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1.
Nat Immunol ; 20(9): 1244-1255, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31431722

RESUMO

Mucosal-associated invariant T cells (MAIT cells) recognize the microbial metabolite 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) presented by the MHC class Ib molecule, MR1. MAIT cells acquire effector functions during thymic development, but the mechanisms involved are unclear. Here we used single-cell RNA-sequencing to characterize the developmental path of 5-OP-RU-specific thymocytes. In addition to the known MAIT1 and MAIT17 effector subsets selected on bone-marrow-derived hematopoietic cells, we identified 5-OP-RU-specific thymocytes that were selected on thymic epithelial cells and differentiated into CD44- naive T cells. MAIT cell positive selection required signaling through the adapter, SAP, that controlled the expression of the transcription factor, ZBTB16. Pseudotemporal ordering of single cells revealed transcriptional trajectories of 5-OP-RU-specific thymocytes selected on either thymic epithelial cells or hematopoietic cells. The resulting model illustrates T cell lineage decisions.


Assuntos
Linhagem da Célula/imunologia , Células T Invariáveis Associadas à Mucosa/citologia , Células T Invariáveis Associadas à Mucosa/imunologia , Timócitos/citologia , Timócitos/imunologia , Animais , Sequência de Bases , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores de Hialuronatos/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Antígenos de Histocompatibilidade Menor/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Análise de Sequência de RNA , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Timo/citologia , Timo/imunologia
2.
Nature ; 571(7764): 265-269, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31207605

RESUMO

Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential1-4. This process, known as T cell exhaustion or dysfunction1, is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1)5-8. The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood9-12. Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein13-15. TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in mice and hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1+ self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Homeodomínio/metabolismo , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Animais , Proliferação de Células , Doença Crônica , Citocinas/imunologia , Citocinas/metabolismo , Epigênese Genética , Feminino , Regulação da Expressão Gênica/imunologia , Hepacivirus/imunologia , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Memória Imunológica , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Fenótipo , Timócitos/citologia , Timócitos/imunologia , Transcrição Genética
3.
Chem Biol Interact ; 302: 143-148, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30779908

RESUMO

N-(3-oxododecanoyl)-l-homoserine-lactone (ODHL), a quorum sensing molecule, affects intracellular Zn2+ concentration ([Zn2+]i) and cellular levels of nonprotein thiols ([NPT]i) of rat thymic lymphocytes, both of which are assumed to affect cell vulnerability to oxidative stress. Therefore, it is interesting to examine the effects of ODHL on the cells under oxidative stress. ODHL augmented the cytotoxicity of H2O2, but not calcium ionophore A23187. ODHL potentiated the H2O2-induced elevation of [Zn2+]i, wherein, it greatly attenuated the H2O2-induced increase in intracellular Ca2+ concentration. ODHL did not affect [NPT]i in the presence of H2O2. Therefore, we conclude that the elevation of [Zn2+]i is involved in the ODHL-induced potentiation of H2O2 cytotoxicity. Our findings suggest that ODHL modifies cell vulnerability to oxidative stress in host cells.


Assuntos
4-Butirolactona/análogos & derivados , Estresse Oxidativo/efeitos dos fármacos , Timócitos/efeitos dos fármacos , 4-Butirolactona/farmacologia , Animais , Calcimicina/farmacologia , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Masculino , Percepção de Quorum/efeitos dos fármacos , Ratos , Ratos Wistar , Compostos de Sulfidrila/metabolismo , Timócitos/citologia , Timócitos/metabolismo , Zinco/metabolismo
4.
Immunity ; 50(2): 348-361.e4, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30737145

RESUMO

NF-κB (nuclear factor κB) signaling is considered critical for single positive (SP) thymocyte development because loss of upstream activators of NF-κB, such as the IKK complex, arrests their development. We found that the compound ablation of RelA, cRel, and p50, required for canonical NF-κB transcription, had no impact upon thymocyte development. While IKK-deficient thymocytes were acutely sensitive to tumor necrosis factor (TNF)-induced cell death, Rel-deficient cells remained resistant, calling into question the importance of NF-κB as the IKK target required for thymocyte survival. Instead, we found that IKK controlled thymocyte survival by repressing cell-death-inducing activity of the serine/threonine kinase RIPK1. We observed that RIPK1 expression was induced during development of SP thymocytes and that IKK was required to prevent RIPK1-kinase-dependent death of SPs in vivo. Finally, we showed that IKK was required to protect Rel-deficient thymocytes from RIPK1-dependent cell death, underscoring the NF-κB-independent function of IKK during thymic development.


