RESUMO
BACKGROUND: Using the FIV model, we reported previously that CD4+CD25+ T regulatory (Treg) cells from FIV+ cats are constitutively activated and suppress CD4+CD25- and CD8+ T cell immune responses. In an effort to further explore Treg-mediated suppression, we asked whether Treg cells induce anergy through the alteration of production of cyclins, cyclin-dependent kinases and their inhibitors. RESULTS: Lymphocytes were obtained from control or FIV+ cats and sorted by FACS into CD4+CD25+ and CD8+ populations. Following co-culture with CD4+CD25+ cells, CD8+ targets were examined by Western blot for changes in cyclins D3, E and A, retinoblastoma (Rb) protein, as well as the cyclin dependent kinase inhibitor p21cip1. Following co-culture with CD4+CD25+cells, we observed up-regulation of p21cip1 and cyclin E, with down-regulation of cyclin D3, in CD8+ cells from FIV+ cats. As expected, CD8+ targets from control cats were quiescent with little up-regulation of p21cip1 and cyclin E. There was also a lack of Rb phosphorylation in CD8+ targets consistent with late G1 cell cycle arrest. Further, IL-2 mRNA was down regulated in CD8+ cells after co-culture with CD4+CD25+ Treg cells. Following CD4+CD25+ co-culture, CD8+ targets from FIV+ cats also had increased Foxp3 mRNA expression; however, these CD8+Foxp3+ cells did not exhibit suppressor function. CONCLUSIONS: Collectively, these data suggest that CD4+CD25+ Treg cells from FIV+ cats induce CD8+ anergy by disruption of normal G1 to S cell cycle progression.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Anergia Clonal , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Vírus da Imunodeficiência Felina , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD4/imunologia , Linfócitos T CD8-Positivos/citologia , Gatos , Ciclo Celular/imunologia , Técnicas de Cocultura , Regulação para Baixo , Subunidade alfa de Receptor de Interleucina-2/imunologiaRESUMO
CD8(+) lymphocytes are critical to the control and elimination of viral pathogens. Impaired CD8(+) responses are well recognized in lentiviral infections; however, the mechanisms underlying CD8(+) impairment remain elusive. Using the feline immunodeficiency virus (FIV) model for human AIDS, we reported previously that CD4(+)CD25(+) Treg cells in both the acute and long-term, asymptomatic phase of infection are constitutively activated and suppress CD4(+)CD25(-) T cell responses. In the current study, we have demonstrated that CD4(+)CD25(+) Treg cells suppress CD8(+) responses to immune stimulation during both the acute and chronic, asymptomatic phase of FIV infection and that the mechanism of suppression may be mediated by membrane-associated TGF-beta (mTGF-beta) on CD4(+)CD25(+) lymphocytes. Depletion of CD4(+)CD25(+) lymphocytes from lymph node suspensions significantly enhanced production of IFN-gamma during the acute phase of infection and coculture of CD8(+) lymphocytes with CD4(+)CD25(+) lymphocytes resulted in suppression of CD8(+) IFN-gamma during both the acute and chronic stages of infection. FACS analysis indicated that there was TGF-betaRII upregulation on CD8(+) cells from FIV(+) cats during the acute and chronic stage of infection. In addition, there was upregulation of mTGF-beta on the CD4(+)CD25(+) subset in chronically infected cats. In support of activation of the TGF-beta signaling pathway, Western blotting showed Smad 2 phosphorylation in CD8(+) targets following CD4(+)CD25(+)/CD8(+) coculture. These results demonstrate the suppressive effect CD4(+)CD25(+) Treg cells have on the CD8(+) immune response during the acute and chronic stages of FIV infection and suggest that the mechanism of suppression may be mediated by mTGF-beta.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Doenças do Gato/imunologia , Vírus da Imunodeficiência Felina/imunologia , Interferon gama/antagonistas & inibidores , Infecções por Lentivirus/veterinária , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Antígenos CD4/análise , Doenças do Gato/virologia , Gatos , Tolerância Imunológica , Subunidade alfa de Receptor de Interleucina-2/análise , Infecções por Lentivirus/virologia , Linfócitos T Reguladores/químicaRESUMO
HIV-induced AIDS may be mediated by the activation of immunosuppressive CD4+CD25+ T regulatory cells (Treg cells). Treg cells have been shown to regulate CD4+ and CD8+ immune responses to HIV and FIV antigens in vitro. We tested the hypothesis that Treg cells become infected and activated during the acute infection with FIV leading to the suppression of CD4+ T helper cell responses. Cats were experimentally infected with FIV-NCSU1 and blood and lymph node cells were collected at weekly intervals following inoculation. Real-time RT-PCR was used to determine plasma viremia and the relative expression of FIV, FoxP3, TGF-beta, and GAPDH mRNA copies in CD4+CD25+ and CD4+CD25- T cell subsets. Flow cytometry was used to assess the absolute numbers of each cell type and the expression of surface TGF-beta and intracellular FoxP3 in CD4+CD25+ and CD4+CD25- T cells at each time-point. Treg suppression of IL-2 production in CD4+ T helper cells was assessed by ELISPOT assays. Our results showed that peak viremia occurred at 2 weeks post infection and correlated with maximal infectivity in CD4+CD25+ T cell populations. FIV-gag-mRNA levels were higher in CD4+CD25+ T cells than CD4+CD25- T cells throughout the acute phase of infection. Induction of FoxP3 and TGF-beta indicated activation of Treg cells during the acute stage infection, which was confirmed by Treg cell suppression of IL-2 production by CD4+ Th cells in an ELISPOT assay. Our findings support the hypothesis that early activation of Treg immunosuppressor function may limit an effective anti-FIV response, contributing to the establishment of chronic infection and the immunodeficiency caused by this virus.
