RESUMO
Natural cytotoxicity receptors (NCRs) were originally identified as specific natural killer cell activating receptors that, on binding to their endogenous ligands, trigger the killing of tumor cell targets. We recently described the differentiation of a novel subset of NCR(+) Vδ1 T cells characterized by a remarkably high cytolytic potential against cancer cells. Here we demonstrate that the engagement of NKp30, one of the NCRs expressed de novo on Vδ1 T cells after stimulation, triggers the production of high levels of CCL3/MIP-1α, CCL4/ MIP-1ß, and CCL5/RANTES but not of CXCL12/SDF-1. In turn, this NKp30-induced secretion of cc-chemokines is able to significantly suppress the replication of a CCR5 tropic strain of HIV-1 in CD4(+)/CCR5(+) infected PM1 cell lines. This experimental evidence disclosing an unanticipated antiviral function of NCR(+) Vδ1 T cells opens new avenues for understanding the pathogenic role and for manipulating the function of γδ T cells in HIV-1 infection.
Assuntos
Quimiocina CCL3/metabolismo , Quimiocina CCL4/metabolismo , Quimiocina CCL5/metabolismo , Infecções por HIV/prevenção & controle , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Replicação Viral/imunologia , Diferenciação Celular , Células Cultivadas , Quimiocina CCL3/imunologia , Quimiocina CCL4/imunologia , Quimiocina CCL5/imunologia , Quimiocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Infecções por HIV/imunologia , Infecções por HIV/metabolismo , HIV-1/patogenicidade , Humanos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
There is disagreement on the optimal timing of HAART initiation based on absolute CD4+ T-cell count (CD4+ count). We investigated if na�ve patients with CD4+ T-cell percentage (%CD4+) <29% or CD4+/CD8+ ratio <1 display signs of immune deterioration notwithstanding CD4+ count ?500 cells/?l. We found that these patients show B-cell aberrations and an impaired control of Torque Teno Virus replication. By contrast, patients with CD4+?500/?l, %CD4+?29% and CD4+/CD8+?1 displayed features of healthy subjects. Results obtained suggest that a combination of these parameters could be an adequate surrogate marker of immunological competence. This will be helpful in deciding when to start HAART.
Assuntos
Biomarcadores/sangue , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Adulto , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Relação CD4-CD8 , Feminino , Infecções por HIV/sangue , HIV-1 , Humanos , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Toll-like receptors (TLRs) play a key role in the cross-talk between the innate and adaptive immune systems. Previous studies investigating associations between certain TLRs and acute graft-versus-host disease (aGVHD) have reported contrasting results, and no studies relating aGVHD to the expression and function of all human TLRs together have been published to date. We prospectively evaluated the expression of 9 TLRs on T lymphocytes and monocytes by flow cytometry in relation to aGVHD in 34 patients. Induction of TNF-α, IL-4, IFN-γ, and monocyte chemotactic protein 1 on TLR activation was assessed by ELISA on cell supernatants. Nineteen patients developed aGVHD, at a median time of 28 days (range, 20-50 days) after transplantation. A 2-step multivariate analysis was performed using principal component analysis and multifactor analysis of variance. The levels of TLR-5 expression on monocytes and T lymphocytes were positively correlated to aGVHD (P = .01), whereas levels of TLR-1 and -9 were negative predictors (P = .03 and .01, respectively). This profile of TLR-1, -5, and -9 can promote an overall immunostimulatory/proinflammatory response. If our findings are confirmed by further studies, this TLR profile could be a useful biomarker of aGVHD.
