RESUMO
Recent studies have shown that the quantity of donor-reactive memory T cells is an important factor in determining the relative heterologous immunity barrier posed during transplantation. Here, we hypothesized that the quality of T cell memory also potently influences the response to costimulation blockade-based immunosuppression. Using a murine skin graft model of CD8(+) memory T cell-mediated costimulation blockade resistance, we elicited donor-reactive memory T cells using three distinct types of pathogen infections. Strikingly, we observed differential efficacy of a costimulation and integrin blockade regimen based on the type of pathogen used to elicit the donor-reactive memory T cell response. Intriguingly, the most immunosuppression-sensitive memory T cell populations were composed primarily of central memory cells that possessed greater recall potential, exhibited a less differentiated phenotype, and contained more multi-cytokine producers. These data, therefore, demonstrate that the memory T cell barrier is dependent on the specific type of pathogen infection via which the donor-reactive memory T cells are elicited, and suggest that the immune stimulation history of a given transplant patient may profoundly influence the relative barrier posed by heterologous immunity during transplantation.
Assuntos
Bactérias/patogenicidade , Infecções Bacterianas/terapia , Linfócitos T CD8-Positivos/imunologia , Rejeição de Enxerto/terapia , Memória Imunológica/imunologia , Integrinas/antagonistas & inibidores , Transplante de Pele , Doadores de Tecidos , Animais , Infecções Bacterianas/etiologia , Rejeição de Enxerto/etiologia , Sobrevivência de Enxerto , Humanos , Terapia de Imunossupressão , Imunossupressores/uso terapêutico , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fatores de Risco , Transplante HomólogoRESUMO
Nephropathy associated with BK polyomavirus causes kidney allograft dysfunction and failure. Understanding the pathogenesis of polyomavirus-associated allograft nephropathy (PVAN) is hampered by the species specificity of Polyomaviridae family members. Using a mouse polyomavirus (MPyV) kidney transplant model, we investigated clinically relevant variables that may contribute to PVAN. We found that the timing and source (i.e. donor vs. recipient) of MPyV infection and the titer of the viral inoculum have significant effects on the extent of allograft injury, with acute infection of the recipient by high-titer MPyV inoculums producing the most profound PVAN. In contrast, altering the degree of MHC matching or increasing ischemia/reperfusion injury by prolonging the cold ischemic time of the allograft did not affect the severity of PVAN. Survival correlated positively with serum creatinine levels, but not with viral loads in the kidney allograft. Using splenectomized alymphoplasia mice, which are unable to mount primary adaptive immune responses, we further demonstrate that persistent high viral loads in the kidney are not sufficient to cause advanced PVAN. These findings suggest that the mechanism of PVAN in mice is not a direct consequence of viral cytopathology, but rather involves interplay between viral infection and the recipient antidonor immune response.
Assuntos
Adaptação Fisiológica , Nefropatias/imunologia , Infecções por Polyomavirus/imunologia , Animais , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Carga ViralRESUMO
HLA-matched bone marrow transplantation (BMT) is a cure for nonmalignant hematological disorders; however, rejection rates are high and correlate with the number of antecedent transfusions. Recently, using murine models, we reported that minor antigens (mHAs) in transfused leukoreduced red blood cell (RBC) or platelet units induce rejection of subsequent BMT. To study RBCs as an immunogen, we utilized transgenic donors that express a model mHA selectively on RBCs (HOD mouse). Transfusion of HOD blood did not induce BMT rejection of marrow that shared mHAs with the HOD RBCs. Similarly, no endogenous anti-HOD CD8(+) T-cell response was detected with antigen-specific tetramer reagents. Adoptively transferred OT-I T cells rapidly expanded after HOD blood transfusion; however, only a semi-effector phenotype was observed (tumor necrosis factor-α and interferon-γ secretion, but essentially no Granzyme B). After initial expansion, OT-I T cells contracted rapidly to very low levels. A similar trend was observed by in vivo CTL assay, with only transient lytic activity. Together, these data indicate that RBCs may not be the component of RBC units that induces BMT rejection, and suggest that contaminating platelets or leukocytes may be responsible.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Transfusão de Eritrócitos , Eritrócitos/imunologia , Animais , Apoptose , Citometria de Fluxo , Rejeição de Enxerto/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Polyomavirus-associated nephropathy is an important cause of dysfunction and failure of renal transplants. BK virus is an ubiquitous human polyoma virus that persistently infects the kidney. This otherwise silent infection can reactivate in immunosuppressed individuals, resulting in renal complications. Because polyoma viruses are highly species-specific, we developed a mouse polyoma virus-renal transplant model in order to investigate the pathogenesis of polyomavirus-associated nephropathy. Using this model, we found that polyoma virus preferentially replicates in the allogeneic kidney grafts, accelerating graft failure; thus, this animal model is able to mimic the polyomavirus-associated nephropathy seen in human renal transplant patients. Acute polyoma virus infection of mouse allograft recipients augmented the alloreactive CD8+ T-cell response, while maintaining the anti-viral CD8+ T-cell response. In addition to the known virus-induced cytopathology, these findings demonstrate a potential role for an enhanced anti-donor T-cell response in the pathogenesis of polyomavirus-associated nephropathy.
