RESUMO
An exclusive feature of dendritic cells (DCs) is their ability to cross-present exogenous antigens in MHC class I molecules. We analyzed the fate of protein antigen in antigen presenting cell (APC) subsets after uptake of naturally formed antigen-antibody complexes in vivo. We observed that murine splenic DC subsets were able to present antigen in vivo for at least a week. After ex vivo isolation of four APC subsets, the presence of antigen in the storage compartments was visualized by confocal microscopy. Although all APC subsets stored antigen for many days, their ability and kinetics in antigen presentation was remarkably different. CD8α+ DCs showed sustained MHC class I-peptide specific CD8+ T-cell activation for more than 4 days. CD8α- DCs also presented antigenic peptides in MHC class I but presentation decreased after 48 h. In contrast, only the CD8α- DCs were able to present antigen in MHC class II to specific CD4+ T cells. Plasmacytoid DCs and macrophages were unable to activate any of the two T-cell types despite detectable antigen uptake. These results indicate that naturally occurring DC subsets have functional antigen storage capacity for prolonged T-cell activation and have distinct roles in antigen presentation to specific T cells in vivo.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada , Células Dendríticas/imunologia , Macrófagos/imunologia , Animais , Apresentação de Antígeno , Antígenos CD8/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Baço/imunologiaRESUMO
Dendritic cells (DCs) are specialized in Ag engulfment via a wide variety of uptake receptors on their cell surface. In the present study we investigated Ag uptake and presentation of in vivo-formed Ag-Ab complexes by i.v. injecting mice with Ag-specific Abs followed by the cognate Ag. We show by this natural Ab-mediated Ag targeting system that uptake by splenic APC subsets is severely hampered in mice lacking complement factor C1q (C1qa-/-). Moreover, no detectable Ag cross-presentation by CD8α+ DCs from C1qa-/- mice was found. On the contrary, Ag uptake was not hampered by APCs in FcγRI/II/III/IV-deficient (FcγR quadruple-/-) mice, and the cross-presentation ability of CD8α+ DCs was not affected. In conclusion, we show that C1q rather than FcγRs controls the Ab-mediated Ag uptake and its presentation by spleen APC subsets to T cells.
Assuntos
Apresentação de Antígeno , Complexo Antígeno-Anticorpo/imunologia , Complemento C1q/imunologia , Células Dendríticas/imunologia , Imunidade Adaptativa , Animais , Antígenos CD8/imunologia , Complemento C1q/deficiência , Complemento C1q/genética , Apresentação Cruzada , Camundongos , Camundongos Endogâmicos C57BL , Receptores de IgG/imunologiaRESUMO
The fate and numbers of hematopoietic stem cells (HSC) and their progeny that seed the thymus constitute a fundamental question with important clinical implications. HSC transplantation is often complicated by limited T-cell reconstitution, especially when HSC from umbilical cord blood are used. Attempts to improve immune reconstitution have until now been unsuccessful, underscoring the need for better insight into thymic reconstitution. Here we made use of the NOD-SCID-IL-2Rγ(-/-) xenograft model and lentiviral cellular barcoding of human HSCs to study T-cell development in the thymus at a clonal level. Barcoded HSCs showed robust (>80% human chimerism) and reproducible myeloid and lymphoid engraftment, with T cells arising 12 wk after transplantation. A very limited number of HSC clones (<10) repopulated the xenografted thymus, with further restriction of the number of clones during subsequent development. Nevertheless, T-cell receptor rearrangements were polyclonal and showed a diverse repertoire, demonstrating that a multitude of T-lymphocyte clones can develop from a single HSC clone. Our data imply that intrathymic clonal fitness is important during T-cell development. As a consequence, immune incompetence after HSC transplantation is not related to the transplantation of limited numbers of HSC but to intrathymic events.