Assuntos
Doenças do Gato/imunologia , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Regulação da Expressão Gênica/imunologia , Vírus da Imunodeficiência Felina/imunologia , Linfócitos T Reguladores/virologia , Animais , Gatos , Citometria de Fluxo/veterinária , Fatores de Transcrição Forkhead/metabolismo , Vírus da Imunodeficiência Felina/genética , Interleucina-2/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Viremia/veterináriaRESUMO
FIV/HIV infections are associated with an early robust humoral and cellular anti-viral immune response followed by a progressive immune suppression that eventually results in AIDS. Several mechanisms responsible for this immune dysfunction have been proposed including cytokine dysregulation, immunologic anergy and apoptosis, and inappropriate activation of immune regulatory cells. Studies on FIV infection provide evidence for all three. Cytokine alterations include decreases in IL2 and IL12 production and increases in IFNgamma and IL10 in FIV(+) cats compared to normal cats. The elevated IL10:IL12 ratio is associated with the inability of FIV(+) cats to mount a successful immune response to secondary pathogens. Additionally, chronic antigenic (FIV) stimulation results in an increase in the percent of activated T cells expressing B7 and CTLA4 co-stimulatory molecules in infected cats. The expression of these molecules is associated with T cells that are undergoing apoptosis in the lymph nodes. As ligation of CTLA4 by B7 transduces a signal for induction of anergy, one can speculate that the activated T cells are capable of T cell-T cell interactions resulting in anergy and apoptosis. The inability of CD4(+) cells from FIV(+) cats to produce IL2 in response to recall antigens and the gradual loss of CD4(+) cell numbers could be due to B7-CTLA4 interactions. The chronic antigenemia may also lead to activation of CD4(+)CD25(+) T regulatory cells. Treg cells from FIV(+) cats are chronically activated and inhibit the mitogen-induced proliferative response of CD4(+)CD25(-) by down-regulating IL2 production. Although Treg cell activation can be antigen-specific, the suppressor function is not, and thus activated Treg cells would suppress responses to secondary pathogens as well as to FIV. Concomitant with the well-known virus-induced immune suppression is a progressive immune hyper-activation. Evidence for immune hyper-activation includes polyclonal B cell responses, gradual replacement of naïve CD4(+) and CD8(+) T cell phenotypes with activation phenotypes (CD62L(-), B7(+), CTLA4(+)), and the chronic activation of CD4(+)CD25(+) Treg cells. Thus lentivirus infections lead to severe immune dysregulation manifested as both chronic immune suppression and chronic immune activation. FIV infection of cats provides a number of advantages over other lentivirus infections as a model to study this immune dysregulation. It is a natural infection that has existed in balance with the cat's immune system for thousands of years. As such, the natural history and pathogenesis provides an excellent model to study the long-term relationships between AIDS lentivirus and host immune system function/dysregulation.