Assuntos
Transplante de Rim/patologia , Infecções por Polyomavirus/patologia , Infecções Tumorais por Vírus/patologia , Animais , Sequência de Bases , Linfócitos T CD8-Positivos/imunologia , Primers do DNA , Camundongos , Modelos Animais , Nefrectomia , Reação em Cadeia da Polimerase , Polyomavirus/genética , Polyomavirus/isolamento & purificação , Complicações Pós-Operatórias/patologia , Carga Viral , Ensaio de Placa ViralRESUMO
Because oncogenic DNA viruses establish persistent infections in humans, continuous immunosurveillance for neoplastic cells is required to prevent virus-induced tumors. Antigen-specific CD8+ T lymphocytes are critical in vivo effectors for eliminating virus-infected and virus-transformed cells. Investigation into the induction, regulation, and maintenance of CD8+ T cells specific for these viruses is hindered by the lack of tractable animal models that mimic natural infection. Resistance to tumors induced by polyoma virus, a persistent natural mouse DNA virus, is mediated by polyoma-specific CD8+ T cells. Mice susceptible to polyoma virus tumorigenesis mount a smaller, albeit still considerable, expansion of anti-polyoma CD8+ T cells; importantly, these antiviral CD8+ T cells lack cytotoxic activity while retaining the phenotype of cytotoxic T lymphocyte (CTL) effectors. In this review, we will discuss potential in vivo mechanisms that regulate the functional competence of anti-polyoma CD8+ T cells, particularly in the context of chronic antigenic stimulation provided by persistent viral infections and tumors.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias Experimentais/imunologia , Infecções por Polyomavirus/imunologia , Polyomavirus/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Humanos , CamundongosRESUMO
Polyoma virus is a potent oncogenic pathogen when inoculated into newborn mice of particular H-2(k) strains. Using D(k) tetramers containing the dominant antipolyoma CD8(+) T cell epitope, middle T protein (MT)389-397, and intracellular interferon gamma staining, we enumerated MT389-specific CD8(+) T cells in infected neonates having opposite susceptibilities to polyoma virus-induced tumors. In resistant mice, MT389-specific CD8(+) T cells dramatically expanded during acute infection in neonates to a frequency rivaling that in adults; furthermore, in both neonatal and adult mice, this antipolyoma CD8(+) T cell response exhibited nearly identical T cell receptor (TCR) functional avidities and TCR functional fingerprints. Susceptible mice mounted an MT389-specific CD8(+) T cell response of only fourfold lower magnitude than resistant mice; but, in clear contrast to resistant mice, these CD8(+) T cells lacked ex vivo MT389-specific cytotoxic activity. However, MT389-specific CD8(+) T cells in resistant and susceptible mice expressed similar TCR avidities, perforin levels, and surface type O-glycan levels indicative of mature CD8(+) T cell effectors. Upon in vitro restimulation with infected antigen-presenting cells, CD8(+) T cells from acutely infected susceptible neonates acquired strong MT389-specific cytotoxicity. These findings indicate that polyoma-specific CD8(+) T cells are armed with, but restrained from deploying, their cytotoxic effector function in mice susceptible to polyoma virus tumorigenesis.