Assuntos
Células da Medula Óssea/citologia , Linfócitos T/citologia , Timo/citologia , Animais , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
To gain more insight into initiation and regulation of T cell receptor (TCR) gene rearrangement during human T cell development, we analyzed TCR gene rearrangements by quantitative PCR analysis in nine consecutive T cell developmental stages, including CD34+ lin- cord blood cells as a reference. The same stages were used for gene expression profiling using DNA microarrays. We show that TCR loci rearrange in a highly ordered way (TCRD-TCRG-TCRB-TCRA) and that the initiating Ddelta2-Ddelta3 rearrangement occurs at the most immature CD34+CD38-CD1a- stage. TCRB rearrangement starts at the CD34+CD38+CD1a- stage and complete in-frame TCRB rearrangements were first detected in the immature single positive stage. TCRB rearrangement data together with the PTCRA (pTalpha) expression pattern show that human TCRbeta-selection occurs at the CD34+CD38+CD1a+ stage. By combining the TCR rearrangement data with gene expression data, we identified candidate factors for the initiation/regulation of TCR recombination. Our data demonstrate that a number of key events occur earlier than assumed previously; therefore, human T cell development is much more similar to murine T cell development than reported before.
Assuntos
Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/imunologia , Rearranjo Gênico da Cadeia delta dos Receptores de Antígenos dos Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Diferenciação Celular/genética , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T/genética , Rearranjo Gênico da Cadeia delta dos Receptores de Antígenos dos Linfócitos T/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/genética , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T/imunologia , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Canonical Wnt signaling has been implicated in various aspects of hematopoiesis. Its role is controversial due to different outcomes between various inducible Wnt-signaling loss-of-function models and also compared with gain-of-function systems. We therefore studied a mouse deficient for a Wnt gene that seemed to play a nonredundant role in hematopoiesis. Mice lacking Wnt3a die prenatally around embryonic day (E) 12.5, allowing fetal hematopoiesis to be studied using in vitro assays and transplantation into irradiated recipient mice. Here we show that Wnt3a deficiency leads to a reduction in the numbers of hematopoietic stem cells (HSCs) and progenitor cells in the fetal liver (FL) and to severely reduced reconstitution capacity as measured in secondary transplantation assays. This deficiency is irreversible and cannot be restored by transplantation into Wnt3a competent mice. The impaired long-term repopulation capacity of Wnt3a(-/-) HSCs could not be explained by altered cell cycle or survival of primitive progenitors. Moreover, Wnt3a deficiency affected myeloid but not B-lymphoid development at the progenitor level, and affected immature thymocyte differentiation. Our results show that Wnt3a signaling not only provides proliferative stimuli, such as for immature thymocytes, but also regulates cell fate decisions of HSC during hematopoiesis.
Assuntos
Diferenciação Celular/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Proteínas Wnt/metabolismo , Animais , Apoptose , Proliferação de Células , Embrião de Mamíferos , Citometria de Fluxo , Imuno-Histoquímica , Camundongos , Camundongos Mutantes , Transdução de Sinais/genética , Linfócitos T/citologia , Linfócitos T/imunologia , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3ARESUMO
Measuring Wnt expression levels is essential when trying to identify or test new Wnt therapeutic targets. Previous studies have shown that canonical Wnt signaling operates via a dosage-driven mechanism, motivating the need to study and measure Wnt signaling in various cell types. Although several reporter models have been proposed to represent physiological Wnt expression, either the genetic context or the reporter protein highly influenced the validity, accuracy, and flexibility of these tools. This paper describes methods for acquiring and analyzing data obtained with the Axin2-mTurquoise2 mouse Wnt reporter model, which contains a mutated Axin2em1Fstl allele. This model facilitates the study of endogenous canonical Wnt signaling in individual cells over a wide range of Wnt activity. This protocol describes how to fully appreciate Axin2-mTurquoise2 reporter activity using cell population analysis of the hematopoietic system, combined with cell surface markers or ß-catenin intracellular staining. These procedures serve as a base for implementation and reproduction in other tissues or cells of interest. By combining fluorescence-activated cell sorting and confocal imaging, distinct canonical Wnt expression levels can be visualized. The recommended measurement and analysis strategies provide quantitative data on the fluorescent expression levels for precise assessment of canonical Wnt signaling. These methods will be useful for researchers who want to use the Axin2-mTurquise2 model for canonical Wnt expression patterns.
