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1.
Annu Rev Immunol ; 33: 787-821, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25706097

RESUMO

Epstein-Barr virus (EBV) is usually acquired silently early in life and carried thereafter as an asymptomatic infection of the B lymphoid system. However, many circumstances disturb the delicate EBV-host balance and cause the virus to display its pathogenic potential. Thus, primary infection in adolescence can manifest as infectious mononucleosis (IM), as a fatal illness that magnifies the immunopathology of IM in boys with the X-linked lymphoproliferative disease trait, and as a chronic active disease leading to life-threatening hemophagocytosis in rare cases of T or natural killer (NK) cell infection. Patients with primary immunodeficiencies affecting the NK and/or T cell systems, as well as immunosuppressed transplant recipients, handle EBV infections poorly, and many are at increased risk of virus-driven B-lymphoproliferative disease. By contrast, a range of other EBV-positive malignancies of lymphoid or epithelial origin arise in individuals with seemingly intact immune systems through mechanisms that remain to be understood.


Assuntos
Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Imunidade Adaptativa , Animais , Portador Sadio , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Humanos , Imunidade Inata , Hospedeiro Imunocomprometido , Síndromes de Imunodeficiência/etiologia , Transtornos Linfoproliferativos/etiologia
2.
PLoS Pathog ; 14(9): e1007110, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30248160

RESUMO

Human herpesviruses are antigenically rich agents that induce strong CD8+T cell responses in primary infection yet persist for life, continually challenging T cell memory through recurrent lytic replication and potentially influencing the spectrum of antigen-specific responses. Here we describe the first lytic proteome-wide analysis of CD8+ T cell responses to a gamma1-herpesvirus, Epstein-Barr virus (EBV), and the first such proteome-wide analysis of primary versus memory CD8+ T cell responses to any human herpesvirus. Primary effector preparations were generated directly from activated CD8+ T cells in the blood of infectious mononucleosis (IM) patients by in vitro mitogenic expansion. For memory preparations, EBV-specific cells in the blood of long-term virus carriers were first re-stimulated in vitro by autologous dendritic cells loaded with a lysate of lytically-infected cells, then expanded as for IM cells. Preparations from 7 donors of each type were screened against each of 70 EBV lytic cycle proteins in combination with the donor's individual HLA class I alleles. Multiple reactivities against immediate early (IE), early (E) and late (L) lytic cycle proteins, including many hitherto unrecognised targets, were detected in both contexts. Interestingly however, the two donor cohorts showed a different balance between IE, E and L reactivities. Primary responses targeted IE and a small group of E proteins preferentially, seemingly in line with their better presentation on the infected cell surface before later-expressed viral evasins take full hold. By contrast, target choice equilibrates in virus carriage with responses to key IE and E antigens still present but with responses to a select subset of L proteins now often prominent. We infer that, for EBV at least, long-term virus carriage with its low level virus replication and lytic antigen release is associated with a re-shaping of the virus-specific response.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/patogenicidade , Mononucleose Infecciosa/imunologia , Proteoma/imunologia , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Portador Sadio/imunologia , Portador Sadio/virologia , Expressão Gênica/genética , Genes Virais , Antígenos HLA/imunologia , Herpesvirus Humano 4/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mononucleose Infecciosa/metabolismo , Mononucleose Infecciosa/virologia , Proteoma/metabolismo
3.
J Virol ; 91(21)2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-28835490

RESUMO

Epstein-Barr virus (EBV) is typically acquired asymptomatically in childhood. In contrast, infection later in life often leads to infectious mononucleosis (IM), a febrile illness characterized by anti-EBV IgM antibody positivity, high loads of circulating latently infected B cells, and a marked lymphocytosis caused by hyperexpansion of EBV-specific CD8+ T cells plus a milder expansion of CD56dim NKG2A+ KIR- natural killer (NK) cells. How the two situations compare is unclear due to the paucity of studies on clinically silent infection. Here we describe five prospectively studied patients with asymptomatic infections identified in a seroepidemiologic survey of university entrants. In each case, the key blood sample had high cell-associated viral loads without a marked CD8 lymphocytosis or NK cell disturbance like those seen in patients during the acute phase of IM. Two of the cases with the highest viral loads showed a coincident expansion of activated EBV-specific CD8+ T cells, but overall CD8+ T cell numbers were either unaffected or only mildly increased. Two cases with slightly lower loads, in whom serology suggests the infection may have been caught earlier in the course of infection, also showed no T or NK cell expansion at the time. Interestingly, in another case with a higher viral load, in which T and NK cell responses were undetectable in the primary blood sample in which infection was detected, EBV-specific T cell responses did not appear until several months later, by which time the viral loads in the blood had already fallen. Thus, some patients with asymptomatic primary infections have very high circulating viral loads similar to those in patients during the acute phase of IM and a cell-mediated immune response that is qualitatively similar to that in IM patients but of a lower magnitude. However, other patients may have quite different immune responses that ultimately could reveal novel mechanisms of host control.IMPORTANCE Epstein-Barr virus (EBV) is transmitted orally, replicates in the throat, and then invades the B lymphocyte pool through a growth-transforming latent infection. While primary infection in childhood is usually asymptomatic, delayed infection is associated with infectious mononucleosis (IM), a febrile illness in which patients have high circulating viral loads and an exaggerated virus-induced immune response involving both CD8+ T cells and natural killer (NK) cells. Here we show that in five cases of asymptomatic infection, viral loads in the blood were as high as those in patients during the acute phase of IM, whereas the cell-mediated responses, even when they resembled those in patients during the acute phase of IM in timing and quality, were never as exaggerated. We infer that IM symptoms arise as a consequence not of the virus infection per se but of the hyperactivated immune response. Interestingly, there were idiosyncratic differences among asymptomatic cases in the relationship between the viral load and the response kinetics, emphasizing how much there is still to learn about primary EBV infection.