Assuntos
Doença Enxerto-Hospedeiro/sangue , Transplante de Células-Tronco , Receptores Toll-Like/sangue , Doença Aguda , Adolescente , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Linfócitos T/metabolismo , Transplante Homólogo , Adulto JovemRESUMO
The success of cancer immunotherapy depends on productive tumor cell recognition by killer lymphocytes. γδ T cells are a population of innate-like lymphocytes endowed with strong, MHC-unrestricted cytotoxicity against tumor cells. This notwithstanding, we recently showed that a large proportion of human hematologic tumors is resistant to γδ peripheral blood lymphocytes (PBLs) activated with specific agonists to the highly prevalent Vγ9Vδ2 TCR. Although this probably constitutes an important limitation to current γδ T cell-mediated immunotherapy strategies, we describe here the differentiation of a novel subset of Vδ2(-) Vδ1(+) PBLs expressing natural cytotoxicity receptors (NCRs) that directly mediate killing of leukemia cell lines and chronic lymphocytic leukemia patient neoplastic cells. We show that Vδ1(+) T cells can be selectively induced to express NKp30, NKp44 and NKp46, through a process that requires functional phosphatidylinositol 3-kinase (PI-3K)/AKT signaling on stimulation with γ(c) cytokines and TCR agonists. The stable expression of NCRs is associated with high levels of granzyme B and enhanced cytotoxicity against lymphoid leukemia cells. Specific gain-of-function and loss-of-function experiments demonstrated that NKp30 makes the most important contribution to TCR-independent leukemia cell recognition. Thus, NKp30(+) Vδ1(+) T cells constitute a novel, inducible and specialized killer lymphocyte population with high potential for immunotherapy of human cancer.
Assuntos
Leucemia Linfoide/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Diferenciação Celular , Separação Celular , Células Cultivadas , Citotoxicidade Imunológica/imunologia , Citometria de Fluxo , Humanos , Ativação Linfocitária/imunologia , Receptor 3 Desencadeador da Citotoxicidade Natural/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismoRESUMO
In this study, we demonstrate that the in vitro interactions between a CD56(neg)/CD16(pos) (CD56(neg)) subset of natural killer (NK) cells and autologous dendritic cells (DCs) from HIV-1-infected viremic but not aviremic individuals are markedly impaired and likely interfere with the development of an effective immune response. Among the defective interactions are abnormalities in the process of reciprocal NK-DC activation and maturation as well as a defect in the NK cell-mediated editing or elimination of immature DCs (iDCs). Notably, the lysis of mature DCs (mDCs) by autologous NK cells was highly impaired even after the complete masking of major histocompatibility complex I molecules, suggesting that the defective elimination of autologous iDCs is at the level of activating NK cell receptors. In this regard, the markedly impaired expression/secretion and function of NKp30 and TNF-related apoptosis-inducing ligand, particularly among the CD56(neg) NK cell subset, largely accounts for the highly defective NK cell-mediated lysis of autologous iDCs. Moreover, mDCs generated from HIV-1 viremic but not aviremic patients are substantially impaired in their ability to secrete interleukin (IL)-10 and -12 and to prime the proliferation of neighboring autologous NK cells, which, in turn, fail to secrete adequate amounts of interferon-gamma.
Assuntos
Células Dendríticas/imunologia , Infecções por HIV/imunologia , HIV-1 , Células Matadoras Naturais/imunologia , Adulto , Anticorpos Monoclonais , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Ativação Linfocitária/imunologia , Microscopia de Fluorescência , Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Alterations of B cell subpopulations have been described up to date as characterizing advanced stage of HIV-1 infection. However, whether such defects are relevant in subjects with a preserved number of CD4⺠T cells (>350 cells/µL) is unclear. In a cross-sectional study, we investigated if signs of B cells exhaustion and impaired viral immune surveillance are present in a cohort of 43 asymptomatic HIV-1-infected patients with preserved CD4⺠T cell counts (>350 cells/µL) and highly active antiretroviral therapy (HAART) untreated. A dramatic expansion of exhausted tissue-like memory B cells (CD10â»CD21(low)CD27â») was observed. B cells alteration was related to an increase in Torque teno virus (TTV) load, used as surrogate marker of immune function. Successfully HAART-treated patients showed normalization of B cell subpopulations frequency and TTV load. These results provide new insights on B cell in HIV-1 infection and show that development of B cell abnormalities precedes CD4⺠T cell decline.