Assuntos
Timócitos , Via de Sinalização Wnt , Animais , Proteína Axina/genética , Citometria de Fluxo , Indicadores e Reagentes , CamundongosRESUMO
Hematopoietic stem cells (HSC) are rare with estimated frequencies of 1 in 10,000 bone marrow cells and 1 in every 100,000 blood cells. The most important characteristic of HSC is their capacity to provide complete restoration of all blood cell lineages after bone marrow ablation. Therefore they are considered as the ideal targets for various clinical applications including stem cell transplantation and gene therapy. In adult mice and men, the main stem cell source is the bone marrow. For clinical applications HSC derived from umbilical cord blood (UCB) and G-CSF mobilized peripheral blood (PB) have been demonstrated to have several advantages compared to bone marrow; therefore, they are slowly replacing BM as alternative source of stem cells. The mouse is the model organism of choice for immunological and hematological research; therefore, studies of murine HSC are an important research topic. Here we described the most often used protocols and methods to isolate human and mouse HSC to high purity.
Assuntos
Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD34/imunologia , Citometria de Fluxo , Células-Tronco Hematopoéticas/imunologia , Humanos , Separação Imunomagnética , CamundongosRESUMO
Overexpression of LMO2 is known to be one of the causes of T-cell acute lymphoblastic leukemia (T-ALL) development; however, the mechanisms behind its oncogenic activity are incompletely understood. LMO2-overexpressing transgenic mouse models suggest an accumulation of immature T-cell progenitors in the thymus as the main preleukemic event. The effects of LMO2 overexpression on human T-cell development in vivo are unknown. Here, we report studies of a humanized mouse model transplanted with LMO2-transduced human hematopoietic stem/progenitor cells. The effects of LMO2 overexpression were confined to the T-cell lineage; however, initially, multipotent cells were transduced. Three effects of LMO2 on human T-cell development were observed: (1) a block at the double-negative/immature single-positive stage, (2) an accumulation of CD4(+)CD8(+) double-positive CD3(-) cells, and (3) an altered CD8/CD4 ratio with enhanced peripheral T lymphocytes. Microarray analysis of sorted double-positive cells overexpressing LMO2 led to the identification of an LMO2 gene set that clustered with human T-ALL patient samples of the described "proliferative" cluster. In this article, we demonstrate previously unrecognized mechanisms by which LMO2 alters human T-cell development in vivo; these mechanisms correlate with human T-ALL leukemogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Expressão Gênica , Proteínas com Domínio LIM/genética , Proteínas Proto-Oncogênicas/genética , Linfócitos T/metabolismo , Animais , Antígenos CD34/metabolismo , Diferenciação Celular/genética , Proliferação de Células , Perfilação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/metabolismo , Camundongos , Linfócitos T/patologia , Transdução GenéticaRESUMO
Canonical Wnt signaling has been implicated in the regulation of hematopoiesis. By employing a Wnt-reporter mouse, we observed that Wnt signaling is differentially activated during hematopoiesis, suggesting an important regulatory role for specific Wnt signaling levels. To investigate whether canonical Wnt signaling regulates hematopoiesis in a dosage-dependent fashion, we analyzed the effect of different mutations in the Adenomatous polyposis coli gene (Apc), a negative modulator of the canonical Wnt pathway. By combining different targeted hypomorphic alleles and a conditional deletion allele of Apc, a gradient of five different Wnt signaling levels was obtained in vivo. We here show that different, lineage-specific Wnt dosages regulate hematopoietic stem cells (HSCs), myeloid precursors, and T lymphoid precursors during hematopoiesis. Differential, lineage-specific optimal Wnt dosages provide a unifying concept that explains the differences reported among inducible gain-of-function approaches, leading to either HSC expansion or depletion of the HSC pool.