Assuntos
Infecções Assintomáticas/epidemiologia , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Vírus Epstein-Barr/virologia , Células Matadoras Naturais/imunologia , Adulto , Anticorpos Antivirais/sangue , DNA Viral/genética , Infecções por Vírus Epstein-Barr/diagnóstico , Infecções por Vírus Epstein-Barr/imunologia , Feminino , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/isolamento & purificação , Humanos , Masculino , Prognóstico , Estudos Prospectivos , Reino Unido/epidemiologia , Carga Viral , Adulto Jovem
4.
PLoS Pathog ; 12(11): e1006042, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27893813

RESUMO

Kaposi sarcoma-associated herpesvirus (KSHV) is linked with the development of Kaposi sarcoma and the B lymphocyte disorders primary effusion lymphoma (PEL) and multi-centric Castleman disease. T cell immunity limits KSHV infection and disease, however the virus employs multiple mechanisms to inhibit efficient control by these effectors. Thus KSHV-specific CD4+ T cells poorly recognize most PEL cells and even where they can, they are unable to kill them. To make KSHV-infected cells more sensitive to T cell control we treated PEL cells with the thymidine analogue azidothymidine (AZT), which sensitizes PEL lines to Fas-ligand and TRAIL challenge; effector mechanisms which T cells use. PELs co-cultured with KSHV-specific CD4+ T cells in the absence of AZT showed no control of PEL outgrowth. However in the presence of AZT PEL outgrowth was controlled in an MHC-restricted manner. To investigate how AZT sensitizes PELs to immune control we first examined BJAB cells transduced with individual KSHV-latent genes for their ability to resist apoptosis mediated by stimuli delivered through Fas and TRAIL receptors. This showed that in addition to the previously described vFLIP protein, expression of vIRF3 also inhibited apoptosis delivered by these stimuli. Importantly vIRF3 mediated protection from these apoptotic stimuli was inhibited in the presence of AZT as was a second vIRF3 associated phenotype, the downregulation of surface MHC class II. Although both vFLIP and vIRF3 are expressed in PELs, we propose that inhibiting vIRF3 function with AZT may be sufficient to restore T cell control of these tumor cells.


Assuntos
Antivirais/farmacologia , Linfócitos T CD4-Positivos/imunologia , Fatores Reguladores de Interferon/metabolismo , Linfoma de Efusão Primária/imunologia , Evasão Tumoral/efeitos dos fármacos , Proteínas Virais/metabolismo , Zidovudina/farmacologia , Linhagem Celular , Infecções por Herpesviridae/imunologia , Herpesvirus Humano 8 , Humanos , Reação em Cadeia da Polimerase , Evasão Tumoral/imunologia
5.
PLoS Pathog ; 12(8): e1005799, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27540722

RESUMO

Disruption of T cell memory during severe immune suppression results in reactivation of chronic viral infections, such as Epstein Barr virus (EBV) and Cytomegalovirus (CMV). How different subsets of memory T cells contribute to the protective immunity against these viruses remains poorly defined. In this study we examined the compartmentalization of virus-specific, tissue resident memory CD8+ T cells in human lymphoid organs. This revealed two distinct populations of memory CD8+ T cells, that were CD69+CD103+ and CD69+CD103-, and were retained within the spleen and tonsils in the absence of recent T cell stimulation. These two types of memory cells were distinct not only in their phenotype and transcriptional profile, but also in their anatomical localization within tonsils and spleen. The EBV-specific, but not CMV-specific, CD8+ memory T cells preferentially accumulated in the tonsils and acquired a phenotype that ensured their retention at the epithelial sites where EBV replicates. In vitro studies revealed that the cytokine IL-15 can potentiate the retention of circulating effector memory CD8+ T cells by down-regulating the expression of sphingosine-1-phosphate receptor, required for T cell exit from tissues, and its transcriptional activator, Kruppel-like factor 2 (KLF2). Within the tonsils the expression of IL-15 was detected in regions where CD8+ T cells localized, further supporting a role for this cytokine in T cell retention. Together this study provides evidence for the compartmentalization of distinct types of resident memory T cells that could contribute to the long-term protection against persisting viral infections.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Memória Imunológica , Antígenos CD/imunologia , Linfócitos T CD8-Positivos/patologia , Infecções por Citomegalovirus/patologia , Infecções por Vírus Epstein-Barr/patologia , Feminino , Humanos , Interleucina-15/imunologia , Fatores de Transcrição Kruppel-Like/imunologia , Masculino , Especificidade de Órgãos/imunologia
6.
J Virol ; 90(8): 3849-3859, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26819313