Assuntos
Síndrome de Imunodeficiência Adquirida Felina/imunologia , Vírus da Imunodeficiência Felina/imunologia , Animais , Formação de Anticorpos/imunologia , Apoptose/imunologia , Gatos , Síndrome de Imunodeficiência Adquirida Felina/virologia , Imunidade Celular/imunologiaRESUMO
We have reported that CD4+CD25+ T-regulatory (Treg) cells are fully activated for suppressor function in feline immunodeficiency virus (FIV)-infected cats. Studies have suggested that surface transforming growth factor-beta (TGFbeta; membrane TGFbeta [mTGFbeta]) is a feature of activated CD4+CD25+ Treg cells and may play a role in Treg homeostasis and suppressor function. Herein, we explore the role of TGFbeta in feline Treg homeostasis and suppressor function and what effect FIV infection of cats might have on these processes. Stimulation of CD4+CD25- T helper (Th) cells with Concanavalin A (ConA) plus TGFbeta converts them to Treg-like cells capable of suppressor function. Reverse-transcription polymerase chain reaction and flow cytometry revealed that these ConA/TGFbeta-converted Treg cells upregulate Foxp3 and mTGFbeta. ConA stimulation of CD4+CD25- T cells upregulates TGFbeta receptor II (RII), and pretreatment of these cells with anti-TGFbeta-RII antibodies blocks the TGFbeta-induced conversion to Treg cells. Pretreatment of ConA/TGFbeta-converted Treg cells with anti-TGFbeta antibodies also abrogates their suppressor function, suggesting that Treg homeostasis and suppressor function may be mediated by mTGFbeta. Finally, we show that treatment of CD4+CD25+ mTGFbeta-positive Treg cells from FIV-infected cats with anti-TGFbeta antibodies or treatment of ConA-stimulated CD4+CD25- Th target cells with anti-TGFbeta-RII antibodies diminishes suppressor function. These data suggest that the recruitment of Treg cells from the Th pool and suppressor function of Treg cells are dependent on the TGFbeta/TGFbeta-RII signaling pathway and that this pathway is constitutively upregulated in asymptomatic chronically FIV-infected cats.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Linfócitos T Reguladores/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Gatos , Células Cultivadas , Citometria de Fluxo , Regulação da Expressão Gênica , Homeostase , Subunidade alfa de Receptor de Interleucina-2/análise , Leucócitos Mononucleares/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo II , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Organismos Livres de Patógenos Específicos , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Many virus infections result in the suppression of one or more functions of the immune system. Multiple mechanisms have been proposed to explain viral-induced immunosuppression, including an imbalance in the cellular Th1/Th2 or cytokine profile, induction of anergy, depletion of effector cells and most recently the activation of CD4+CD25+ regulatory T (T reg) cells. CD4+CD25+ T reg cells are a subset of circulating CD4+ T cells with suppressive properties. CD4+CD25+ T reg cells were first identified in mice as cells capable of maintaining self-tolerance by suppressing autoreactive T cells. This review focuses on interactions between CD4+CD25+ T reg cells and viral pathogens. Most cases in which CD4+CD25+ T reg cells participate in response to infection reported so far involve chronic or persistent viral infections. Examples have been growing recently and include members of different viral families including retroviridae, herpesviridae and picornaviridae. It is currently not known how microbes are recognized by CD4+CD25+ T reg cells and whether exoantigen-specific T reg cells are of the same lineage as self-reacting natural T reg cells or represent peripherally induced counterparts derived from CD4+CD25- T cells. The findings that T reg cells influence the functional immunity during viral infections, however, might indicate that, in some cases, virus-specific T reg cells not only influence immune pathology or prevent pathogen elimination but also can promote a generalized state of immunosuppression in vivo such that the host is more susceptible to secondary infections with other pathogens or has reduced resistance to tumors. Conceivably, the activities of T reg cells might be one of the contributing reasons why it has been difficult so far to produce effective vaccines against some persisting viral infections.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Viroses/imunologia , Animais , Infecções por Herpesviridae/imunologia , Tolerância Imunológica , Ativação Linfocitária , Camundongos , Infecções por Picornaviridae/imunologia , Receptores de Interleucina-2/metabolismo , Infecções por Retroviridae/imunologia , Subpopulações de Linfócitos T/imunologiaRESUMO
Cellular activation plays an important role in retroviral replication. Previously, we have shown that CD4(+)CD25(+) T cells by the virtue of their partially activated phenotype represent ideal candidates for a productive feline immunodeficiency virus (FIV) infection. In the present study, we extended our previous observations with regard to FIV replication in CD4(+)CD25(+) and CD4(+)CD25(-) cells under different stimulation conditions. Both CD4(+)CD25(+) and CD4(+)CD25(-) cells remain latently infected in the absence of IL-2 or concanvalinA (ConA), respectively; harboring a replication competent provirus capable of reactivation several days post-infection. While CD4(+)CD25(+) cells require low levels of exogenous IL-2 and virus inputs for an efficient FIV replication, CD4(+)CD25(-) T cells can only be productively infected in the presence of either high concentrations of IL-2 or high virus titers, even in the absence of mitogenic stimulation. Interestingly, while high virus input activates CD4(+)CD25(-) cells to replicate FIV, it induces apoptosis in a high percentage of CD4(+)CD25(+) T cells. High IL-2 concentrations but not high virus inputs lead to surface upregulation of CD25 and significant cellular proliferation in CD4(+)CD25(-) cells. These results suggest that CD4(+)CD25(+) and CD4(+)CD25(-) T cells have different activation requirements which can be modulated by both viral and cytokine stimuli to reach threshold activation levels in order to harbor a productive FIV infection. This holds implications in vivo for CD4(+)CD25(+) and