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1.
Cell ; 165(2): 449-63, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26949186

RESUMO

Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Linfócitos B/imunologia , Anticorpos Anti-HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , HIV-1/imunologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Alinhamento de Sequência
2.
J Immunol ; 204(1): 112-121, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31818981

RESUMO

CMV is an obligate and persistent intracellular pathogen that continually drives the production of highly differentiated virus-specific CD8+ T cells in an Ag-dependent manner, a phenomenon known as memory inflation. Extensive proliferation is required to generate and maintain inflationary CD8+ T cell populations, which are counterintuitively short-lived and typically exposed to limited amounts of Ag during the chronic phase of infection. An apparent discrepancy therefore exists between the magnitude of expansion and the requirement for ongoing immunogenic stimulation. To address this issue, we explored the clonal dynamics of memory inflation. First, we tracked congenically marked OT-I cell populations in recipient mice infected with murine CMV (MCMV) expressing the cognate Ag OVA. Irrespective of numerical dominance, stochastic expansions were observed in each population, such that dominant and subdominant OT-I cells were maintained at stable frequencies over time. Second, we characterized endogenous CD8+ T cell populations specific for two classic inflationary epitopes, M38 and IE3. Multiple clonotypes simultaneously underwent Ag-driven proliferation during latent infection with MCMV. In addition, the corresponding CD8+ T cell repertoires were stable over time and dominated by persistent clonotypes, many of which also occurred in more than one mouse. Collectively, these data suggest that stochastic encounters with Ag occur frequently enough to maintain oligoclonal populations of inflationary CD8+ T cells, despite intrinsic constraints on epitope display at individual sites of infection with MCMV.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Seleção Clonal Mediada por Antígeno/imunologia , Memória Imunológica/imunologia , Muromegalovirus/imunologia , Animais , Proliferação de Células , Epitopos/imunologia , Infecções por Herpesviridae/imunologia , Infecções por Herpesviridae/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologia
3.
J Virol ; 93(21)2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31434738

RESUMO

Adoptive cell transfer (ACT) is a powerful experimental approach to directly study T-cell-mediated immunity in vivo In the rhesus macaque AIDS virus model, infusing simian immunodeficiency virus (SIV)-infected animals with CD8 T cells engineered to express anti-SIV T-cell receptor specificities enables direct experimentation to better understand antiviral T-cell immunity in vivo Limiting factors in ACT experiments include suboptimal trafficking to, and poor persistence in, the secondary lymphoid tissues targeted by AIDS viruses. Previously, we redirected CD8 T cells to B-cell follicles by ectopic expression of the CXCR5 homing protein. Here, we modify peripheral blood mononuclear cell (PBMC)-derived CD8 T cells to express the CCR9 chemokine receptor, which induces preferential homing of the engineered cells to the small intestine, a site of intense early AIDS virus replication and pathology in rhesus macaques. Additionally, we increase in vivo persistence and overall systemic distribution of infused CD8 T cells, especially in secondary lymphoid tissues, by minimizing ex vivo culture/manipulation, thereby avoiding the loss of CD28+/CD95+ central memory T cells by differentiation in culture. These proof-of-principle results establish the feasibility of preferentially localizing PBMC-derived CD8 T cells to the small intestine and enables the direct experimental ACT-based assessment of the potential role of the quality and timing of effective antiviral CD8 T-cell responses to inhibit viral infection and subsequent replication in small intestine CD4 T cells. More broadly, these results support the engineered expression of homing proteins to direct CD8 T cells to target tissues as a means for both experimental and potential therapeutic advances in T-cell immunotherapies, including cancer.IMPORTANCEAdoptive cell transfer (ACT) of T cells engineered with antigen-specific effector properties can deliver targeted immune responses against malignancies and infectious diseases. Current T-cell-based therapeutic ACT relies on circulatory distribution to deliver engineered T cells to their targets, an approach which has proven effective for some leukemias but provided only limited efficacy against solid tumors. Here, engineered expression of the CCR9 homing receptor redirected CD8 T cells to the small intestine in rhesus macaque ACT experiments. Targeted homing of engineered T-cell immunotherapies holds promise to increase the effectiveness of adoptively transferred cells in both experimental and clinical settings.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Quimiotaxia de Leucócito/imunologia , Intestino Delgado/imunologia , Receptores CCR/metabolismo , Transferência Adotiva , Animais , Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Quimiocinas CC/metabolismo , Memória Imunológica , Intestino Delgado/virologia , Leucócitos Mononucleares/imunologia , Linfonodos/imunologia , Macaca mulatta , Transdução de Sinais , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia
4.
Nature ; 511(7511): 601-5, 2014 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-25043006