RESUMO

UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) has tropism for B lymphocytes, in which it establishes latency, and can also cause lymphoproliferative disorders of these cells manifesting as primary effusion lymphoma (PEL) and multicentric Castleman disease (MCD). T cell immunity is vital for the control of KSHV infection and disease; however, few models of B lymphocyte infection exist to study immune recognition of such cells. Here, we developed a model of B lymphocyte infection with KSHV in which infected tonsillar B lymphocytes were expanded by providing mitogenic stimuli and then challenged with KSHV-specific CD4(+)T cells. The infected cells expressed viral proteins found in PELs, namely, LANA and viral IRF3 (vIRF3), albeit at lower levels, with similar patterns of gene expression for the major latency, viral interleukin 6 (vIL-6), and vIRF3 transcripts. Despite low-level expression of open reading frame 50 (ORF50), transcripts for the immune evasion genes K3 and K5 were detected, with some downregulation of cell surface-expressed CD86 and ICAM. The vast majority of infected lymphocytes expressed IgM heavy chains with Igλ light chains, recapitulating the features seen in infected cells in MCD. We assessed the ability of the infected lymphocytes to be targeted by a panel of major histocompatibility complex (MHC) class II-matched CD4(+)T cells and found that LANA-specific T cells restricted to different epitopes recognized these infected cells. Given that at least some KSHV latent antigens are thought to be poor targets for CD8(+)T cells, we suggest that CD4(+)T cells are potentially important effectors for thein vivocontrol of KSHV-infected B lymphocytes. IMPORTANCE: KSHV establishes a latent reservoir within B lymphocytes, but few models exist to study KSHV-infected B cells other than the transformed PEL cell lines, which have likely accrued mutations during the transformation process. We developed a model of KSHV-infected primary B lymphocytes that recapitulates features seen in PEL and MCD by gene expression and cell phenotype analysis, allowing the study of T cell recognition of these cells. Challenge of KSHV-infected B cells with CD4(+)T cells specific for LANA, a protein expressed in all KSHV-infected cells and malignanciesin vivo, showed that these effectors could efficiently recognize such targets. Given that the virus expresses immune evasion genes or uses proteins with intrinsic properties, such as LANA, that minimize epitope recognition by CD8(+)T cells, CD4(+)T cell immunity to KSHV may be important for maintaining the virus-host balance.


Assuntos
Antígenos Virais/imunologia , Linfócitos B/virologia , Linfócitos T CD4-Positivos/imunologia , Transformação Celular Viral , Herpesvirus Humano 8/fisiologia , Proteínas Nucleares/imunologia , Antígenos de Superfície/imunologia , Proliferação de Células , Células Cultivadas , Expressão Gênica , Genes Virais , Herpesvirus Humano 8/genética , Humanos , Fatores Reguladores de Interferon/imunologia , Modelos Biológicos , Tonsila Palatina/citologia , Fenótipo , Receptores Imunológicos/biossíntese , Proteínas Virais/imunologia
7.
PLoS Pathog ; 11(3): e1004746, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25816224

RESUMO

Epstein-Barr virus (EBV) infection often occurs in early childhood and is asymptomatic. However, if delayed until adolescence, primary infection may manifest as acute infectious mononucleosis (AIM), a febrile illness characterised by global CD8+ T-cell lymphocytosis, much of it reflecting a huge expansion of activated EBV-specific CD8+ T-cells. While the events of AIM have been intensely studied, little is known about how these relate to asymptomatic primary infection. Here Gambian children (14-18 months old, an age at which many acquire the virus) were followed for the ensuing six months, monitoring circulating EBV loads, antibody status against virus capsid antigen (VCA) and both total and virus-specific CD8+ T-cell numbers. Many children were IgG anti-VCA-positive and, though no longer IgM-positive, still retained high virus loads comparable to AIM patients and had detectable EBV-specific T-cells, some still expressing activation markers. Virus loads and the frequency/activation status of specific T-cells decreased over time, consistent with resolution of a relatively recent primary infection. Six children with similarly high EBV loads were IgM anti-VCA-positive, indicating very recent infection. In three of these donors with HLA types allowing MHC-tetramer analysis, highly activated EBV-specific T-cells were detectable in the blood with one individual epitope response reaching 15% of all CD8+ T-cells. That response was culled and the cells lost activation markers over time, just as seen in AIM. However, unlike AIM, these events occurred without marked expansion of total CD8+ numbers. Thus asymptomatic EBV infection in children elicits a virus-specific CD8+ T-cell response that can control the infection without over-expansion; conversely, in AIM it appears the CD8 over-expansion, rather than virus load per se, is the cause of disease symptoms.


