RESUMO
Changes in mean telomeric terminal restriction fragment (TRF) length were examined as a marker for cellular replicative history in HIV-1-infected individuals after institution of anti-retroviral therapy (ART). Increases in mean T cell TRF lengths were observed in most patients following therapy; however, the contribution of individual T cell subsets was complex. An elongation of CD8+ T cell TRF was nearly uniformly observed while changes in mean TRF length in CD4+ T cells were heterogeneous as, despite potent suppression of viral replication, CD4 cell telomeres recovered in some patients, yet continued to decline in others. Increases in CD8 cell TRF correlated with decreased memory cells, suggesting a negative selection in the periphery for CD8 cells with extensive replicative history. In contrast, increases in CD4+ T cell TRF length correlated with increases in naive cell subsets, suggesting that the CD4+ T cell TRF increase may reflect a thymic contribution in some patients. These are the first increases in somatic cell telomere length in a population of cells observed in vivo, and the findings are compatible with therapy-induced reconstitution of the lymphoid compartment with cells having a more extensive replicative potential. These findings further distinguish lymphocytes from other somatic cell populations where only decreases in TRF over time have been noted. Thus, institution of ART in persons with moderately advanced HIV-1 disease reveals distinct population dynamics of CD4 and CD8 T cell subsets and also shows that the lymphocyte replicative history is dynamic.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Infecções por HIV/tratamento farmacológico , Telômero/genética , Adulto , Divisão Celular , Estudos de Coortes , Humanos , Imunofenotipagem , Contagem de Linfócitos/efeitos dos fármacos , Pessoa de Meia-Idade , Mapeamento por Restrição , Retroviridae/efeitos dos fármacos , Linfócitos T/citologiaRESUMO
Intracellular signals that mediate differentiation of pluripotent hemopoietic progenitors to dendritic cells (DC) are largely undefined. We have previously shown that protein kinase C (PKC) activation (with phorbol ester (PMA) alone) specifically induces differentiation of primary human CD34+ hemopoietic progenitor cells (HPC) to mature DC. We now find that cytokine-driven (granulocyte-macrophage CSF and TNF-alpha) CD34+ HPC-->DC differentiation is preferentially blocked by inhibitors of PKC activation. To further identify intracellular signals and downstream events important in CD34+ HPC-->DC differentiation we have characterized a human leukemic cell line model of this process. The CD34+ myelomonocytic cell line KG1 differentiates into dendritic-like cells in response to granulocyte-macrophage CSF plus TNF-alpha, or PMA (with or without the calcium ionophore ionomycin, or TNF-alpha), with different stimuli mediating different aspects of the process. Phenotypic DC characteristics of KG1 dendritic-like cells include morphology (loosely adherent cells with long neurite processes), MHC I+/MHC IIbright/CD83+/CD86+/CD14- surface Ag expression, and RelB and DC-CK1 gene expression. Functional DC characteristics include fluid phase macromolecule uptake (FITC-dextran) and activation of resting T cells. Comparison of KG1 to the PMA-unresponsive subline KG1a reveals differences in expression of TNF receptors 1 and 2; PKC isoforms alpha, beta I, beta II, and mu; and RelB, suggesting that these components/pathways are important for DC differentiation. Together, these findings demonstrate that cytokine or phorbol ester stimulation of KG1 is a model of human CD34+ HPC to DC differentiation and suggest that specific intracellular signaling pathways mediate specific events in DC lineage commitment.
Assuntos
Antígenos CD34/imunologia , Células Dendríticas/citologia , Células-Tronco Hematopoéticas/citologia , Líquido Intracelular/imunologia , Transdução de Sinais/imunologia , Adulto , Antígenos de Superfície/biossíntese , Apoptose/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Divisão Celular/imunologia , Linhagem Celular , Citocinas/fisiologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Ativação Enzimática/imunologia , Regulação da Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Imunofenotipagem , Ativação Linfocitária/efeitos dos fármacos , Substâncias Macromoleculares , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas/biossíntese , Linfócitos T/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição RelB , Fatores de Transcrição/biossínteseRESUMO
Fusion and entry of the human immunodeficiency virus (HIV) into CD4(+) T lymphocytes requires expression of CD4 and a coreceptor. At least eight chemokine receptors can serve as coreceptors for HIV. Accumulating evidence indicates that multiple factors, including the state of cellular differentia- tion and activation, regulate the expression of alpha- and beta-chemokine receptors on lymphocytes. For example, binding of antibodies to the CD28 coreceptor can downregulate expression of beta-chemokine receptors, and this appears to have important consequences on the susceptibility of CD4(+) T lymphocytes to infection by HIV-1. In contrast, binding of the natural CD28 ligand B7 or antibodies to the CD28 homologue CTLA-4 can upregulate CCR5 expression, sug- gesting a reciprocal interaction between CD28 and CTLA-4 and the regulation of beta-chemokine receptor expression. Thus, the CD28/CTLA-4/B7 co-stimulation pathway is identi- fied as a potential novel target for the control of susceptibility to some strains of HIV-1 infection.