Assuntos
Antígenos Virais de Tumores/imunologia , Linfócitos T CD8-Positivos/imunologia , Epitopos/imunologia , Neoplasias Experimentais/imunologia , Polyomavirus/imunologia , Fatores Etários , Animais , Animais Recém-Nascidos , Antígenos Transformantes de Poliomavirus/imunologia , Linfócitos T CD8-Positivos/citologia , Linhagem Celular , Testes Imunológicos de Citotoxicidade , Suscetibilidade a Doenças/imunologia , Imunidade Celular/imunologia , Epitopos Imunodominantes/imunologia , Interferon gama/biossíntese , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos CBA , Neoplasias Experimentais/virologia , Infecções por Papillomavirus/imunologia , Fragmentos de Peptídeos/imunologia , Polyomavirus/patogenicidade , Baço/citologia , Baço/imunologia , Baço/virologia , Linfócitos T Citotóxicos/imunologia , Infecções Tumorais por Vírus/imunologiaRESUMO
Dendritic cells are pivotal antigen-presenting cells for generating adaptive T-cell responses. Here, we show that dendritic cells belonging to either the myeloid-related or lymphoid-related subset are permissive for infection by mouse polyomavirus and, when loaded with a peptide corresponding to the immunodominant anti-polyomavirus CD8(+) T-cell epitope or infected by polyomavirus, are each capable of driving expansion of primary polyomavirus-specific CD8(+) T-cell responses in vivo.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/fisiologia , Polyomavirus/imunologia , Animais , Antígenos CD11/análise , Células CHO , Cricetinae , Feminino , Camundongos , Camundongos Endogâmicos C3HRESUMO
CD8 serves both as an adhesion molecule for class I MHC molecules and as a coreceptor with the TCR for T cell activation. Here we study the developmental regulation of CD8-mediated binding to noncognate peptide/MHC ligands (i.e., those not bound by the TCR). We show that CD8's ability to bind soluble class I MHC tetramers and to mediate T cell adhesion under shear flow conditions diminishes as double-positive thymocytes mature into CD8(+) T cells. Furthermore, we provide evidence that this decreased CD8 binding results from increased T cell sialylation upon T cell maturation. These data suggest that CD8's ability to interact with class I MHC is not fixed and is developmentally regulated through the T cell's glycosylation state.
Assuntos
Antígenos CD8/metabolismo , Antígenos H-2/metabolismo , Glicoproteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Subpopulações de Linfócitos T/citologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Adesão Celular , Diferenciação Celular , Senescência Celular , Glicosilação , Antígenos H-2/imunologia , Antígeno de Histocompatibilidade H-2D , Ligantes , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/farmacologia , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Ligação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/deficiência , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Reologia , Solubilidade , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/citologia , Timo/crescimento & desenvolvimentoRESUMO
CD8(+) cytotoxic T lymphocytes are critical for clearance of infection and prevention of tumors caused by mouse polyoma virus. High susceptibility to polyoma-induced tumors is manifested by neonatal inoculation of mice belonging to particular H-2(k) haplotype inbred strains. We previously reported that tumor-susceptible mice generate polyoma-specific CD8(+) T cells, but at a frequency approximately 20-fold lower than tumor-resistant H-2(k) mice. To determine whether susceptibility or resistance may also be associated with a cytokine microenvironment conducive for promoting cell-mediated (i.e., type 1 cytokines) or humoral (i.e., type 2 cytokines) immune responses, we used quantitative bioluminescence RT-PCR to measure in vivo message levels for viral proteins and cytokines during infection of neonatal mice. We found that the level of polyoma viral transcripts peaked higher and fell with significantly slower kinetics in tumor-susceptible mice than in tumor-resistant mice. Interestingly, message for VP1, the major viral capsid protein, persisted in multiple organs of mice of both susceptible and resistant strains, indicating chronic productive infection regardless of tumor susceptibility. IL-1beta, IL-12, IL-2, IFN-&gama; and IL-4 message levels were all higher in infected susceptible than resistant mice. Although both susceptible and resistant mice expressed transcripts for IFN-&gama; and IL-4, the signature type 1 and type 2 cytokines, respectively, a dominance of IL-4 message, with concomitant drop in IFN-&gama; message, was seen only in the susceptible mice. These results suggest that a type 2 pattern of cytokine expression may contribute to susceptibility to polyoma virus tumorigenesis.