Assuntos
Anticorpos Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Imunoglobulina M/imunologia , Anticorpos Antivirais/sangue , Linfócitos T CD8-Positivos/metabolismo , Infecções por Vírus Epstein-Barr/sangue , Infecções por Vírus Epstein-Barr/epidemiologia , Feminino , Gâmbia/epidemiologia , Humanos , Imunoglobulina M/sangue , Lactente , Contagem de Linfócitos , Masculino
8.
Trends Immunol ; 35(4): 159-69, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24589417

RESUMO

Epstein-Barr virus (EBV), a human herpesvirus with potent B cell growth transforming ability, induces multiple cellular immune responses in the infected host. How these host responses work together to prevent virus pathogenicity, and how immune imbalance predisposes to disease, remain poorly understood. Here, we describe three ongoing lines of enquiry that are shedding new light on these issues. These focus on: (i) patients with infectious mononucleosis or its fatal equivalent, X-linked lymphoproliferative disease; (ii) EBV infection in a range of new, genetically defined, primary immune deficiency states; and (iii) experimental infection in two complementary animal models, the rhesus macaque and the human haemopoietic stem cell reconstituted mouse.


Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Imunidade Celular/imunologia , Animais , Modelos Animais de Doenças , Humanos
9.
PLoS Pathog ; 10(8): e1004322, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25144360

RESUMO

CD8+ T cell responses to Epstein-Barr virus (EBV) lytic cycle expressed antigens display a hierarchy of immunodominance, in which responses to epitopes of immediate-early (IE) and some early (E) antigens are more frequently observed than responses to epitopes of late (L) expressed antigens. It has been proposed that this hierarchy, which correlates with the phase-specific efficiency of antigen presentation, may be due to the influence of viral immune-evasion genes. At least three EBV-encoded genes, BNLF2a, BGLF5 and BILF1, have the potential to inhibit processing and presentation of CD8+ T cell epitopes. Here we examined the relative contribution of these genes to modulation of CD8+ T cell recognition of EBV lytic antigens expressed at different phases of the replication cycle in EBV-transformed B-cells (LCLs) which spontaneously reactivate lytic cycle. Selective shRNA-mediated knockdown of BNLF2a expression led to more efficient recognition of immediate-early (IE)- and early (E)-derived epitopes by CD8+ T cells, while knock down of BILF1 increased recognition of epitopes from E and late (L)-expressed antigens. Contrary to what might have been predicted from previous ectopic expression studies in EBV-negative model cell lines, the shRNA-mediated inhibition of BGLF5 expression in LCLs showed only modest, if any, increase in recognition of epitopes expressed in any phase of lytic cycle. These data indicate that whilst BNLF2a interferes with antigen presentation with diminishing efficiency as lytic cycle progresses (IE>E>>L), interference by BILF1 increases with progression through lytic cycle (IE

Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Evasão da Resposta Imune/imunologia , Western Blotting , Desoxirribonucleases/imunologia , Técnicas de Silenciamento de Genes , Herpesvirus Humano 4/imunologia , Humanos , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/imunologia , Proteínas da Matriz Viral/imunologia , Proteínas Virais/imunologia
10.
Curr Top Microbiol Immunol ; 391: 325-53, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26428380

RESUMO

Epstein-Barr virus (EBV) is arguably one of the most successful pathogens of humans, persistently infecting over ninety percent of the world's population. Despite this high frequency of carriage, the virus causes apparently few adverse effects in the vast majority of infected individuals. Nevertheless, the potent growth transforming ability of EBV means the virus has the potential to cause malignancies in infected individuals. Indeed, EBV is thought to cause 1% of human malignancies, equating to 200,000 malignancies each year. A clear factor as to why virus-induced disease is relatively infrequent in healthy infected individuals is the presence of a potent immune response to EBV, in particular, that mediated by T cells. Thus, patient groups with immunodeficiencies or whose cellular immune response is suppressed have much higher frequencies of EBV-induced disease and, in at least some cases, these diseases can be controlled by restoration of the T-cell compartment. In this chapter, we will primarily review the role the αß subset of T cells in the control of EBV in healthy and diseased individuals.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Animais , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/genética , Humanos
11.
PLoS Pathog ; 9(10): e1003751, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24204280