Assuntos
Subpopulações de Linfócitos B/imunologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , HIV-1/imunologia , Linfócitos T/imunologia , Adulto , Idoso , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Doenças Assintomáticas , Subpopulações de Linfócitos B/patologia , Contagem de Linfócito CD4 , Estudos de Casos e Controles , Estudos de Coortes , Coinfecção , Estudos Transversais , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/virologia , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , Humanos , Vigilância Imunológica , Itália , Masculino , Pessoa de Meia-Idade , Torque teno virus/efeitos dos fármacos , Torque teno virus/imunologia , Carga Viral/efeitos dos fármacos , Carga Viral/imunologiaRESUMO
HIV-1 infection in humans results in an early and progressive NK cell dysfunction and an accumulation of an "anergic" CD56- CD16+ NK subset, which is characterised by low natural cytotoxicity receptor expression and low cytokine producing capacity. In contrast to humans, chimpanzee NK cells do not display a distinguishable CD56(bright) and CD56(dim) subset but, as shown here, could be subdivided into functionally different CD8+ and CD8- subsets. The CD8+ NK cells expressed significantly higher levels of triggering receptors including NKp46 and, upon in vitro activation, produced more IFN-gamma, TNF-alpha and CD107 than their CD8- counterparts. In addition, chimpanzee CD8- NK cells had relatively high levels of HLA-DR expression, suggestive of an activated state. Killing inhibitory receptors were expressed only at low levels; however, upon in vitro stimulation, they were up-regulated in CD8+ but not in CD8- NK cells and were functionally capable of inhibiting NKp30-triggered killing. In contrast to HIV-1-infected humans, infected chimpanzees maintained their dominant CD8+ NK cell population, with high expression of natural cytotoxicity receptors.
Assuntos
Citocinas/biossíntese , Infecções por HIV/imunologia , HIV-1 , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/imunologia , Pan troglodytes/imunologia , Receptores de Células Matadoras Naturais/análise , Animais , Antígenos CD/análise , Antígenos CD/biossíntese , Antígenos CD/genética , Antígeno CD56/análise , Antígenos CD8/análise , Células Cultivadas/imunologia , Citocinas/genética , Citotoxicidade Imunológica , Humanos , Imunofenotipagem , Interferon gama/biossíntese , Interferon gama/genética , Células Matadoras Naturais/química , Subpopulações de Linfócitos/química , Subfamília C de Receptores Semelhantes a Lectina de Células NK/análise , Subfamília C de Receptores Semelhantes a Lectina de Células NK/biossíntese , Subfamília C de Receptores Semelhantes a Lectina de Células NK/genética , Receptores de Células Matadoras Naturais/biossíntese , Receptores de Células Matadoras Naturais/genética , Regulação para CimaRESUMO
HIV-1 has developed several strategies to evade natural killer (NK)-cell antiviral functions. One of these mechanisms is the HIV-1-induced expansion of highly dysfunctional NK-cell subsets. Here, we analyze a large cohort of HIV-1-infected patients in early or chronic phases of infection, both cross-sectionally and longitudinally. We demonstrate that a striking decrease in the surface expression of sialic acid-binding immunoglobulin-like lectin 7 (Siglec-7) represents the earliest marker of the aberrant NK-cell dysregulation, which precedes the down-modulation of CD56 mostly occurring in patients with chronic HIV-1 viremia. The combined detection of Siglec-7 and CD56 allows the identification of 2 new pathologic NK-cell subsets expanded preferentially in early (Siglec-7-/CD56+) or chronic (Siglec-7-/CD56-) stages of HIV-1 infection. Remarkably, these phenotypic abnormalities were directly associated with progressive and distinct impairments of NK-cell functions. The aforementioned NK-cell aberrancies could be observed only in the presence of high levels of viral replication and not in patients with low or undetectable HIV-1 viremia, such as long-term nonprogressors or patients having undergone antiretroviral therapy. High frequencies of Siglec-7-/CD56+ and Siglec-7-/CD56- pathologic NK cells reflect the immune and clinical status of HIV-1 infection and can also track the effectiveness of therapy.