RESUMO

Inflammation in HIV infection is predictive of non-AIDS morbidity and death, higher set point plasma virus load and virus acquisition; thus, therapeutic agents are in development to reduce its causes and consequences. However, inflammation may simultaneously confer both detrimental and beneficial effects. This dichotomy is particularly applicable to type I interferons (IFN-I) which, while contributing to innate control of infection, also provide target cells for the virus during acute infection, impair CD4 T-cell recovery, and are associated with disease progression. Here we manipulated IFN-I signalling in rhesus macaques (Macaca mulatta) during simian immunodeficiency virus (SIV) transmission and acute infection with two complementary in vivo interventions. We show that blockade of the IFN-I receptor caused reduced antiviral gene expression, increased SIV reservoir size and accelerated CD4 T-cell depletion with progression to AIDS despite decreased T-cell activation. In contrast, IFN-α2a administration initially upregulated expression of antiviral genes and prevented systemic infection. However, continued IFN-α2a treatment induced IFN-I desensitization and decreased antiviral gene expression, enabling infection with increased SIV reservoir size and accelerated CD4 T-cell loss. Thus, the timing of IFN-induced innate responses in acute SIV infection profoundly affects overall disease course and outweighs the detrimental consequences of increased immune activation. Yet, the clinical consequences of manipulation of IFN signalling are difficult to predict in vivo and therapeutic interventions in human studies should be approached with caution.


Assuntos
Progressão da Doença , Interferon-alfa/uso terapêutico , Macaca mulatta/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia/imunologia , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Interferon-alfa/farmacologia , Estimativa de Kaplan-Meier , Transdução de Sinais/efeitos dos fármacos , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle
5.
Immunol Cell Biol ; 97(6): 586-596, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30875134

RESUMO

The peripheral maturation of human CD1d-restricted natural killer T (NKT) cells has not been well described. In this study, we identified four major subsets of NKT cells in adults, distinguished by the expression of CD4, CD8 and CCR5. Phenotypic analysis suggested a hierarchical pattern of differentiation, whereby immature CD4+ CD8- CCR5- cells progressed to an intermediate CD4+ CD8- CCR5+ stage, which remained less differentiated than the CD4- CD8- and CD4- CD8+ subsets, both of which expressed CCR5. This interpretation was supported by functional data, including clonogenic potential and cytokine secretion profiles, as well as T-cell receptor (TCR) excision circle analysis. Moreover, conventional and high-throughput sequencing of the corresponding TCR repertoires demonstrated significant clonotypic overlap within individuals, especially between the more differentiated CD4- CD8- and CD4- CD8+ subsets. Collectively, these results mapped a linear differentiation pathway across the post-thymic landscape of human CD1d-restricted NKT cells.


Assuntos
Subpopulações de Linfócitos/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/genética , Antígenos CD1d/metabolismo , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Diferenciação Celular , Células Cultivadas , Células Clonais , Citocinas/metabolismo , Citometria de Fluxo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunofenotipagem , Receptores CCR5/metabolismo
6.
J Immunol ; 193(11): 5626-36, 2014 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-25348625

RESUMO

Despite progress toward understanding the correlates of protective T cell immunity in HIV infection, the optimal approach to Ag delivery by vaccination remains uncertain. We characterized two immunodominant CD8 T cell populations generated in response to immunization of BALB/c mice with a replication-deficient adenovirus serotype 5 vector expressing the HIV-derived Gag and Pol proteins at equivalent levels. The Gag-AI9/H-2K(d) epitope elicited high-avidity CD8 T cell populations with architecturally diverse clonotypic repertoires that displayed potent lytic activity in vivo. In contrast, the Pol-LI9/H-2D(d) epitope elicited motif-constrained CD8 T cell repertoires that displayed lower levels of physical avidity and lytic activity despite equivalent measures of overall clonality. Although low-dose vaccination enhanced the functional profiles of both epitope-specific CD8 T cell populations, greater polyfunctionality was apparent within the Pol-LI9/H-2D(d) specificity. Higher proportions of central memory-like cells were present after low-dose vaccination and at later time points. However, there were no noteworthy phenotypic differences between epitope-specific CD8 T cell populations across vaccine doses or time points. Collectively, these data indicate that the functional and phenotypic properties of vaccine-induced CD8 T cell populations are sensitive to dose manipulation, yet constrained by epitope specificity in a clonotype-dependent manner.