RESUMO

Peptides presentation to T cells by MHC class II molecules is of importance in initiation of immune response to a pathogen. The level of MHC II expression directly influences T lymphocyte activation and is often targeted by various viruses. Kaposi's sarcoma-associated herpesvirus (KSHV) encoded LANA is known to evade MHC class I peptide processing, however, the effect of LANA on MHC class II remains unclear. Here, we report that LANA down-regulates MHC II expression and presentation by inhibiting the transcription of MHC II transactivator (CIITA) promoter pIII and pIV in a dose-dependent manner. Strikingly, although LANA knockdown efficiently disrupts the inhibition of CIITA transcripts from its pIII and pIV promoter region, the expression of HLA-DQß but no other MHC II molecules was significantly restored. Moreover, we revealed that the presentation of HLA-DQß enhanced by LANA knockdown did not help LANA-specific CD4+ T cell recognition of PEL cells, and the inhibition of CIITA by LANA is independent of IL-4 or IFN-γ signaling but dependent on the direct interaction of LANA with IRF-4 (an activator of both the pIII and pIV CIITA promoters). This interaction dramatically blocked the DNA-binding ability of IRF-4 on both pIII and pIV promoters. Thus, our data implies that LANA can evade MHC II presentation and suppress CIITA transcription to provide a unique strategy of KSHV escape from immune surveillance by cytotoxic T cells.


Assuntos
Apresentação de Antígeno , Antígenos Virais/imunologia , Regulação para Baixo , Cadeias beta de HLA-DQ/imunologia , Herpesvirus Humano 8/imunologia , Fatores Reguladores de Interferon/imunologia , Proteínas Nucleares/imunologia , Transativadores/imunologia , Antígenos Virais/genética , Antígenos Virais/metabolismo , Feminino , Células HEK293 , Cadeias beta de HLA-DQ/genética , Cadeias beta de HLA-DQ/metabolismo , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-4/metabolismo , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Elementos de Resposta/genética , Elementos de Resposta/imunologia , Transativadores/genética , Transativadores/metabolismo , Transcrição Gênica/genética , Transcrição Gênica/imunologia
12.
J Virol ; 87(10): 5340-50, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23449805

RESUMO

Evasion of immune T cell responses is crucial for persistent viruses to establish a normal carrier state. Most studies on active immune modulation mechanisms have focused on the stage of virus production in infected cells, when large numbers of viral antigens and potential immune modulators are expressed. For oncogenic viruses such as Kaposi's sarcoma-associated herpesvirus (KSHV), which is carried as a lifelong infection, usually with little harmful effect, but can cause various tumors, the immune evasion strategies can also be relevant in the context of tumorigenesis. Here we report that the virus-encoded interferon regulatory factor 3 (vIRF3) latent viral gene expressed in KSHV-related tumors functions as a potent immunevasin. Expression of vIRF3 downregulates surface major histocompatibility complex class II (MHC-II) DR expression with slow kinetics but, more importantly, can substantially inhibit recognition by KSHV-specific CD4 T cells prior to its effects on MHC-II DR downregulation in model cell systems. This property of vIRF3 is only partly due to its ability to inhibit the transcription of CIITA and, thus, MHC-II expression; CIITA-independent inhibition of MHC-II transcripts and another as yet unidentified posttranscriptional mechanism are also involved in qualitatively modulating the availability of specific peptide/MHC-II complexes at the cell surface. Consistent with these observations, the vIRF3-expressing KSHV-associated primary effusion lymphoma (PEL) lines are generally resistant to recognition by KSHV-specific CD4 T cells. Interestingly, some PEL lines exhibit small subpopulations with lower vIRF3 expression that can be recognized. These data implicate vIRF3 as a critical determinant of the MHC-II antigen presentation function in KSHV-associated PELs that is likely to be important in the pathogenesis of these tumors.


Assuntos
Apresentação de Antígeno , Herpesvirus Humano 8/patogenicidade , Antígenos de Histocompatibilidade Classe II/metabolismo , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Fatores Reguladores de Interferon/metabolismo , Transativadores/antagonistas & inibidores , Proteínas Virais/metabolismo , Transformação Celular Neoplásica , Regulação para Baixo , Herpesvirus Humano 8/imunologia , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos
13.
Blood ; 119(9): 2083-92, 2012 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-22234686

RESUMO

T-cell immunity is important for controlling Kaposi sarcoma-associated herpesvirus (KSHV) diseases such as the endothelial cell malignancy Kaposi sarcoma, or the B-cell malignancy, primary effusion lymphoma (PEL). However, little is known about KSHV-specific T-cell immunity in healthy donors and immune control of disease. Using PBMCs from healthy KSHV-infected donors, we found weak ex vivo responses to the KSHV latent antigens LANA, vFLIP, vCyclin, and Kaposin, with LANA most frequently recognized. CD4(+) T-cell clones specific to LANA, a protein expressed in all KSHV-infected cells and malignancies, were established to determine whether they could recognize LANA-expressing cells. B-cell targets expressing or fed LANA protein were consistently recognized by the clones; however, most PEL cell lines were not. PELs express the KSHV protein vIRF3 that inhibits promoter function of the HLA class II transactivator, decreasing expression of genes controlled by this transactivator. Re-expressing the class II transactivator in the PELs increased expression of downstream targets such as HLA class II and restored recognition but not killing by the LANA-specific clones. We suggest that PELs are poorly controlled in vivo because of inefficient recognition and killing by T cells.


