RESUMO
The determinants of HIV-1-associated lymphadenopathy are poorly understood. We hypothesized that lymphocytes could be sequestered in the HIV-1+ lymph node (LN) through impairments in sphingosine-1-phosphate (S1P) responsiveness. To test this hypothesis, we developed novel assays for S1P-induced Akt phosphorylation and actin polymerization. In the HIV-1+ LN, naïve CD4 T cells and central memory CD4 and CD8 T cells had impaired Akt phosphorylation in response to S1P, whereas actin polymerization responses to S1P were impaired dramatically in all LN maturation subsets. These defects were improved with antiretroviral therapy. LN T cells expressing CD69 were unable to respond to S1P in either assay, yet impaired S1P responses were also seen in HIV-1+ LN T cells lacking CD69 expression. Microbial elements, HIV-1, and interferon α - putative drivers of HIV-1 associated immune activation all tended to increase CD69 expression and reduce T-cell responses to S1P in vitro. Impairment in T-cell egress from lymph nodes through decreased S1P responsiveness may contribute to HIV-1-associated LN enlargement and to immune dysregulation in a key organ of immune homeostasis.
Assuntos
Linfa/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Esfingosina/análogos & derivados , Linfócitos T/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antirretrovirais/uso terapêutico , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Citometria de Fluxo , Expressão Gênica/imunologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Linfa/imunologia , Linfa/metabolismo , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/imunologia , Receptores de Lisoesfingolipídeo/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Esfingosina/farmacologia , Receptores de Esfingosina-1-Fosfato , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
CpG oligodeoxynucleotides (ODNs) signal through TLR9 to induce type I IFN (IFN-alphabeta) in dendritic cells (DCs). CpG-A ODNs are more efficacious than CpG-B ODNs for induction of IFN-alphabeta. Because IFN-alphabeta may contribute to autoimmunity, it is important to identify mechanisms to inhibit induction of IFN-alphabeta. In our studies, CpG-B ODN inhibited induction of IFN-alphabeta by CpG-A ODN, whereas induction of TNF-alpha and IL-12p40 by CpG-A ODN was not affected. CpG-B inhibition of IFN-alphabeta was observed in FLT3 ligand-induced murine DCs, purified murine myeloid DCs, plasmacytoid DCs, and human PBMCs. CpG-B ODN inhibited induction of IFN-alphabeta by agonists of multiple receptors, including MyD88-dependent TLRs (CpG-A ODN signaling via TLR9, or R837 or Sendai virus signaling via TLR7) and MyD88-independent receptors (polyinosinic:polycytidylic acid signaling via TLR3 or ds break-DNA signaling via a cytosolic pathway). CpG-B ODN did not inhibit the IFN-alphabeta positive feedback loop second-wave IFN-alphabeta, because IFN-alphabeta-induced expression of IFN-alphabeta was unaffected, and CpG-B inhibition of IFN-alphabeta was manifested in IFN-alphabetaR(-/-) DCs, which lack the positive feedback mechanism. Rather, CpG-B ODN inhibited early TLR-induced first wave IFN-alpha4 and IFN-beta. Chromatin immunoprecipitation revealed that association of IFN regulatory factor 1 with the IFN-alpha4 and IFN-beta promoters was induced by CpG-A ODN but not CpG-B ODN. Moreover, CpG-A-induced association of IFN regulatory factor 1 with these promoters was inhibited by CpG-B ODN. Our studies demonstrate a novel mechanism of transcriptional regulation of first-wave IFN-alphabeta that selectively inhibits induction of IFN-alphabeta downstream of multiple receptors and may provide targets for future therapeutic inhibition of IFN-alphabeta expression in vivo.
