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1.
Commun Biol ; 5(1): 590, 2022 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-35710943

RESUMO

Aging is a major risk factor for developing severe COVID-19, but few detailed data are available concerning immunological changes after infection in aged individuals. Here we describe main immune characteristics in 31 patients with severe SARS-CoV-2 infection who were >70 years old, compared to 33 subjects <60 years of age. Differences in plasma levels of 62 cytokines, landscape of peripheral blood mononuclear cells, T cell repertoire, transcriptome of central memory CD4+ T cells, specific antibodies are reported along with features of lung macrophages. Elderly subjects have higher levels of pro-inflammatory cytokines, more circulating plasmablasts, reduced plasmatic level of anti-S and anti-RBD IgG3 antibodies, lower proportions of central memory CD4+ T cells, more immature monocytes and CD56+ pro-inflammatory monocytes, lower percentages of circulating follicular helper T cells (cTfh), antigen-specific cTfh cells with a less activated transcriptomic profile, lung resident activated macrophages that promote collagen deposition and fibrosis. Our study underlines the importance of inflammation in the response to SARS-CoV-2 and suggests that inflammaging, coupled with the inability to mount a proper anti-viral response, could exacerbate disease severity and the worst clinical outcome in old patients.


Assuntos
COVID-19 , Idoso , Citocinas , Humanos , Leucócitos Mononucleares , SARS-CoV-2 , Células T Auxiliares Foliculares
3.
Nat Commun ; 12(1): 4677, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34326336

RESUMO

SARS-CoV-2 infection can affect all human beings, including pregnant women. Thus, understanding the immunological changes induced by the virus during pregnancy is nowadays of pivotal importance. Here, using peripheral blood from 14 pregnant women with asymptomatic or mild SARS-CoV-2 infection, we investigate cell proliferation and cytokine production, measure plasma levels of 62 cytokines, and perform a 38-parameter mass cytometry analysis. Our results show an increase in low density neutrophils but no lymphopenia or gross alterations of white blood cells, which display normal levels of differentiation, activation or exhaustion markers and show well preserved functionality. Meanwhile, the plasma levels of anti-inflammatory cytokines such as interleukin (IL)-1RA, IL-10 and IL-19 are increased, those of IL-17, PD-L1 and D-dimer are decreased, but IL-6 and other inflammatory molecules remain unchanged. Our profiling of antiviral immune responses may thus help develop therapeutic strategies to avoid virus-induced damages during pregnancy.


Assuntos
COVID-19/imunologia , Citocinas/sangue , Inflamação/imunologia , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/virologia , SARS-CoV-2/imunologia , Adolescente , Adulto , Infecções Assintomáticas , Biomarcadores/sangue , COVID-19/sangue , COVID-19/virologia , Estudos de Casos e Controles , Estudos Transversais , Citocinas/imunologia , Feminino , Humanos , Inflamação/sangue , Inflamação/prevenção & controle , Inflamação/virologia , Pessoa de Meia-Idade , Gravidez , Complicações Infecciosas na Gravidez/sangue , SARS-CoV-2/isolamento & purificação , Adulto Jovem
4.
Blood ; 128(6): 783-93, 2016 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-27301863