Assuntos
Proteínas do Capsídeo , Citocinas/genética , Infecções por Papillomavirus/genética , Polyomavirus/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Infecções Tumorais por Vírus/genética , Animais , Antígenos Transformantes de Poliomavirus/genética , Capsídeo/genética , Feminino , Regulação Viral da Expressão Gênica , Haplótipos/genética , Interferon gama/genética , Interleucina-4/genética , Medições Luminescentes , Camundongos , Camundongos Endogâmicos ICR , Camundongos Endogâmicos , Infecções por Papillomavirus/virologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th1/virologia , Células Th2/imunologia , Células Th2/metabolismo , Células Th2/virologia , Infecções Tumorais por Vírus/virologia , Carga ViralRESUMO
CD8(+) T cells are critical for the clearance of acute polyomavirus infection and the prevention of polyomavirus-induced tumors, but the antigen-presenting cell(s) involved in generating polyomavirus-specific CD8(+) T cells have not been defined. We investigated whether dendritic cells and macrophages are permissive for polyomavirus infection and examined their potential for inducing antiviral CD8(+) T cells. Although dendritic cells and macrophages both supported productive polyomavirus infection, dendritic cells were markedly more efficient at presenting the immunodominant viral epitope to CD8(+) T cells. Additionally, infected dendritic cells, but not infected macrophages, primed anti-polyomavirus CD8(+) T cells in vivo. Treatment with Flt3 ligand, a hematopoietic growth factor that dramatically expands the number of dendritic cells, markedly enhanced the magnitude of virus-specific CD8(+) T-cell responses during acute infection and the pool of memory anti-polyomavirus CD8(+) T cells. These findings suggest that virus-infected dendritic cells induce polyomavirus-specific CD8(+) T cells in vivo and raise the potential for their use as cellular adjuvants to promote CD8(+) T cell surveillance against polyomavirus-induced tumors.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Polyomavirus/imunologia , Células 3T3 , Adjuvantes Imunológicos , Animais , Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/virologia , Cricetinae , Células Dendríticas/virologia , Epitopos de Linfócito T/imunologia , Humanos , Epitopos Imunodominantes/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/virologia , Proteínas de Membrana/administração & dosagem , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C3HRESUMO
T cells are critical for clearing infection and preventing tumors induced by polyoma virus, a natural murine papovavirus. We previously identified the immunodominant epitope for polyoma virus-specific CTL in tumor-resistant H-2k mice as the Dk-restricted peptide, MT389-397, derived from the polyoma middle T oncoprotein. In this study, we developed tetrameric Dk complexes containing the MT389-397 peptide to directly visualize and enumerate MT389-397-specific CTL during polyoma virus infection. We found that Dk/MT389 tetramer+CD8+ T cells undergo a massive expansion during primary infection such that by day 7 postinfection these Ag-specific CD8+ T cells constitute approximately 20% of the total and approximately 40% of the activated CD8+ T cells in the spleen. This expansion of Dk/MT389 tetramer+CD8+ T cells parallels the emergence of MT389-397-specific ex vivo cytolytic activity and clearance of polyoma virus. Notably, Dk/MT389 tetramer+CD8+ T cells are maintained in memory at very high levels. The frequencies of Dk/MT389 tetramer+CD8+ effector and memory T cells in vivo match those of CD8+ T cells producing intracellular IFN-gamma after 6-h in vitro stimulation by MT389-397 peptide. Consistent with preferential Vbeta6 expression by MT389-397-specific CD8+CTL lines and clones, Dk/MT389 tetramer+CD8+ T cells exhibit biased expression of this Vbeta gene segment. Finally, we show that Dk/MT389 tetramer+CD8+ T cells efficiently infiltrate a polyoma tumor challenge to virus-immune mice. Taken together, these findings strongly implicate virus-induced MT389-397-specific CD8+ T cells as essential effectors in eliminating polyoma-infected and polyoma-transformed cells in vivo.