Assuntos
Dosagem de Genes , Hematopoese , Via de Sinalização Wnt , Animais , Diferenciação Celular , Células Clonais , Citometria de Fluxo , Marcação de Genes , Sistema Hematopoético/metabolismo , Camundongos , Modelos Biológicos , Mutação/genética , Células Mieloides/citologia , Células Mieloides/metabolismo , Linfócitos T/citologiaRESUMO
Wnt signaling is essential for T cell development in the thymus, but the stages in which it occurs and the molecular mechanisms underlying Wnt responsiveness have remained elusive. Here we examined Wnt signaling activity in both human and murine thymocyte populations by determining beta-catenin levels, Tcf-reporter activation and expression of Wnt-target genes. We demonstrate that Wnt signaling occurs in all thymocyte subsets, including the more mature populations, but most prominently in the double negative (DN) subsets. This differential sensitivity to Wnt signaling was not caused by differences in the presence of Wnts or Wnt receptors, as these appeared to be expressed at comparable levels in all thymocyte subsets. Rather, it can be explained by high expression of activating signaling molecules in DN cells, e.g., beta-catenin, plakoglobin, and long forms of Tcf-1, and by low levels of inhibitory molecules. By blocking Wnt signaling from the earliest stage onwards using overexpression of Dickkopf, we show that inhibition of the canonical Wnt pathway blocks development at the most immature DN1 stage. Thus, responsiveness to developmental signals can be regulated by differential expression of intracellular mediators rather than by abundance of receptors or ligands.
Assuntos
Transdução de Sinais , Timo/metabolismo , Proteínas Wnt/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Humanos , Timo/citologia , Timo/imunologia , Fatores de Tempo , beta Catenina/metabolismoRESUMO
It is a longstanding question which bone marrow-derived cell seeds the thymus and to what level this cell is committed to the T-cell lineage. We sought to elucidate this issue by examining gene expression, lineage potential, and self-renewal capacity of the 2 most immature subsets in the human thymus, namely CD34+ CD1a- and CD34+ CD1a+ thymocytes. DNA microarrays revealed the presence of several myeloid and erythroid transcripts in CD34+ CD1a- thymocytes but not in CD34+ CD1a+ thymocytes. Lineage potential of both subpopulations was assessed using in vitro colony assays, bone marrow stroma cultures, and in vivo transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The CD34+ CD1a- subset contained progenitors with lymphoid (both T and B), myeloid, and erythroid lineage potential. Remarkably, development of CD34+ CD1a- thymocytes toward the T-cell lineage, as shown by T-cell receptor delta gene rearrangements, could be reversed into a myeloid-cell fate. In contrast, the CD34+ CD1a+ cells yielded only T-cell progenitors, demonstrating their irreversible commitment to the T-cell lineage. Both CD34+ CD1a- and CD34+ CD1a+ thymocytes failed to repopulate NOD/SCID mice. We conclude that the human thymus is seeded by multipotent progenitors with a much broader lineage potential than previously assumed. These cells resemble hematopoietic stem cells but, by analogy with murine thymocytes, apparently lack sufficient self-renewal capacity.
Assuntos
Linhagem da Célula/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Multipotentes/fisiologia , Timo/fisiologia , Animais , Antígenos CD1/metabolismo , Antígenos CD34/metabolismo , Linfócitos B/citologia , Linfócitos B/fisiologia , Ensaio de Unidades Formadoras de Colônias/métodos , Rearranjo Gênico do Linfócito T/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Multipotentes/citologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Linfócitos T/citologia , Linfócitos T/fisiologia , Timo/citologiaRESUMO
BACKGROUND: T-cell development in the thymus is an extensively studied subject, mainly in mice. Nevertheless, the normal composition and cell numbers of the noninvoluted human thymus are largely unknown. OBJECTIVE: We aimed to gain insight into age-related changes in different thymic subpopulations and to provide reference values for the distribution of thymocyte subsets. The composition of the normal thymus may serve as a reference for thymi in pathological conditions and may aid diagnoses of immunodeficiency diseases. METHODS: Thymic lobes of 70 children (58 immunologically normal and 12 diseased), ranging in age from 8 days to 8 years old, were studied by 4-color flow-cytometric analysis. Detailed staining and gating strategies allowed us to dissect small subsets, including immature CD4(-) CD8(-) populations and thymic B, natural killer, and T-cell receptor gammadelta + cells. RESULTS: We demonstrate that distribution of thymocyte subsets changes with age and correlates with age-related fluctuations of T-lymphocyte counts in peripheral blood. Thymi of children 3 to 6 months old appear to be the most active: they have high numbers of total thymocytes, the highest percentage of double-positive cells, and large numbers of CD34 + progenitors in their thymi. Furthermore, we show that the human thymus is a site for B-cell development, because all B-cell progenitor stages that can be found in the bone marrow are also present in the thymus. CONCLUSION: We conclude that T-cell development in children is a dynamic process, answering the demands of a maturing and expanding immune system.