Assuntos
Antígenos de Diferenciação Mielomonocítica/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Células Matadoras Naturais/imunologia , Lectinas/imunologia , Viremia/imunologia , Replicação Viral/imunologia , Antígeno CD56/imunologia , Estudos de Coortes , Feminino , Infecções por HIV/patologia , Humanos , Células Matadoras Naturais/patologia , Masculino , Viremia/patologiaRESUMO
Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24(pos) blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24(neg) blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and -B alleles and against heterologous MHC-I(neg) cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56(neg)/CD16(pos) subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24(pos) blasts derived from primary T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Citotoxicidade Imunológica , Infecções por HIV/imunologia , Interleucina-2/imunologia , Células Matadoras Naturais/imunologia , Monócitos Matadores Ativados/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Proliferação de Células , Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/sangue , Antígenos HLA-A/metabolismo , Antígenos HLA-B/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Monócitos Matadores Ativados/metabolismo , Monócitos Matadores Ativados/virologia , Proteínas Recombinantes , Replicação ViralRESUMO
Human Metapneumovirus (HMPV) is a major cause of lower respiratory tract infections. HMPV infection has been hypothesized to alter dendritic cell (DC) immune response; however, many questions regarding HMPV pathogenesis within the infected lung remain unanswered. Here, we show that HMPV productively infects human lung microvascular endothelial cells (L-HMVECs). The release of infectious virus occurs for up to more than 30 days of culture without producing overt cytopathic effects and medium derived from persistently HMPV-infected L-HMVECs (secretome) induced monocyte-derived DCs to prime naïve CD4 T-cells toward a Th2 phenotype. Moreover, we demonstrated that infected secretomes trigger DCs to up-regulate OX40L expression and OX40L neutralization abolished the pro-Th2 effect that is induced by HMPV-secretome. We clarified secretome from HMPV by size exclusion and ultracentrifugation with the aim to characterize the role of viral particles in the observed pro-Th2 effect. In both cases, the percentage of IL-4-producing cells and expression of OX40L returned at basal levels. Finally, we showed that HMPV, per se, could reproduce the ability of secretome to prime pro-Th2 DCs. These results suggest that HMPV, persistently released by L-HMVECs, might take part in the development of a skewed, pro-Th2 lung microenvironment.
RESUMO
We investigated whether a different pattern of HLA-specific inhibitory natural killer receptor (iNKR) expression on peripheral blood mononuclear cells (PBMC) of long-term non-progressor (LTNP) patients explained their benign course of HIV-1 infection. The surface expression of p70, p140 and CD94/NKG2A in their CD3+CD8+ PBMC was comparable to that of uninfected donors. The lack of iNKR-mediated functional inhibition of HIV-1-specific cytotoxic T lymphocytes in vitro could provide an additional mechanism for the efficient control of virus spread in LTNP patients.
