RESUMO
The gp120 CD4 binding site (CD4bs) and coreceptor binding site (CoRbs) are two functionally conserved elements of the HIV-1 envelope glycoproteins (Env). We previously defined the presence of CD4bs-neutralizing antibodies in the serum of an HIV-1-infected individual and subsequently isolated the CD4bs-specific monoclonal antibodies (MAbs) VRC01 and VRC03 from the memory B cell population. Since this donor's serum also appeared to contain neutralizing antibodies to the CoRbs, we employed a differential fluorescence-activated cell sorter (FACS)-based sorting strategy using an Env trimer possessing a CoRbs knockout mutation (I420R) to isolate specific B cells. The MAb VRC06 was recovered from these cells, and its genetic sequence allowed us to identify a clonal relative termed VRC06b, which was isolated from a prior cell sort using a resurfaced core gp120 probe and its cognate CD4bs knockout mutant. VRC06 and VRC06b neutralized 22% and 44% of viruses tested, respectively. Epitope mapping studies revealed that the two MAbs were sensitive to mutations in both the gp120 CoRbs and the CD4bs and could cross-block binding of both CD4bs and CoRbs MAbs to gp120. Fine mapping indicated contacts within the gp120 bridging sheet and the base of the third major variable region (V3), which are elements of the CoRbs. Cell surface binding assays demonstrated preferential recognition of fully cleaved Env trimers over uncleaved trimers. Thus, VRC06 and VRC06b are Env trimer precursor cleavage-sensitive neutralizing MAbs that bind to a region of gp120 that overlaps both the primary and the secondary HIV-1 receptor binding sites.
Assuntos
Anticorpos Neutralizantes/imunologia , Sítios de Ligação de Anticorpos , Antígenos CD4/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Receptores de HIV/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/metabolismo , Linfócitos B/imunologia , Antígenos CD4/metabolismo , Células Cultivadas , Mapeamento de Epitopos , Epitopos/imunologia , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Mutação , Receptores de HIV/metabolismoRESUMO
The surface HIV-1 exterior envelope glycoprotein, gp120, binds to CD4 on the target cell surface to induce the co-receptor binding site on gp120 as the initial step in the entry process. The binding site is comprised of a highly conserved region on the gp120 core, as well as elements of the third variable region (V3). Antibodies against the co-receptor binding site are abundantly elicited during natural infection of humans, but the mechanism of elicitation has remained undefined. In this study, we investigate the requirements for elicitation of co-receptor binding site antibodies by inoculating rabbits, monkeys and human-CD4 transgenic (huCD4) rabbits with envelope glycoprotein (Env) trimers possessing high affinity for primate CD4. A cross-species comparison of the antibody responses showed that similar HIV-1 neutralization breadth was elicited by Env trimers in monkeys relative to wild-type (WT) rabbits. In contrast, antibodies against the co-receptor site on gp120 were elicited only in monkeys and huCD4 rabbits, but not in the WT rabbits. This was supported by the detection of high-titer co-receptor antibodies in all sera from a set derived from human volunteers inoculated with recombinant gp120. These findings strongly suggest that complexes between Env and (high-affinity) primate CD4 formed in vivo are responsible for the elicitation of the co-receptor-site-directed antibodies. They also imply that the naïve B cell receptor repertoire does not recognize the gp120 co-receptor site in the absence of CD4 and illustrate that conformational stabilization, imparted by primary receptor interaction, can alter the immunogenicity of a type 1 viral membrane protein.