Assuntos
Antígenos CD28/imunologia , Quimiocinas/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Imunoconjugados , Receptores de Quimiocinas/imunologia , Linfócitos T/imunologia , Linfócitos T/virologia , Abatacepte , Antígenos CD , Antígenos de Diferenciação/imunologia , Antígeno CTLA-4 , Humanos , Receptores CCR5/imunologia , Receptores Virais/imunologia , Transdução de Sinais/imunologiaRESUMO
Recombinant forms of human immunodeficiency virus type 1 (HIV-1) have been shown to be of major importance in the global AIDS pandemic. Viral RNA dimer formation mediated by the dimerization initiation sequence (DIS) is believed to be essential for viral genomic RNA packaging and therefore for RNA recombination. Here, we demonstrate that HIV-1 recombination and replication are not restricted by variant DIS loop sequences. Three DIS loop forms found among HIV-1 isolates, DIS (CG), DIS (TA), and DIS (TG), when introduced into deletion mutants of HIV-1 recombined efficiently, and the progeny virions replicated with comparable kinetics. A fourth DIS loop form, containing an artificial AAAAAA sequence disrupting the putative DIS loop-loop interactions [DIS (A6)], supported efficient recombination with DIS loop variants; however, DIS (A6) progeny virions exhibited a modest replication disadvantage in mixed cultures. Our studies indicate that the nonhomologous DIS sequences found in different HIV-1 subtypes are not a primary obstacle to intersubtype recombination.
Assuntos
HIV-1/genética , Recombinação Genética , Clonagem Molecular , Dimerização , Variação Genética , HIV-1/isolamento & purificação , HIV-1/fisiologia , Humanos , Células Tumorais Cultivadas , Replicação ViralRESUMO
Immunization with plasmids expressing specific genes (DNA or nucleic acid vaccination (NAV)) elicits robust humoral and cell-mediated immune responses. The mechanisms involved in T cell activation by NAV are incompletely characterized. We have examined the costimulatory requirements of NAV. CD28-deficient mice did not mount Ab or CTL responses following i.m. immunization with eukaryotic expression plasmids encoding the bacterial gene beta-galactosidase (beta gal). Because these mice retained their ability to up-regulate the CTLA4 receptor (a negative regulator of T cell activation), we examined CTLA4's role in the response of wild-type BALB/c mice to NAV. Intact anti-CTLA4 mAb but not Fab fragments suppressed the primary humoral response to pCIA/beta gal without affecting recall responses, indicating CTLA4 activation inhibited Ab production but not T cell priming. Blockade of the ligands for CD28 and CTLA4, CD80 (B7-1) and CD86 (B7-2), revealed distinct and nonoverlapping function. Blockade of CD80 at initial immunization completely abrogated primary and secondary Ab responses, whereas blockade of CD86 suppressed primary but not secondary responses. Simultaneous blockade of CD80 + CD86 was less effective at suppressing Ab responses than either alone. Enhancement of costimulation via coinjection of B7-expressing plasmids augmented CTL responses but not Ab responses, and without evidence of Th1 to Th2 skewing. These findings suggest complex and distinct roles for CD28, CTLA4, CD80, and CD86 in T cell costimulation following nucleic acid vaccination.