RESUMO

Primary immunodeficiency diseases comprise a group of heterogeneous genetic defects that affect immune system development and/or function. Here we use in vitro differentiation of human induced pluripotent stem cells (iPSCs) generated from patients with different recombination-activating gene 1 (RAG1) mutations to assess T-cell development and T-cell receptor (TCR) V(D)J recombination. RAG1-mutants from severe combined immunodeficient (SCID) patient cells showed a failure to sustain progression beyond the CD3(--)CD4(-)CD8(-)CD7(+)CD5(+)CD38(-)CD31(-/lo)CD45RA(+) stage of T-cell development to reach the CD3(-/+)CD4(+)CD8(+)CD7(+)CD5(+)CD38(+)CD31(+)CD45RA(-) stage. Despite residual mutant RAG1 recombination activity from an Omenn syndrome (OS) patient, similar impaired T-cell differentiation was observed, due to increased single-strand DNA breaks that likely occur due to heterodimers consisting of both an N-terminal truncated and a catalytically dead RAG1. Furthermore, deep-sequencing analysis of TCR-ß (TRB) and TCR-α (TRA) rearrangements of CD3(-)CD4(+)CD8(-) immature single-positive and CD3(+)CD4(+)CD8(+) double-positive cells showed severe restriction of repertoire diversity with preferential usage of few Variable, Diversity, and Joining genes, and skewed length distribution of the TRB and TRA complementary determining region 3 sequences from SCID and OS iPSC-derived cells, whereas control iPSCs yielded T-cell progenitors with a broadly diversified repertoire. Finally, no TRA/δ excision circles (TRECs), a marker of TRA/δ locus rearrangements, were detected in SCID and OS-derived T-lineage cells, consistent with a pre-TCR block in T-cell development. This study compares human T-cell development of SCID vs OS patients, and elucidates important differences that help to explain the wide range of immunologic phenotypes that result from different mutations within the same gene of various patients.


Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes Induzidas/patologia , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/patologia , Linfócitos T/patologia , Células Cultivadas , Quebras de DNA , Genes RAG-1 , Humanos , Lactente , Mutação , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Recombinação V(D)J
5.
Mol Ther ; 24(3): 582-91, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26444081

RESUMO

Many applications of pluripotent stem cells (PSCs) require efficient editing of silent chromosomal genes. Here, we show that a major limitation in isolating edited clones is silencing of the selectable marker cassette after homologous recombination and that this can be overcome by using a ubiquitous chromatin opening element (UCOE) promoter-driven transgene. We use this strategy to edit the silent IL2RG locus in human PSCs with a recombinant adeno-associated virus (rAAV)-targeting vector in the absence of potentially genotoxic, site-specific nucleases and show that IL2RG is required for natural killer and T-cell differentiation of human PSCs. Insertion of an active UCOE promoter into a silent locus altered the histone modification and cytosine methylation pattern of surrounding chromatin, but these changes resolved when the UCOE promoter was removed. This same approach could be used to correct IL2RG mutations in X-linked severe combined immunodeficiency patient-derived induced PSCs (iPSCs), to prevent graft versus host disease in regenerative medicine applications, or to edit other silent genes.


Assuntos
Edição de Genes , Inativação Gênica , Subunidade gama Comum de Receptores de Interleucina/genética , Células-Tronco Pluripotentes/metabolismo , Diferenciação Celular , Sobrevivência Celular/genética , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Epigênese Genética , Técnicas de Inativação de Genes , Marcação de Genes , Loci Gênicos , Humanos , Células Matadoras Naturais/citologia , Células-Tronco Pluripotentes/citologia , Regiões Promotoras Genéticas , Subpopulações de Linfócitos T/citologia , Transgenes , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética
6.
Nat Cell Biol ; 17(5): 580-91, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25915127

RESUMO

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34(+)CD73(-)CD184(-) fraction of day 8 embryoid bodies and it undergoes a NOTCH-dependent EHT to generate RUNX1C(+) cells with multilineage potential. Arterial and venous VE progenitors, in contrast, segregate to the CD34(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.


Assuntos
Artérias/fisiologia , Diferenciação Celular , Linhagem da Célula , Células Progenitoras Endoteliais/fisiologia , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Multipotentes/fisiologia , Células-Tronco Pluripotentes/fisiologia , 5'-Nucleotidase/deficiência , Antígenos CD34/metabolismo , Artérias/citologia , Artérias/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Separação Celular/métodos , Técnicas de Cocultura , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Células Progenitoras Endoteliais/metabolismo , Proteínas Ligadas por GPI/deficiência , Células-Tronco Hematopoéticas/metabolismo , Humanos , Microscopia de Vídeo , Células-Tronco Multipotentes/metabolismo , Fenótipo , Células-Tronco Pluripotentes/metabolismo , Células Precursoras de Linfócitos T/fisiologia , Receptores CXCR5/deficiência , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Tempo , Veias/citologia , Veias/fisiologia
7.
Nat Biotechnol ; 32(6): 554-61, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24837661

