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1.
J Immunol ; 195(1): 156-65, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-26026063

RESUMO

Extrathymic T cell precursors can be detected in many tissues and represent an immediately competent population for rapid T cell reconstitution in the event of immunodeficiencies. Blood T cell progenitors have been detected, but their source in the bone marrow (BM) remains unclear. Prospective purification of BM-resident and circulating progenitors, together with RT-PCR single-cell analysis, was used to evaluate and compare multipotent progenitors (MPPs) and common lymphoid progenitors (CLPs). Molecular analysis of circulating progenitors in comparison with BM-resident progenitors revealed that CCR9(+) progenitors are more abundant in the blood than CCR7(+) progenitors. Second, although Flt3(-) CLPs are less common in the BM, they are abundant in the blood and have reduced Cd25(+)-expressing cells and downregulated c-Kit and IL-7Rα intensities. Third, in contrast, stage 3 MPP (MPP3) cells, the unique circulating MPP subset, have upregulated Il7r, Gata3, and Notch1 in comparison with BM-resident counterparts. Evaluation of the populations' respective abilities to generate splenic T cell precursors (Lin(-)Thy1.2(+)CD25(+)IL7Rα(+)) after grafting recipient nude mice revealed that MPP3 cells were the most effective subset (relative to CLPs). Although several lymphoid genes are expressed by MPP3 cells and Flt3(-) CLPs, the latter only give rise to B cells in the spleen, and Notch1 expression level is not modulated in the blood, as for MPP3 cells. We conclude that CLPs have reached the point where they cannot be a Notch1 target, a limiting condition on the path to T cell engagement.


Assuntos
Linfócitos B/metabolismo , Células da Medula Óssea/metabolismo , Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Células-Tronco Multipotentes/metabolismo , Linfócitos T/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Diferenciação Celular , Linhagem da Célula/imunologia , Proliferação de Células , Feminino , Perfilação da Expressão Gênica , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/imunologia , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/imunologia , Receptor Notch1/genética , Receptor Notch1/imunologia , Receptores CCR/genética , Receptores CCR/imunologia , Receptores CCR7/genética , Receptores CCR7/imunologia , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/imunologia , Análise de Célula Única , Linfócitos T/citologia , Linfócitos T/imunologia , Tirosina Quinase 3 Semelhante a fms/deficiência , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/imunologia
2.
Biol Cell ; 105(12): 549-60, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24024612

RESUMO

BACKGROUND INFORMATION: In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single-cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel. RESULTS: Compared with population analysis, using a single-cell approach, we were able to evaluate not only the number of cells that maintained the expression of a specific gene but, most importantly, how many cells co-expressed different markers. We found that micropatterned hydrogel seems to represent a good alternative to MEF, as the expression of stemness markers is better preserved than in Matrigel culture. CONCLUSIONS: This single-cell assay allows for the assessment of the stemness maintenance at a single-cell level in terms of gene expression profile, and can be applied in stem cell research to characterise freshly isolated and cultured cells, or to standardise, for instance, the method of culture closely linked to the transcriptional activity and the differentiation potential.


Assuntos
Biomarcadores/metabolismo , Técnicas de Cultura de Células/instrumentação , Células-Tronco Embrionárias/citologia , Reação em Cadeia da Polimerase/métodos , Análise de Célula Única/métodos , Animais , Diferenciação Celular , Células Cultivadas , Meios de Cultura/química , Meios de Cultura/metabolismo , Primers do DNA/genética , Primers do DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Células Alimentadoras/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Masculino , Camundongos
3.
J Immunol ; 184(9): 4907-17, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20368277