CD4(+)CD25(-) cells to serve as potential reservoirs of a productive and latent FIV infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Vírus da Imunodeficiência Felina/fisiologia , Ativação Linfocitária/imunologia , Receptores de Interleucina-2/metabolismo , Animais , Apoptose , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Gatos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Concanavalina A/farmacologia , Relação Dose-Resposta a Droga , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Vírus da Imunodeficiência Felina/imunologia , Interleucina-2/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Mitógenos/farmacologia , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/imunologia , Regulação para Cima/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Previously, we have characterized feline CD4+ CD25+ T-regulatory (Treg) cells with regard to their immune regulatory properties and ability to support feline immunodeficiency virus (FIV) replication in vitro and in vivo. Our studies showed that while CD4+ CD25+ cells were capable of replicating FIV in the presence of interleukin-2 (IL-2) alone, CD4+ CD25- cells harbored a latent infection that required a strong mitogenic stimulus to activate virus replication. In the present study, we investigated the mechanisms governing the preferential replication of FIV in highly purified CD4+ CD25+ Treg cells compared to their CD4+ CD25+ counterparts. Studies aimed at elucidating mechanisms regulating infection of these cells revealed that CD4+ CD25- cells were less susceptible to FIV binding and entry than CD4+ CD25+ cells, which correlated with increased surface expression of FIV coreceptor CXCR4. In addition, the number of CD4+ CD25+ cells that expressed the primary receptor CD134 was greater than for CD4+ CD25- cells. Although increased permissiveness to FIV infection of CD4+ CD25- cells following mitogenic stimulation correlated strongly with upregulation of surface CXCR4, it did not correlate with CD134 expression. Further, study of intracellular factors regulating FIV replication revealed that CD4+ CD25+ but not CD4+ CD25- T cells showed constitutive and IL-2-responsive transactivation of activating transcription factor, CAAT enhancer binding protein, and activating protein 1 transcription factors that are important for FIV replication. These factors were upregulated in CD4+ CD25- T cells following ConA stimulation, which correlated with FIV replication. This is the first report elucidating the mechanisms that allow for productive lentiviral infection of CD4+ CD25+ Treg cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Vírus da Imunodeficiência Felina/fisiologia , Receptores CXCR4/genética , Receptores de Interleucina-2/imunologia , Linfócitos T/imunologia , Animais , Benzilaminas , Linfócitos T CD4-Positivos/virologia , Gatos , Células Cultivadas , Ciclamos , Compostos Heterocíclicos/farmacologia , Vírus da Imunodeficiência Felina/imunologia , Linfonodos/imunologia , Linfonodos/virologia , Receptores CXCR4/antagonistas & inibidores , Linfócitos T/virologia , Replicação ViralRESUMO
Feline Immunodeficiency Virus (FIV) is a lentivirus that causes immunodeficiency in cats, which parallels HIV-1-induced immunodeficiency in humans. It has been established that HIV envelope (Env) glycoprotein mediates T cell loss via a mechanism that requires CXCR4 binding. The Env glycoprotein of FIV, similar to HIV, requires CXCR4 binding for viral entry, as well as inducing membrane fusion leading to syncytia formation. However, the role of FIV Env in T cell loss and the molecular mechanisms governing this process have not been elucidated. We studied the role of Env glycoprotein in FIV-mediated T cell apoptosis in an in vitro model. Our studies demonstrate that membrane-expressed FIV Env induces apoptosis in activated feline peripheral blood mononuclear cells (PBMC) by a mechanism that requires CXCR4 binding, as the process was inhibited by CXCR4 antagonist AMD3100 in a dose-dependent manner. Interestingly, studies regarding the role of CD134, the recently identified primary receptor of FIV, suggest that binding to CD134 may not be important for induction of apoptosis in PBMC. However, inhibiting Env-mediated fusion post CXCR4 binding by FIV gp41-specific fusion inhibitor also inhibited apoptosis. Under similar conditions, a fusion-defective gp41 mutant was unable to induce apoptosis in activated PBMC. Our findings are the first report suggesting the potential of FIV Env to mediate apoptosis in bystander cells by a process that is dependent on gp41 function.
Assuntos
Apoptose , Glicoproteínas/fisiologia , Vírus da Imunodeficiência Felina/patogenicidade , Leucócitos Mononucleares/virologia , Proteínas do Envelope Viral/fisiologia , Proteínas Virais de Fusão/fisiologia , Animais , Fármacos Anti-HIV/farmacologia , Benzilaminas , Gatos , Fusão Celular , Sobrevivência Celular , Células Cultivadas , Ciclamos , Glicoproteínas/genética , Compostos Heterocíclicos/farmacologia , Leucócitos Mononucleares/citologia , Fusão de Membrana , Mutação/genética , Mutação/fisiologia , Receptores CXCR4/antagonistas & inibidores , Receptores OX40 , Receptores do Fator de Necrose Tumoral , Proteínas do Envelope Viral/genética , Proteínas Virais de Fusão/genéticaRESUMO
Increased susceptibility to intracellular pathogens in HIV-infected individuals and FIV-infected cats is attributed to a defective T-helper 1 (Th1) immune response. However, little is known about specific cytokine responses to secondary pathogens. To address this question, control and FIV-infected cats were challenged with Toxoplasma gondii, and lymph node cells analyzed for cytokine mRNA expression. Twenty-four weeks post-FIV infection, prior to T. gondii challenge, IL2 and IL12 mRNAs were depressed, whereas IL10 and IFNgamma mRNAs were increased in CD4+ and CD8+ subsets. Following T. gondii challenge, control cats showed increased expression of IL2, IFNgamma, IL10, IL12, and IL6 mRNAs. In contrast, IL2, IL6, IFNgamma, and IL12 mRNAs were suppressed in FIV-T. gondii co-infected cats, whereas IL10 remained at the high prechallenge levels. IFNgamma and IL10 mRNAs were produced by both CD4+ and CD8+ cells in FIV-T. gondii cats. Elevated IL10 may suppress a Th1 cytokine response to T. gondii challenge.
