RESUMO
Infection with human immunodeficiency virus 1 (HIV-1) results in the dissemination of virus to gut-associated lymphoid tissue. Subsequently, HIV-1 mediates massive depletion of gut CD4+ T cells, which contributes to HIV-1-induced immune dysfunction. The migration of lymphocytes to gut-associated lymphoid tissue is mediated by integrin alpha4beta7. We demonstrate here that the HIV-1 envelope protein gp120 bound to an activated form of alpha4beta7. This interaction was mediated by a tripeptide in the V2 loop of gp120, a peptide motif that mimics structures presented by the natural ligands of alpha4beta7. On CD4+ T cells, engagement of alpha4beta7 by gp120 resulted in rapid activation of LFA-1, the central integrin involved in the establishment of virological synapses, which facilitate efficient cell-to-cell spreading of HIV-1.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , HIV-1/imunologia , Integrinas/metabolismo , Mucosa Intestinal/imunologia , Linfócitos T CD4-Positivos/virologia , Movimento Celular/imunologia , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/virologia , Citometria de Fluxo , Humanos , Mucosa Intestinal/virologia , Células Matadoras Naturais/imunologia , Ligantes , Antígeno-1 Associado à Função Linfocitária/metabolismo , Ligação Proteica/imunologia , Transdução de Sinais/imunologiaRESUMO
Antigen-activated lymphocytes undergo extraordinarily rapid cell division in the course of immune responses. We hypothesized that this unique aspect of lymphocyte biology leads to unusual genomic stress in recently antigen-activated lymphocytes and that targeted manipulation of DNA damage-response (DDR) signaling pathways would allow for selective therapeutic targeting of pathological T cells in disease contexts. Consistent with these hypotheses, we found that activated mouse and human T cells display a pronounced DDR in vitro and in vivo. Upon screening a variety of small-molecule compounds, we found that potentiation of p53 (via inhibition of MDM2) or impairment of cell cycle checkpoints (via inhibition of CHK1/2 or WEE1) led to the selective elimination of activated, pathological T cells in vivo. The combination of these strategies [which we termed "p53 potentiation with checkpoint abrogation" (PPCA)] displayed therapeutic benefits in preclinical disease models of hemophagocytic lymphohistiocytosis and multiple sclerosis, which are driven by foreign antigens or self-antigens, respectively. PPCA therapy targeted pathological T cells but did not compromise naive, regulatory, or quiescent memory T-cell pools, and had a modest nonimmune toxicity profile. Thus, PPCA is a therapeutic modality for selective, antigen-specific immune modulation with significant translational potential for diverse immune-mediated diseases.
Assuntos
Dano ao DNA/imunologia , Doenças do Sistema Imunitário/terapia , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/imunologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Etoposídeo/administração & dosagem , Humanos , Doenças do Sistema Imunitário/imunologia , Ativação Linfocitária , Linfo-Histiocitose Hemofagocítica/imunologia , Linfo-Histiocitose Hemofagocítica/terapia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/imunologia , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologia , Proteína Supressora de Tumor p53/imunologiaRESUMO
The current clinical approach for treating autoimmune diseases is to broadly blunt immune responses as a means of preventing autoimmune pathology. Among the major side effects of this strategy are depressed beneficial immunity and increased rates of infections and tumors. Using the experimental autoimmune encephalomyelitis model for human multiple sclerosis, we report a novel alternative approach for purging autoreactive T cells that spares beneficial immunity. The moderate and temporally limited use of etoposide, a topoisomerase inhibitor, to eliminate encephalitogenic T cells significantly reduces the onset and severity of experimental autoimmune encephalomyelitis, dampens cytokine production and overall pathology, while dramatically limiting the off-target effects on naive and memory adaptive immunity. Etoposide-treated mice show no or significantly ameliorated pathology with reduced antigenic spread, yet have normal T cell and T-dependent B cell responses to de novo antigenic challenges as well as unimpaired memory T cell responses to viral rechallenge. Thus, etoposide therapy can selectively ablate effector T cells and limit pathology in an animal model of autoimmunity while sparing protective immune responses. This strategy could lead to novel approaches for the treatment of autoimmune diseases with both enhanced efficacy and decreased treatment-associated morbidities.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Depleção Linfocítica , Linfócitos T/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Progressão da Doença , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/patologia , Etoposídeo/administração & dosagem , Etoposídeo/farmacologia , Feminino , Memória Imunológica , Ativação Linfocitária/efeitos dos fármacos , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Recidiva , Linfócitos T/efeitos dos fármacosRESUMO