Assuntos
Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Timócitos/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Quinase I-kappa B/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Timócitos/citologia , Timócitos/efeitos dos fármacos , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
5.
Chem Biol Interact ; 300: 35-39, 2019 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-30629953

RESUMO

Azoxystrobin, a broad-spectrum fungicide, has been increasingly used in the agricultural industry. In Japan in 2018, azoxystrobin at five times the normal limit was detected in a shipment of Australian barley that had been used in food products. Therefore, the effects of azoxystrobin need to be carefully examined to predict potential adverse reactions in humans. In this study, the effects of azoxystrobin on the membrane potential and intracellular Ca2+ levels of thymocytes have been photochemically examined using flow cytometry. Azoxystrobin hyperpolarized plasma membrane potential. This hyperpolarization appeared to be due to the activation of Ca2+-dependent K+ channels, as both the removal of extracellular Ca2+ and addition of charybdotoxin attenuated the observed hyperpolarization. In the presence of quinine, an anti-malarial drug that blocks Ca2+-dependent K+ channels, azoxystrobin depolarized the membranes instead. Azoxystrobin increased intracellular Ca2+ levels in a concentration-dependent manner through the influx of extracellular Ca2+ and intracellular release of Ca2+, as confirmed by reduction in azoxystrobin-induced response in the absence of extracellular Ca2+. It appears likely that azoxystrobin at micromolar concentrations modifies membrane ion permeability in thymocytes. Since changes in membrane potential and intracellular Ca2+ levels occur during typical physiological lymphocyte responses, azoxystrobin may disturb lymphocyte function.


Assuntos
Fungicidas Industriais/farmacologia , Pirimidinas/farmacologia , Estrobilurinas/farmacologia , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Masculino , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio Cálcio-Ativados/antagonistas & inibidores , Canais de Potássio Cálcio-Ativados/metabolismo , Quinina/farmacologia , Ratos , Ratos Wistar , Timócitos/citologia , Timócitos/efeitos dos fármacos , Timócitos/metabolismo
6.
J Immunol ; 202(4): 1033-1038, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626694

RESUMO

Intricate life-versus-death decisions are programmed during T cell development, and the regulatory mechanisms that coordinate their activation and repression are still under investigation. In this study, HDAC3-deficient double-positive (DP) thymocytes exhibit a severe decrease in numbers. The thymic cortex is rich in ATP, which is released by macrophages that clear apoptotic DP thymocytes that fail to undergo positive selection. We demonstrate that HDAC3 is required to repress expression of the purinergic receptor P2X7 to prevent DP cell death. HDAC3-deficient DP thymocytes upregulate the P2X7 receptor, increasing sensitivity to ATP-induced cell death. P2rx7/HDAC3-double knockout mice show a partial rescue in DP cell number. HDAC3 directly binds to the P2rx7 enhancer, which is hyperacetylated in the absence of HDAC3. In addition, RORγt binds to the P2rx7 enhancer and promotes P2X7 receptor expression in the absence of HDAC3. Therefore, HDAC3 is a critical regulator of DP thymocyte survival and is required to suppress P2X7 receptor expression.