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Rejeição de Enxerto/imunologia , Infecções por Papillomavirus/imunologia , Polyomavirus/imunologia , Infecções Tumorais por Vírus/imunologia , Doença Aguda , Animais , Antígenos Transformantes de Poliomavirus/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Movimento Celular/imunologia , Citotoxicidade Imunológica , Epitopos de Linfócito T/imunologia , Feminino , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Rejeição de Enxerto/virologia , Memória Imunológica , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Infecções por Papillomavirus/virologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Infecções Tumorais por Vírus/virologiaRESUMO
We recently identified the immunodominant epitope for polyoma virus-specific CTL as the Dk-associated peptide MT389-397 derived from the middle T (MT) viral oncoprotein. Another Dk-restricted peptide corresponding to residues 236-244 of MT was recognized by nearly all MT389-397-reactive CTL clones, but required concentrations at least 2 logs higher to sensitize syngeneic target cells for lysis. Except for identity at the three putative Dk-peptide anchor residues, MT236-244 shares no homology with MT389-397. Using a novel europium-based class I MHC-peptide binding immunoassay, we determined that MT236-244 bound Dk 2-3 logs less well than MT389-397. Infection with a mutant polyoma virus whose MT is truncated just before the MT389-397 epitope or immunization with MT389-397 or MT236-244 peptides elicited CTL that recognized both MT389-397 and MT236-244. Importantly, infection with a polyoma virus lacking MT389-397 and mutated in an MT236-244 Dk anchor position induced polyoma virus-specific CTL recognizing neither MT389-397 nor MT236-244 epitopes. Despite predominant usage of the Vbeta6 gene segment, MT389-397/MT236-244 cross-reactive CTL clones possess diverse complementarity-determining region 3beta domains; this is functionally reflected in their heterogeneous recognition patterns of alanine-monosubstituted MT389-397 peptides. Using Dk/MT389-397 tetramers, we directly visualized MT236-244 peptide-induced TCR down-modulation of virtually all MT389-397-specific CD8+ T cells freshly explanted from polyoma-infected mice, suggesting that a single TCR recognizes both Dk-restricted epitopes. The availability of immunodominant epitope-specific CTL capable of recognizing a second epitope in MT, a viral protein essential for tumorigenesis, may serve to amplify the CTL response to the immunodominant epitope and prevent the emergence of immunodominant epitope-loss viruses and virus-induced tumors.
Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , Epitopos de Linfócito T/metabolismo , Epitopos Imunodominantes/metabolismo , Polyomavirus/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos Transformantes de Poliomavirus/química , Antígenos Transformantes de Poliomavirus/genética , Epitopos Imunodominantes/genética , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mimetismo Molecular/imunologia , Fragmentos de Peptídeos/imunologia , Polyomavirus/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologiaRESUMO
A fluoroimmunoassay employing europium-streptavidin and time-resolved fluorimetry was used to measure binding of biotin-labeled peptides to H-2Dk molecules. A fluorescein-labeled octameric peptide from the middle T (MT) protein of mouse polyoma virus was identified that specifically binds to purified Dk using a previously-established assay based on size exclusion chromatography. A europium immunoassay was adapted to measure binding of a biotin-derivative of this peptide to purified Dk. The assay was found to be sensitive and highly specific. Binding was optimal at pH 5.0 and 24-27 degrees C, and it was not enhanced in the presence of additional beta2-microglobulin (beta2m). Excellent results were also obtained in experiments with fixed cells that express Dk. This assay is expected to be useful for high volume, routine analysis of peptide binding to MHC class I molecules.