RESUMO

Efforts to derive hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) are complicated by the fact that embryonic hematopoiesis consists of two programs, primitive and definitive, that differ in developmental potential. As only definitive hematopoiesis generates HSCs, understanding how this program develops is essential for being able to produce this cell population in vitro. Here we show that both hematopoietic programs transition through hemogenic endothelial intermediates and develop from KDR(+)CD34(-)CD144(-) progenitors that are distinguished by CD235a expression. Generation of primitive progenitors (KDR(+)CD235a(+)) depends on stage-specific activin-nodal signaling and inhibition of the Wnt-ß-catenin pathway, whereas specification of definitive progenitors (KDR(+)CD235a(-)) requires Wnt-ß-catenin signaling during this same time frame. Together, these findings establish simple selective differentiation strategies for the generation of primitive or definitive hematopoietic progenitors by Wnt-ß-catenin manipulation, and in doing so provide access to enriched populations for future studies on hPSC-derived hematopoietic development.


Assuntos
Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Camundongos
8.
Blood ; 122(26): 4210-9, 2013 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-24215033

RESUMO

Hematopoietic stem cell transplantation (HSCT) is followed by a period of immune deficiency due to a paucity in T-cell reconstitution. Underlying causes are a severely dysfunctional thymus and an impaired production of thymus-seeding progenitors in the host. Here, we addressed whether in vitro-derived human progenitor T (proT)-cells could not only represent a source of thymus-seeding progenitors, but also able to influence the recovery of the thymic microenvironment. We examined whether co-transplantation of in vitro-derived human proT-cells with hematopoietic stem cells (HSCs) was able to facilitate HSC-derived T-lymphopoiesis posttransplant. A competitive transfer approach was used to define the optimal proT subset capable of reconstituting immunodeficient mice. Although the 2 subsets tested (proT1, CD34(+)CD7(+)CD5(-); proT2, CD34(+)CD7(+)CD5(+)) showed thymus engrafting function, proT2-cells exhibited superior engrafting capacity. Based on this, when proT2-cells were coinjected with HSCs, a significantly improved and accelerated HSC-derived T-lymphopoiesis was observed. Furthermore, we uncovered a potential mechanism by which receptor activator of nuclear factor κb (RANK) ligand-expressing proT2-cells induce changes in both the function and architecture of the thymus microenvironment, which favors the recruitment of bone marrow-derived lymphoid progenitors. Our findings provide further support for the use of Notch-expanded progenitors in cell-based therapies to aid in the recovery of T-cells in patients undergoing HSCT.


Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/citologia , Síndromes de Imunodeficiência/terapia , Linfopoese/imunologia , Linfócitos T/citologia , Timo/citologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Modelos Animais de Doenças , Humanos , Síndromes de Imunodeficiência/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Regeneração/imunologia , Células Estromais/citologia , Células Estromais/imunologia , Linfócitos T/imunologia , Timo/imunologia
9.
Int Immunol ; 25(10): 601-11, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23988616