Assuntos
Infecções por HIV/imunologia , Sobreviventes de Longo Prazo ao HIV , HIV-1 , Receptores Imunológicos/sangue , Linfócitos T Citotóxicos/imunologia , Adulto , Citotoxicidade Imunológica , Progressão da Doença , Humanos , Células Matadoras Naturais/imunologia , Células Tumorais CultivadasRESUMO
BACKGROUND: The HIV-1-induced expansion of highly dysfunctional natural killer (NK) cell subsets represents a strategy to evade NK cell antiviral functions. In this context, the loss of NKG2A NK cells in chronic viremic HIV-1-infected individuals has also been associated with a dramatic expansion of NKG2C NK cells. The viral trigger associated with high frequencies of NK cell subsets expressing NKG2C is still being debated. OBJECTIVE: To confirm that human cytomegalovirus (HCMV) infection is necessary for the expansion of NKG2C NK cells and to assess whether this phenomenon affects NKG2A/NKG2C ratio on NK cells in patients coinfected with HIV-1 and HCMV. DESIGN: We measured the expression of NKG2A and NKG2C on NK cells from 70 healthy donors, 21 early, 96 chronic and 27 long-term nonprogressor's (LTNPs) HIV-1-infected patients using a multicolor flow cytometric approach. HCMV infection was detected by titrating the serum levels of specific circulating antibodies. RESULTS: A significant expansion of NKG2C NK cells could be detected only in HCMV-infected patients. This phenotypic feature, together with the HIV-1-mediated downmodulation of NKG2A, pathologically reverses the ratio of NKG2A/NKG2C uniquely on NK cells from chronic viremic HIV-1-infected patients with a concomitant HCMV infection. The normalization of NKG2A/NKG2C ratio to values more than one occurred only after 24 months of suppression of HIV-1 replication following antiretroviral therapy. CONCLUSION: The inversion of NKG2A/NKG2C ratio characterizes advanced stages of HIV-1 disease in patients showing a concomitant HCMV infection. This NK cell immune parameter renders this cohort of patients distinguishable from LTNPs and early HIV-1-infected individuals.
Assuntos
Infecções por Citomegalovirus/imunologia , Regulação Viral da Expressão Gênica/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Células Matadoras Naturais/imunologia , Viremia/imunologia , Infecções por Citomegalovirus/virologia , Feminino , Citometria de Fluxo , Infecções por HIV/complicações , Infecções por HIV/virologia , Humanos , Células Matadoras Naturais/fisiologia , Células Matadoras Naturais/virologia , Masculino , Subfamília C de Receptores Semelhantes a Lectina de Células NK/sangue , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Viremia/virologiaRESUMO
BACKGROUND: HIV-1 Protease Inhibitors, namely PIs, originally designed to inhibit HIV-1 aspartic protease, can modulate the immune response by mechanisms largely unknown, and independent from their activity on viral replication. Here, we analyzed the ability of PIs to interfere with differentiation program of monocytes toward dendritic cell (DCs) lineage, a key process in the inflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: Monocytes from healthy donors were isolated and induced to differentiate in vitro in the presence or absence of saquinavir, ritonavir, nelfinavir, indinavir or amprenavir (sqv, rtv, nlfv, idv, apv, respectively). These drugs demonstrated a differential ability to sustain the generation of immature DCs (iDCs) with an altered phenotype, including low levels of CD1a, CD86, CD36 and CD209. DCs generated in the presence of rtv also failed to acquire the typical phenotype of mature DCs (mDCs), and secreted lower amounts of IL-12 and IL-15. Accordingly, these aberrant mDCs failed to support activation of autologous Natural Killer (NK) cells, and resulted highly susceptible to NK cell-mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: Our findings uncover novel functional properties of PIs within the DC-NK cell cross-talk, unveiling the heterogeneous ability of members of this class drugs to drive the generation of atypical monocyte-derived DCs (MDDCs) showing an aberrant phenotype, a failure to respond appropriately to bacterial endotoxin, a weak ability to prime autologous NK cells, and a high susceptibility to NK cell killing. These unexpected properties might contribute to limit inflammation and viral spreading in HIV-1 infected patients under PIs treatment, and open novel therapeutical perspectives for this class drugs as immunomodulators in autoimmunity and cancer.