Assuntos
Vacinas contra a AIDS , Linfócitos T CD8-Positivos/imunologia , Epitopos Imunodominantes/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene pol do Vírus da Imunodeficiência Humana/metabolismo , Adenoviridae/genética , Animais , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Citotoxicidade Imunológica , Feminino , Vetores Genéticos , Antígenos H-2/metabolismo , Antígeno de Histocompatibilidade H-2D/metabolismo , Humanos , Epitopos Imunodominantes/genética , Memória Imunológica , Camundongos , Camundongos Endogâmicos BALB C , Vacinação , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genética
7.
J Allergy Clin Immunol ; 133(6): 1667-75, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24797421

RESUMO

BACKGROUND: Autosomal recessive loss-of-function mutations in dedicator of cytokinesis 8 (DOCK8) cause a combined immunodeficiency characterized by atopy, recurrent infections, and cancer susceptibility. A genotype-phenotype explanation for the variable disease expression is lacking. OBJECTIVE: We investigated whether reversions contributed to the variable disease expression. METHODS: Patients followed at the National Institutes of Health's Clinical Center were studied. We performed detailed genetic analyses and intracellular flow cytometry to detect DOCK8 protein expression within lymphocyte subsets. RESULTS: We identified 17 of 34 DOCK8-deficient patients who had germline mutations with variable degrees of reversion caused by somatic repair. Somatic repair of the DOCK8 mutations resulted from second-site mutation, original-site mutation, gene conversion, and intragenic crossover. Higher degrees of reversion were associated with recombination-mediated repair. DOCK8 expression was restored primarily within antigen-experienced T cells or natural killer cells but less so in naive T or B cells. Several patients exhibited multiple different repair events. Patients who had reversions were older and had less severe allergic disease, although infection susceptibility persisted. No patients were cured without hematopoietic cell transplantation. CONCLUSIONS: In patients with DOCK8 deficiency, only certain combinations of germline mutations supported secondary somatic repair. Those patients had an ameliorated disease course with longer survival but still had fatal complications or required hematopoietic cell transplantation. These observations support the concept that some DOCK8-immunodeficient patients have mutable mosaic genomes that can modulate disease phenotype over time.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Síndromes de Imunodeficiência/genética , Mutação , Fenótipo , Adolescente , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Criança , Pré-Escolar , Análise Mutacional de DNA , Reparo do DNA , Genótipo , Mutação em Linhagem Germinativa , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/mortalidade , Imunofenotipagem , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo
8.
Nature ; 452(7188): 773-6, 2008 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-18337720

RESUMO

The autosomal dominant hyper-IgE syndrome (HIES, 'Job's syndrome') is characterized by recurrent and often severe pulmonary infections, pneumatoceles, eczema, staphylococcal abscesses, mucocutaneous candidiasis, and abnormalities of bone and connective tissue. Mutations presumed to underlie HIES have recently been identified in stat3, the gene encoding STAT3 (signal transducer and activator of transcription 3) (refs 3, 4). Although impaired production of interferon-gamma and tumour-necrosis factor by T cells, diminished memory T-cell populations, decreased delayed-type-hypersensitivity responses and decreased in vitro lymphoproliferation in response to specific antigens have variably been described, specific immunological abnormalities that can explain the unique susceptibility to particular infections seen in HIES have not yet been defined. Here we show that interleukin (IL)-17 production by T cells is absent in HIES individuals. We observed that ex vivo T cells from subjects with HIES failed to produce IL-17, but not IL-2, tumour-necrosis factor or interferon-gamma, on mitogenic stimulation with staphylococcal enterotoxin B or on antigenic stimulation with Candida albicans or streptokinase. Purified naive T cells were unable to differentiate into IL-17-producing (T(H)17) T helper cells in vitro and had lower expression of retinoid-related orphan receptor (ROR)-gammat, which is consistent with a crucial role for STAT3 signalling in the generation of T(H)17 cells. T(H)17 cells have emerged as an important subset of helper T cells that are believed to be critical in the clearance of fungal and extracellular bacterial infections. Thus, our data suggest that the inability to produce T(H)17 cells is a mechanism underlying the susceptibility to the recurrent infections commonly seen in HIES.