Assuntos
Európio , Fluorimunoensaio/métodos , Antígenos de Histocompatibilidade Classe I/metabolismo , Oligopeptídeos/metabolismo , Animais , Biotina , Meios de Contraste , Fluoresceína , Fluorometria , Antígenos H-2/metabolismo , Antígeno de Histocompatibilidade H-2D , Camundongos , Ligação ProteicaRESUMO
The lipogenic enzyme fatty acid synthase (FAS) is elevated in various human primary cancers and certain human cancer cell lines. FAS overexpression in human neoplasia has clinical relevance because of its association with tumor aggression and potential chemotherapeutic intervention. Here, we surveyed FAS in cell lines established from normal murine mammary epithelium (NMuMG) and from mammary tumors induced by either rodent polyoma (Py) virus or murine mammary tumor virus (MMTV). Western blotting revealed greater content of FAS in Py-transformed A1-1 and T1 than NMuMG or MMTV-transformed Mm5MT, RIIIMT and MMT060562. These data suggest that signaling events mediated by Py transformation may increase cellular amounts of FAS. Although FAS content was elevated to similar levels in A1-1 and T1, specific activities were significantly different as enzyme activity in T1 was 3-fold higher than A1-1. Likewise, FAS activity in NMuMG was about 0.5-fold higher than the MMTV-transformed lines, even though enzyme content was similar. Immunoprecipitation studies employing anti-phosphoamino acid antibodies followed by immunoblot analysis with anti-FAS antisera (and vice versa) were used to characterize the constitutive phosphorylation state of the enzyme. Phosphoserine and phosphothreonine residues were detected in the more active FAS from T1 and NMuMG, but not in the less active FAS from Mm5MT or A1-1. Discovery of phosphorylated FAS suggests that the enzyme may have more immediate control over lipogenesis than previously thought. High-dose (10-4 M) dexamethasone induced FAS content and activity in NMuMG and MMTV-transformed lines but not Py-transformed cells. Lower concentrations (10-8, 10-6 M) of dexamethasone also activated FAS but without concomitant elevation of its protein content, which was consistent with a phosphorylated form of FAS. Finally, cell lines were treated with the FAS inhibitor cerulenin: almost all breast cancer lines were growth inhibited at significantly lower amounts of drug than normal cell lineages, suggesting that FAS plays a greater role in viability of tumor cells than normal cells. Pretreatment with palmitate (a primary end-product of FAS) prior to cerulenin rescued A1-1 cells only slightly from growth inhibition, whereas pretreatment with oleate (a monounsaturated fatty acid synthesized from palmitate) synergized cerulenin's cytotoxic effects.
Assuntos
Ácido Graxo Sintases/análise , Neoplasias Mamárias Experimentais/enzimologia , Animais , Linhagem Celular Transformada , Transformação Celular Neoplásica , Transformação Celular Viral , Cerulenina/farmacologia , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Feminino , Glucocorticoides/farmacologia , Vírus do Tumor Mamário do Camundongo , Camundongos , Ácido Oleico/farmacologia , Palmitatos/farmacologia , Fosforilação , Polyomavirus , Progestinas/farmacologia , Promegestona/farmacologiaRESUMO
The natural mouse pathogen polyoma virus is highly oncogenic in H-2k mice carrying the endogenous superantigen encoded by the mouse mammary tumor provirus Mtv-7. This superantigen results in deletion of Vbeta6 TCR-expressing polyoma-specific CD8+ CTL, which appear to be critical effectors against polyoma tumorigenesis. Here we have isolated cloned lines of CD8+ T cells from resistant (i.e., Mtv-7-) H-2k mice that specifically lyse syngeneic polyoma virus-infected cells and polyoma tumor cells. Nearly all these CTL clones express Vbeta6 and are restricted in their recognition of virus-infected cells by H-2Dk. Screening a panel of synthetic peptides predicted to bind to Dk, for which no consensus peptide binding motif is known, we identified a peptide corresponding to a nine-amino acid sequence in the carboxyl-terminus of the middle T (MT) protein (amino acids 389-397) that was recognized by all the Vbeta6+ CD8+ CTL clones. The inability of MT(389-397)-reactive CTL to recognize cells infected with a mutant polyoma virus encoding a MT truncated just proximal to this sequence indicates that MT(389-397) is a naturally processed peptide. The frequencies of precursor CTL specific for polyoma virus and MT(389-397) peptide were similar, indicating that MT(389-397) is the immunodominant epitope in H-2k mice. In addition, polyoma-infected resistant mice possess a 10- to 20-fold higher MT(389-397)-specific precursor CTL frequency than susceptible mice. This highly focused CTL response to polyoma virus provides a valuable animal model to investigate the in vivo activity of CTL against virus-induced neoplasia.