Assuntos
Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Antígenos CD4/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Animais , Anticorpos Antivirais/genética , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Antígenos CD4/genética , Linhagem Celular , Feminino , Proteína gp120 do Envelope de HIV/genética , Infecções por HIV/genética , HIV-1/genética , Humanos , Macaca fascicularis , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Coelhos , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Especificidade da Espécie , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
Transcription factors (TFs) are critical for B-cell differentiation, affecting gene expression both by repression and transcriptional activation. Still, this information is not used for classification of B-cell lymphomas (BCLs). Traditionally, BCLs are diagnosed based on a phenotypic resemblance to normal B-cells; assessed by immunohistochemistry or flow cytometry, by using a handful of phenotypic markers. In the last decade, diagnostic and prognostic evaluation has been facilitated by global gene expression profiling (GEP), providing a new powerful means for the classification, prediction of survival, and response to treatment of lymphomas. However, most GEP studies have typically been performed on whole tissue samples, containing varying degrees of tumor cell content, which results in uncertainties in data analysis. In this study, global GEP analyses were performed on highly purified, flow-cytometry sorted tumor-cells from eight subgroups of BCLs. This enabled identification of TFs that can be uniquely associated to the tumor cells of chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), hairy cell leukemia (HCL), and mantle cell lymphoma (MCL). The identified transcription factors influence both the global and specific gene expression of the BCLs and have possible implications for diagnosis and treatment.
Assuntos
Subpopulações de Linfócitos B/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Linfoma de Células B/genética , Proteínas de Neoplasias/genética , Fatores de Transcrição/genética , Subpopulações de Linfócitos B/patologia , Separação Celular , Análise por Conglomerados , Citometria de Fluxo , Humanos , Linfoma de Células B/classificação , Linfoma de Células B/patologia , Proteínas de Neoplasias/biossíntese , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Fatores de Transcrição/biossíntese , Transcrição GênicaRESUMO
Multiparameter flow cytometry has matured tremendously since the 1990s, giving rise to a technology that allows us to study the immune system in unprecedented detail. In this article, we review the development of hardware, reagents, and data analysis tools for multiparameter flow cytometry and discuss future advances in the field. Finally, we highlight new applications that use this technology to reveal previously unappreciated aspects of cell biology and immunity.
Assuntos
Citometria de Fluxo/métodos , Estatística como Assunto , Animais , Corantes , Desenho de Equipamento , Citometria de Fluxo/tendências , Previsões , Humanos , Doenças do Sistema Imunitário/diagnósticoRESUMO
Differential gene expression analysis, using high-density microarray chips, demonstrated 300-400 genes to be deregulated in mantle cell lymphomas (MCLs) compared with normal B-cell populations. To investigate the significance of this genetic signature in lymphoma etiology and diagnostics, we selected 90 annotated genes involved in a number of cellular functions for further analysis. Our findings demonstrated a normal gene expression of CCR7, which indicated a normal homing to primary follicles, which was in contrast to other receptors for B-cell trafficking, such as a significant down-regulation for CXCR5 and CCR6, as well as down-regulation of IL4R involved in differentiation. This indicated that the malignant transformation of a normal B cell could have appeared during the transition of a primary follicle to a germinal center, i.e., after an initial B-cell activation. Genes involved in blockage of antiproliferative signals in normal cells were also deregulated, e.g., gene expression of TGF beta 2 and Smad3 was suppressed in MCLs. Furthermore, lymphoproliferative signal pathways were active in MCLs compared with normal B cells, because genes encoding, e.g., IL10R alpha and IL18 were up-regulated, as were oncogenes like Bcl-2 and MERTK. Genes encoding receptors for different neurotransmitters mediating B-cell stimulation, such as norepinephrine and cannabinoids were also up-regulated, again illustrating deregulation of a complex network of genes involved in growth and differentiation. Furthermore, hierarchical cluster analysis revealed two subpopulations of MCLs, which indicates that despite the homogeneous and strong overexpression of cyclin D1, further subtyping might be possible.