Assuntos
Células Dendríticas/imunologia , Regulação da Expressão Gênica/imunologia , Interferon Tipo I/biossíntese , Oligodesoxirribonucleotídeos/imunologia , Transdução de Sinais/imunologia , Adjuvantes Imunológicos/farmacologia , Animais , Western Blotting , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Regulação da Expressão Gênica/genética , Humanos , Interferon Tipo I/genética , Interferon Tipo I/imunologia , Camundongos , Oligodesoxirribonucleotídeos/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição GênicaRESUMO
Type I interferon (IFNalpha/beta) plays a complex role in HIV-1 infection and has been proposed alternately to have roles in either disease protection or progression. Although IFNalpha/beta plays crucial roles in regulating monocytes and dendritic cells, responsiveness of these cells to IFNalpha/beta in HIV-1 infection is poorly understood. We report significant defects in IFNalpha/beta receptor (IFNalpha/betaR) expression, IFNalpha signaling, and IFNalpha-induced gene expression in monocytes from HIV-1-infected subjects. IFNalpha/betaR expression correlated directly with CD4+ T-cell count and inversely with HIV-1 RNA level and expression of CD38 by memory (CD45RO+) CD8+ T cells, a measure of pathologic immune activation in HIV-1 infection associated with disease progression. In addition, monocytes from HIV-1-infected persons showed diminished responses to IFNalpha, including decreased induction of phosphorylated STAT1 and the classical interferon-stimulated gene produces MxA and OAS. These IFNalpha responses were decreased regardless of IFNalpha/betaR expression, suggesting that regulation of intracellular signaling may contribute to unresponsiveness to IFNalpha/beta in HIV-1 disease. Defective monocyte responses to IFNalpha/beta may play an important role in the pathogenesis of HIV-1 infection, and decreased IFNalpha/betaR expression may serve as a novel marker of disease progression.
Assuntos
Farmacorresistência Viral/imunologia , Infecções por HIV/tratamento farmacológico , HIV-1 , Imunidade Ativa , Interferon Tipo I/uso terapêutico , Adulto , Antivirais/uso terapêutico , Biomarcadores/sangue , Estudos de Casos e Controles , Estudos Transversais , Progressão da Doença , Feminino , Infecções por HIV/sangue , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Imunidade Ativa/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Monócitos/patologia , Receptores de Interferon/metabolismoRESUMO
CTL are endowed with the ability to eliminate pathogens through perforin-mediated cytotoxic activity. The mechanism for perforin-mediated Ag-specific killing has been solely attributed to cytotoxic granule exocytosis from activated CD8(+) T cells. In this study, we redefine this mechanism, demonstrating that virus-specific CD8(+) T cells rapidly up-regulate perforin in response to stimulation temporally with IFN-gamma and CD107a expression. Following Ag-specific activation, newly synthesized perforin rapidly appears at the immunological synapse, both in association with and independent of cytotoxic granules, where it functions to promote cytotoxicity. Our work suggests a novel mechanism of CTL cytotoxicity and identifies a novel correlate of CD8(+) T cell-mediated immunity.
Assuntos
Grânulos Citoplasmáticos/imunologia , Testes Imunológicos de Citotoxicidade , Epitopos de Linfócito T/imunologia , Sinapses Imunológicas/imunologia , Perforina/biossíntese , Perforina/fisiologia , Linfócitos T Citotóxicos/imunologia , Regulação para Cima/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Degranulação Celular/imunologia , Linhagem Celular Transformada , Grânulos Citoplasmáticos/metabolismo , Testes Imunológicos de Citotoxicidade/métodos , Humanos , Ativação Linfocitária/imunologia , Perforina/metabolismo , Transporte Proteico/imunologia , Transdução de Sinais/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologiaRESUMO
Intratumoural heterogeneity of pulmonary adenocarcinoma challenges the accurate interpretation of programmed death ligand 1 (PD-L1) immunohistochemistry, which is the only validated predictive marker for successful anti-PD-1/PD-L1 immunotherapy. The aim of this study was to determine whether PD-L1 expression is related to adenocarcinoma histological differentiation in a retrospective analysis of tumour biopsies with intratumoural histological heterogeneity. Adenocarcinomas with high intratumoural heterogeneity were categorised as 'mixed adenocarcinomas'. PD-L1 expression was determined immunohistochemically using tumour proportion scores (TPS). In 'mixed adenocarcinomas' PD-L1 scores were assessed across tumour areas with specific histological patterns. Comparisons were performed between histologically distinct differentiated tumours and/or histological areas. Poorly differentiated adenocarcinomas, represented by predominantly solid or micropapillary histological patterns, showed significantly higher expression of PD-L1 than other subtypes (p<0.001). Differentiation of intra-adenocarcinoma components was inversely correlated with PD-L1 expression: there were more PD-L1 positive cells in poorly differentiated areas than less differentiated (p<0.001), or than well differentiated areas (p<0.001), and in less differentiated more than well differentiated areas (p=0.001). In conclusion, PD-L1 expression is associated with poorly differentiated morphology in adenocarcinomas with intratumoural histological heterogeneity. Consequently, a TPS approach may not account for the contribution of more aggressive tumour components with higher levels of PD-L1 expression in within the tumour. Performing spectral analyses of PD-L1 expression across tumours is likely to be more accurate.