Assuntos
Diferenciação Celular , Genes Dominantes , Interleucina-17/biossíntese , Síndrome de Job/imunologia , Síndrome de Job/patologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/patologia , Adolescente , Adulto , Candida albicans/imunologia , Criança , Pré-Escolar , Enterotoxinas/imunologia , Feminino , Humanos , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-2/biossíntese , Interleucina-2/imunologia , Síndrome de Job/genética , Síndrome de Job/metabolismo , Masculino , Pessoa de Meia-Idade , Estreptoquinase/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia
9.
Adv Mater ; 36(15): e2309843, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38302823

RESUMO

Injectable scaffold delivery is a strategy to enhance the efficacy of cancer vaccine immunotherapy. The choice of scaffold biomaterial is crucial, impacting both vaccine release kinetics and immune stimulation via the host response. Extracellular matrix (ECM) scaffolds prepared from decellularized tissues facilitate a pro-healing inflammatory response that promotes local cancer immune surveillance. Here, an ECM scaffold-assisted therapeutic cancer vaccine that maintains an immune microenvironment consistent with tissue reconstruction is engineered. Several immune-stimulating adjuvants are screened to develop a cancer vaccine formulated with decellularized small intestinal submucosa (SIS) ECM scaffold co-delivery. It is found that the STING pathway agonist cyclic di-AMP most effectively induces cytotoxic immunity in an ECM scaffold vaccine, without compromising key interleukin 4 (IL-4) mediated immune pathways associated with healing. ECM scaffold delivery enhances therapeutic vaccine efficacy, curing 50-75% of established E.G-7OVA lymphoma tumors in mice, while none are cured with soluble vaccine. SIS-ECM scaffold-assisted vaccination prolonged antigen exposure is dependent on CD8+ cytotoxic T cells and generates long-term antigen-specific immune memory for at least 10 months post-vaccination. This study shows that an ECM scaffold is a promising delivery vehicle to enhance cancer vaccine efficacy while being orthogonal to characteristics of pro-healing immune hallmarks.


Assuntos
Vacinas Anticâncer , Neoplasias , Animais , Camundongos , Matriz Extracelular/metabolismo , Memória Imunológica , Neoplasias/metabolismo , Alicerces Teciduais , Microambiente Tumoral , Interleucina-4/química , Interleucina-4/metabolismo
10.
J Immunol ; 187(6): 3111-20, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21849680

RESUMO

Each of the three Th2 cytokine genes, IL-4, IL-5, and IL-13, has different functions. We hypothesized that Th2 heterogeneity could yield Th2 subpopulations with different cytokine expression and effector functions. Using multiple approaches, we demonstrate that human Th2 cells are composed of two major subpopulations: a minority IL-5(+) (IL-5(+), IL-4(+), IL-13(+)) and majority IL-5(-) Th2 (IL-5(-), IL-4(+), IL-13(+)) population. IL-5(+) Th2 cells comprised only 20% of all Th2 cells. Serial rounds of in vitro differentiation initially yielded IL-5(-) Th2, but required multiple rounds of differentiation to generate IL-5(+) Th2 cells. IL-5(+) Th2 cells expressed less CD27 and greater programmed cell death-1 than IL-5(-) Th2 cells, consistent with their being more highly differentiated, Ag-exposed memory cells. IL-5(+) Th2 cells expressed greater IL-4, IL-13, and GATA-3 relative to IL-5(-) Th2 cells. GATA-3 and H3K4me(3) binding to the IL5 promoter (IL5p) was greater in IL-5(+) relative to IL-5(-) Th2 cells, whereas there was no difference in their binding to the IL4p and IL13p. Conversely, H3K27me(3) binding to the IL5p was greater in IL-5(-) Th2 cells. These findings demonstrate Th2 lineage heterogeneity, in which the IL5 gene is regulated in a hierarchical manner relative to other Th2 genes. IL-5(+) Th2 cells are phenotypically distinct and have epigenetic changes consistent with greater IL5p accessibility. Recurrent antigenic exposure preferentially drives the differentiation of IL-5(+) Th2 cells. These results demonstrate that IL-5(+) and IL-5(-) Th2 cells, respectively, represent more and less highly differentiated Th2 cell subpopulations. Such Th2 subpopulations may differentially contribute to Th2-driven pathology.


Assuntos
Diferenciação Celular/imunologia , Interleucina-5/imunologia , Subpopulações de Linfócitos T/citologia , Células Th2/citologia , Adolescente , Adulto , Separação Celular , Imunoprecipitação da Cromatina , Citometria de Fluxo , Humanos , Interleucina-5/metabolismo , Pessoa de Meia-Idade , Fenótipo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Adulto Jovem
11.
J Exp Med ; 203(13): 2865-77, 2006 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-17158960