Assuntos
Subpopulações de Linfócitos B/fisiologia , Linfócitos B/imunologia , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/imunologia , Apoptose/genética , Apoptose/imunologia , Apoptose/fisiologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/citologia , Regulação para Baixo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/imunologia , Substâncias de Crescimento/biossíntese , Substâncias de Crescimento/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Oncogenes , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento/genética , Receptores de Neurotransmissores/biossíntese , Receptores de Neurotransmissores/genéticaRESUMO
BACKGROUND: Flow cytometry-based receptor occupancy (RO) assessments for pharmacodynamic (PD) response measurements along with drug pharmacokinetic (PK) measurements represent a cornerstone in mechanism based PK/PD modeling of drugs against cell surface targets. This report describes the utility of using a "Free" and a "Bound" assay in combination to derive RO estimations through a weighted calculation method. METHODS: Data from a RO assay validation study in human samples was used to explore the performance of various RO data calculation methods. The calculation methods were subsequently applied to investigate the best method to generate RO data in a first in human phase 1 clinical trial. Finally, the outcome of the analysis was used for PK/PD modeling of a prospective phase 2a trial. RESULTS: The validation data assessment demonstrated that a weighted RO calculation method had a better performance in terms of precision, accuracy and dynamic range. In the phase 1 clinical trial data analysis the weighted method again demonstrated a better performance resulting in a more robust RO estimation, and subsequently also generating a more reliable PK/PD simulation for the phase 2a trial. CONCLUSIONS: This report demonstrated the utility of using a combined "Free" and "Bound" RO assessment together with a weighted calculation method to better support mechanism-based PK/PD modeling activities.
Assuntos
Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos/métodos , Citometria de Fluxo/métodos , Humanos , FarmacocinéticaRESUMO
The C5a/C5a receptor (C5aR) pathway, a key component in the proinflammatory immune response, is an attractive therapeutic target since its dysregulation is implicated in a variety of autoimmune and inflammatory disorders. The objective of the present study was to validate a receptor occupancy (RO) assay for a human anti-C5aR monoclonal antibody drug candidate, NNC0215-0384 (NN0384). This flow cytometry-based assay measures the percentage (%), median fluorescence intensity (MFI), and molecules of equivalent soluble fluorochrome (MESF) of NN0384 binding to its target cells, neutrophils and monocytes, in whole blood from normal healthy donors and rheumatoid arthritis (RA) patients with clinically active disease. The validation parameters assessed included postcollection and postprocessing sample stability, intra- and interassay precision, an analyst-to-analyst comparison, a comparison of normal healthy donor and RA patient sample postcollection stability, and a laboratory-to-laboratory comparison and assay transfer. The cumulative results indicate that the assay was reproducible, met the clearly defined acceptance criteria for the validation parameters tested, and was transferable to another laboratory. In conclusion, this RO assay is suitable for use to accrue pharmacodynamic biomarker data in a multicenter, global clinical trial.
Assuntos
Anticorpos Monoclonais/imunologia , Artrite Reumatoide/tratamento farmacológico , Citometria de Fluxo , Receptor da Anafilatoxina C5a/isolamento & purificação , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Humanos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Receptor da Anafilatoxina C5a/imunologiaRESUMO
Receptor occupancy measurements demonstrate the binding of a biotherapeutic agent to its extra-cellular target and represent an integral component of the pharmacodynamic (PD) portfolio utilized to advance the development and commercialization of a therapeutic agent. Coupled with traditional pharmacokinetic (PK) assessments derived from serum drug concentration, receptor occupancy data can be used to model PK/PD relationships and validate dose selection decisions throughout the drug development lifecycle. Receptor occupancy assays can be even more challenging to develop than other flow cytometric methods (e.g. surface immunophenotyping). In addition to typical considerations regarding stability of the cell type of interest, stability of the target-bound therapeutic agent and stability of the target receptor must be taken into account. Reagent selection is also challenging as reagents need to be evaluated for the potential to compete with the therapeutic agent and bind with comparable affinity. This article provides technical guidance for the development and validation of cytometry-based receptor occupancy assays.
Assuntos
Anticorpos Monoclonais/imunologia , Descoberta de Drogas , Citometria de Fluxo , Anticorpos Monoclonais/uso terapêutico , Corantes Fluorescentes/uso terapêutico , HumanosRESUMO
The measurement of the binding of a biotherapeutic to its cellular target, receptor occupancy (RO), is increasingly important in development of biologically-based therapeutic agents. Receptor occupancy (RO) assays by flow cytometry describe the qualitative and/or quantitative assessment of the binding of a therapeutic agent to its cell surface target. Such RO assays can be as simple as measuring the number of cell surface receptors bound by an antireceptor therapeutic agent or can be designed to address more complicated scenarios such as internalization or shedding events once a receptor engages the administered therapeutic agent. Data generated from RO assays can also be used to model whether given doses of an experimental therapeutic agent and their administration schedules lead to predicted levels of receptor occupancy and whether the receptor is modulated (up or down) on cells engaged by the therapeutic agent. There are a variety of approaches that can be used when undertaking RO assays and with the ability to measure distinct subsets in heterogeneous populations, flow cytometry is ideally suited to RO measurements. This article highlights the importance of RO assays on the flow cytometric platform in the development of biotherapeutic agents.