RESUMO

The thymus provides a unique environment for the induction of T-cell lineage commitment and differentiation, which is predicted by specific Notch ligand-receptor interactions on epithelial cells and lymphoid progenitors, respectively. Accordingly, a bone marrow-derived stromal cell line (OP9) ectopically expressing the Notch ligand Delta-like 1 (Dll1) or Dll4 (OP9-DL1 and OP9-DL4, respectively) gains the ability to recapitulate thymus-like function, supporting T-cell differentiation of both mouse and human progenitors. In this study, we extend these findings by demonstrating that, unlike the NIH3T3 cell line, mouse primary fibroblasts made to express Dll4 (mFibro-DL4) acquire the capacity to promote and support T-cell development from hematopoietic stem cells (HSCs) into TCRαß(+), CD4(+) and CD8(+) T-lineage cells. However, mFibro-DL4 cells showed a lower efficiency of T-cell generation than OP9-DL4 cells did. Nevertheless, progenitor T-cells (CD117(+) CD44(+) CD25(+)) generated in HSC/mFibro-DL4 co-cultures, when intravenously transferred into immunodeficient (Rag2(-/-) γc(-/-)) mice, home to the thymus, undergo differentiation, and give rise to mature T-cells that go on to populate the periphery. Surprisingly, primary human fibroblast cells expressing Dll4 showed very low efficiency in supporting human T-lineage differentiation, which could not be improved by blocking myelopoiesis. Nevertheless, mFibro-DL4 cells could support human T-cell lineage differentiation. Our results provide a functional framework for the induction of T-cell development using easily accessible fibroblasts made to express Dll4. These cells, which are amenable for in vitro applications, can be further utilized in the design of individualized systems for T-cell production, with implications for the treatment of immunodeficiencies.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Fibroblastos/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Timo/imunologia , Transferência Adotiva , Animais , Proteínas de Ligação ao Cálcio , Diferenciação Celular , Linhagem Celular , Seleção Clonal Mediada por Antígeno , Técnicas de Cocultura , Proteínas de Ligação a DNA/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transgenes/genética
10.
Methods Mol Biol ; 946: 103-13, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23179828

RESUMO

T cells typically differentiate via a series of coordinated steps within the highly specialized microenvironment of the thymus. Traditionally, human T-lymphopoiesis in vitro has been studied using the hybrid human/mouse fetal thymic organ culture system. Pioneering work by McCune et al. devised a method to examine human T cell development in vivo in relation to HIV-1 using the SCID/hu (thy/liv) model. This was followed by models that better reflected the ability of human hematopoietic cells to home and differentiate within the mouse host without human fetal tissues; however, human T cell development in these animals was poor. In this chapter, we outline a procedure to generate human progenitor T (proT) cells in vitro from umbilical cord blood-derived hematopoietic stem cells using the OP9-DL1 cell system; in addition, we describe the method used to examine the engraftment of in vitro-derived proT cells into immunodeficient mouse strains.


Assuntos
Separação Celular/métodos , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/transplante , Transplante de Células-Tronco/métodos , Linfócitos T/citologia , Animais , Linhagem Celular , Técnicas de Cocultura , Citometria de Fluxo , Humanos , Recém-Nascido , Fígado/citologia , Camundongos , Células Estromais/citologia , Cordão Umbilical/citologia
11.
Cell Rep ; 2(6): 1722-35, 2012 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-23219550

RESUMO

The efficient generation of hematopoietic stem cells from human pluripotent stem cells is dependent on the appropriate specification of the definitive hematopoietic program during differentiation. In this study, we used T lymphocyte potential to track the onset of definitive hematopoiesis from human embryonic and induced pluripotent stem cells differentiated with specific morphogens in serum- and stromal-free cultures. We show that this program develops from a progenitor population with characteristics of hemogenic endothelium, including the expression of CD34, VE-cadherin, GATA2, LMO2, and RUNX1. Along with T cells, these progenitors display the capacity to generate myeloid and erythroid cells. Manipulation of Activin/Nodal signaling during early stages of differentiation revealed that development of the definitive hematopoietic progenitor population is not dependent on this pathway, distinguishing it from primitive hematopoiesis. Collectively, these findings demonstrate that it is possible to generate T lymphoid progenitors from pluripotent stem cells and that this lineage develops from a population whose emergence marks the onset of human definitive hematopoiesis.