Assuntos
Células Dendríticas/efeitos dos fármacos , Inibidores da Protease de HIV/farmacologia , Protease de HIV/efeitos dos fármacos , Células Matadoras Naturais/efeitos dos fármacos , Proliferação de Células , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imunofenotipagem , Células Matadoras Naturais/imunologiaRESUMO
The ability of natural killer (NK) cells to kill virus-infected cells depends on the presence of ligands for activation receptors on the target cells. We found the presence of few, if any, NKp30 and NK46 ligands on T cell blasts infected with HIV, although NKp44 ligands were found on infected cells. HIV does induce the NKG2D ligands ULBP-1, -2, and -3. These ligands are involved in triggering NK cells to kill autologous HIV-infected cells, because interfering with the interaction between NKG2D, but not NKp46, on NK cells and its ligands on HIV-infected cells drastically reduced the lysis of infected cells. Interfering with the binding of the NK-cell coreceptors NTB-A and 2B4 to their ligands also decreased destruction by NK cells. The coreceptor ligands, NTB-A and CD48, were also found to be down-regulated during the course of HIV infection. Thus, ligands for NK-cell receptors are modulated during the course of HIV infection, which may greatly alter NK cells' ability to kill the infected cells.
Assuntos
Citotoxicidade Imunológica/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Células Matadoras Naturais/imunologia , Receptores Imunológicos/metabolismo , Linfócitos T/virologia , Antígenos CD/metabolismo , Antígeno CD48 , Proteínas Ligadas por GPI , Regulação da Expressão Gênica , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Soropositividade para HIV , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/virologia , Ligantes , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptor 3 Desencadeador da Citotoxicidade Natural , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/imunologia , Receptores de Células Matadoras Naturais , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Linfócitos T/imunologiaRESUMO
Hepatitis C virus (HCV) readily establishes high-level lifelong persistent infection in the majority of immunocompetent adults with failure of HCV-specific CD8+ CTL to clear viral replication. Virus-induced conditioning of innate immune responses is a possible mechanism that may contribute to the impairment of virus-specific CD8+ CTL responses. Here, we analyzed whether triggering of NK cell receptor expression and function is affected during chronic viremic HCV infection. Flow cytometric analysis of purified resting peripheral NK cells showed no evidence of NK cell activation, while analysis of natural cytotoxicity receptors (NCR) showed that NK cells from HCV-infected patients had selective increased expression of NKp30 and NKp46. NK cells had corresponding conserved cytotoxic activity against all targets with the exception of HepG2 hepatoma cells. Freshly separated NK cells from HCV patients showed significant production of IL-10 and normal concentrations of IFN-gamma upon cell-mediated triggering. Thus, increased expression of NKp30 during HCV infection with increased IL-10 production could contribute, once NK cells localize in the liver, to a NK-DC crosstalk leading to skewing of subsequent adaptive immune responses and lack of virus control.
Assuntos
Hepatite C Crônica/imunologia , Interleucina-10/biossíntese , Células Matadoras Naturais/imunologia , Glicoproteínas de Membrana/biossíntese , Receptores Imunológicos/biossíntese , Citotoxicidade Imunológica , Citometria de Fluxo , Imunofluorescência , Humanos , Interferon gama/biossíntese , Glicoproteínas de Membrana/imunologia , Receptor 1 Desencadeador da Citotoxicidade Natural , Receptor 3 Desencadeador da Citotoxicidade Natural , Receptores Imunológicos/imunologia , ViremiaRESUMO
HIV-1 infection in chimpanzees, the closest human relative, rarely leads to disease progression. NK cells contribute to the shaping of adaptive immune responses in humans and show perturbed phenotype and function during HIV-1 infection. In this study, we provide full phenotypic, molecular, and functional characterization for triggering molecules (NKp46, NKp30 NKp80, and NKG2D) on Pan troglodytes NK cells. We demonstrate that, in this AIDS-resistant species, relevant differences to human NK cells involve NKp80 and particularly NKp30, which is primarily involved in NK-dendritic cell interactions. Resting peripheral chimpanzee NK cells have low or absent NKp30 molecule expression due to posttranscriptional regulation and increase its levels upon in vitro activation. Following long-standing HIV-1 infection, peripheral NK cells in chimpanzees have conserved triggering receptor expression and display moderate phenotypic and functional decreases only once activated and cultured in vitro. These data suggest that one of the keys to successful lentivirus control may reside in part in a different regulation of NK cell-triggering receptor expression.