RESUMO

Progressive restriction to a differentiation pathway results from both activation and silencing of particular gene expression programs. To identify the coexpression and the expression levels of regulatory genes during hematopoietic stem cell (HSC) differentiation toward the T cell branch, we applied a new single-cell RT-PCR technique to analyze the simultaneous expression of 13 genes in 9 functionally purified populations from the bone marrow and the thymus. We report in this paper that Lin(-)Sca1(+)ckit(+) HSCs display, at the single-cell level, a homogeneous and high transcriptional activity as do early thymic progenitors. Moreover, the coexpression of lymphoid and myeloid genes is an early event detected in approximately 30% of short-term HSC and most multipotent progenitors, suggesting novel sources for the generation of early thymic progenitors, common lymphoid progenitors (CLPs), and common myeloid progenitors. Loss of multipotency in Lin(-)Sca1(+)ckit(+) cells directed to the lymphoid branch is characterized by Lmo2 and Gata2 gene expression downregulation. Indeed, highest levels of Gata2 expression are detected only in long-term and short-term HSC populations. Complete shutdown of Pu1 gene expression in all triple-negative (TN)3 stage thymic pre-T cells is indicative of total T cell commitment. Interestingly, this is also observed in 30% of TN2 cells and 25% of CLP in the bone marrow, suggesting a possible initiation of T cell engagement in TN2 and CLP. Also, our strategy highlights similar gene patterns among HSCs and intrathymic progenitors, proposing, therefore, that identical activation signals are maintained until further maturation and generation of CD4 and CD8 coreceptors bearing thymocytes.


Assuntos
Diferenciação Celular/genética , Diferenciação Celular/imunologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Células-Tronco Hematopoéticas/imunologia , Subpopulações de Linfócitos/imunologia , Células-Tronco Multipotentes/imunologia , Células Mieloides/imunologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Feminino , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Timo/citologia , Timo/imunologia , Timo/metabolismo
4.
J Leukoc Biol ; 78(5): 1106-17, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16266974

RESUMO

The Fas/Fas ligand (FasL) pathway has been largely implicated in the homeostasis of mature cells. However, it is still unclear whether it plays a role at the progenitor level. To address this issue, we created chimeric mice by transferring C57BL/6 bone marrow (BM) cells of the lpr (Fas-FasL+) or gld (Fas+FasL-) genotype into Rag-2-/- hosts of the same genetic background. In this model, the consequences of a deficient Fas/FasL pathway on lymphoid differentiation could be evaluated without endogenous competition. Analysis of the chimerism revealed a differential sensitivity of hematopoietic lineages to the lack of Fas receptor signaling. While donor-derived myelo-monocytic cells were similarly distributed in all chimeric mice, mature B cells were deleted in the BM and the spleen of lpr chimera, leading to the absence of the marginal zone (MZ) as detected by immunohistology. In contrast, B cell hematopoiesis was complete in gld chimera but MZ macrophages undetectable. These defects suggest a direct and determinant dual role of FasL regulation in negative selection of B cells and in maintenance of the MZ.


Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Glicoproteínas de Membrana/imunologia , Transdução de Sinais/imunologia , Fatores de Necrose Tumoral/imunologia , Receptor fas/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Proliferação de Células , Proteína Ligante Fas , Genótipo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Quimera por Radiação/genética , Quimera por Radiação/imunologia , Baço/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fatores de Necrose Tumoral/genética , Receptor fas/genética
5.
PLoS One ; 8(10): e73098, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24098325

RESUMO

T cell commitment and αß/γδ lineage specification in the thymus involves interactions between many different genes. Characterization of these interactions thus requires a multiparameter analysis of individual thymocytes. We developed two efficient single-cell methods: (i) the quantitative evaluation of the co-expression levels of nine different genes, with a plating efficiency of 99-100% and a detection limit of 2 mRNA molecules/cell; and (ii) single-cell differentiation cultures, in the presence of OP9 cells transfected with the thymus Notch1 ligand DeltaL4. We show that during T cell commitment, Gata3 has a fundamental, dose-dependent role in maintaining Notch1 expression, with thymocytes becoming T-cell-committed when they co-express Notch1, Gata3 and Bc11b. Of the transcription factor expression patterns studied here, only that of Bcl11b was suggestive of a role in Pu1 down-regulation. Individual thymocytes became αß/γδ lineage-committed at very different stages (from the TN2a stage onwards). However, 20% of TN3 cells are not αß/γδ-lineage committed and TN4 cells comprise two main subpopulations with different degrees of maturity. The existence of a correlation between differentiation potential and expression of the pre-TCR showed that 83% of αß-committed cells do not express the pre-TCR and revealed a major stochastic component in αß-lineage specification.