RESUMO

The role of CD4+ T cells in the control of persistent viral infections beyond the provision of cognate help remains unclear. We used polychromatic flow cytometry to evaluate the production of the cytokines interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-2, the chemokine macrophage inflammatory protein (MIP)-1beta, and surface mobilization of the degranulation marker CD107a by CD4+ T cells in response to stimulation with cytomegalovirus (CMV)-specific major histocompatibility complex class II peptide epitopes. Surface expression of CD45RO, CD27, and CD57 on responding cells was used to classify CD4+ T cell maturation. The functional profile of virus-specific CD4+ T cells in chronic CMV infection was unique compared with that observed in other viral infections. Salient features of this profile were: (a) the simultaneous production of MIP-1beta, TNF-alpha, and IFN-gamma in the absence of IL-2; and (b) direct cytolytic activity associated with surface mobilization of CD107a and intracellular expression of perforin and granzymes. This polyfunctional profile was associated with a terminally differentiated phenotype that was not characterized by a distinct clonotypic composition. Thus, mature CMV-specific CD4+ T cells exhibit distinct functional properties reminiscent of antiviral CD8+ T lymphocytes.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Citomegalovirus/imunologia , Citotoxicidade Imunológica/imunologia , Adulto , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Ligante de CD40/metabolismo , Antígenos CD57/imunologia , Quimiocina CCL4 , Epitopos de Linfócito T/imunologia , Feminino , Produtos do Gene gag/imunologia , Granzimas/metabolismo , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Proteínas Inflamatórias de Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Perforina , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Vaccinia virus/imunologia
12.
J Immunol ; 185(11): 6646-63, 2010 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-20980630

RESUMO

Because T cells act primarily through short-distance interactions, homing receptors can identify colocalizing cells that serve common functions. Expression patterns for multiple chemokine receptors on CD4(+) T cells from human blood suggested a hierarchy of receptors that are induced and accumulate during effector/memory cell differentiation. We characterized CD4(+)CD45RO(+) T cells based on expression of two of these receptors, CCR5 and CCR2, the principal subsets being CCR5(-)CCR2(-) (∼70%), CCR5(+)CCR2(-) (∼25%), and CCR5(+)CCR2(+) (∼5%). Relationships among expression of CCR5 and CCR2 and CD62L, and the subsets' proliferation histories, suggested a pathway of progressive effector/memory differentiation from the CCR5(-)CCR2(-) to CCR5(+)CCR2(-) to CCR5(+)CCR2(+) cells. Sensitivity and rapidity of TCR-mediated activation, TCR signaling, and effector cytokine production by the subsets were consistent with such a pathway. The subsets also showed increasing responsiveness to IL-7, and the CCR5(+)CCR2(+) cells were CD127(bright) and invariably showed the greatest response to tetanus toxoid. CCR5(+)CCR2(+) cells also expressed the largest repertoire of chemokine receptors and migrated to the greatest number of chemokines. By contrast, the CCR5(+)CCR2(-) cells had the greatest percentages of regulatory T cells, activated/cycling cells, and CMV-reactive cells, and were most susceptible to apoptosis. Our results indicate that increasing memory cell differentiation can be uncoupled from susceptibility to death, and is associated with an increase in chemokine responsiveness, suggesting that vaccination (or infection) can produce a stable population of effector-capable memory cells that are highly enriched in the CCR5(+)CCR2(+) subset and ideally equipped for rapid recall responses in tissue.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Memória Imunológica , Receptores CCR2/fisiologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular/imunologia , Células Cultivadas , Humanos , Imunofenotipagem , Receptores CCR2/biossíntese , Receptores CCR5/biossíntese , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Fase de Repouso do Ciclo Celular/imunologia , Subpopulações de Linfócitos T/citologia
13.
Nat Med ; 11(11): 1238-43, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16227988

RESUMO

CD4(+)CD25(+) regulatory T (T(reg)) cells have a crucial role in maintaining immune tolerance. Mice and humans born lacking T(reg) cells develop severe autoimmune disease, and depletion of T(reg) cells in lymphopenic mice induces autoimmunity. Interleukin (IL)-2 signaling is required for thymic development, peripheral expansion and suppressive activity of T(reg) cells. Animals lacking IL-2 die of autoimmunity, which is prevented by administration of IL-2-responsive T(reg) cells. In light of the emerging evidence that one of the primary physiologic roles of IL-2 is to generate and maintain T(reg) cells, the question arises as to the effects of IL-2 therapy on them. We monitored T(reg) cells during immune reconstitution in individuals with cancer who did or did not receive IL-2 therapy. CD4(+)CD25(hi) cells underwent homeostatic peripheral expansion during immune reconstitution, and in lymphopenic individuals receiving IL-2, the T(reg) cell compartment was markedly increased. Mouse studies showed that IL-2 therapy induced expansion of existent T(reg) cells in normal hosts, and IL-2-induced T(reg) cell expansion was further augmented by lymphopenia. On a per-cell basis, T(reg) cells generated by IL-2 therapy expressed similar levels of FOXP3 and had similar potency for suppression compared to T(reg) cells present in normal hosts. These studies suggest that IL-2 and lymphopenia are primary modulators of CD4(+)CD25(+) T(reg) cell homeostasis.


Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Interleucina-2/uso terapêutico , Linfopenia/tratamento farmacológico , Receptores de Interleucina-2/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Adolescente , Adulto , Animais , Linfócitos T CD4-Positivos/imunologia , Criança , Feminino , Fatores de Transcrição Forkhead/análise , Homeostase/imunologia , Humanos , Interleucina-2/administração & dosagem , Interleucina-2/imunologia , Transfusão de Linfócitos , Linfopenia/induzido quimicamente , Linfopenia/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes/uso terapêutico , Sarcoma/complicações , Sarcoma/tratamento farmacológico , Linfócitos T Reguladores/imunologia
14.
J Exp Med ; 202(10): 1349-61, 2005 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-16287711

RESUMO

The forces that govern clonal selection during the genesis and maintenance of specific T cell responses are complex, but amenable to decryption by interrogation of constituent clonotypes within the antigen-experienced T cell pools. Here, we used point-mutated peptide-major histocompatibility complex class I (pMHCI) antigens, unbiased TCRB gene usage analysis, and polychromatic flow cytometry to probe directly ex vivo the clonal architecture of antigen-specific CD8(+) T cell populations under conditions of persistent exposure to structurally stable virus-derived epitopes. During chronic infection with cytomegalovirus and Epstein-Barr virus, CD8(+) T cell responses to immunodominant viral antigens were oligoclonal, highly skewed, and exhibited diverse clonotypic configurations; TCRB CDR3 sequence analysis indicated positive selection at the protein level. Dominant clonotypes demonstrated high intrinsic antigen avidity, defined strictly as a physical parameter, and were preferentially driven toward terminal differentiation in phenotypically heterogeneous populations. In contrast, subdominant clonotypes were characterized by lower intrinsic avidities and proportionately greater dependency on the pMHCI-CD8 interaction for antigen uptake and functional sensitivity. These findings provide evidence that interclonal competition for antigen operates in human T cell populations, while preferential CD8 coreceptor compensation mitigates this process to maintain clonotypic diversity. Vaccine strategies that reconstruct these biological processes could generate T cell populations that mediate optimal delivery of antiviral effector function.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Vírus de DNA/imunologia , Epitopos de Linfócito T/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Epitopos Imunodominantes/metabolismo , Subpopulações de Linfócitos T/metabolismo , Latência Viral/imunologia , Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Células Cultivadas , Células Clonais , Citomegalovirus/imunologia , Epitopos de Linfócito T/fisiologia , Herpesvirus Humano 4/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Epitopos Imunodominantes/fisiologia , Imunofenotipagem , Dados de Sequência Molecular , Mutação Puntual , Receptores de Antígenos de Linfócitos T/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/imunologia
15.
Eur J Immunol ; 40(7): 1973-84, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20468055

RESUMO

A novel T-cell vaccine strategy designed to deal with the enormity of HIV-1 variation is described and tested for the first time in macaques to inform and complement approaching clinical trials. T-cell immunogen HIVconsv, which directs vaccine-induced responses to the most conserved regions of the HIV-1, proteome and thus both targets diverse clades in the population and reduces the chance of escape in infected individuals, was delivered using six different vaccine modalities: plasmid DNA (D), attenuated human (A) and chimpanzee (C) adenoviruses, modified vaccinia virus Ankara (M), synthetic long peptides, and Semliki Forest virus replicons. We confirmed that the initial DDDAM regimen, which mimics one of the clinical schedules (DDDCM), is highly immunogenic in macaques. Furthermore, adjuvanted synthetic long peptides divided into sub-pools and delivered into anatomically separate sites induced T-cell responses that were markedly broader than those elicited by traditional single-open-reading-frame genetic vaccines and increased by 30% the overall response magnitude compared with DDDAM. Thus, by improving both the HIV-1-derived immunogen and vector regimen/delivery, this approach could induce stronger, broader, and theoretically more protective T-cell responses than vaccines previously used in humans.