Assuntos
Doenças do Gato/imunologia , Vírus da Imunodeficiência Felina , Interleucinas/genética , Infecções por Lentivirus/veterinária , Toxoplasmose Animal/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Doenças do Gato/genética , Gatos , Expressão Gênica , Interleucina-10/genética , Interleucina-12/genética , Interleucina-2/genética , Interleucina-6/genética , Infecções por Lentivirus/complicações , Infecções por Lentivirus/genética , Infecções por Lentivirus/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Toxoplasmose Animal/complicações , Toxoplasmose Animal/genéticaRESUMO
Disease progression of feline immunodeficiency virus (FIV) infection is characterized by up-regulation of B7.1 and B7.2 costimulatory molecules and their ligand CTLA4 on CD4(+) and CD8(+) T cells. The CD4(+)CTLA4(+)B7(+) phenotype described in FIV(+) cats is reminiscent of CD4(+)CD25(+)CTLA4(+) cells, a phenotype described for immunosuppressive T regulatory (Treg) cells. In the present study, we describe the phenotypic and functional characteristics of CD4(+)CD25(+) T cells in PBMC and lymph nodes (LN) of FIV(+) and control cats. Similar to Treg cells, feline CD4(+)CD25(+) but not CD4(+)CD25(-) T cells directly isolated from LN of FIV(+) cats do not produce IL-2 and fail to proliferate in response to mitogen stimulation. Unstimulated CD4(+)CD25(+) T cells from FIV(+) cats significantly suppress the proliferative response and the IL-2 production of Con A-stimulated autologous CD4(+)CD25(-) T cells compared with unstimulated CD4(+)CD25(+) T cells from FIV(-) cats. Flow-cytometric analysis confirmed the apparent activation phenotype of the CD4(+)CD25(+) cells in LN of chronically FIV(+) cats, because these cells showed significant up-regulation of expression of costimulatory molecules B7.1, B7.2, and CTLA4. These FIV-activated, anergic, immunosuppressive CD25(+)CTLA4(+)B7(+)CD4(+) Treg-like cells may contribute to the progressive loss of T cell immune function that is characteristic of FIV infection.
Assuntos
Síndrome de Imunodeficiência Adquirida Felina/imunologia , Vírus da Imunodeficiência Felina/imunologia , Imunofenotipagem , Ativação Linfocitária/imunologia , Receptores de Interleucina-2/biossíntese , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD/biossíntese , Antígenos CD/metabolismo , Antígenos de Diferenciação/biossíntese , Antígenos Virais/biossíntese , Antígeno B7-1/biossíntese , Antígeno B7-1/metabolismo , Antígeno B7-2 , Antígeno CTLA-4 , Gatos , Divisão Celular/imunologia , Separação Celular , Células Cultivadas , Doença Crônica , Técnicas de Cocultura , Concanavalina A/farmacologia , Síndrome de Imunodeficiência Adquirida Felina/virologia , Tolerância Imunológica , Interleucina-2/biossíntese , Interleucina-2/deficiência , Ligantes , Lipopolissacarídeos/farmacologia , Linfonodos/citologia , Linfonodos/imunologia , Contagem de Linfócitos , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/metabolismo , Receptores de Interleucina-2/sangue , Receptores de Interleucina-2/metabolismo , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/virologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/virologia , Regulação para Cima/imunologiaRESUMO
Lymph node (LN) T cells from feline immunodeficiency virus (FIV)-infected cats have an increased expression of B7 co-stimulatory molecules as well as their ligand CTLA4, resembling an activation phenotype shown to induce anergy and apoptosis in activated T cells. In addition, LN T cells from FIV-infected cats also show increased spontaneous apoptosis compared to uninfected animals. The apoptosis observed in these animals occurs primarily in T cells expressing B7 and CTLA4, suggesting a role for B7 and CTLA4 interactions in the induction of anergy/apoptosis. In order to investigate the role of B7 and CTLA4 interactions on T cell apoptosis in LN T cells from FIV-infected cats, we performed blocking experiments by measuring T cell apoptosis in LN T cell cultures treated with anti-feline B7.1, B7.2, and CTLA4 specific antibodies, as well as interleukin (IL)-2. The addition of IL2, the primary cytokine produced by B7/CD28 interactions, resulted in a significant decrease of T cell apoptosis in cultured LN cells as assessed by two-color flow cytometry and TUNEL assay. The addition of anti-B7.1 antibodies significantly inhibited T cell apoptosis in FIV-infected cats with low-level plasma viremia, while addition of anti-B7.2 and anti-CTLA4 antibodies had no affect. These results suggest a role of B7 signaling in the increased spontaneous apoptosis observed in LN T cells from FIV-infected animals.