Assuntos
Antígenos de Diferenciação/fisiologia , Antígenos CD28/fisiologia , Imunoconjugados , Ativação Linfocitária , Linfócitos T/imunologia , Vacinas de DNA/imunologia , Abatacepte , Animais , Formação de Anticorpos , Antígenos CD/fisiologia , Antígeno B7-1/fisiologia , Antígeno B7-2 , Antígeno CTLA-4 , DNA Complementar/imunologia , Imunização , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Citotóxicos/fisiologiaRESUMO
When HIV-infected leukocytes are activated by the CD28 costimulatory receptor, HIV-1 is rapidly cleared from cultures, suggesting that costimulation can render T cells resistant to HIV-1 infection. In this study we tested the hypothesis that enhanced secretion of cytokines or chemokines could account for CD28-induced antiviral effects. In an acute infection system, resistance to infection with macrophage-tropic strains of HIV-1 was shown to be comprised of both soluble and cell-associated components. Induction of HIV-1 resistance was specific for CD28 costimulation, in that a variety of other accessory receptors, such as CD2, CD4, CD5, and MHC class I, failed to confer the antiviral resistance. The soluble component was secreted by both CD4 and CD8 T cells, was not unique to CD28 costimulation, and could be neutralized by removal of C-C chemokines (RANTES (regulated upon activation, normal T cell expressed and secreted) and macrophage inflammatory protein-1alpha and -1beta) from the culture supernatants of costimulated CD4 T cells. In contrast, CD28 stimulation of CD4 cells resulted in the specific induction of a pronounced intrinsic resistance to HIV-1 infection by macrophage tropic isolates of HIV-1.
Assuntos
Antígenos CD28/imunologia , Infecções por HIV/imunologia , HIV-1 , Imunidade Inata , Linfócitos T/imunologia , Células Cultivadas , Antígenos de Histocompatibilidade Classe I , Humanos , Linfócitos T/virologiaAssuntos
Linfócitos T CD4-Positivos/citologia , Adulto , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Divisão Celular , Células Cultivadas , Humanos , Interleucina-2/farmacologia , Ionomicina/farmacologia , Ativação Linfocitária , Telômero/efeitos dos fármacos , Telômero/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoAssuntos
Antígenos CD28/fisiologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais , Linfócitos T/imunologia , Animais , Citocinas/biossíntese , Humanos , Inflamação , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Choque Séptico/imunologia , Transcrição GênicaRESUMO
Progression to AIDS in asymptomatic HIV-infected individuals is characterized by a gradual but progressive loss of CD4+ T cells. While the mechanisms underlying this decline are currently unknown, recent evidence suggests that these cells are abnormally sensitive to apoptosis in response to activation signals. Recent work has implicated downregulation of Bcl-2 with the increased spontaneous apoptosis in lymphocytes from HIV-infected patients. We have evaluated the roles of the apoptosis-protective proteins Bcl-2 and Bcl-x in stimulated PBMC from asymptomatic HIV-infected and HIV-uninfected individuals. We found that Bcl-2 was constitutively expressed in PBMC from both HIV-infected and uninfected samples. However, Bcl-x induction was delayed and responses were decreased in stimulated HIV-infected samples. Additionally, single-cell intracellular staining of Bcl-x revealed a significant inverse correlation between PWM-induced Bcl-x expression and apoptosis (r = -0.695, P = 0.05). This was confirmed at the single-cell level in direct experiments when stimulated cells were sorted based on Bcl-x induction and then measured for apoptosis. Furthermore, low Bcl-x expression was not due to reduced lymphocyte activation following PWM stimulation. Our data indicate that the induction of Bcl-x is markedly impaired in asymptomatic HIV-infected patients and that stimuli which induce inadequate expression of Bcl-x are associated with increased levels of apoptosis in these cells.
Assuntos
Apoptose/imunologia , Infecções por HIV/imunologia , Leucócitos Mononucleares/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Antígenos CD/biossíntese , Antígenos de Diferenciação de Linfócitos T/biossíntese , Apoptose/efeitos dos fármacos , Feminino , Infecções por HIV/metabolismo , Humanos , Células Jurkat , Lectinas Tipo C , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Mitógenos de Phytolacca americana/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/efeitos dos fármacos , Proteína bcl-XRESUMO
Activation of CD4(+) T lymphocytes from human immunodeficiency virus-type 1 (HIV-1)-infected donors with immobilized antibodies to CD3 and CD28 induces a virus-resistant state. This effect is specific for macrophage-tropic HIV-1. Transcripts encoding CXCR4/Fusin, the fusion cofactor used by T cell line-tropic isolates, were abundant in CD3/CD28-stimulated cells, but transcripts encoding CCR5, the fusion cofactor used by macrophage-tropic viruses, were not detectable. Thus, CD3/CD28 costimulation induces an HIV-1-resistant phenotype similar to that seen in some highly exposed and HIV-uninfected individuals.