Assuntos
Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Herpesvirus Humano 8/imunologia , Imunidade Celular/imunologia , Linfoma de Efusão Primária/imunologia , Proteínas Nucleares/imunologia , Antígenos Virais/genética , Células Cultivadas , Herpesvirus Humano 8/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Linfoma de Efusão Primária/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Sarcoma de Kaposi/imunologia , Doadores de Tecidos , Transativadores/metabolismo
14.
PLoS Biol ; 9(11): e1001187, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22069374

RESUMO

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency caused by mutations in SH2D1A which encodes SAP. SAP functions in signalling pathways elicited by the SLAM family of leukocyte receptors. A defining feature of XLP is exquisite sensitivity to infection with EBV, a B-lymphotropic virus, but not other viruses. Although previous studies have identified defects in lymphocytes from XLP patients, the unique role of SAP in controlling EBV infection remains unresolved. We describe a novel approach to this question using female XLP carriers who, due to random X-inactivation, contain both SAP(+) and SAP(-) cells. This represents the human equivalent of a mixed bone marrow chimera in mice. While memory CD8(+) T cells specific for CMV and influenza were distributed across SAP(+) and SAP(-) populations, EBV-specific cells were exclusively SAP(+). The preferential recruitment of SAP(+) cells by EBV reflected the tropism of EBV for B cells, and the requirement for SAP expression in CD8(+) T cells for them to respond to Ag-presentation by B cells, but not other cell types. The inability of SAP(-) clones to respond to Ag-presenting B cells was overcome by blocking the SLAM receptors NTB-A and 2B4, while ectopic expression of NTB-A on fibroblasts inhibited cytotoxicity of SAP(-) CD8(+) T cells, thereby demonstrating that SLAM receptors acquire inhibitory function in the absence of SAP. The innovative XLP carrier model allowed us to unravel the mechanisms underlying the unique susceptibility of XLP patients to EBV infection in the absence of a relevant animal model. We found that this reflected the nature of the Ag-presenting cell, rather than EBV itself. Our data also identified a pathological signalling pathway that could be targeted to treat patients with severe EBV infection. This system may allow the study of other human diseases where heterozygous gene expression from random X-chromosome inactivation can be exploited.


Assuntos
Herpesvirus Humano 4/patogenicidade , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Transtornos Linfoproliferativos/patologia , Antígenos CD/imunologia , Linfócitos B/patologia , Linfócitos B/virologia , Antígeno CD48 , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Células Dendríticas/imunologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Feminino , Genótipo , Herpesvirus Humano 4/imunologia , Humanos , Switching de Imunoglobulina , Influenza Humana/imunologia , Influenza Humana/virologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/virologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/virologia , Orthomyxoviridae/imunologia , Orthomyxoviridae/patogenicidade , Receptores de Superfície Celular/imunologia , Receptores Imunológicos/imunologia , Transdução de Sinais , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Inativação do Cromossomo X
15.
J Exp Med ; 204(8): 1863-73, 2007 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-17620360

RESUMO

gamma 1-Herpesviruses such as Epstein-Barr virus (EBV) have a unique ability to amplify virus loads in vivo through latent growth-transforming infection. Whether they, like alpha- and beta-herpesviruses, have been driven to actively evade immune detection of replicative (lytic) infection remains a moot point. We were prompted to readdress this question by recent work (Pudney, V.A., A.M. Leese, A.B. Rickinson, and A.D. Hislop. 2005. J. Exp. Med. 201:349-360; Ressing, M.E., S.E. Keating, D. van Leeuwen, D. Koppers-Lalic, I.Y. Pappworth, E.J.H.J. Wiertz, and M. Rowe. 2005. J. Immunol. 174:6829-6838) showing that, as EBV-infected cells move through the lytic cycle, their susceptibility to EBV-specific CD8(+) T cell recognition falls dramatically, concomitant with a reductions in transporter associated with antigen processing (TAP) function and surface human histocompatibility leukocyte antigen (HLA) class I expression. Screening of genes that are unique to EBV and closely related gamma 1-herpesviruses of Old World primates identified an early EBV lytic cycle gene, BNLF2a, which efficiently blocks antigen-specific CD8(+) T cell recognition through HLA-A-, HLA-B-, and HLA-C-restricting alleles when expressed in target cells in vitro. The small (60-amino acid) BNLF2a protein mediated its effects through interacting with the TAP complex and inhibiting both its peptide- and ATP-binding functions. Furthermore, this targeting of the major histocompatibility complex class I pathway appears to be conserved among the BNLF2a homologues of Old World primate gamma 1-herpesviruses. Thus, even the acquisition of latent cycle genes endowing unique growth-transforming ability has not liberated these agents from evolutionary pressure to evade CD8(+) T cell control over virus replicative foci.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Regulação da Expressão Gênica , Herpesvirus Humano 4/metabolismo , Sequência de Aminoácidos , Animais , Linfócitos T CD8-Positivos/metabolismo , Cercopithecidae , Clonagem Molecular , Citometria de Fluxo , Antígenos HLA/química , Antígenos HLA/metabolismo , Herpesviridae/metabolismo , Humanos , Sistema Imunitário/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Homologia de Sequência de Aminoácidos
16.
J Virol ; 86(11): 6246-57, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22491458