Assuntos
Antígenos de Diferenciação/biossíntese , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Células-Tronco Pluripotentes/metabolismo , Células Precursoras de Linfócitos T/metabolismo , Transdução de Sinais/fisiologia , Linfócitos T/metabolismo , Linhagem Celular , Humanos , Células-Tronco Pluripotentes/citologia , Células Precursoras de Linfócitos T/citologia , Linfócitos T/citologia
12.
Front Biosci (Schol Ed) ; 3(3): 961-9, 2011 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-21622245

RESUMO

T cells are unique in that they begin their development as a progenitor within the bone marrow but complete their differentiation within the thymus. Furthermore, long-term T-lymphopoiesis requires a continuous supply of thymus-bound progenitors derived from the bone marrow. The critical role for T cells is clearly observed in individuals with genetic or acquired immunodeficiencies or those having undergone hematopoietic stem cell transplantation. Here, we review the work done by several groups aimed at characterizing the earliest T-lineage progenitors (ETPs), in mouse and human, found within the thymus, in addition to the long-sought after thymus-colonizing progenitor, which makes its journey from the bone marrow via the bloodstream into thymus. The characterization of these progenitors may herald therapeutic insight into the restoration of T cells in immunodeficient individuals.


Assuntos
Hematopoese/fisiologia , Células Precursoras de Linfócitos T/citologia , Timo/citologia , Animais , Diferenciação Celular/fisiologia , Movimento Celular/fisiologia , Humanos , Camundongos , Especificidade da Espécie
13.
BMC Immunol ; 12: 22, 2011 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-21429219

RESUMO

BACKGROUND: T cell development occurs within the highly specialized thymus. Cytotoxic CD8 T cells are critical in adaptive immunity by targeting virally infected or tumor cells. In this study, we addressed whether functional CD8 T cells can be generated fully in vitro using human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells. RESULTS: HSC/OP9-DL1 cocultures supported the differentiation of CD8 T cells, which were TCR/CD3(hi) CD27(hi) CD1a(neg) and thus phenotypically resembled mature functional CD8 single positive thymocytes. These in vitro-generated T cells also appeared to be conventional CD8 cells, as they expressed high levels of Eomes and low levels of Plzf, albeit not identical to ex vivo UCB CD8 T cells. Consistent with the phenotypic and molecular characterization, upon TCR-stimulation, in vitro-generated CD8 T cells proliferated, expressed activation markers (MHC-II, CD25, CD38), secreted IFN-γ and expressed Granzyme B, a cytotoxic T-cell effector molecule. CONCLUSION: Taken together, the ability to direct human hematopoietic stem cell or T-progenitor cells towards a mature functional phenotype raises the possibility of establishing cell-based treatments for T-immunodeficiencies by rapidly restoring CD8 effector function, thereby mitigating the risks associated with opportunistic infections.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Ativação Linfocitária , Linfopoese , Proteínas com Domínio T/metabolismo , Antígenos CD/biossíntese , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Técnicas de Cocultura , Sangue Fetal/citologia , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Granzimas/genética , Granzimas/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Imunofenotipagem , Interferon gama/genética , Interferon gama/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/imunologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores de Antígenos de Linfócitos T/imunologia , Células Estromais/citologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/imunologia
14.
Eur J Immunol ; 40(8): 2289-95, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20540115

RESUMO

Human peripheral blood NK cells may be divided into two main subsets: CD56(bright)CD16(-) and CD56(dim)CD16(+). Since TGF-ß is known to influence the development of many leukocyte lineages, its effects on NK cell differentiation either from human CD34(+)Lin(-) hematopoietic progenitor/stem cells in vitro or from peripheral blood NK cells were investigated. TGF-ß represses development of NK cells from CD34(+) progenitors and inhibits differentiation of CD16(+) NK cells. Moreover, TGF-ß also results in conversion of a minor fraction of CD56(bright)CD16(+) cells found in peripheral blood into CD56(bright)CD16(-) cells, highlighting a possible role of the former as a developmental intermediate and of TGF-ß in influencing the genesis of NK subsets found in blood.


Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Anticorpos Bloqueadores/farmacologia , Antígenos CD/biossíntese , Antígenos CD/genética , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Medula Óssea/patologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/imunologia , Células Cultivadas , Citocinas/metabolismo , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/patologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Subpopulações de Linfócitos/efeitos dos fármacos , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/patologia , Fator de Crescimento Transformador beta/imunologia
15.
Curr Opin Hematol ; 17(4): 327-32, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20571395

RESUMO

PURPOSE OF REVIEW: The thymus provides a unique and essential microenvironment for T-cell precursors to develop into mature functionally competent T lymphocytes. Ageing causes architectural changes in the thymus resulting in a loss of thymic epithelial space required for thymopoiesis - a process known as thymic involution. Additionally, cytoablative regimens used to treat malignancies also destroy thymic architecture. The net result of both processes is diminished thymic output and function that may lead to impaired immunity. Thus, immunocompromised individuals would benefit from strategies aimed at enhancing T-cell reconstitution. RECENT FINDINGS: Here we discuss strategies such as the use of sex steroid ablation, keratinocyte growth factor, interleukin-7, and in-vitro-generated progenitor T cells as candidates for restoring T-cell immunity. Using various animal models of ageing or hematopoietic stem cell transplantation, these strategies have been shown to restore thymic architecture and cellularity, resulting in increased output and T-cell function in the periphery. SUMMARY: These candidate approaches are currently being tested in clinical trials, with preliminary evidence showing encouraging effects on T-cell reconstitution. Nevertheless, although these strategies show clear promise in animal models, and in early human trials, further data are needed to determine their efficacy in patients.


Assuntos
Regeneração/fisiologia , Linfócitos T/fisiologia , Timo/fisiologia , Animais , Ensaios Clínicos como Assunto , Fator 7 de Crescimento de Fibroblastos/farmacologia , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Interleucina-7/farmacologia , Regeneração/efeitos dos fármacos , Regeneração/imunologia , Linfócitos T/imunologia , Timo/citologia , Timo/imunologia
16.
Blood ; 114(5): 972-82, 2009 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-19491395

RESUMO

T-cell development follows a defined set of stage-specific differentiation steps. However, molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. To address this, human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T lineage in OP9-DL1 cocultures. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. Quantitative clonal analyses demonstrated that CD34(+)CD38(-) and CD34(+)CD38(lo) subsets of UCB contain a similarly high T-lineage progenitor frequency, whereas the frequency in CD34(+)CD38(+/hi) cells was 5-fold lower. Delta-like/Notch-induced signals increased the T-cell progenitor frequency of CD34(+)CD38(-/lo) cells differentiated on OP9-DL1, and 2 distinct progenitor subsets, CD34(+)CD45RA(+)CD7(++)CD5(-)CD1a(-) (proT1) and CD34(+)CD45RA(+)CD7(++)CD5(+)CD1a(-) (proT2), were identified and their thymus engrafting capacity was examined, with proT2 cells showing a 3-fold enhanced reconstituting capacity compared with the proT1 subset. Furthermore, in vitro-generated CD34(+)CD7(++) progenitors effectively engrafted the thymus of immunodeficient mice, which was enhanced by the addition of an IL-7/IL-7 antibody complex. Taken together, the identification of T-progenitor subsets readily generated in vitro may offer important avenues to improve cellular-based immune-reconstitution approaches.


Assuntos
Células-Tronco Hematopoéticas/citologia , Subpopulações de Linfócitos T/citologia , Animais , Complexo Antígeno-Anticorpo/farmacologia , Antígenos CD/análise , Linhagem da Célula , Células Cultivadas/citologia , Células Cultivadas/transplante , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Sangue Fetal/citologia , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/cirurgia , Recém-Nascido , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Interleucina-7/imunologia , Interleucina-7/farmacologia , Linfopoese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Técnicas de Cultura de Órgãos , Organismos Livres de Patógenos Específicos , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/transplante , Timo/citologia , Timo/embriologia , Transplante Heterólogo
17.
Methods Mol Biol ; 430: 135-42, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18370296