Assuntos
Antígenos Transformantes de Poliomavirus/imunologia , Epitopos de Linfócito T/imunologia , Antígenos H-2/imunologia , Epitopos Imunodominantes/imunologia , Infecções por Papillomavirus/imunologia , Polyomavirus/imunologia , Linfócitos T Citotóxicos/imunologia , Infecções Tumorais por Vírus/imunologia , Animais , Linhagem Celular , Separação Celular , Células Clonais , Suscetibilidade a Doenças , Epitopos de Linfócito T/metabolismo , Feminino , Antígenos H-2/metabolismo , Imunidade Inata , Epitopos Imunodominantes/metabolismo , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/imunologia , Ratos , Ratos Sprague-Dawley , Células-Tronco/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
Polyoma virus is highly oncogenic when inoculated into immunocompromised adult mice and neonatal mice of specific inbred strains. Although T lymphocytes are known to be essential in controlling polyoma virus tumorigenesis, the importance of class I MHC-restricted CD8+ T cells in mediating tumor resistance remains unclear. Here, we investigated the tumorigenicity of polyoma virus in adult mice rendered CD8+ T cell-deficient by homozygous (-/-) disruption of the beta 2-microglobulin (beta 2m) or CD8 alpha (CD8) genes. Nearly all (94%) of the virus-infected adult C57BL/6 beta 2m-/- mice developed tumors, and 20% of the virus-inoculated adult C57BL/6CD8-/- mice developed hindlimb paralysis, which is indicative of vertebral tumors. Only 2 of 20 virus-inoculated adult normal C57BL/6 mice developed tumors. Despite these different tumor susceptibilities, persistent viral DNA was detected in multiple organs of mice of all three strains. Multifocal lymphoplasmacytic interstitial infiltrates were present in the kidneys and lungs of virus-infected C57BL/6 beta 2m-/- and in the lungs of virus-inoculated C57BL/6CD8-/- mice. These infiltrates were composed primarily of B cells and colocalized with staining for the major viral capsid protein, VP1. No infiltrates or VP1 staining was detected in the kidneys of infected C57BL/6 mice. Using a highly sensitive RT-PCR bioluminescence immunoassay, we investigated and detected persistent polyoma T protein and VP1 messages in both C57BL/6 beta 2m-/- and C57BL/6 mice. C57BL/6 beta 2m-/- and C57BL/6 mice had equivalent serum virus-neutralizing antibody titers. These results provide in vivo evidence that class I MHC-restricted CD8+ T cells are involved in mediating protection against polyoma virus tumor development.
Assuntos
Predisposição Genética para Doença/patologia , Infecções por Papillomavirus/patologia , Polyomavirus/patogenicidade , Infecções Tumorais por Vírus/patologia , Microglobulina beta-2/fisiologia , Animais , Anticorpos Antivirais/biossíntese , Southern Blotting , Western Blotting , DNA Viral/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Polyomavirus/imunologia , Microglobulina beta-2/genéticaRESUMO
A dominant gene carried in certain inbred mouse strains confers susceptibility to tumors induced by polyoma virus. This gene, designated Pyvs, was defined in crosses between the highly susceptible C3H/BiDa strain and the highly resistant but H-2k-identical C57BR/cdJ strain. The resistance of C57BR/cdJ mice is overcome by irradiation, indicating an immunological basis. In F1 x C57BR/cdJ backcross mice, tumor susceptibility cosegregates with Mtv-7, a mouse mammary tumor provirus carried by the C3H/BiDa strain. This suggests that Pyvs might encode the Mtv-7 superantigen (SAG) and abrogate polyoma tumor immunosurveillance through elimination of T cells bearing specific V beta domains. DNA typing of 110 backcross mice showed no evidence of recombination between Pyvs and Mtv-7. Strongly biased usage of V beta 6 by polyoma virus-specific CD8+ cytotoxic T lymphocytes in C57BR/cdJ mice implicates T cells bearing this Mtv-7 SAG-reactive V beta domain as critical anti-polyoma tumor effector cells in vivo. These results indicate identity between Pyvs and Mtv-7 sag, and demonstrate a novel mechanism of inherited susceptibility to virus-induced tumors based on effects of an endogenous superantigen on the host's T cell repertoire.