Mucosal transmission of HIV is inefficient. The virus must breach physical barriers before it infects mucosal CD4+ T cells. Low-level viral replication occurs initially in mucosal CD4+ T cells, but within days high-level replication occurs in Peyer's patches, the gut lamina propria and mesenteric lymph nodes. Understanding the early events in HIV transmission may provide valuable information relevant to the development of an HIV vaccine. The viral quasispecies in a donor contracts through a genetic bottleneck in the recipient, such that, in low-risk settings, infection is frequently established by a single founder virus. Early-transmitting viruses in subtypes A and C mucosal transmission tend to encode gp120s with reduced numbers of N-linked glycosylation sites at specific positions throughout the V1-V4 domains, relative to typical chronically replicating isolates in the donor quasispecies. The transmission advantage gained by the absence of these N-linked glycosylation sites is unknown. Using primary α4ß7/CD4+ T cells and a flow-cytometry based steady-state binding assay we show that the removal of transmission-associated N-linked glycosylation sites results in large increases in the specific reactivity of gp120 for integrin-α4ß7. High-affinity for integrin α4ß7, although not found in many gp120s, was observed in early-transmitting gp120s that we analyzed. Increased α4ß7 affinity is mediated by sequences encoded in gp120 V1/V2. α4ß7-reactivity was also influenced by N-linked glycosylation sites located in C3/V4. These results suggest that the genetic bottleneck that occurs after transmission may frequently involve a relative requirement for the productive infection of α4ß7+/CD4+ T cells. Early-transmitting gp120s were further distinguished by their dependence on avidity-effects to interact with CD4, suggesting that these gp120s bear unusual structural features not present in many well-characterized gp120s derived from chronically replicating viruses. Understanding the structural features that characterize early-transmitting gp120s may aid in the design of an effective gp120-based subunit vaccine.
Assuntos
Linfócitos T CD4-Positivos/virologia , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/transmissão , HIV/genética , Integrinas/metabolismo , Mucosa Intestinal/virologia , Sequência de Aminoácidos , Anticorpos Neutralizantes , Células Cultivadas , Citometria de Fluxo , Genótipo , Glicosilação , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/virologia , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Ressonância de Plasmônio de SuperfícieRESUMO
Both activated and resting CD4(+) T cells in mucosal tissues play important roles in the earliest phases of infection after sexual transmission of HIV-1, a process that is inefficient. HIV-1 gp120 binds to integrin alpha(4)beta(7) (alpha(4)beta(7)), the gut mucosal homing receptor. We find that alpha(4)beta(7)(high) CD4(+) T cells are more susceptible to productive infection than are alpha(4)beta(7)(low-neg) CD4(+) T cells in part because this cellular subset is enriched with metabolically active CD4(+) T cells. alpha(4)beta(7)(high) CD4(+) T cells are CCR5(high) and CXCR4(low); on these cells, alpha(4)beta(7) appears in a complex with CD4. The specific affinity of gp120 for alpha(4)beta(7) provides a mechanism for HIV-1 to target activated cells that are critical for efficient virus propagation and dissemination following sexual transmission.
Assuntos
Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Integrinas/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Anticorpos Monoclonais/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Membrana Celular/virologia , Feminino , Genitália Feminina/efeitos dos fármacos , Genitália Feminina/imunologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Imunoprecipitação , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Receptores CCR5/metabolismo , Subpopulações de Linfócitos T/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
The programmed death (PD)-1 molecule and its ligands (PD-L1 and PD-L2), negative regulatory members of the B7 family, play an important role in peripheral tolerance. Previous studies have demonstrated that PD-1 is up-regulated on T cells following TCR-mediated activation; however, little is known regarding PD-1 and Ag-independent, cytokine-induced T cell activation. The common gamma-chain (gamma c) cytokines IL-2, IL-7, IL-15, and IL-21, which play an important role in peripheral T cell expansion and survival, were found to up-regulate PD-1 and, with the exception of IL-21, PD-L1 on purified T cells in vitro. This effect was most prominent on memory T cells. Furthermore, these cytokines induced, indirectly, the expression of PD-L1 and PD-L2 on monocytes/macrophages in PBMC. The in vivo correlate of these observations was confirmed on PBMC isolated from HIV-infected individuals receiving IL-2 immunotherapy. Exposure of gamma c cytokine pretreated T cells to PD-1 ligand-IgG had no effect on STAT5 activation, T cell proliferation, or survival driven by gamma c cytokines. However, PD-1 ligand-IgG dramatically inhibited anti-CD3/CD28-driven proliferation and Lck activation. Furthermore, following restimulation with anti-CD3/CD28, cytokine secretion by both gamma c cytokine and anti-CD3/CD28 pretreated T cells was suppressed. These data suggest that gamma c cytokine-induced PD-1 does not interfere with cytokine-driven peripheral T cell expansion/survival, but may act to suppress certain effector functions of cytokine-stimulated cells upon TCR engagement, thereby minimizing immune-mediated damage to the host.