Assuntos
Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Vírus da Imunodeficiência Felina/imunologia , Interleucina-2/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Animais , Anticorpos Bloqueadores/imunologia , Antígenos CD/imunologia , Apoptose/efeitos dos fármacos , Antígeno B7-2 , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Gatos , Síndrome de Imunodeficiência Adquirida Felina/sangue , Vírus da Imunodeficiência Felina/genética , Marcação In Situ das Extremidades Cortadas/veterinária , Interleucina-2/imunologia , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/imunologia , RNA Viral/química , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Organismos Livres de Patógenos EspecíficosRESUMO
Apoptosis in lymph node (LN) T cells of feline immunodeficiency virus (FIV)-infected cats is associated with cells co-expressing B7.1 and B7.2 costimulatory molecules, and their ligand CTLA4. To study the possibility of B7.1/B7.2-CTLA4 mediated T-T interactions and the predicted induction of T cell apoptosis in vitro, costimulatory molecules were up-regulated on CD4+ and CD8+ T cells by mitogen stimulation. B7.1 expression on in vitro stimulated CD4+ and CD8+ cells increased within 24h; B7.2 and CTLA4 expression increased after 48-72 h. Apoptosis, as analyzed by terminal deoxynucleotidyl transferase (transferase nick end labeling, TUNEL)-based staining followed by three color flow cytometric analysis, correlated to the cells expressing B7 and/or CTLA4. Blocking experiments revealed that CD4+ and CD8+ T cell apoptosis could be significantly inhibited with anti-B7 antibodies. As FIV infection results in immune activation with a T cell phenotype similar to that of the in vitro activated T cells, the data support the hypothesis that the chronic expansion of B7+CTLA4+ LN T cells in infected cats allows for T-T cell interactions resulting in T cell depletion and eventually the development of AIDS.
Assuntos
Antígenos de Diferenciação/imunologia , Apoptose/imunologia , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Vírus da Imunodeficiência Felina/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD/biossíntese , Antígenos CD/imunologia , Antígenos de Diferenciação/biossíntese , Antígeno B7-1/biossíntese , Antígeno B7-1/imunologia , Antígeno B7-2 , Antígeno CTLA-4 , Gatos , Comunicação Celular/imunologia , Concanavalina A/imunologia , Citometria de Fluxo/veterinária , Marcação In Situ das Extremidades Cortadas/veterinária , Ionomicina/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/imunologia , Organismos Livres de Patógenos Específicos , Linfócitos T/virologia , Acetato de Tetradecanoilforbol/imunologiaRESUMO
Feline immunodeficiency virus (FIV) shares remarkable homology to primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The process of lentiviral env glycoprotein-mediated fusion of membranes is essential for viral entry and syncytia formation. A detailed understanding of this phenomenon has helped identify new targets for antiviral drug development. Using a model based on syncytia formation between FIV env-expressing cells and a feline CD4+ T cell line we have studied the mechanism of FIV env-mediated fusion. Using this model we show that FIV env-mediated fusion mechanism and kinetics are similar to HIV env. Syncytia formation could be blocked by CXCR4 antagonist AMD3100, establishing the importance of this receptor in FIV gp120 binding. Interestingly, CXCR4 alone was not sufficient to allow fusion by a primary isolate of FIV, as env glycoprotein from FIV-NCSU(1) failed to induce syncytia in several feline cell lines expressing CXCR4. Syncytia formation could be inhibited at a post-CXCR4 binding step by synthetic peptide T1971, which inhibits interaction of heptad repeat regions of gp41 and formation of the hairpin structure. Finally, using site-directed mutagenesis, we also show that a conserved tryptophan-rich region in the membrane proximal ectodomain of gp41 is critical for fusion, possibly at steps post hairpin structure formation.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Vírus da Imunodeficiência Felina/fisiologia , Proteínas Virais de Fusão/fisiologia , Sequência de Aminoácidos , Animais , Benzilaminas , Linfócitos T CD4-Positivos/virologia , Gatos , Linhagem Celular , Ciclamos , Células Gigantes , Compostos Heterocíclicos/farmacologia , Vírus da Imunodeficiência Felina/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/farmacologia , Conformação Proteica , Estrutura Terciária de Proteína , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/fisiologia , Alinhamento de Sequência , Triptofano , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismoRESUMO
Studies attempting to identify reservoirs of HIV-1 latency have documented that the virus persists as both a latent and productive infection in subsets of CD4(+) cells. Reports regarding establishment of a stable HIV-1 infection in quiescent T cells in vitro, however, are controversial. In the present study, we investigated the susceptibility of naive and activated CD4(+) cell subsets (distinguished by differential expression of CD25) to feline immunodeficiency virus (FIV) infection, their ability to replicate the virus, and potentially act as a reservoir for virus persistence in infected animals. While both CD4(+)CD25(+) and CD4(+)CD25(-) cells are susceptible to FIV infection in vitro and in vivo, only CD4(+)CD25(+) cells produce infectious virions when cultured with interleukin-2 (IL-2). Latently infected CD4(+)CD25(-) cells produce infectious virions following ConcanvalinA (ConA) stimulation, which correlates with upregulated surface expression of CD25. In contrast to CD4(+)CD25(-) cells, CD4(+)CD25(+) cells remain unresponsive to mitogen stimulation and are relatively resistant to apoptosis whether or not infected with FIV. The ability of CD4(+)CD25(+) cells to replicate FIV efficiently in the presence of IL-2 but remain anergic and unresponsive to apoptotic signaling suggests that these cells may provide a reservoir of productive FIV infection. On the contrary, CD4(+)CD25(-) cells seem to establish as latent viral reservoirs capable of being reactivated after stimulation.
Assuntos
Linfócitos T CD4-Positivos/virologia , Síndrome de Imunodeficiência Adquirida Felina/virologia , Vírus da Imunodeficiência Felina/fisiologia , Animais , Apoptose , Linfócitos T CD4-Positivos/imunologia , Gatos , Divisão Celular/imunologia , Modelos Animais de Doenças , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Ativação Linfocitária , Receptores de Interleucina-2/análise , Latência Viral , Replicação ViralRESUMO
OBJECTIVE: To determine whether FIV infection in captive African lions is associated with changes in immune cell variables similar to those detected in domestic cats infected with FIV. ANIMALS: 5 captive African lions naturally infected with FIV (FIV+) and 5 lions not infected with FIV (FIV-). PROCEDURE: Peripheral blood samples were collected from FIV+ lions during annual examinations conducted during a 7-year period and at a single time point from the FIV- lions. From results of CBC and flow cytometry, lymphocyte subsets were characterized and compared. RESULTS: Flow cytometric analysis revealed that the percentage and absolute number of CD4+ and CD8+ T cells were significantly lower in FIV+ lions, compared with these values in FIV- lions. In FIV+ lions, severe depletion in the absolute number of CD4+ and CD8+ T cells was detected, although this did not correlate with clinical signs. Muscle wasting was the most consistent clinical sign of infection. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that FIV+ African lions develop lymphocyte deficiencies, including significant decreases in the absolute number of CD4+ and CD8+ T cells; these findings of immune dysfunction are similar to those defined for FIV+ domestic cats. It is important to monitor the number of CD4+ T cells in infected animals as a measure of disease progression.
Assuntos
Síndrome de Imunodeficiência Adquirida Felina/imunologia , Vírus da Imunodeficiência Felina , Leões/imunologia , Linfócitos T/imunologia , Animais , Animais de Zoológico , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Gatos , Feminino , Citometria de Fluxo , North CarolinaRESUMO
Venezuelan equine encephalitis (VEE) virus-replicon particles (VRP) were used to generate feline immunodeficiency virus (FIV) Gag- and ENV-expressing vaccine vectors. Serum and mucosal FIV-specific antibody was detected in cats immunized subcutaneously, once monthly for 5 months, with FIV-expressing VRP. Expansion of the CD8+ L-selectin negative phenotype and transient CD8+ noncytolytic suppressor activity were seen in cats immunized with FIV-expressing or control VRP. Despite induction of FIV-specific immune responses and nonspecific suppressor responses, all cats became infected following vaginal challenge with high dose, pathogenic cell-associated FIV-NCSU(1) although relative early maintenance of CD4+ cells was seen in FIV-immunized cats.