Assuntos
Adenocarcinoma de Pulmão/metabolismo , Antígeno B7-H1/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Antígeno B7-H1/genética , Humanos , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Estudos RetrospectivosRESUMO
The human immunodeficiency virus (HIV) accesses the central nervous system (CNS) early during infection, leading to HIV-associated cognitive impairment and establishment of a viral reservoir. Here, we describe a dichotomy in inflammatory responses in different CNS regions in simian immunodeficiency virus (SIV)-infected macaques, a model for HIV infection. We found increased expression of inflammatory genes and perivascular leukocyte infiltration in the midbrain of SIV-infected macaques. Conversely, the frontal lobe showed downregulation of inflammatory genes associated with interferon-γ and interleukin-6 pathways, and absence of perivascular cuffing. These immunologic alterations were not accompanied by differences in SIV transcriptional activity within the tissue. Altered expression of genes associated with neurotoxicity was observed in both midbrain and frontal lobe. The segregation of inflammatory responses to specific regions of the CNS may both account for HIV-associated neurological symptoms and constitute a critical hurdle for HIV eradication by shielding the CNS viral reservoir from antiviral immunity.
RESUMO
Persistent type I IFN production occurs during chronic viral infections, such as HIV disease. As type I IFNs have antiproliferative activity, it is possible that chronic exposure to these cytokines could adversely affect T cell homeostasis. We investigated the capacity of IFN-α to impair T cell proliferation induced by the homeostatic cytokine, IL-7, or another common γ-chain cytokine, IL-2, in cells from healthy human donors. We found that IL-7- or IL-2-induced proliferation of CD4(+) T cells was partially inhibited in the presence of IFN-α. The CD4(+) T cells that were exposed to IFN-α also displayed attenuated induction of IL-2 and CD40L following TCR stimulation. Analyses of signaling pathways indicated that IL-7 and IL-2 induced a delayed and sustained P-Akt signal that lasted for several days and was partially inhibited by IFN-α. In contrast, IL-7-induced P-STAT5 was not affected by IFN-α. Furthermore, IFN-α had no detectable effect on P-Akt that was induced by the chemokine SDF-1. Both inhibitors of P-Akt and P-STAT5 blocked IL-7-induced T cell proliferation, confirming that both signaling pathways are important for IL-7-induced T cell proliferation. These results demonstrate that IFN-α can selectively inhibit cytokine-induced P-Akt as a potential mechanism to disrupt homeostasis of T lymphocytes.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interferon-alfa/imunologia , Interleucina-2/imunologia , Interleucina-7/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Antígenos CD4/genética , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Ligante de CD40/genética , Ligante de CD40/imunologia , Proliferação de Células , Regulação da Expressão Gênica , Humanos , Interferon-alfa/farmacologia , Interleucina-2/farmacologia , Interleucina-7/farmacologia , Fosforilação , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Transdução de SinaisRESUMO
Combination antiretroviral therapy during primary human immunodeficiency virus-1 infection may enable long-term drug-free virological control in rare individuals. We describe a female who maintained aviremia and a normal CD4(+)/CD8(+) T cell ratio for 10 years after stopping therapy, despite a persistent viral reservoir. Cellular immune responses may have contributed to this outcome.