Assuntos
Morte Celular , Histona Desacetilases/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Timócitos/citologia , Timócitos/enzimologia , Animais , Histona Desacetilases/deficiência , Camundongos , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Receptores Purinérgicos P2X7/genética , Timócitos/metabolismo
7.
J Exp Med ; 216(1): 231-243, 2019 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-30545902

RESUMO

Expression of Rag1 and Rag2 is tightly regulated in developing T cells to mediate TCR gene assembly. Here we have investigated the molecular mechanisms governing the assembly and disassembly of a transcriptionally active RAG locus chromatin hub in CD4+CD8+ thymocytes. Rag1 and Rag2 gene expression in CD4+CD8+ thymocytes depends on Rag1 and Rag2 promoter activation by a distant antisilencer element (ASE). We identify GATA3 and E2A as critical regulators of the ASE, and Runx1 and E2A as critical regulators of the Rag1 promoter. We reveal hierarchical assembly of a transcriptionally active chromatin hub containing the ASE and RAG promoters, with Rag2 recruitment and expression dependent on assembly of a functional ASE-Rag1 framework. Finally, we show that signal-dependent down-regulation of RAG gene expression in CD4+CD8+ thymocytes depends on Ikaros and occurs with disassembly of the RAG locus chromatin hub. Our results provide important new insights into the molecular mechanisms that orchestrate RAG gene expression in developing T cells.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Regulação da Expressão Gênica/fisiologia , Loci Gênicos/fisiologia , Proteínas de Homeodomínio/biossíntese , Timócitos/metabolismo , Transcrição Genética/fisiologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Antígenos CD4/genética , Antígenos CD4/metabolismo , Antígenos CD8/genética , Antígenos CD8/metabolismo , Proteínas de Ligação a DNA/genética , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/metabolismo , Proteínas de Homeodomínio/genética , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Camundongos , Elementos de Resposta/fisiologia , Timócitos/citologia
8.
J Immunol ; 202(3): 966-978, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30567730

RESUMO

T cell development depends on sequential interactions of thymocytes with cortical thymic epithelial cells (cTECs) and medullary thymic epithelial cells. PSMB11 is a catalytic proteasomal subunit present exclusively in cTECs. Because proteasomes regulate transcriptional activity, we asked whether PSMB11 might affect gene expression in cTECs. We report that PSMB11 regulates the expression of 850 cTEC genes that modulate lymphostromal interactions primarily via the WNT signaling pathway. cTECs from Psmb11 -/- mice 1) acquire features of medullary thymic epithelial cells and 2) retain CD8 thymocytes in the thymic cortex, thereby impairing phase 2 of positive selection, 3) perturbing CD8 T cell development, and 4) causing dramatic oxidative stress leading to apoptosis of CD8 thymocytes. Deletion of Psmb11 also causes major oxidative stress in CD4 thymocytes. However, CD4 thymocytes do not undergo apoptosis because, unlike CD8 thymocytes, they upregulate expression of chaperones and inhibitors of apoptosis. We conclude that PSMB11 has pervasive effects on both CD4 and CD8 thymocytes via regulation of gene expression in cTECs.


Assuntos
Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Células Epiteliais/citologia , Complexo de Endopeptidases do Proteassoma/genética , Timócitos/citologia , Animais , Apoptose , Diferenciação Celular , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/imunologia , Timo/imunologia , Via de Sinalização Wnt
9.
J Immunol ; 202(3): 760-769, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30567733

RESUMO

SRC3, a highly conserved member of the steroid receptor coactivator (SRC) family, is recruited by transcription factors to regulate cellular function. Previously, we demonstrated that SRC1, another highly conserved member of the SRC family, interacts with RORγt to regulate Th17 differentiation. However, the relationship between SRC1 and SRC3 in the regulation of Th17 cell function remains unknown. In this study, we demonstrate that mouse SRC3 interacts with RORγt in Th17 cells but not in thymocytes. In addition, Src3-/- mice exhibited defective Th17 differentiation and induction of experimental autoimmune encephalomyelitis but normal thymocyte development. Furthermore, a K313 to arginine mutation of RORγt (RORγt-K313R), which disrupts the interaction of RORγt with SRC3 but not with SRC1, impairs Th17 differentiation but not thymocyte development. These data suggest that SRC3 works with SRC1 to regulate RORγt-dependent Th17 differentiation but is not essential for RORγt-dependent thymocyte development.