Assuntos
Vacinas contra a AIDS , Antígenos HIV/administração & dosagem , HIV-1/imunologia , Fragmentos de Peptídeos/administração & dosagem , Linfócitos T/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Sequência Conservada/genética , Sistemas de Liberação de Medicamentos , Mapeamento de Epitopos/métodos , Epitopos de Linfócito T/genética , Vetores Genéticos , Antígenos HIV/genética , Humanos , Imunização , Ativação Linfocitária/efeitos dos fármacos , Macaca mulatta , Fragmentos de Peptídeos/genética , Biblioteca de Peptídeos , Especificidade do Receptor de Antígeno de Linfócitos T/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologia
16.
PLoS Pathog ; 5(10): e1000646, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19876388

RESUMO

Induction of a functional subset of HIV-specific CD4+ T cells that is resistant to HIV infection could enhance immune protection and decrease the rate of HIV disease progression. CMV-specific CD4+ T cells, which are less frequently infected than HIV-specific CD4+ T cells, are a model for such an effect. To determine the mechanism of this protection, we compared the functional response of HIV gag-specific and CMV pp65-specific CD4+ T cells in individuals co-infected with CMV and HIV. We found that CMV-specific CD4+ T cells rapidly up-regulated production of MIP-1alpha and MIP-1beta mRNA, resulting in a rapid increase in production of MIP-1alpha and MIP-1beta after cognate antigen stimulation. Production of beta-chemokines was associated with maturational phenotype and was rarely seen in HIV-specific CD4+ T cells. To test whether production of beta-chemokines by CD4+ T cells lowers their susceptibility to HIV infection, we measured cell-associated Gag DNA to assess the in vivo infection history of CMV-specific CD4+ T cells. We found that CMV-specific CD4+ T cells which produced MIP-1beta contained 10 times less Gag DNA than did those which failed to produce MIP-1beta. These data suggest that CD4+ T cells which produce MIP-1alpha and MIP-1beta bind these chemokines in an autocrine fashion which decreases the risk of in vivo HIV infection.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Quimiocinas CC/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Adulto , Células Cultivadas , Quimiocina CCL3/metabolismo , Quimiocina CCL4/metabolismo , Quimiocinas CC/biossíntese , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Feminino , Citometria de Fluxo , Humanos , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/metabolismo , Receptores CCR5/metabolismo , Proteínas da Matriz Viral/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo
17.
Blood ; 114(24): 5071-80, 2009 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-19776383

RESUMO

The successful reconstitution of adaptive immunity to human cytomegalovirus (CMV) in hematopoietic stem cell transplantation (HSCT) recipients is central to the reduction of viral reactivation-related morbidity and mortality. Here, we characterized the magnitude, specificity, phenotype, function, and clonotypic composition of CMV-specific T-cell responses in 18 donor-recipient pairs both before and after HSCT. The principal findings were: (1) the specificity of CMV-specific T-cell responses in the recipient after HSCT mirrors that in the donor; (2) the maintenance of these targeting patterns reflects the transfer of epitope-specific T-cell clonotypes from donor to recipient; (3) less differentiated CD27(+)CD57(-) CMV-specific memory T cells are more likely to persist in the recipient after HSCT compared with more terminally differentiated CD27(-) CD57(+) CMV-specific memory T cells; (4) the presence of greater numbers of less differentiated CD8(+) CMV-specific T cells in the donor appears to confer protection against viral reactivation in the recipient after HSCT; and (5) CMV-specific T cells acquire a more differentiated phenotype and a restricted functional profile after HSCT. Overall, these findings define the immunologic factors that influence the successful adoptive transfer of antigen-specific T-cell immunity during HSCT, which enables the identification of recipients at particular risk of CMV reactivation after HSCT.


Assuntos
Imunidade Adaptativa , Citomegalovirus/imunologia , Transplante de Células-Tronco Hematopoéticas , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Diferenciação Celular/imunologia , Cromatografia Líquida de Alta Pressão , Infecções por Citomegalovirus/imunologia , Citometria de Fluxo , Humanos , Memória Imunológica/imunologia , Fenótipo , Subpopulações de Linfócitos T/citologia , Adulto Jovem
18.
J Immunol ; 183(2): 1120-32, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19564339