RESUMO

Regulating appropriate activation of the immune response in the healthy host despite continual immune surveillance dictates that immune responses must be either self-limiting and therefore negatively regulated following their activation or prevented from developing inappropriately. In the case of antigen-specific T cells, their response is attenuated by several mechanisms, including ligation of CTLA-4 and PD-1. Through the study of the viral OX2 (vOX2) immunoregulator encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), we have identified a T cell-attenuating role both for this protein and for CD200, a cellular orthologue of the viral vOX2 protein. In vitro, antigen-presenting cells (APC) expressing either native vOX2 or CD200 suppressed two functions of cognate antigen-specific T cell clones: gamma interferon (IFN-γ) production and mobilization of CD107a, a cytolytic granule component and measure of target cell killing ability. Mechanistically, vOX2 and CD200 expression on APC suppressed the phosphorylation of ERK1/2 mitogen-activated protein kinase in responding T cells. These data provide the first evidence for a role of both KSHV vOX2 and cellular CD200 in the negative regulation of antigen-specific T cell responses. They suggest that KSHV has evolved to harness the host CD200-based mechanism of attenuation of T cell responses to facilitate virus persistence and dissemination within the infected individual. Moreover, our studies define a new paradigm in immune modulation by viruses: the provision of a negative costimulatory signal to T cells by a virus-encoded orthologue of CD200.


Assuntos
Antígenos CD/metabolismo , Herpesvirus Humano 8/imunologia , Herpesvirus Humano 8/patogenicidade , Tolerância Imunológica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neuropeptídeos/metabolismo , Linfócitos T/imunologia , Proteínas Virais/metabolismo , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/virologia , Antígenos CD/imunologia , Humanos , Interferon gama/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Receptores de Orexina , Receptores Acoplados a Proteínas G/imunologia , Receptores de Neuropeptídeos/imunologia , Proteínas Virais/imunologia , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo
17.
PLoS Pathog ; 7(12): e1002455, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22216005

RESUMO

Evasion of immune T cell responses is crucial for viruses to establish persistence in the infected host. Immune evasion mechanisms of Epstein-Barr virus (EBV) in the context of MHC-I antigen presentation have been well studied. In contrast, viral interference with MHC-II antigen presentation is less well understood, not only for EBV but also for other persistent viruses. Here we show that the EBV encoded BZLF1 can interfere with recognition by immune CD4+ effector T cells. This impaired T cell recognition occurred in the absence of a reduction in the expression of surface MHC-II, but correlated with a marked downregulation of surface CD74 on the target cells. Furthermore, impaired CD4+ T cell recognition was also observed with target cells where CD74 expression was downregulated by shRNA-mediated inhibition. BZLF1 downregulated surface CD74 via a post-transcriptional mechanism distinct from its previously reported effect on the CIITA promoter. In addition to being a chaperone for MHC-II αß dimers, CD74 also functions as a surface receptor for macrophage Migration Inhibitory Factor and enhances cell survival through transcriptional upregulation of Bcl-2 family members. The immune-evasion function of BZLF1 therefore comes at a cost of induced toxicity. However, during EBV lytic cycle induced by BZLF1 expression, this toxicity can be overcome by expression of the vBcl-2, BHRF1, at an early stage of lytic infection. We conclude that by inhibiting apoptosis, the vBcl-2 not only maintains cell viability to allow sufficient time for synthesis and accumulation of infectious virus progeny, but also enables BZLF1 to effect its immune evasion function.


Assuntos
Antígenos de Diferenciação de Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Regulação para Baixo/imunologia , Herpesvirus Humano 4/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Evasão da Resposta Imune , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Transativadores/imunologia , Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígenos de Diferenciação de Linfócitos B/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Regulação para Baixo/genética , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/metabolismo , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transativadores/genética , Transativadores/metabolismo
18.
J Immunol ; 186(6): 3594-605, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21296983