RESUMO

Traditionally, the study of human T cell development has relied on the availability of human and mouse thymic tissue. In this chapter, we outline a simple in vitro protocol for generating large numbers of human T-lineage cells from umbilical cord blood (CB)- derived hematopoietic stem cells (HSCs) using a bone marrow stromal cell line. This protocol is broken into three major steps: (1) the maintenance of a working stock of OP9 bone marrow stromal cells expressing the Notch receptor ligand Delta-like 1 (OP9- DL1), (2) the purification of human HSCs from umbilical CB, and (3) the initiation and maintenance/expansion of OP9-DL1 cocultures over time (see Fig. 1). The use of this system opens avenues for basic research as it equips us with a simple in vitro method for studying human T cell development.


Assuntos
Receptores Notch/metabolismo , Linfócitos T/citologia , Linhagem Celular , Técnicas de Cocultura , Citometria de Fluxo , Células-Tronco Hematopoéticas/citologia , Humanos , Ligantes
18.
Semin Immunol ; 19(5): 341-9, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17997108

RESUMO

Immunodeficient individuals are susceptible to opportunistic infection. While stem cell transplantation can restore a functional immune system, T cells are slow to recover and limited in eliciting adaptive immune responses. Approaches to selectively enhance T cell function have focused on boosting thymopoiesis to generate new T cells or expanding existing T cells. By taking advantage of the role of Notch signaling in T cell development, we have developed an in vitro system able to generate large numbers of progenitor T cells from human hematopoietic stem cells. Here, we discuss this in vitro system and its implications for the potential treatment of T cell immunodeficiency.


Assuntos
Células-Tronco Hematopoéticas/fisiologia , Linfopoese , Linfócitos T/fisiologia , Técnicas de Cocultura , Transplante de Células-Tronco Hematopoéticas , Humanos , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores Notch/fisiologia , Transdução de Sinais , Células Estromais/fisiologia
19.
J Leukoc Biol ; 74(3): 420-7, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12949246

RESUMO

Interleukin-4 (IL-4) is a multifunctional cytokine, which is involved in numerous disease states, including atopic asthma. IL-4 not only induces direct responses in cells but can also prime for secondary responses to stimuli. Little is known about the priming effects of IL-4 on endothelial cells; therefore, we chose to examine the ability of IL-4 to prime endothelial cells for platelet-activating factor (PAF) synthesis and prostaglandin E(2) (PGE(2)) release. IL-4 alone did not enhance PAF synthesis or PGE(2) release; however, pretreatment with IL-4 primed for PAF synthesis and PGE(2) release in response to subsequent stimulation with histamine. In contrast, tumor necrosis factor alpha (TNF-alpha), oncostatin M (OSM), and IL-1beta did not prime endothelial cells for PAF synthesis in response to histamine. The priming effects of IL-4 occurred without any detectable changes in the requirement for signaling pathways upstream of PGE(2) release. IL-4 treatment increased the expression of mRNA for histamine receptor 1 (HR1) and shifted the inhibition curve for pyrilamine, a specific HR1 antagonist. In addition, the dose-response curve for histamine-induced elevations in intracellular calcium was shifted following IL-4 stimulation. Together, these data indicate that HR1 is up-regulated in IL-4-stimulated human umbilical vein endothelial cells (HUVEC) and suggest that this up-regulation may contribute to the enhanced responsiveness of IL-4-stimulated HUVEC to histamine.


Assuntos
Dinoprostona/metabolismo , Endotélio Vascular/efeitos dos fármacos , Histamina/farmacologia , Interleucina-4/farmacologia , Fator de Ativação de Plaquetas/metabolismo , Cálcio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Endotélio Vascular/metabolismo , Humanos , Interleucina-1/farmacologia , Oncostatina M , Ocitócicos/metabolismo , Peptídeos/farmacologia , Receptores Histamínicos/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/citologia , Regulação para Cima
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