RESUMO

Recent studies have revealed the critical role of programmed death-1 (PD-1) in exhaustion of HIV- and SIV-specific CD8(+) T cells. In this study, we show that high expression of PD-1 correlates with increased ex vivo spontaneous and CD95/Fas-induced apoptosis, particularly in the "effector-memory" CD8(+) T cell population from HIV(+) donors. High expression of PD-1 was linked to a proapoptotic phenotype characterized by low expression of Bcl-2 and IL7-R alpha, high expression of CD95/Fas and high mitochondrial mass. Expression of PD-1 and CD57 was differentially associated with the maturation status of CD8(+) T cells in HIV infection. CD57 was linked to higher apoptosis resistance, with cells expressing a PD-1(L)CD57(H) phenotype exhibiting lower levels of cell death. The majority of HIV-specific CD8(+) T cells were found to express a PD-1(H)CD57(L) or PD-1(H)CD57(H) phenotype. No correlation was found between PD-1 expression and ex vivo polyfunctionality of either HIV- or CMV-specific CD8(+) T cells. Contrary to CD57, high expression of PD-1 was characterized by translocation of PD-1 into the area of CD95/Fas-capping, an early necessary step of CD95/Fas-induced apoptosis. Thus, our data further support the role of PD-1 as a preapoptotic factor for CD8(+) T cells in HIV infection.


Assuntos
Antígenos CD/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Apoptose , Antígenos CD57/fisiologia , Linfócitos T CD8-Positivos/patologia , Infecções por HIV/patologia , Antígenos CD/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Sobrevivência Celular , Células Cultivadas , Citomegalovirus/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Receptor de Morte Celular Programada 1 , Transporte Proteico , Receptor fas
19.
BMC Microbiol ; 10: 267, 2010 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-20961439

RESUMO

BACKGROUND: Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA)-accepted assay to detect and type botulinum neurotoxins (BoNTs) is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of C. botulinum regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR) technology to determine the specific serotype of the neurotoxin. RESULTS: We assayed purified C. botulinum DNA and crude toxin preparations, as well as food and stool from healthy individuals spiked with purified BoNT DNA, and one stool sample from a case of infant botulism for the presence of the NTNH gene, which is part of the BoNT gene cluster, and for the presence of serotype-specific BoNT genes. The PCR surpassed the mouse bioassay both in specificity and sensitivity, detecting positive signals in BoNT preparations containing well below the 1 LD50 required for detection via the mouse bioassay. These results were type-specific and we were reliably able to quantify as few as 10 genomic copies. CONCLUSIONS: While other studies have reported conventional or quantitative PCR-based assays for the detection of C. botulinum genes, our procedure's high-throughput capability and its portability allows most laboratories to quickly assess the possible presence of BoNTs either in food processing samples or in suspected cases of botulism. Thus, this assay provides rapid and specific detection of BoNT and toxin complex genes and would enable the targeting of appropriate therapeutics to infected individuals in a timely manner.


Assuntos
Toxinas Botulínicas/isolamento & purificação , Clostridium botulinum/genética , Neurotoxinas/isolamento & purificação , Bioensaio , Toxinas Botulínicas/genética , DNA Bacteriano/análise , Microbiologia de Alimentos , Genes Bacterianos , Humanos , Lactente , Neurotoxinas/genética , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade
20.
J Immunol Methods ; 321(1-2): 107-20, 2007 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-17316678

RESUMO

The characterization of the T-cell receptor (TCR) repertoire of CD4+ regulatory T cells (T(R)) has been limited due to the RNA degradation that results following permeabilization and fixation as routinely used for intracellular staining of Foxp3. In the present study the clonal composition of human umbilical cord blood (UCB) and adult peripheral blood mononuclear cell (PBMC) CD4+ T(R) and non-T(R) was characterized by a DNA-based multiplex PCR which allowed for the consistent clonotypic characterization of cells that have undergone fixation and permeabilization. To validate this method, CD8+ T cells from two HLA A()0201 individuals were sorted and compared clonotypically based upon their ability either to secrete interferon-gamma in response to a CMV pp65 epitope or to bind to the corresponding pMHC I tetramer. Clonotypes shared between the CD4+CD25+Foxp3+ and CD4+CD25+Foxp3- subsets were observed in all 3 UCB and in one adult PBMCs, suggesting that naïve and memory CD4+ T(R) can share the same clonotypes as CD4+ non-T(R) in humans.


Assuntos
Primers do DNA , Fatores de Transcrição Forkhead/análise , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Subunidade alfa de Receptor de Interleucina-2/análise , Leucócitos Mononucleares/imunologia , Reação em Cadeia da Polimerase/métodos , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Adulto , Sequência de Aminoácidos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Células Clonais/imunologia , Sangue Fetal/citologia , Sangue Fetal/imunologia , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunofenotipagem/métodos , Subunidade alfa de Receptor de Interleucina-2/genética , Leucócitos Mononucleares/virologia , Dados de Sequência Molecular , Fosfoproteínas/imunologia , Estabilidade de RNA , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Reprodutibilidade dos Testes , Linfócitos T Reguladores/imunologia , Proteínas da Matriz Viral/imunologia
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