RESUMO
The combination of intermittent subcutaneous IL-2 and highly active antiretroviral therapy in individuals infected with HIV-1 has been shown to have a beneficial quantitative effect on the CD4 T cell count. We observed IL-2-associated viral load 'blips' inducing HIV-1-specific lymphoproliferative responses at 24 weeks in such individuals. This immunotherapeutic approach, utilizing autologous virus as autovaccination, may be a viable, safer alternative to structured treatment interruption and potentially more efficacious than therapeutic vaccines.
Assuntos
Terapia Antirretroviral de Alta Atividade , Infecções por HIV/tratamento farmacológico , HIV-1 , Imunoterapia Ativa/métodos , Interleucina-2/uso terapêutico , Contagem de Linfócito CD4 , Infecções por HIV/imunologia , Humanos , Interleucina-2/imunologia , Carga ViralRESUMO
HIV-1 infection results in profound dysfunction of the CD4 T-cell population. Although highly active antiretroviral therapy allows the reconstitution of CD4 T-cell numbers, functional abnormalities remain, including inadequate responses to vaccination. IL-2 increases CD4 T-cell numbers and function. We report the effects of IL-2 given with tetanus vaccination in two patients in a larger immunotherapy study. In one case IL-2 induced sustained responses, which may have been caused by the timing of IL-2 with vaccination.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1 , Interleucina-2/administração & dosagem , Toxoide Tetânico/administração & dosagem , Terapia Antirretroviral de Alta Atividade , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Terapia Combinada , Infecções por HIV/imunologia , HumanosRESUMO
Type-I interferon (IFN-I) has been increasingly implicated in HIV-1 pathogenesis. Various studies have shown elevated IFN-I and an IFN-I-induced gene and protein expression signature in HIV-1 infection, yet the elevated IFN-I species has not been conclusively identified, its source remains obscure and its role in driving HIV-1 pathogenesis is controversial. We assessed IFN-I species in plasma by ELISAs and bioassay, and we investigated potential sources of IFN-I in blood and lymph node tissue by qRT-PCR. Furthermore, we measured the effect of therapeutic administration of IFNα in HCV-infected subjects to model the effect of IFNα on chronic immune activation. IFN-I bioactivity was significantly increased in plasma of untreated HIV-1-infected subjects relative to uninfected subjects (p = 0.012), and IFNα was the predominant IFN-I subtype correlating with IFN-I bioactivity (r = 0.658, p<0.001). IFNα was not detectable in plasma of subjects receiving anti-retroviral therapy. Elevated expression of IFNα mRNA was limited to lymph node tissue cells, suggesting that peripheral blood leukocytes are not a major source of IFNα in untreated chronic HIV-1 infection. Plasma IFN-I levels correlated inversely with CD4 T cell count (p = 0.003) and positively with levels of plasma HIV-1 RNA and CD38 expression on CD8 T cells (p = 0.009). In hepatitis C virus-infected subjects, treatment with IFN-I and ribavirin increased expression of CD38 on CD8 T cells (p = 0.003). These studies identify IFNα derived from lymph nodes, rather than blood leukocytes, as a possible source of the IFN-I signature that contributes to immune activation in HIV-1 infection.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/sangue , Imunidade Ativa , Interferon-alfa/imunologia , Antivirais/administração & dosagem , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/virologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , HIV-1/imunologia , HIV-1/patogenicidade , Hepacivirus/efeitos dos fármacos , Hepacivirus/patogenicidade , Hepatite C/sangue , Hepatite C/tratamento farmacológico , Humanos , Interferon-alfa/administração & dosagem , Interferon-alfa/sangue , Linfonodos/efeitos dos fármacos , Linfonodos/metabolismo , Ribavirina/administração & dosagemRESUMO