Assuntos
Síndrome de Imunodeficiência Adquirida Felina/imunologia , Vírus da Imunodeficiência Felina/imunologia , Linfócitos T Reguladores/imunologia , Vacinas Virais/administração & dosagem , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Gatos , Vírus da Encefalite Equina Venezuelana/genética , Estudos de Avaliação como Assunto , Feminino , Vetores Genéticos/genética , Glicoproteínas , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vagina/imunologia , Vagina/virologia , Proteínas do Envelope Viral , Vacinas Virais/imunologia , Viremia/sangueRESUMO
Natural peptide antibiotics are part of host innate immunity against a wide range of microbes, including some viruses. Synthetic peptides modeled after natural peptide antibiotics interfere with microbial membranes and are termed peptidyl membrane-interactive molecules (peptidyl-MIM [Demegen Inc, Pittsburgh, Pa.]). Sixteen peptidyl-MIM candidates were tested for activity against feline immunodeficiency virus (FIV) on infected CrFK cells. Three of them (D4E1, DC1, and D1D6) showed potent anti-FIV activity in chronically infected CrFK cells as measured by decreased reverse transcriptase (RT) activity, having 50% inhibitory concentrations of 0.46, 0.75, and 0.94 micro M, respectively, which were approximately 10 times lower than their direct cytotoxic concentrations. Treatment of chronically infected CrFK cells with 2 micro M D4E1 for 3 days completely reversed virus-induced cytopathic effect. Immunofluorescence revealed reduced p26 staining in these cells. Treatment of chronically infected CrFK cells with 2 micro M D4E1 suppressed virus production ( approximately 50%) for up to 7 days, The virions from the D4E1-treated culture had impaired infectivity, as measured by the 50% tissue culture infectious dose and nested PCR analysis of proviral DNA. However, these noninfectious virions were able to bind and internalize, suggesting a defect at some postentry step. After chronically infected CrFK cells were treated with D4E1 for 24 h, increased cell-associated mature p26 Gag and decreased extracellular virus-associated p26 Gag were observed by Western blot analysis, suggesting that virus assembly and/or release may be blocked by D4E1 treatment, whereas virus binding, penetration, RNA synthesis, and protein synthesis appear to be unaffected. Synthetic peptide antibiotics may be useful tools in the search for antiviral drugs having a wide therapeutic window for host cells.
Assuntos
Antivirais/farmacologia , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Gatos , Produtos do Gene gag/análise , Antígenos HIV/análise , Dados de Sequência Molecular , Estrutura Secundária de Proteína , RNA Mensageiro/análise , Inibidores da Transcriptase Reversa/farmacologia , Montagem de Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência HumanaRESUMO
The B7.1 and B7.2 costimulatory molecules on antigen-presenting cells provide second signals for regulating T cell immune responses via CD28 and cytotoxic T lymphocyte antigen 4 (CTLA4) on T cells. CD28 signals cell proliferation, whereas CTLA4 signals for anergy or apoptosis, terminating the immune response. Because T cell apoptosis and immunodeficiency is a characteristic of feline immunodeficiency virus (FIV)-infected cats, it is possible that negative T cell signaling via B7 and CTLA4 may be favored in these cats. Flow cytometry revealed high percentages of CD8+ and CD4+ cells expressing B7.1, B7.2, and CTLA4 in lymph nodes of FIV-positive cats and a large fraction of CTLA4+ T cells coexpressing B7.1 and B7.2. Three-color analysis with anti-B7.1, anti-B7.2, or anti-CTLA4 and TUNEL (terminal deoxynucleotidyl transferase nick-end-labeling) analysis revealed that apoptosis was a characteristic of B7.1+ B7.2+ CTLA4+ T cells. These data support the hypothesis that lymph node apoptosis and immune deterioration in FIV-infected cats results from chronic B7.1- and/or B7.2-CTLA4-mediated T-T interactions.
Assuntos
Antígenos CD/análise , Antígenos de Diferenciação/análise , Apoptose , Antígeno B7-1/análise , Síndrome de Imunodeficiência Adquirida Felina/imunologia , Imunoconjugados , Glicoproteínas de Membrana/análise , Linfócitos T/fisiologia , Abatacepte , Animais , Antígeno B7-2 , Antígeno CTLA-4 , Gatos , Marcação In Situ das Extremidades CortadasRESUMO
Free-ranging African lion (Panthera leo) peripheral blood mononuclear cells (PBMC) were examined using flow cytometry and antibodies developed for use in the domestic cat to determine if phenotypic changes occurred in lion lymphocytes as a result of feline immunodeficiency virus (FIV) infection. The percentage of CD8 cells from lion peripheral blood was considerably lower than in the domestic cat. Lions with elevated levels of CD8+ cells were typically infected with FIV, similar to observations in the domestic cat. Antibodies against the alpha chain of the CD8 receptor (monoclonal antibody (mAb) 3.357) did not react consistently in all lions examined. Flow cytometric analysis determined that approximately 82 and 80% of the animals from Kruger and Hluhluwe-Umfolozi National Parks in South Africa reacted with the monoclonal antibody against the alpha chain of CD8 receptor, while only 17% of the lions in Etosha National Park in Namibia cross-reacted with the CD8alpha chain. There was no apparent correlation between FIV status and CD8alpha chain reactivity. The relative isolation of Etosha from the other two parks could explain the marked difference in CD8alpha chain expression and suggests that lions similar to other mammalian species demonstrate polymorphic expression of the CD8alpha chain (197).