Assuntos
Diferenciação Celular , Coativador 3 de Receptor Nuclear/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Células Th17/imunologia , Timócitos/citologia , Animais , Encefalomielite Autoimune Experimental/imunologia , Regulação da Expressão Gênica , Ativação Linfocitária , Camundongos , Camundongos Knockout , Células Th17/citologia , Timócitos/imunologia
10.
Biochem Cell Biol ; 97(2): 201-213, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30352171

RESUMO

B-cell lymphoma/leukemia 11B (Bcl11b) is a transcription factor critical for thymocyte development. We have previously characterized the kinetic post-translational modifications (PTMs) of Bcl11b in double-positive (DP) thymocytes during stimulation of the T cell receptor-activated MAP kinase pathway. However, the PTMs of Bcl11b in thymocytes from other developmental stages in the thymus, primarily double-negative (DN) cells, have not been previously identified. We found that kinetic modifications of Bcl11b in DN cells are somewhat different than the patterns observed in DP cells. Distinct from DP thymocytes, phosphorylation and sumoylation of Bcl11b in DN cells were not oppositely regulated in response to activation of MAP kinase, even though hyper-phosphorylation of Bcl11b coincided with near complete desumoylation. Additionally, prolonged stimulation of the MAP kinase pathway in DN cells, unlike DP thymocytes, did not alter Bcl11b levels of sumoylation or ubiquitinylation, or stability. On the other hand, activation of Wnt-Gsk3-dependent signaling in DN cells resulted in composite dephosphorylation and sumoylation of Bcl11b. Moreover, stimulation of MAP kinase and (or) Wnt signaling pathways differentially affects gene expression of some Bcl11b target and maturation-associated genes. Defining the signaling pathways and regulation of sequence-specific transcription factors by PTMs at various stages of thymopoiesis may improve our understanding of leukemogenesis.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Repressoras/metabolismo , Timócitos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Via de Sinalização Wnt , Animais , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/genética , Quinase 3 da Glicogênio Sintase/genética , Ativação Linfocitária , Camundongos , Proteínas Repressoras/genética , Timócitos/citologia , Proteínas Supressoras de Tumor/genética
11.
Cell Mol Biol (Noisy-le-grand) ; 64(14): 25-30, 2018 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30511618

RESUMO

Melatonin, produced mainly by the pineal gland, has an immunomodulatory role. However, the effects of the pineal gland and/or melatonin on thymus cytokine levels such as interferon-gamma (IFN-γ), interleukin (IL)-4, and IL-10 are not well known. Twenty-one male Wistar rats (220-250 gr) were randomly divided into three groups (n=7): intact control, sham, and pinealectomy. Primary thymocyte cultures were prepared from each group and dispensed into well plates as Control, DMSO (or vehicle), Sham-pinealectomy, Pinealectomy, Pinealectomy+10µM melatonin, and Pinealectomy+100µM melatonin. IFN-γ, IL-4, and IL-10 concentrations were measured in the thymocytes (as nonstimulated and Concanavalin A-stimulated) after 24 h. IFN-γ levels significantly increased and IL-10 levels significantly decreased in both media prepared from pinealectomized rats. There was no significant difference between the groups in terms of IL-4. In the pinealectomy+10µM melatonin group, IFN-γ and IL-10 levels did not differ from the pinealectomy group. However, the dose of 100µM melatonin caused a decrease in levels of IFN-γ in both thymocyte media and an increase in the concentration of IL-10 in Concanavalin A-stimulated thymocytes. In conclusion, pineal gland and/or melatonin affect IFN-γ and IL-10 levels in the thymus gland.


Assuntos
Interferon gama/metabolismo , Interleucina-10/metabolismo , Glândula Pineal/cirurgia , Pinealectomia , Timócitos/citologia , Timócitos/metabolismo , Animais , Células Cultivadas , Interleucina-4/metabolismo , Masculino , Melatonina/farmacologia , Ratos Wistar
12.
Cell Rep ; 25(11): 3059-3073.e10, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30540939

RESUMO

Mitochondria are key players in the regulation of T cell biology by dynamically responding to cell needs, but how these dynamics integrate in T cells is still poorly understood. We show here that the mitochondrial pro-fission protein Drp1 fosters migration and expansion of developing thymocytes both in vitro and in vivo. In addition, we find that Drp1 sustains in vitro clonal expansion and cMyc-dependent metabolic reprogramming upon activation, also regulating effector T cell numbers in vivo. Migration and extravasation defects are also exhibited in Drp1-deficient mature T cells, unveiling its crucial role in controlling both T cell recirculation in secondary lymphoid organs and accumulation at tumor sites. Moreover, the observed Drp1-dependent imbalance toward a memory-like phenotype favors T cell exhaustion in the tumor microenvironment. All of these findings support a crucial role for Drp1 in several processes during T cell development and in anti-tumor immune-surveillance.


Assuntos
Movimento Celular , Dinaminas/metabolismo , Vigilância Imunológica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Contagem de Células , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Homeostase , Ativação Linfocitária/imunologia , Tecido Linfoide/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos Knockout , Fenótipo , Receptores de Antígenos de Linfócitos T , Timócitos/citologia , Timócitos/metabolismo
13.
Front Immunol ; 9: 2426, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30483245

RESUMO

Functional antigen receptor genes are assembled by somatic rearrangements that are largely lymphocyte lineage specific. The immunoglobulin heavy chain (IgH) gene locus is unique amongst the seven antigen receptor loci in undergoing partial gene rearrangements in the wrong lineage. Here we demonstrate that breakdown of lineage-specificity is associated with inappropriate activation of the Eµ enhancer during T cell development by a different constellation of transcription factors than those used in developing B cells. This is reflected in reduced enhancer-induced epigenetic changes, eRNAs, formation of the RAG1/2-rich recombination center, attenuated chromatin looping and markedly different utilization of DH gene segments in CD4+CD8+ (DP) thymocytes. Additionally, CTCF-dependent VH locus compaction is disrupted in DP cells despite comparable transcription factor binding in both lineages. These observations identify multiple mechanisms that contribute to lineage-specific antigen receptor gene assembly.


Assuntos
Regulação da Expressão Gênica , Loci Gênicos , Cadeias Pesadas de Imunoglobulinas/genética , Timócitos/imunologia , Timócitos/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Cromatina/genética , Elementos Facilitadores Genéticos , Íntrons , Camundongos , Curva ROC , Timócitos/citologia , Recombinação V(D)J
14.
J Exp Med ; 215(12): 2984-2993, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30425120

RESUMO

The emigration of mature thymocytes from the thymus is critical for establishing peripheral T cell compartments. However, the pathways controlling this process and the timing of egress in relation to postselection developmental stages are poorly defined. Here, we reexamine thymocyte egress and test current and opposing models in relation to the requirement for LTßR, a regulator of thymic microenvironments and thymocyte emigration. Using cell-specific gene targeting, we show that the requirement for LTßR in thymocyte egress is distinct from its control of thymic epithelium and instead maps to expression by endothelial cells. By separating emigration into sequential phases of perivascular space (PVS) entry and transendothelial migration, we reveal a developmentally ordered program of egress where LTßR operates to rate limit access to the PVS. Collectively, we show the process of thymic emigration ensures only the most mature thymocytes leave the thymus and demonstrate a role for LTßR in the initiation of thymus emigration that segregates from its control of medulla organization.


Assuntos
Movimento Celular/imunologia , Células Endoteliais/imunologia , Receptor beta de Linfotoxina/imunologia , Timócitos/imunologia , Timo/imunologia , Animais , Movimento Celular/genética , Células Endoteliais/citologia , Receptor beta de Linfotoxina/genética , Camundongos , Camundongos Knockout , Timócitos/citologia , Timo/citologia
15.
Nat Commun ; 9(1): 4685, 2018 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-30410062

RESUMO

The ligand for the c-Kit receptor, KitL, exists as a membrane-associated (mKitL) and a soluble form (sKitL). KitL functions outside c-Kit activation have not been identified. We show that co-culture of c-Kit- and mKitL-expressing NIH3T3 cells results in signaling through mKitL: c-Kit-bound mKitL recruits calcium-modulating cyclophilin ligand (CAML) to selectively activate Akt, leading to CREB phosphorylation, mTOR pathway activation, and increased cell proliferation. Activation of mKitL in thymic vascular endothelial cells (VECs) induces mKitL- and Akt-dependent proliferation, and genetic ablation of mKitL in thymic VECs blocks their c-Kit responsiveness and proliferation during neonatal thymic expansion. Therefore, mKitL-c-Kit form a bi-directional signaling complex that acts in the developing thymus to coordinate thymic VEC and early thymic progenitor (ETP) expansion by simultaneously promoting ETP survival and VEC proliferation. This mechanism may be relevant to both normal tissues and malignant tumors that depend on KitL-c-Kit signaling for their proliferation.


Assuntos
Membrana Celular/metabolismo , Células Endoteliais/citologia , Transdução de Sinais , Fator de Células-Tronco/metabolismo , Timócitos/citologia , Timo/citologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proliferação de Células , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Endoteliais/metabolismo , Camundongos , Células NIH 3T3 , Ligação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína S6 Ribossômica/metabolismo , Fator de Células-Tronco/química , Timócitos/metabolismo
16.
Proc Natl Acad Sci U S A ; 115(48): E11331-E11340, 2018 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-30413615

RESUMO

Thymocyte-expressed molecule involved in selection (Themis) has been shown to be important for T cell selection by setting the threshold for positive versus negative selection. Themis interacts with the protein tyrosine phosphatase (PTP) Src-homology domain containing phosphatase-1 (Shp1), a negative regulator of the T cell receptor (TCR) signaling cascade. However, how Themis regulates Shp1 is still not clear. Here, using a very sensitive phosphatase assay on ex vivo thymocytes, we have found that Themis enhances Shp1 phosphatase activity by increasing its phosphorylation. This positive regulation of Shp1 activity by Themis is found in thymocytes, but not in peripheral T cells. Shp1 activity is modulated by different affinity peptide MHC ligand binding in thymocytes. Themis is also associated with phosphatase activity, due to its constitutive interaction with Shp1. In the absence of Shp1 in thymocytes, Themis interacts with Shp2, which leads to almost normal thymic development in Shp1 conditional knockout (cKO) mice. Double deletion of both Themis and Shp1 leads to a thymic phenotype similar to that of Themis KO. These findings demonstrate unequivocally that Themis positively regulates Shp1 phosphatase activity in TCR-mediated signaling in developing thymocytes.


Assuntos
Diferenciação Celular , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas/metabolismo , Linfócitos T/enzimologia , Animais , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Knockout , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteínas/genética , Transdução de Sinais , Linfócitos T/citologia , Timócitos/citologia , Timócitos/enzimologia
17.
Immunol Lett ; 204: 9-15, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30308217

RESUMO

Hematopoietic precursors entering the thymus undergo a maturation process leading to the generation of a variety of T cell subsets that migrate to the periphery to perform their effector functions. This maturation process is strictly regulated by multiple interactions of developing T cells with thymic stroma cells. Signals received via the T cell receptor for antigen, co-stimulatory molecules and cytokines will determine, through thymic selection and lineage choice, thymocyte-fate. Recently, different populations of peripheral antigen presenting cells and T cells have been reported to enter the thymus. Here we review how these cells migrating from the periphery to the thymus modulate T cell development.


Assuntos
Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Diferenciação Celular/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Timo/citologia , Timo/imunologia , Animais , Células Apresentadoras de Antígenos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Movimento Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Tolerância Imunológica , Subpopulações de Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/imunologia , Timócitos/metabolismo , Timo/metabolismo
18.
J Immunol ; 201(10): 2947-2958, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30291166

RESUMO

Recently we reported that IL-4 and IL-13 signaling in murine early thymic progenitors (ETPs) expressing the heteroreceptor (HR) comprising IL-4 receptor α (IL-4Rα) and IL-13 receptor α 1 (IL-13Rα1) activate STAT6 and inhibit ETP maturation potential toward T cells. In this study, we asked whether IL-4 and IL-13 signaling through the HR mobilizes other STAT molecules to shape ETP fate decision. The findings indicate that HR+ ETPs undergoing cytokine signaling display increased STAT1, but not STAT3, phosphorylation in addition to STAT6 activation. In parallel, the ETPs had a STAT1-dependent heightened expression of IRF-8, a transcription factor essential for development of CD8α+ dendritic cells (DCs). Interestingly, STAT1 phosphorylation and IRF-8 upregulation, which are independent of STAT6 activation, guided ETP maturation toward myeloid cells with a CD8α+ DC phenotype. Furthermore, these CD8α+ DCs display a thymic resident phenotype, as they did not express SIRPα, a molecule presumed to be involved in cell migration. These findings suggest that IL-4 and IL-13 cytokine-induced HR signaling provides a double-edged sword that simultaneously blocks T cell lineage potential but advances myeloid maturation that could impact T cell selection and central tolerance.


Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/citologia , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Timócitos/citologia , Animais , Tolerância Central/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Fatores Reguladores de Interferon/imunologia , Fatores Reguladores de Interferon/metabolismo , Interleucina-13/imunologia , Interleucina-4/imunologia , Camundongos , Camundongos Knockout , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT6/imunologia , Fator de Transcrição STAT6/metabolismo , Timócitos/imunologia , Timócitos/metabolismo
19.
J Exp Med ; 215(11): 2887-2900, 2018 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-30287480

RESUMO

Natural killer T (NKT) cells expressing the invariant T cell receptor (iTCR) serve an essential function in clearance of certain pathogens and have been implicated in autoimmune and allergic diseases. Complex effector programs of these iNKT cells are wired in the thymus, and upon thymic egress, they can respond within hours of antigenic challenges, classifying iNKT cells as innate-like. It has been assumed that the successful rearrangement of the invariant iTCRα chain is the central event in the divergence of immature thymocytes to the NKT cell lineage, but molecular properties that render the iTCR signaling distinct to permit the T cell lineage diversification remain obscure. Here we show that the High Mobility Group (HMG) transcription factor (TF) SOX4 controls the production of iNKT cells by inducing MicroRNA-181 (Mir181) to enhance TCR signaling and Ca2+ fluxes in precursors. These results suggest the existence of tailored, permissive gene circuits in iNKT precursors for innate-like T cell development.


Assuntos
Sinalização do Cálcio/imunologia , MicroRNAs/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Fatores de Transcrição SOXC/imunologia , Timócitos/imunologia , Animais , Sinalização do Cálcio/genética , Rearranjo Gênico do Linfócito T/imunologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , Células T Matadoras Naturais/citologia , Receptores de Antígenos de Linfócitos T/genética , Fatores de Transcrição SOXC/genética , Timócitos/citologia
20.
Nat Commun ; 9(1): 3749, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30218105

RESUMO

While CD69 may regulate thymocyte egress by inhibiting S1P1 expression, CD69 expression is not thought to be required for normal thymocyte development. Here we show that CD69 is in fact specifically required for the differentiation of mature NKT2 cells, which do not themselves express CD69. Mechanistically, CD69 expression is required on CD24+ PLZFhi innate precursors for their retention in the thymus and completion of their differentiation into mature NKT2 cells. By contrast, CD69-deficient CD24+ PLZFhi innate precursors express S1P1 and prematurely exit the thymus, while S1P1 inhibitor treatment of CD69-deficient mice retains CD24+ PLZFhi innate precursors in the thymus and restores NKT2 cell differentiation. Thus, CD69 prevents S1P1 expression on CD24+ PLZFhi innate precursor cells from aborting NKT2 differentiation in the thymus. This study reveals the importance of CD69 to prolong the thymic residency time of developing immature precursors for proper differentiation of a T cell subset.


Assuntos
Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos T/genética , Lectinas Tipo C/genética , Linfopoese/genética , Células T Matadoras Naturais/citologia , Receptores de Lisoesfingolipídeo/genética , Subpopulações de Linfócitos T/citologia , Timócitos/citologia , Animais , Antígeno CD24/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Subpopulações de Linfócitos T/metabolismo , Timócitos/metabolismo
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