Assuntos
Regulação da Expressão Gênica/imunologia , Infecções por HIV/imunologia , Células Matadoras Naturais/metabolismo , Glicoproteínas de Membrana/genética , Receptores Imunológicos/genética , Animais , HIV-1 , Humanos , Imunidade Celular , Células Matadoras Naturais/imunologia , Lectinas Tipo C , Receptor 3 Desencadeador da Citotoxicidade Natural , Pan troglodytes , Fenótipo , Receptores de Células Matadoras Naturais , Transcrição GênicaRESUMO
An involvement of innate immunity and of NK cells during the priming of adaptive immune responses has been recently suggested in normal and disease conditions such as HIV infection and acute myelogenous leukemia. The analysis of NK cell-triggering receptor expression has been so far restricted to only NKp46 and NKp30 in Macaca fascicularis. In this study, we extended the molecular and functional characterization to the various NK cell-triggering receptors using PBMC and to the in vitro-derived NK cell populations by cytofluorometry and by cytolytic activity assays. In addition, RT-PCR strategy, cDNA cloning/sequencing, and transient transfections were used to identify and characterize NKp80, NKG2D, CD94/NKG2C, and CD94/NKG2A in M. fascicularis and Macaca mulatta as well as in the signal transducing polypeptide DNAX-activating protein DAP-10. Both M. fascicularis and M. mulatta NK cells express NKp80, NKG2D, and NKG2C molecules, which displayed a high degree of sequence homology with their human counterpart. Analysis of NK cells in simian HIV-infected M. fascicularis revealed reduced surface expression of selected NK cell-triggering receptors associated with a decreased NK cell function only in some animals. Overall surface density of NK cell-triggering receptors on peripheral blood cells and their triggering function on NK cell populations derived in vitro was not decreased compared with uninfected animals. Thus, triggering NK cell receptor monitoring on macaque NK cells is possible and could provide a valuable tool for assessing NK cell function during experimental infections and for exploring possible differences in immune correlates of protection in humans compared with cynomolgus and rhesus macaques undergoing different vaccination strategies.
Assuntos
Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptores Imunológicos/fisiologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Antígenos CD/imunologia , Antígenos CD/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Clonagem Molecular , Testes Imunológicos de Citotoxicidade/métodos , Citotoxicidade Imunológica/genética , Dimerização , Humanos , Células K562 , Células Matadoras Naturais/virologia , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Macaca fascicularis , Macaca mulatta , Proteínas de Membrana/genética , Proteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Subfamília C de Receptores Semelhantes a Lectina de Células NK , Subfamília D de Receptores Semelhantes a Lectina de Células NK , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/isolamento & purificação , Receptores Imunológicos/metabolismo , Receptores de Células Matadoras Naturais , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismoRESUMO
Membrane-bound and soluble interleukin-15 (IL-15)/IL-15 receptor alpha (Ralpha) complexes trigger differential transcription factor activation and functions on human hematopoietic progenitors. Indeed, human spleen myofibroblasts (SMFs) are characterized by a novel mechanism of IL-15 trans-presentation (SMFmb [membrane-bound]-IL-15), based on the association of an endogenous IL-15/IL-15Ralpha complex with the IL-15Rbetagamma c chains. SMFmb-IL-15 (1) induces lineage-specific signaling pathways that differ from those controlled by soluble IL-15 in unprimed and committed normal progenitors; (2) triggers survival and proliferation of leukemic progenitors expressing low-affinity IL-15R (M07Sb cells); (3) causes only an antiapoptotic effect on leukemic cells expressing high-affinity receptors (TF1beta cells). This behavior is likely due to the IL-15Ralpha chain present on these cells that interact with the SMFmb-IL-15, inhibiting signal transducer and transcriptional activator 5 (STAT5) activation. On the other hand, the soluble IL-15/IL-15Ralpha complex (hyper IL-15) displays a dominant pattern of action, activating only those cells expressing low-affinity IL-15R (IL-15Rbetagamma c). Thus, hyper IL-15 induces antiapoptotic effects on M075b cells and the up-regulation of STAT6 activation on adult peripheral blood (PB) pre-natural killer (NK) committed progenitors. The latter effect using 100-fold concentrations of recombinant (r)-IL-15. In conclusion, SMFmb-IL-15 and soluble IL-15Ralpha/IL-15 complexes seem to play a pivotal role in the control of the survival, proliferation and differentiation of both normal and leukemic circulating progenitors, highlighting new functions of IL-15 and of IL-15Ralpha.