Assuntos
Vírus do Tumor Mamário do Camundongo/imunologia , Infecções por Papillomavirus/imunologia , Polyomavirus , Superantígenos/fisiologia , Infecções Tumorais por Vírus/imunologia , Animais , Cruzamentos Genéticos , Feminino , Vírus do Tumor Mamário do Camundongo/isolamento & purificação , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos CBA , Infecções por Papillomavirus/genética , Provírus/isolamento & purificação , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Superantígenos/genética , Linfócitos T Citotóxicos/imunologia , Infecções Tumorais por Vírus/genéticaRESUMO
Results in a previous report established the presence of dominant genetic factor(s) in C3H/BiDa mice which contributed to the unusually high susceptibility of this strain to polyoma virus-induced tumors (Freund et al. (1992) Virology 191, 724-731). Here we show that C57BR/cdJ mice, while identical to C3H/BiDa at the H-2 locus, are almost completely lacking in susceptibility. Analysis of crosses between these two H-2k strains has shown that susceptibility is due to a single dominant autosomal gene, designated Pyvs. On an H-2k background, Pyvs acts in a highly penetrant manner to confer susceptibility to induction of a broad range of tumors by the virus. Analysis of F1 mice between C3H/BiDa and the highly resistant C57BL/6 (H-2b) strain shows that Pyvs can partially overcome the immunologically mediated resistance associated with an H-2b haplotype. Pyvs does not encode cell receptors for the virus, nor does it affect levels of virus replication or anti-viral humoral responses in the host. Studies with early passage embryo fibroblasts in culture show that Pyvs does not affect intracellular events essential for either productive infection or cell transformation by the virus. Pyvs thus determines a generalized susceptibility of the host to polyoma-induced tumors but apparently does not act at the level of target cells for tumor induction.
Assuntos
Transformação Celular Viral , Genes Dominantes , Polyomavirus/fisiologia , Infecções Tumorais por Vírus/imunologia , Células 3T3 , Animais , Células Cultivadas , Cruzamentos Genéticos , Predisposição Genética para Doença , Imuno-Histoquímica , Rim/microbiologia , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Receptores Virais/genética , Infecções Tumorais por Vírus/microbiologiaRESUMO
Cloned populations of antigen-specific, murine T-lymphocytes, maintained in long-term continuous culture, have been reported to exhibit functional activity upon adoptive transfer. Few data are currently available on the in vivo distribution of these homogeneous, functional cell populations. Such information is important to understanding the mechanisms by which T-lymphocytes exert their effector activity. We examined 2 cloned populations of influenza virus-specific T-lymphocytes. One clone was a Lyt-2+, L3T4-, class I MHC-restricted, cytolytic T-lymphocyte subset. The other clone was a Lyt-2-, L3T4+, Class II MHC-restricted clone of the helper/amplifier T-cell subset. Both clones promote recovery from lethal pulmonary influenza infection upon adoptive transfer. Using cell populations labeled with [3H]thymidine, we examined the distribution of these cells in tissue sections from various organs. These cloned cell populations were preferentially retained in the lungs of uninfected and influenza-virus-infected animals. This retention is independent of the cell's viral antigenic specificity, but may be dependent on the phenotype of the clone. Once retained in the lungs, these cells migrated across the bronchial lamina propria and entered the epithelium and pulmonary lumen. The significance of these observations for in vivo T-lymphocyte functions is discussed.
Assuntos
Vírus da Influenza A/imunologia , Sistema Respiratório/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos Virais/imunologia , Células Clonais/imunologia , Epitopos/imunologia , Imunização Passiva , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA-specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II-restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets.