RESUMO
The requirement for Notch signaling during T-cell development has been extensively studied. Nevertheless, the developmental stage at which it is required and whether additional signaling pathways are needed are still poorly understood. By using a stromal-cell-free culture system, we show that sorted double-negative 3 (DN3) thymocytes only require a Delta-like-4-induced Notch signal to differentiate into double-positive (DP) cells. This differentiation process is preTCR-α dependent. DN3 cells undergo 4-5 proliferation cycles, and the addition of the chemokine CXCL12 improves proliferation. IL-7 blocks the differentiation of DN3 cells to DP cells but not the Notch-induced proliferation of cultured DN3 cells. The impaired differentiation correlates with an inhibition of Rag-2 up-regulation. Overall, the in vitro stromal-cell-free culture system presented here also provides a powerful and unique tool for studying the mechanisms involved in the positive and negative selection of T cells.
Assuntos
Diferenciação Celular/imunologia , Quimiocina CXCL12/imunologia , Interleucina-7/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Proteínas de Membrana/imunologia , Transdução de Sinais/imunologia , Timócitos/citologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Ligação ao Cálcio , Separação Celular , Quimiocina CXCL12/metabolismo , Feminino , Citometria de Fluxo , Interleucina-7/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Timócitos/imunologia , Timócitos/metabolismoRESUMO
In young adult mice, the thymus produces about a million newly formed T cells every day that colonize peripheral lymphoid tissues. Mostly regarded as a primary lymphoid organ only, the relationship between the thymus and peripheral lymphoid organs is considered unidirectional. However, this perception has been challenged by reports showing that peripheral lymphocytes, mostly T cells, can migrate back into the thymus. The presence of recirculating T cells in the thymus is rather incongruous and raises the question: is the presence of 'peripheral' T cells in the thymus superfluous or do these cells fulfill some relevant physiologic functions? There is now evidence that cells of the hematopoietic lineage, including T cells, can play an active role during thymocyte selection, a role generally considered the exclusive property of thymic epithelial cells and dendritic cells. Although, on a per cell basis, peripheral T cells in the thymus may be less efficient than thymus epithelial cells or dendritic cells at thymocyte positive and negative selection, they may nevertheless contribute to selection by influencing the selectable TCR repertoire and post-selection T cell functionality. Here, peripheral lymphocytes re-entering the thymus may be envisioned as Trojan horses as these cells may introduce antigens necessary for both positive and negative selection of T cells.
Assuntos
Movimento Celular/imunologia , Linfócitos T/imunologia , Timo/imunologia , Animais , Deleção Clonal , Humanos , CamundongosRESUMO
The thymus continuously produces T lymphocytes that contribute to the maintenance of the peripheral T cell pool. Since peripheral recirculating T cells represent a very minor population among total thymocytes in normal animals, the relationship between the thymus and secondary lymphoid organs is generally considered unidirectional. Recently, several reports have described the presence of recirculating T cells in the thymus, raising issues regarding their possible function. In this article, we show that the niche for recirculating T cells in the thymus, i.e., their absolute number, is the same in lymphopenic and normal mice. Using a novel combination of TCR-transgenic mice in which the ligand necessary for positive selection of host T cells is only expressed by transferred donor T cells, we show that mature T cells recirculating back to the thymus can mediate positive selection.
Assuntos
Linfócitos T/imunologia , Timo/imunologia , Animais , Diferenciação Celular , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Parabiose , Linfócitos T/metabolismo , Timo/fisiologiaRESUMO
T cell homeostasis is essential for the functioning of the vertebrate immune system, but the intracellular signals required for T cell homeostasis are largely unknown. We here report that the WD-repeat protein family member coronin-1, encoded by the gene Coro1a, is essential in the mouse for T cell survival through its promotion of Ca2+ mobilization from intracellular stores. Upon T cell receptor triggering, coronin-1 was essential for the generation of inositol-1,4,5-trisphosphate from phosphatidylinositol-4,5-bisphosphate. The absence of coronin-1, although it did not affect T cell development, resulted in a profound defect in Ca2+ mobilization, interleukin-2 production, T cell proliferation and T cell survival. We conclude that coronin-1, through activation of Ca2+ release from intracellular stores, is an essential regulator of peripheral lymphocyte survival.
Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/biossíntese , Proteínas dos Microfilamentos/fisiologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Actinas/metabolismo , Animais , Sinalização do Cálcio/genética , Sinalização do Cálcio/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Líquido Intracelular/imunologia , Líquido Intracelular/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
Haematopoietic stem cells (HSCs), a very rare cell type in the bone marrow, are responsible for the life-long production of all cells of the blood including T and B cells. Until recently, it was thought that the differentiation of HSCs into the various haematopoietic cells was rather hierarchical in that differentiation along a given lineage was associated with a progressive loss of potential to give rise to other blood cell lineages. The recent development of very sensitive and quantitative in vitro assays, together with the identification of new progenitor subpopulations, has challenged this idea. Thus, lymphocyte progenitors can be shown to keep their developmental potential to give rise to myeloid, dendritic and NK cells until just prior to their final commitment stage. Here we review these new findings and concepts.
RESUMO
Analysis of hematopoietic development has for decades been central to understanding lineage diversification. Some models consider hematopoietic commitment to be random, and branching lineage maps often include an early myeloid or lymphoid bifurcation. However, the existence of joint lymphoid or myeloid intermediate progenitors argues against both. One of us earlier proposed the sequential determination (SD) model, which features a limited and stepwise set of binary choices across the full hematopoietic spectrum. This model arose from observations that hematopoietic progenitors show preferences for particular associations of lineage potentials--indicating that these linked fates are neighbours developmentally. An updated SD model complemented by several recently recognized processes--spatiotemporal fluctuations in transcription factor concentrations, asymmetric cell division, and Notch signalling--still offers a sound summary of hematopoiesis.
Assuntos
Linhagem da Célula , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Modelos Biológicos , Animais , Divisão Celular , HumanosRESUMO
Haematopoietic stem cells (HSCs), a very rare cell type in the bone marrow, are responsible for the life-long production of all cells of the blood including T and B cells. Until recently, it was thought that the differentiation of HSCs into the various haematopoietic cells was rather hierarchical in that differentiation along a given lineage was associated with a progressive loss of potential to give rise to other blood cell lineages. The recent development of very sensitive and quantitative in vitro assays, together with the identification of new progenitor subpopulations, has challenged this idea. Thus, lymphocyte progenitors can be shown to keep their developmental potential to give rise to myeloid, dendritic and NK cells until just prior to their final commitment stage. Here we review these new findings and concepts.
Assuntos
Linfócitos B/fisiologia , Células da Medula Óssea/citologia , Linfócitos T/citologia , Timo/citologia , Animais , Antígenos CD/fisiologia , Diferenciação Celular , Proliferação de Células , Células-Tronco Hematopoéticas/citologia , HumanosRESUMO
The early events in T lineage commitment are difficult to study because of the rarity of these cells. We have therefore used cloned Pax5-/- pre-BI cell lines as a model system to study this. Stimulation in vitro of Pax5-/- pre-BI cells with stromal cells expressing the Notch ligand Delta-like 1 results in them coincidently undergoing some of the phenotypic and functional changes associated with early T cell commitment. Kinetic analysis indicated that there was a rapid induction of transcripts for the two chemokine receptors CCR4 and CXCR6. Transcripts for CCR8 increased with slower kinetics. Migration assays indicated that Delta-like 1 signalling of Pax5-/- pre-BI cells had induced responsiveness to the chemokines MDC and MIP-1beta, ligands for the receptors CCR4 and CCR8, respectively. Importantly, following Delta-like 1 signalling, similar increases in chemokine receptor transcripts were seen in a recently described bone marrow progenitor subpopulation having significant T cell progenitor activity and being phenotypically and functionally similar to Pax5-/- pre-BI cells. The relevance of these findings to studies of early T cell development will be discussed.
Assuntos
Linhagem da Célula/genética , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Quimiocinas/genética , Linfócitos T/citologia , Movimento Celular/genética , Movimento Celular/imunologia , Células Cultivadas , Quimiocinas/farmacologia , Técnicas de Cocultura , Regulação da Expressão Gênica , Humanos , Mutação , Fator de Transcrição PAX5/genética , Receptores de Quimiocinas/metabolismo , Células Estromais/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Transcrição Gênica , Regulação para CimaRESUMO
The peripheral B-cell pool is in dynamic equilibrium and is controlled by a variety of factors. The rate of generation of B cells can influence both the composition and size of the peripheral B-cell compartment. Mice deficient for lambda5 gene expression have a block in early B-cell development leading to a markedly reduced number of peripheral B cells. To address the question of how this early developmental block influences the composition of the B-cell pool, we have analyzed mature B-cell subpopulations in lambda5-deficient mice. In analogy with other situations of B lymphopenia, the proportion was increased but not the absolute number of marginal-zone B cells, whereas those of follicular B cells were decreased. Immunohistology revealed that B-cell follicles were smaller in overall size and contained a prominent B-cell replete marginal zone. BrdU labelling kinetics showed slower turnover of follicular as well as of marginal-zone B cells. Functionally, lambda5(-/-) mice were able to mount not only primary but also secondary thymus-dependent as well as thymus-independent responses, albeit mostly at reduced levels.
Assuntos
Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular , Deleção de Genes , Homeostase , Cadeias Leves de Imunoglobulina/genética , Animais , Linfócitos B/metabolismo , Medula Óssea/metabolismo , Quimera/genética , Quimera/metabolismo , Cadeias Leves de Imunoglobulina/imunologia , Cadeias Leves de Imunoglobulina/metabolismo , Camundongos , Camundongos KnockoutRESUMO
In this report, we identify in the bone marrow (BM) of normal mice a subpopulation of B220+ CD117+ CD19- NK1.1- cells with potent lymphoid and myeloid developmental potential. These cells represent 0.1-0.2% of nucleated BM cells. By limiting dilution analysis in the presence of the appropriate combination of stromal cells and cytokines, 1 in 5-10 sorted cells formed B cells, 1 in 10-15 formed T cells and 1 in 5-10 generated macrophages. When cultured on a mixture of OP9 stroma and OP9 stromal cells expressing the Notch ligand Delta-like-1, single cells generated both T and B cells. Following intravenous infusion, freshly sorted cells transiently reconstituted both the T and B cell progenitor compartments, generating cohorts of mature T and B lymphocytes. The relationship between B220+ CD117+ CD19- NK1.1- cells of wild-type mice and other multi-lineage BM progenitors is discussed.
Assuntos
Antígenos CD19/biossíntese , Células-Tronco Hematopoéticas/imunologia , Antígenos Comuns de Leucócito/biossíntese , Proteínas Proto-Oncogênicas c-kit/biossíntese , Animais , Antígenos/imunologia , Antígenos Ly , Antígenos de Superfície , Linfócitos B/citologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Feminino , Células-Tronco Hematopoéticas/citologia , Lectinas Tipo C , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Mieloides/citologia , Células Mieloides/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK , Proteínas/imunologia , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
The immune response of T lymphocytes to pathogens is initiated in draining secondary lymphoid organs, and activated cells then migrate to the site of infection. Thus, control of naive and regulatory CD4+ T-cell migration is crucial; however, it is poorly understood in physiological and pathological conditions. We found that CD4+ subpopulations displayed characteristic regulator of G-protein signalling (RGS) gene expression profiles. Regulatory T cells express higher levels of RGS1, RGS9 and RGS16 than naive cells. These genes are up-regulated upon cell activation and their level of expression correlates with in vivo cell migration. Using parabiosis, we showed that regulatory T lymphocytes migrate less than naive T cells and that migrant naive T cells express even lower RGS levels than their static counterparts. Our results show an inverse correlation between the capacity to migrate and the levels of RGS1, RGS9 and RGS16 for both naive and regulatory T cells. Taken together, these results suggest a role for RGS molecules in chemokine-induced lymphocyte migration and demonstrate the peculiarity of regulatory T cells in terms of phenotype and migration ability, providing new insights into their function.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Quimiotaxia de Leucócito/imunologia , Proteínas RGS/imunologia , Animais , Feminino , Expressão Gênica/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Parabiose , Reação em Cadeia da Polimerase/métodos , Proteínas RGS/genética , Proteínas RGS/metabolismo , Receptores de Interleucina-2/análise , Transdução de Sinais/imunologiaRESUMO
In some respects, our understanding of the cellular and molecular aspects of early T-cell differentiation is lagging behind that of B cells. Papers describing gene-knockout and reporter-transgenic mice in which thymocyte development is affected are often difficult to interpret. Progress in this field will be hampered unless a more detailed phenotypic and molecular analysis of progenitor thymocytes at the single-cell level is carried out.
Assuntos
Antígenos CD4/análise , Antígenos CD8/análise , Linfócitos T/fisiologia , Animais , Rearranjo Gênico do Linfócito T , Células-Tronco Hematopoéticas/fisiologia , Humanos , Imunofenotipagem , Contagem de Linfócitos , Proteínas Proto-Oncogênicas c-kit/análise , Linfócitos T/imunologiaRESUMO
Knowledge of the complete nucleotide sequence of the mouse TCRAD locus allows an accurate determination V-J rearrangement status. Using multiplex genomic PCR assays and real time PCR analysis, we report a comprehensive and systematic analysis of the V-J recombination of TCR alpha chain in normal mouse thymocytes during development. These respective qualitative and quantitative approaches give rise to four major points describing the control of gene rearrangements. (a) The V-J recombination pattern is not random during ontogeny and generates a limited TCR alpha repertoire; (b) V-J rearrangement control is intrinsic to the thymus; (c) each V gene rearranges to a set of contiguous J segments with a gaussian-like frequency; (d) there are more rearrangements involving V genes at the 3' side than 5' end of V region. Taken together, this reflects a preferential association of V and J gene segments according to their respective positions in the locus, indicating that accessibility of both V and J regions is coordinately regulated, but in different ways. These results provide a new insight into TCR alpha repertoire size and suggest a scenario for V usage during differentiation.