Assuntos
Células-Tronco Hematopoéticas/citologia , Interleucina-15/metabolismo , Receptores de Interleucina-2/metabolismo , Antígenos CD34/biossíntese , Apoptose , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Linhagem da Célula , Membrana Celular/metabolismo , Proliferação de Células , Separação Celular , Citocinas/metabolismo , Citosol/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Immunoblotting , Imunoprecipitação , Microscopia Confocal , Receptores de Interleucina-15 , Proteínas Recombinantes de Fusão/química , Transdução de Sinais , Baço/citologia , Fatores de Tempo , Fatores de TranscriçãoRESUMO
Signals leading to NK cell triggering are primarily mediated by natural cytotoxicity receptors (NCR) upon binding to as-yet-undefined cell surface ligand(s) on normal hematopoietic cells, pathogen-infected cells or tumor cells. In this study we tried to determine whether the decreased NK cell cytolytic function that is observed in HIV-1-infected patients may be related to a decreased expression of NCR. In HIV-1-infected patients, freshly drawn, purified NK cells expressed significantly decreased surface densities of NKp46 and NKp30 NCR. The low surface density of NKp46, NKp30 and NKp44 was also confirmed in in-vitro-activated NK cell populations and NK cell clones derived from HIV-1 patients compared with uninfected donors. This defective NCR expression in HIV-1 patients was associated with a parallel decrease of NCR-mediated killing of different tumor target cells. Thus, the present study indicates that the defective expression of NCR represents at least one of the possible mechanisms leading to the impaired NK cell function in HIV-1 infection and it can contribute to explain the relatively high frequency of opportunistic tumors reported in cohorts of untreated patients before the occurrence of profound immunosuppression (<200 CD4(+) cells/mm(3)).
Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Células Matadoras Naturais/imunologia , Receptores Imunológicos/metabolismo , Regulação para Baixo/imunologia , Regulação para Baixo/fisiologia , Humanos , Células Matadoras Naturais/fisiologia , Receptor 1 Desencadeador da Citotoxicidade Natural , Receptor 2 Desencadeador da Citotoxicidade Natural , Receptor 3 Desencadeador da Citotoxicidade NaturalRESUMO
One of the hallmarks of HIV-1 infection is represented by the finding of massive T cell activation in peripheral blood lymphocytes of infected patients. An impairment of NK cell function during HIV-1 infection is also detected, and is associated with decreased expression of natural cytotoxicity receptors (NCR). In this study we tried to determine whether also NK cells are affected by relevant activation and whether this could be associated with decreased NK cell function. In 18 viremic HIV-1-infected patients, freshly drawn purified peripheral NK cells displayed significant levels of activation with an incomplete pattern (HLA-DR(+)CD69(+)CD25(-)NKp44(-)). Activated (HLA-DR(+)CD69(+)) peripheral NK cells expressed an NCR(dull) phenotype as determined by cytofluorometric analysis in all the patients, and did not derive from a homogeneous/oligoclonal expansion in vivo as analyzed by expression of HLA-specific inhibitory NK cell receptors. As determined by cytotoxicity assays, activated NK cells showed a decreased cytolytic function in HIV-1-infected patients. Thus, the decrease in NK cell function observed during HIV-1 infection is associated not only with decreased NCR expression, but also with significant and incomplete NK cell activation in vivo. These results suggest a consistent continuous involvement of the innate immune response in the failure to control viral replication.