TB remains uncontrolled. In resource-rich countries, only approximately 60% of diagnoses are confirmed by culture. The number is lower in resource-poor environments. Huge scope therefore exists for alternative diagnostic strategies. Counting antigen-specific lymphocytes by virtue of cytokine production following 8-16 h stimulation with tuberculosis antigens is currently the strategy of choice. Several methods exist, including ELISA, ELISpots, and flow cytometry. Although it is clear that blood samples stimulated by ESAT-6 and CFP-10 antigens discriminate between TB infection and BCG vaccination, it is flow-cytometry that seems to be able to distinguish active TB disease from mere TB exposure. Of the various flow-protocols including four-color tests (CD45-CD3-CD4-IFNgamma), three-color tests (CD3-CD4-IFNgamma) and two-color tests (CD4-IFNgamma), even the simplest is performing well, provided that the results are expressed as percentage of IFN-gamma+ cells per CD4+ lymphocytes (%IFNgamma/CD4+). Studies using broncho-alveloar lavage (BAL) and Induced-Sputum (ISp) show that TB-specific CD4+IFN-gamma+ T cells accumulate in the lung in pulmonary and extra-pulmonary TB at frequencies >5-20-fold more frequent than in blood. This pulmonary homing is absent following BCG immunization. The use of PPD to stimulate CD4+IFN-gamma+ cells in the lung in active TB leads to >3-12-fold greater responses than seen with CFP-10 or ESAT-6, and any interference from BCG vaccination is absent. This method is unaffected by HIV coinfection, which has always been the problem for other immune-based diagnostics. Further, lung-based samples provide material for rapid tests of both the IFN-gamma assay and bacteriology, and importantly, these tests are amenable for future simplification with automated fluorescence-image cytometers.Another development of the multiparameter analytical power of flow-cytometry is to use markers for "lung-seeking" populations of CD4+ T cells in blood, obviating lung sampling. In active TB, but not in BCG vaccinees, TB-specific memory CD4+ T cells can be found in blood that are dominantly CD27-negative and probably lung seeking and can be diagnostically useful.
Assuntos
Citometria de Fluxo/métodos , Infecções por HIV/complicações , Interferon gama , Tuberculose Pulmonar/complicações , Tuberculose Pulmonar/diagnóstico , Antígenos de Bactérias , Proteínas de Bactérias , Infecções por HIV/diagnóstico , Humanos , Tuberculina/sangue , Tuberculose Pulmonar/patologiaRESUMO
RATIONALE AND OBJECTIVES: Blood-based studies have demonstrated the potential of immunological assays to detect tuberculosis. However lung fluid sampling may prove superior as it enables simultaneous microbiological detection of mycobacteria to be performed. Until now this has only been possible using the expensive and invasive technique of broncho-alveolar lavage. We sought to evaluate an immunoassay using non-invasive induced-sputum to diagnose active tuberculosis. METHODS AND RESULTS: Prospective cohort study of forty-two spontaneous sputum smear-negative or sputum non-producing adults under investigation for tuberculosis. CD4 lymphocytes specific to purified-protein-derivative of Mycobacterium tuberculosis actively synthesising interferon-gamma were measured by flow cytometry and final diagnosis compared to immunoassay using a cut-off of 0.5%. Sixteen subjects (38%) were HIV-infected (median CD4 count [range] = 332 cells/microl [103-748]). Thirty-eight (90%) were BCG-vaccinated. In 27 subjects diagnosed with active tuberculosis, the median [range] percentage of interferon-gamma synthetic CD4+ lymphocytes was 2.77% [0-23.93%] versus 0% [0-2.10%] in 15 negative for active infection (p<0.0001). Sensitivity and specificity of the immunoassay versus final diagnosis of active tuberculosis were 89% (24 of 27) and 80% (12 of 15) respectively. The 3 positive assays in the latter group occurred in subjects diagnosed with quiescent/latent tuberculosis. Assay performance was unaffected by HIV-status, BCG-vaccination or disease site. Combining this approach with traditional microbiological methods increased the diagnostic yield to 93% (25 of 27) alongside acid-fast bacilli smear and 96% (26 of 27) alongside tuberculosis culture. CONCLUSIONS: These data demonstrate for the first time that a rapid immunological assay to diagnose active tuberculosis can be performed successfully in combination with microbiological methods on a single induced-sputum sample.