinfection. Specific lysis of SV40-infected TC-7/H-2Db cells by H-2Db-restricted SV40 T-antigen-specific CTL clones, Y-1 and Y-3, was strongly reduced by coinfection of the target cells with Ad2 but not with Ad2+ND1. Lysis of SV40-infected TC-7/H-2Kb cells by a H-2Kb-restricted SV40 T-antigen-specific CTL clone Y-4 was also reduced significantly by Ad2 infection, but not Ad2+ND1. These results indicate that the E3/19K protein affects cell-surface expression of H-2Db antigen but not H-2Kb antigen.