Assuntos
Anticorpos/imunologia , Descoberta de Drogas , Citometria de Fluxo/métodos , Anticorpos/uso terapêutico , Citometria de Fluxo/tendências , HumanosRESUMO
Intestinal M play an important role in maintaining gut homeostasis. However, little is known about these cells, their precursors, and their role in intestinal inflammation. Here, we characterize the CD14(+) mononuclear cell populations in intestinal mucosa and blood in patients with CD. Among the LP CD14(+) M, we identified three distinct HLA-DR(+)-expressing subsets. Compared with uninflamed, inflamed mucosa contained a marked increase in the proportion of the CD14(hi)HLA-DR(dim) cellular subset. This subset resembled the classical blood monocytes with low CD16, HLA-DR, and CX3CR1 expression. Classical monocytes migrated efficiently toward CCL2 and released the highest levels of MMP-1 and proinflammatory cytokines when stimulated with immune complexes or LPS. Our findings strongly suggest that it is the classical and not the intermediate or nonclassical monocytes that are the precursors to the dominating intestinal CD14(hi)HLA-DR(dim) subset. This enhances our understanding of CD pathology and may provide new options in treatment.
Assuntos
Doença de Crohn/imunologia , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Adolescente , Adulto , Idoso , Antígenos de Diferenciação Mielomonocítica/imunologia , Doença de Crohn/sangue , Doença de Crohn/patologia , Feminino , Citometria de Fluxo , Antígenos HLA-DR/biossíntese , Antígenos HLA-DR/imunologia , Humanos , Mucosa Intestinal/patologia , Receptores de Lipopolissacarídeos/biossíntese , Receptores de Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Adulto JovemRESUMO
A massive and selective loss of CD4+ memory T cells occurs during the acute phase of immunodeficiency virus infections. The mechanism of this depletion is poorly understood but constitutes a key event with implications for progression. We assessed gene expression of purified T cells in Rhesus Macaques during acute SIVmac239 infection in order to define mechanisms of pathogenesis. We observe a general transcriptional program of over 1,600 interferon-stimulated genes induced in all T cells by the infection. Furthermore, we identify 113 transcriptional changes that are specific to virally infected cells. A striking downregulation of several key cell cycle regulator genes was observed and shared promotor-region E2F binding sites in downregulated genes suggested a targeted transcriptional control of an E2F regulated cell cycle program. In addition, the upregulation of the gene for the fundamental regulator of RNA polymerase II, TAF7, demonstrates that viral interference with the cell cycle and transcriptional regulation programs may be critical components during the establishment of a pathogenic infection in vivo.