Assuntos
Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , HIV-1/fisiologia , Ativação Linfocitária , Proteínas de Membrana/genética , Receptores de HIV/genética , Anticorpos Monoclonais/imunologia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Interleucina-2/imunologia , Fusão de Membrana , Muromonab-CD3/imunologia , Fito-Hemaglutininas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores CCR5 , Receptores CXCR4 , Receptores de Citocinas/genética , Regulação para Cima , Replicação ViralRESUMO
Because stimulation of CD4+ lymphocytes leads to activation of human immunodeficiency virus-type 1 (HIV-1) replication, viral spread, and cell death, adoptive CD4+ T cell therapy has not been possible. When antigen and CD28 receptors on cultured T cells were stimulated by monoclonal antibodies (mAbs) to CD3 and CD28 that had been immobilized, there was an increase in the number of polyclonal CD4+ T cells from HIV-infected donors. Activated cells predominantly secreted cytokines associated with T helper cell type 1 function. The HIV-1 viral load declined in the absence of antiretroviral agents. Moreover, CD28 stimulation of CD4+ T cells from uninfected donors rendered these cells highly resistant to HIV-1 infection. Immobilization of CD28 mAb was crucial to the development of HIV resistance, as cells stimulated with soluble CD28 mAb were highly susceptible to HIV infection. The CD28-mediated antiviral effect occurred early in the viral life cycle, before HIV-1 DNA integration. These data may facilitate immune reconstitution and gene therapy approaches in persons with HIV infection.
Assuntos
Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Ativação Linfocitária , Anticorpos Monoclonais/imunologia , Complexo CD3/imunologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/citologia , Divisão Celular , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Interleucina-2/farmacologia , Fito-Hemaglutininas/farmacologia , Integração Viral , Replicação ViralRESUMO
A variety of deficiencies in T cell activation have been described in HIV-1 infection. To determine whether one component of Ag receptor signal transduction might be impaired and contribute to the immunopathology of HIV infection, we tested CD4 cells from patients with early to mid-stage HIV infection for TCR-induced calcium mobilization. There was no detectable difference between patients and controls in the mean CD4 cell calcium response or in the fraction of responding CD4 cells after cross-linking the TCR with OKT3 Ab. In addition, in HIV-infected patients, there was no correlation between calcium mobilization and the CD4 cell count. These results indicate that there are no intrinsic impairments of Ag receptor calcium signaling in circulating CD4 cells from HIV-infected patients with more than 400 CD4 cells/mm3, although abnormalities in patients with later stage infections cannot be excluded.
Assuntos
Cálcio/fisiologia , Infecções por HIV/imunologia , HIV-1/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Adulto , Linfócitos T CD4-Positivos/metabolismo , Feminino , HIV-1/metabolismo , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Our objective was to determine the role that bone marrow-derived stromal cells have on human hematopoiesis in HIV infection. In particular, we dissected the heterogeneous bone marrow microenvironment to study the effect HIV expression might have on the cell population capable of producing the cytokines which will support human CD34+ cell differentiation. A stromal cell line, Lof(11-10), was established from human bone marrow by transfecting a plasmid containing the SV40 large T-antigen and isolating foci exhibiting a transformed phenotype. The Lof(11-10) cell line was characterized to determine its susceptibility to HIV infection, to identify its cytokine production profile, and to test the ability of conditioned media from this line to support CD34+ cell differentiation in the presence and absence of HIV expression. Nine cytokines were detected by RT-PCR and ELISA analysis. Conditioned media obtained from the Lof(11-10) cell line was able to support CD34+ celle differentiation. However, because the Lof(11-10) cells are not infectible by HIV, molecular clones of HIV were introduced into these cells by transfection. There was no qualitative difference in the levels of cytokine production between HIV-expressing and control Lof(11-10) cells. Furthermore, conditioned media derived from HIV-expressing and control Lof(11-10) cells added to bone marrow-derived CD34+ progenitor cells yielded similar colony formation in methylcellulose assays. Our data suggest that HIV infection of the cytokine-producing cells within the bone marrow microenvironment, as represented by the Lof(11-10) cell line, results in both normal cytokine production and hematopoiesis in spite of HIV expression. This report adds to the evidence against stromal cells being a significant target of HIV and establishes a system for comparison with more relevant models. Copyright 1995 S. Karger AG, Basel
RESUMO
We have constructed stable human immunodeficiency virus (HIV) packaging cell lines that when transfected with an HIV-based retroviral vector produce packaged vectors capable of transducing susceptible CD4+ cells. This HIV-1-based retroviral vector system has the potential for providing targeted delivery and regulated expression of immunogens or antiviral agents in CD4+ cells.