Assuntos
Envelhecimento , Sistema Imunitário/crescimento & desenvolvimento , Linfócitos/citologia , Timo/citologia , Timo/crescimento & desenvolvimento , Criança , Pré-Escolar , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Imunofenotipagem , Lactente , Recém-NascidoRESUMO
The role of specific transcription factors in the initiation and regulation of Ig gene rearrangements has been studied extensively in mouse models, but data on normal human precursor B cell differentiation are limited. We purified five human precursor B cell subsets, and assessed and quantified their IGH, IGK, and IGL gene rearrangement patterns and gene expression profiles. Pro-B cells already massively initiate D(H)-J(H) rearrangements, which are completed with V(H)-DJ(H) rearrangements in pre-B-I cells. Large cycling pre-B-II cells are selected for in-frame IGH gene rearrangements. The first IGK/IGL gene rearrangements were initiated in pre-B-I cells, but their frequency increased enormously in small pre-B-II cells, and in-frame selection was found in immature B cells. Transcripts of the RAG1 and RAG2 genes and earlier defined transcription factors, such as E2A, early B cell factor, E2-2, PAX5, and IRF4, were specifically up-regulated at stages undergoing Ig gene rearrangements. Based on the combined Ig gene rearrangement status and gene expression profiles of consecutive precursor B cell subsets, we identified 16 candidate genes involved in initiation and/or regulation of Ig gene rearrangements. These analyses provide new insights into early human precursor B cell differentiation steps and represent an excellent template for studies on oncogenic transformation in precursor B acute lymphoblastic leukemia and B cell differentiation blocks in primary Ab deficiencies.
Assuntos
Subpopulações de Linfócitos B/metabolismo , Perfilação da Expressão Gênica , Rearranjo Gênico do Linfócito B , Genes de Imunoglobulinas , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/genética , Adolescente , Separação Celular , Criança , Pré-Escolar , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Rearranjo Gênico de Cadeia Leve de Linfócito B , HumanosRESUMO
The thymus is seeded by very small numbers of progenitor cells that undergo massive proliferation before differentiation and rearrangement of TCR genes occurs. Various signals mediate proliferation and differentiation of these cells, including Wnt signals. Wnt signals induce the interaction of the cytoplasmic cofactor beta-catenin with nuclear T cell factor (TCF) transcription factors. We identified target genes of the Wnt/beta-catenin/TCF pathway in the most immature (CD4-CD8-CD34+) thymocytes using Affymetrix DNA microarrays in combination with three different functional assays for in vitro induction of Wnt signaling. A relatively small number (approximately 30) of genes changed expression, including several proliferation-inducing transcription factors such as c-fos and c-jun, protein phosphatases, and adhesion molecules, but no genes involved in differentiation to mature T cell stages. The adhesion molecules likely confine the proliferating immature thymocytes to the appropriate anatomical sites in the thymus. For several of these target genes, we validated that they are true Wnt/beta-catenin/TCF target genes using real-time quantitative PCR and reporter gene assays. The same core set of genes was repressed in Tcf-1-null mice, explaining the block in early thymocyte development in these mice. In conclusion, Wnt signals mediate proliferation and cell adhesion, but not differentiation of the immature thymic progenitor pool.