Assuntos
Antígenos CD/biossíntese , Proteínas Reguladoras de Apoptose/biossíntese , Interleucina-15/imunologia , Interleucina-2/imunologia , Interleucina-7/imunologia , Interleucinas/imunologia , Ativação Linfocitária/imunologia , Antígenos CD/imunologia , Proteínas Reguladoras de Apoptose/imunologia , Antígeno B7-H1 , Células Cultivadas , Citocinas , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Interleucina-2/uso terapêutico , Proteína 2 Ligante de Morte Celular Programada 1 , Receptor de Morte Celular Programada 1 , Linfócitos T/imunologiaRESUMO
Preserving endogenous insulin production is clinically advantageous and remains a vital unmet challenge in the treatment and reversal of type 1 diabetes. Although broad immunosuppression has had limited success in prolonging the so-called remission period, it comes at the cost of compromising beneficial immunity. Here, we used a novel strategy to specifically deplete the activated diabetogenic T cells that drive pathogenesis while preserving not only endogenous insulin production but also protective immunity. Effector T (Teff) cells, such as diabetogenic T cells, are naturally poised on the edge of apoptosis because of activation-induced DNA damage that stresses the p53 regulation of the cell cycle. We have found that using small molecular inhibitors that further potentiate p53 while inhibiting the G2/M cell cycle checkpoint control drives apoptosis of activated T cells in vivo. When delivered at the onset of disease, these inhibitors significantly reduce diabetogenic Teff cells, prolong remission, preserve functional islets, and protect islet allografts while leaving naive, memory, and regulatory T-cell populations functionally untouched. Thus, the targeted manipulation of p53 and cell cycle checkpoints represents a new therapeutic modality for the preservation of islet ß-cells in new-onset type 1 diabetes or after islet transplant.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Pâncreas/imunologia , Linfócitos T/imunologia , Animais , Diabetes Mellitus Tipo 1/imunologia , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Camundongos , Camundongos Endogâmicos NOD , Pâncreas/efeitos dos fármacos , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Pirimidinonas , Linfócitos T/efeitos dos fármacos , Tiofenos/farmacologia , Tiofenos/uso terapêutico , Ureia/análogos & derivados , Ureia/farmacologia , Ureia/uso terapêuticoRESUMO
HIV infection is characterized by CD4(+) T cell depletion and progressive immune dysfunction; particularly impacted are HIV-specific T cell responses. An important component of immune-mediated control of HIV replication, killing of infected cells, appears to be impaired, in part due to poor cytolytic activity of HIV-specific cytotoxic T cells (CTL). In vitro, several functions of HIV-specific T cells, such as cytokine production, can be enhanced by the depletion of the immunosuppressive CD25(+) FoxP3(+) CD4(+) regulatory (Treg) cell subset. However, the effect of CD25(+) Treg cells on virus-specific cytolytic activity in the context of HIV or any human viral infection has not been investigated. The present study demonstrates that CD25(+) Treg cells isolated from the peripheral blood of HIV-infected subjects significantly suppress HIV Gag-specific cytolytic activity in vitro. In addition, CD25(+) Treg cells suppress effector function (coexpression of TNF-alpha and IFN-gamma) of HIV-specific CD8(+) T cells that proliferate in response to HIV antigen. Finally, the secretion of HIV-inhibitory CC-chemokines by HIV-specific and nonspecific CD8(+) T cells is significantly reduced in the presence of CD25(+) Treg cells. These data suggest that CD25(+) Treg-mediated suppression of the antiviral activity of HIV-specific CD8(+) T cells could impact the ability of HIV-infected individuals to control HIV replication in vivo.