RESUMO

EBV, the prototypic human γ(1)-herpesvirus, persists for life in infected individuals, despite the presence of vigorous antiviral immunity. CTLs play an important role in the protection against viral infections, which they detect through recognition of virus-encoded peptides presented in the context of HLA class I molecules at the cell surface. The viral peptides are generated in the cytosol and are transported into the endoplasmic reticulum (ER) by TAP. The EBV-encoded lytic-phase protein BNLF2a acts as a powerful inhibitor of TAP. Consequently, loading of antigenic peptides onto HLA class I molecules is hampered, and recognition of BNLF2a-expressing cells by cytotoxic T cells is avoided. In this study, we characterize BNLF2a as a tail-anchored (TA) protein and elucidate its mode of action. Its hydrophilic N-terminal domain is located in the cytosol, whereas its hydrophobic C-terminal domain is inserted into membranes posttranslationally. TAP has no role in membrane insertion of BNLF2a. Instead, Asna1 (also named TRC40), a cellular protein involved in posttranslational membrane insertion of TA proteins, is responsible for integration of BNLF2a into the ER membrane. Asna1 is thereby required for efficient BNLF2a-mediated HLA class I downregulation. To optimally accomplish immune evasion, BNLF2a is composed of two specialized domains: its C-terminal tail anchor ensures membrane integration and ER retention, whereas its cytosolic N terminus accomplishes inhibition of TAP function. These results illustrate how EBV exploits a cellular pathway for TA protein biogenesis to achieve immune evasion, and they highlight the exquisite adaptation of this virus to its host.


Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Regulação para Baixo/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/imunologia , Proteínas da Matriz Viral/fisiologia , Integração Viral/imunologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/fisiologia , Sequência de Aminoácidos , ATPases Transportadoras de Arsenito/fisiologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Células HEK293 , Células HeLa , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/fisiologia , Proteínas da Matriz Viral/química
19.
J Virol ; 85(4): 1604-14, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21123379

RESUMO

Despite triggering strong immune responses, Epstein-Barr virus (EBV) has colonized more than 90% of the adult human population. Successful persistence of EBV depends on the establishment of a balance between host immune responses and viral immune evasion. Here we have extended our studies on the EBV-encoded BILF1 protein, which was recently identified as an immunoevasin that functions by enhancing degradation of major histocompatibility complex class I (MHC-I) antigens via lysosomes. We now demonstrate that disruption of the EKT signaling motif of BILF1 by a K122A mutation impairs the ability of BILF1 to enhance endocytosis of surface MHC-I molecules, while subsequent lysosomal degradation was impaired by deletion of the 21-residue C-terminal tail of BILF1. Furthermore, we identified another mechanism of BILF1 immunomodulation: it targets newly synthesized MHC-I/peptide complexes en route to the cell surface. Importantly, although the diversion of MHC-I on the exocytic pathway caused a relatively modest reduction in cell surface MHC-I, presentation of endogenously processed target peptides to immune CD8(+) effector T cells was reduced by around 65%. The immune-modulating functions of BILF1 in the context of the whole virus were confirmed in cells lytically infected with a recombinant EBV in which BILF1 was deleted. This study therefore extends our initial observations on BILF1 to show that this immunoevasin can target MHC-I antigen presentation via both the exocytic and endocytic trafficking pathways. The results also emphasize the merits of including functional T cell recognition assays to gain a more complete picture of immunoevasin effects on the antigen presentation pathway.


Assuntos
Apresentação de Antígeno/imunologia , Endocitose/imunologia , Exocitose/imunologia , Herpesvirus Humano 4/patogenicidade , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica/imunologia , Células HEK293 , Herpesvirus Humano 4/fisiologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Camundongos , Transdução de Sinais , Linfócitos T Citotóxicos/imunologia
20.
Blood ; 116(17): 3249-57, 2010 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-20644117

RESUMO

X-linked lymphoproliferative disease (XLP) is a condition associated with mutations in the signaling lymphocytic activation molecule (SLAM)-associated protein (SAP; SH2D1A). SAP functions as an adaptor, binding to and recruiting signaling molecules to SLAM family receptors expressed on T and natural killer cells. XLP is associated with extreme sensitivity to primary Epstein-Barr virus (EBV) infection, often leading to a lethal infectious mononucleosis. To investigate EBV-specific immunity in XLP patients, we studied 5 individuals who had survived EBV infection and found CD8(+) T-cell responses numerically comparable with healthy donors. However, further investigation of in vitro-derived CD8(+) T-cell clones established from 2 of these donors showed they efficiently recognized SLAM ligand-negative target cells expressing EBV antigens, but showed impaired recognition of EBV-transformed, SLAM ligand-positive, lymphoblastoid cell lines (LCLs). Importantly, LCL recognition was restored when interactions between the SLAM receptors CD244 and natural killer-, T-, and B-cell antigen (NTBA) and their ligands on LCLs were blocked. We propose that XLP patients' particular sensitivity to EBV, and not to other viruses, reflects at least in part EBV's strict tropism for B lymphocytes and the often inability of the CD8(+) T-cell response to contain the primary infection of SLAM ligand-expressing target cells.


Assuntos
Antígenos CD/imunologia , Linfócitos B/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/imunologia , Transtornos Linfoproliferativos/imunologia , Receptores de Superfície Celular/imunologia , Células Apresentadoras de Antígenos/imunologia , Células Cultivadas , Infecções por Vírus Epstein-Barr/complicações , Fibroblastos/imunologia , Humanos , Transtornos Linfoproliferativos/complicações , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Linfócitos T/imunologia
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