Assuntos
Diferenciação Celular , Linhagem da Célula , Análise de Célula Única , Timócitos/citologia , Timócitos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Camundongos , Processos Estocásticos
6.
J Immunol ; 179(5): 2925-35, 2007 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17709507

RESUMO

Several extrathymic T cell progenitors have been described but their various contributions to the T cell lineage puzzle are unclear. In this study, we provide evidence for a splenic Lin(-)Thy1.2(+) T cell-committed population, rare in B6 mice, abundant in TCRalpha(-/-), CD3epsilon(-/-), and nude mice, and absent in IL-7- and Rag-2-deficient mice. Neither B nor myeloid cells are generated in vivo and in vitro. The incidence of these pre-T cells is under the control of thymus and/or mature T cells, as revealed by graft experiments. Indeed, IL-7 consumption by mature T cells inhibits the growth of these pre-T cells. Moreover, the nude spleen contains an additional Lin(-)Thy1.2(+)CD25(+) subset which is detected in B6 mice only after thymectomy. We establish that the full pre-T cell potential and proliferation capacity are only present in the c-kit(low) fraction of progenitors. We also show that most CCR9(+) progenitors are retained in the spleen of nude mice, but present in the blood of B6 mice. Thus, our data describe a new T cell lineage restricted subset that accumulates in the spleen before migration to the thymus.


Assuntos
Linhagem da Célula , Interleucina-7/metabolismo , Baço/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos B/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Camundongos , Camundongos Transgênicos , Células Mieloides/imunologia , Baço/citologia , Timo/imunologia
7.
Nat Immunol ; 7(1): 76-82, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16341216

RESUMO

Thymic export of cells is believed to be restricted to mature T cells. Here we show that the thymus also exports fully committed T cell precursors that colonize primary lymphoid organs. These precursor cells exited the thymus before T cell receptor rearrangements and colonized lymphoid organs such as the thymus and the gut. Migration of the thymic T cell-committed precursors led to permanent colonization of the gut precursor compartment, improved the capacity of gut precursors to further differentiate into T cells and was sufficient for the generation of 'euthymic like' CD8alphaalpha(+) intraepithelial lymphocytes. These data demonstrate a new function for the thymus in peripheral seeding with T cell precursors that become long lived after thymus export.


Assuntos
Movimento Celular/imunologia , Células-Tronco Hematopoéticas/citologia , Tecido Linfoide/citologia , Linfócitos T/citologia , Timo/citologia , Animais , Diferenciação Celular/imunologia , Células-Tronco Hematopoéticas/imunologia , Camundongos , Microscopia Confocal , Linfócitos T/imunologia , Timo/imunologia
8.
Nat Immunol ; 7(11): 1217-24, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17013389

RESUMO

Natural killer (NK) cell development is thought to occur in the bone marrow. Here we identify the transcription factor GATA-3 and CD127 (IL-7R alpha) as molecular markers of a pathway of mouse NK cell development that originates in the thymus. Thymus-derived CD127+ NK cells repopulated peripheral lymphoid organs, and their homeostasis was strictly dependent on GATA-3 and interleukin 7. The CD127+ NK cells had a distinct phenotype (CD11b(lo) CD16- CD69(hi) Ly49(lo)) and unusual functional attributes, including reduced cytotoxicity but considerable cytokine production. Those characteristics are reminiscent of human CD56(hi) CD16- NK cells, which we found expressed CD127 and had more GATA-3 expression than human CD56+ CD16+ NK cells. We propose that bone marrow and thymic NK cell pathways generate distinct mouse NK cells with properties similar to those of the two human CD56 NK cell subsets.


Assuntos
Diferenciação Celular/imunologia , Fator de Transcrição GATA3/biossíntese , Subunidade alfa de Receptor de Interleucina-7/biossíntese , Células Matadoras Naturais/citologia , Subpopulações de Linfócitos/citologia , Transdução de Sinais/imunologia , Timo/imunologia , Animais , Citocinas/biossíntese , Citotoxicidade Imunológica , Fator de Transcrição GATA3/genética , Humanos , Imunofenotipagem , Interleucina-7/fisiologia , Subunidade alfa de Receptor de Interleucina-7/genética , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Subpopulações de Linfócitos/imunologia , Subpopulações de Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Timo/citologia , Timo/metabolismo
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