Assuntos
Linfócitos T CD8-Positivos/virologia , Comunicação Celular/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Linfócitos T Reguladores , Linfócitos T CD8-Positivos/imunologia , Produtos do Gene gag/imunologia , Humanos , Hospedeiro Imunocomprometido/imunologia , Interferon gama/metabolismo , Subunidade alfa de Receptor de Interleucina-2 , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/virologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/virologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Natural killer (NK) cells are important in immune defense against virus infections. This is predominantly considered a function of rapid, innate NK-cell killing of virus-infected cells. However, NK cells also prime other immune cells through the release of interferon gamma (IFN-γ) and other cytokines. Additionally, NK cells share features with long-lived adaptive immune cells and can impact disease pathogenesis through the inhibition of adaptive immune responses by virus-specific T and B cells. The relative contributions of these diverse and conflicting functions of NK cells in humans are poorly defined and likely context-dependent, thereby complicating the development of therapeutic interventions. Here we focus on the contributions of NK cells to disease in diverse virus infections germane to human health.
Assuntos
Interações Hospedeiro-Patógeno/imunologia , Células Matadoras Naturais/imunologia , Viroses/imunologia , Viroses/virologia , Vírus/imunologia , Imunidade Adaptativa , Animais , Humanos , Imunidade Inata , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Viroses/metabolismo , Vírus/classificaçãoRESUMO
Focal adhesion kinase (FAK) and CD4 fulfil vital functions in cellular signal transduction: FAK is a central component in integrin signalling, whereas CD4 plays essential roles in the immune defence. In T lymphocytes, FAK and CD4 localise to the same signalling complexes after stimulation by either the human immunodeficiency virus (HIV) gp120 glycoprotein or an antigen, suggesting the concerted action of FAK and CD4 in these cells. Using crystallography and microcalorimetry, we here show that the focal adhesion targeting (FAT) domain of FAK binds specifically to the CD4 endocytosis motif in vitro. This FAT-CD4 complex is structurally and thermodynamically similar to the one FAT forms with paxillin LD motifs. The CD4 binding site on FAT presents the same features as the established CD4 binding site on the HIV-1 Nef protein. The binding of FAT to CD4 is incompatible with the binding of Lck to CD4. We further show that HIV-1 gp120 triggers the association of CD4 with FAK in T cells, under conditions that are known to dissociate Lck from CD4. Our results suggest that the FAK-CD4 complex represents an alternative route for eliciting T-cell-specific signals and that it links gp120 engagement to distinctive T-cell signalling during HIV infection. In infected cells, HIV-1 Nef may displace FAK from CD4 to protect the cells from apoptosis.
Assuntos
Antígenos CD4/química , Antígenos CD4/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Antígenos CD4/biossíntese , Antígenos CD4/genética , Calorimetria , Linhagem Celular , Sequência Consenso , Sequência Conservada , Cristalografia por Raios X , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/isolamento & purificação , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Análise dos Mínimos Quadrados , Microscopia Confocal , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Paxilina/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Receptores CCR5/química , Receptores CCR5/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Linfócitos T/metabolismo , Termodinâmica , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismoRESUMO
CD25(+) CD4(+) FoxP3(+) regulatory T (Treg) cells isolated from the peripheral blood of asymptomatic HIV-infected individuals have been demonstrated to significantly suppress HIV-specific immune responses in vitro. CD25(+) Treg cell suppressor activity in the peripheral blood seems to diminish with progression of HIV disease, and it has been suggested that loss of Treg cells contributes to aberrant immune activation and disease progression. However, phenotypic studies suggest that Treg cells may migrate to, and be maintained or even expanded in, tissue sites of HIV replication. Currently, it is not known whether tissue-associated Treg cells maintain suppressive activity in the context of HIV infection, particularly in individuals with advanced disease. The present study demonstrates that CD25(+) Treg cells isolated from lymph nodes and peripheral blood of HIV(+) subjects, even those with high viral loads and/or low CD4(+) T cell counts, maintain potent suppressive activity against HIV-specific cytolytic T cell function. This activity was better in lymph node as compared with peripheral blood, particularly in patients with high levels of plasma viremia. In addition, the expression of certain CD25(+) Treg-associated markers on CD4(+) T cells isolated from lymph nodes differed significantly from those on CD4(+) T cell subsets isolated from the peripheral blood. These data suggest that CD25(+) Treg cell-mediated suppression of HIV-specific responses continues throughout the course of HIV disease and, because of their particularly potent suppression of HIV-specific CTL activity in lymphoid tissue, may considerably impact the ability to control HIV replication in vivo.