Assuntos
Ciclo Celular/genética , Interações Hospedeiro-Patógeno/genética , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Transcrição Gênica , Animais , Linfócitos T CD4-Positivos/virologia , Regulação para Baixo , Perfilação da Expressão Gênica , Macaca mulatta , Proteínas RepressorasRESUMO
Cross-reactive neutralizing antibodies (NAbs) are found in the sera of many HIV-1-infected individuals, but the virologic basis of their neutralization remains poorly understood. We used knowledge of HIV-1 envelope structure to develop antigenically resurfaced glycoproteins specific for the structurally conserved site of initial CD4 receptor binding. These probes were used to identify sera with NAbs to the CD4-binding site (CD4bs) and to isolate individual B cells from such an HIV-1-infected donor. By expressing immunoglobulin genes from individual cells, we identified three monoclonal antibodies, including a pair of somatic variants that neutralized over 90% of circulating HIV-1 isolates. Exceptionally broad HIV-1 neutralization can be achieved with individual antibodies targeted to the functionally conserved CD4bs of glycoprotein 120, an important insight for future HIV-1 vaccine design.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/isolamento & purificação , Especificidade de Anticorpos , Linfócitos B/imunologia , Sítios de Ligação de Anticorpos , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Reações Cruzadas , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Genes de Cadeia Pesada de Imunoglobulina , Genes de Cadeia Leve de Imunoglobulina , Anticorpos Anti-HIV/isolamento & purificação , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/genética , Humanos , Dados de Sequência Molecular , Testes de Neutralização , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
The process of becoming an Ig-producing plasma cell takes the mature B cell through the germinal center, where Ig genes are diversified through somatic hypermutation and class switch recombination. To more clearly define functional characteristics of the germinal center dark zone centroblasts and the light zone centrocytes, we have performed expression analysis of the CD77(+) and CD77(-) populations, because CD77 has been accepted as a discriminator of centroblasts and centrocytes. Our results demonstrated that the CD77(+) and the CD77(-) populations lack functional associated expression programs discriminating the two populations. Both populations are shown to be actively cycling and to share common features associated with cell cycle regulation and DNA maintenance. They are also shown to have an equally active DNA repair program, as well as components involved in somatic hypermutation and class switch recombination. Moreover, the data also demonstrated that the CD77(-) population comprises cells with an already initiated plasma cell differentiation program. Together this demonstrates that CD77 does not discriminate centroblasts and centrocytes and that the CD77(-) population represents a heterogeneous subset of cells, comprising centroblasts, centrocytes, and plasmablast.
Assuntos
Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/metabolismo , Centro Germinativo/citologia , Triexosilceramidas/metabolismo , Subpopulações de Linfócitos B/imunologia , Ciclo Celular , Diferenciação Celular/imunologia , Proliferação de Células , Reparo do DNA/genética , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Hibridização In Situ , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Plasmócitos/citologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , RNA Mensageiro/análise , Células-Tronco/citologia , Células-Tronco/imunologia , Células-Tronco/metabolismoRESUMO
We have analyzed mantle cell lymphomas (MCLs), using high-density DNA microarrays, and confirmed the expression of differentially regulated antigens, using flow cytometry and immunohistochemistry. The results show that MCLs acquire expression of molecules that normally are involved in interaction with other immune cells and, thus, might affect the ability of the tumor to survive. The MCL signature is represented by the overexpression of the chemokine CCL4 (MIP-1beta), implicated in the recruitment of regulatory T cells, as well as CCL5 and 4-1BB-L. The latter molecules are normally involved in chemotaxis of T cells and B cell activation, respectively. Signaling through 4-1BB-L allows B cells to proliferate and the expression of its ligand, by the intra-tumoral mesh of follicular dendritic cells (FDC), could thus serve as a paracrine loop facilitating growth and survival of MCL cells.
Assuntos
Sobrevivência Celular , Quimiocinas CC/biossíntese , Linfoma de Célula do Manto/genética , Proteínas Inflamatórias de Macrófagos/biossíntese , Fatores de Necrose Tumoral/biossíntese , Ligante 4-1BB , Quimiocina CCL4 , Quimiocina CCL5 , Quimiocinas CC/genética , Quimiotaxia , Células Dendríticas , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Linfoma de Célula do Manto/patologia , Proteínas Inflamatórias de Macrófagos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Necrose Tumoral/genéticaRESUMO
A number of studies have implicated a role for the cell surface glycoprotein CD44 in several biologic events, such as lymphopoiesis, homing, lymphocyte activation, and apoptosis. We have earlier reported that signaling via CD44 on naive B cells in addition to B-cell receptor (BCR) and CD40 engagement generated a germinal center-like phenotype. To further characterize the global role of CD44 in B differentiation, we examined the expression profile of human B cells cultured in vitro in the presence or absence of CD44 ligation, together with anti-immunoglobulin (anti-Ig) and anti-CD40 antibodies. The data sets derived from DNA microarrays were analyzed using a novel statistical analysis scheme created to retrieve the most likely expression pattern of CD44 ligation. Our results show that genes such as interleukin-6 (IL-6), IL-1alpha, and beta(2)-adrenergic receptor (beta(2)-AR) were specifically up-regulated by CD44 ligation, suggesting a novel role for CD44 in immunoregulation and inflammation.