RESUMO
During normal T cell development in the thymus, αß TCRs signal immature thymocytes to differentiate into mature T cells by binding to peptide-MHC ligands together with CD4/CD8 coreceptors. Conversely, in MHC and CD4/CD8 coreceptor-deficient mice, the thymus generates mature T cells expressing MHC-independent TCRs that recognize native conformational epitopes rather than linear antigenic-peptides presented by MHC. To date, no structural information of MHC-independent TCRs is available, and their structural recognition of non-MHC ligand remains unknown. To our knowledge in this study, we determined the first structures of two murine MHC-independent TCRs (A11 and B12A) that bind with high nanomolar affinities to mouse adhesion receptor CD155. Solution binding demonstrated the Vαß-domain is responsible for MHC-independent B12A recognition of its ligand. Analysis of A11 and B12A sequences against various MHC-restricted and -independent TCR sequence repertoires showed that individual V-genes of A11 and B12A did not exhibit preference against MHC-restriction. Likewise, CDR3 alone did not discriminate against MHC binding, suggesting VDJ recombination together with Vα/Vß pairing determine their MHC-independent specificity for CD155. The structures of A11 and B12A TCR are nearly identical to those of MHC-restricted TCR, including the conformations of CDR1 and 2. Mutational analysis, together with negative-staining electron microscopy images, showed that the CDR regions of A11 and B12A recognized epitopes on D1 domain of CD155, a region also involved in CD155 binding to poliovirus and Tactile in human. Taken together, MHC-independent TCRs adopt canonical TCR structures to recognize native Ags, highlighting the importance of thymic selection in determining TCR ligand specificity.
Assuntos
Complexo Principal de Histocompatibilidade/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores Virais/metabolismo , Animais , Células HEK293 , Humanos , Ligantes , Camundongos , Peptídeos/metabolismo , Poliovirus/metabolismo , Ligação Proteica , Domínios Proteicos , Timócitos/metabolismo , Recombinação V(D)J/fisiologiaRESUMO
B cell development requires the coordinated action of transcription factors and cytokines, in particular interleukin-7 (IL-7). We report that mice lacking the POZ (Poxvirus and zinc finger) domain of the transcription factor Miz-1 (Zbtb17(ΔPOZ/ΔPOZ)) almost entirely lacked follicular B cells, as shown by the fact that their progenitors failed to activate the Jak-Stat5 pathway and to upregulate the antiapoptotic gene Bcl2 upon IL-7 stimulation. We show that Miz-1 exerted a dual role in the interleukin-7 receptor (IL-7R) pathway by directly repressing the Janus kinase (Jak) inhibitor suppressor of cytokine signaling 1 (Socs1) and by activating Bcl2 expression. Zbtb17(ΔPOZ/ΔPOZ) (Miz-1-deficient) B cell progenitors had low expression of early B cell genes as transcription factor 3 (Tcf3) and early B cell factor 1 (Ebf1) and showed a propensity for apoptosis. Only the combined re-expression of Bcl2 and Ebf1 could reconstitute the ability of Miz-1-deficient precursors to develop into CD19(+) B cells.
Assuntos
Linfócitos B/metabolismo , Medula Óssea/patologia , Proteínas Nucleares/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Receptores de Interleucina-7/metabolismo , Proteína de Morte Celular Associada a bcl/biossíntese , Animais , Linfócitos B/imunologia , Linfócitos B/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Sobrevivência Celular/genética , Células Cultivadas , Camundongos , Camundongos Mutantes , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/imunologia , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/imunologia , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Ubiquitina-Proteína Ligases , Proteína de Morte Celular Associada a bcl/genética , Proteína de Morte Celular Associada a bcl/imunologiaRESUMO
Growth factor independent 1 (Gfi1) is a transcriptional repressor originally identified as a gene activated in T-cell leukemias induced by Moloney-murine-leukemia virus infection. Notch1 is a transmembrane receptor that is frequently mutated in human T-cell acute lymphoblastic leukemia (T-ALL). Gfi1 is an important factor in the initiation and maintenance of lymphoid leukemias and its deficiency significantly impedes Notch dependent initiation of T-ALL in animal models. Here, we show that immature hematopoietic cells require Gfi1 to competently integrate Notch-activated signaling. Notch1 activation coupled with Gfi1 deficiency early in T-lineage specification leads to a dramatic loss of T-cells, whereas activation in later stages leaves development unaffected. In Gfi1 deficient multipotent precursors, Notch activation induces lethality and is cell autonomous. Further, without Gfi1, multipotent progenitors do not maintain Notch1-activated global expression profiles typical for T-lineage precursors. In agreement with this, we find that both lymphoid-primed multipotent progenitors (LMPP) and early T lineage progenitors (ETP) do not properly form or function in Gfi1(-/-) mice. These defects correlate with an inability of Gfi1(-/-) progenitors to activate lymphoid genes, including IL7R, Rag1, Flt3 and Notch1. Our data indicate that Gfi1 is required for hematopoietic precursors to withstand Notch1 activation and to maintain Notch1 dependent transcriptional programming to determine early T-lymphoid lineage identity.
Assuntos
Proteínas de Ligação a DNA/genética , Receptor Notch1/genética , Transdução de Sinais/genética , Linfócitos T/citologia , Fatores de Transcrição/genética , Animais , Linhagem da Célula , Regulação Leucêmica da Expressão Gênica/imunologia , Redes Reguladoras de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfócitos/citologia , Linfócitos/metabolismo , Camundongos , Linfócitos T/imunologiaRESUMO
The Myc interacting zinc finger protein 1 (Miz-1) is a BTB/POZ domain containing transcription factor that can function as an activator or repressor depending on its binding partners. In a complex with co-factors such as nuclophosmin or p300, Miz-1 stimulates transcription of genes that encode regulators of cell cycle progression such as p21(Cip1) or p15(Ink4b) or inhibitors of apoptosis such as Bcl-2. In contrast, Miz-1 becomes a transcriptional repressor when it binds to c-Myc or Bcl-6, which replace nucleophosmin or p300. During lymphocyte development, Miz-1 functions as a regulator of the IL-7 signaling pathway at very early steps in the bone marrow and thymus. When the IL-7 receptor (IL-7R) recognizes its cognate cytokine, a cascade of events is initiated that involves the recruitment of janus kinases (JAK) to the cytoplasmic part of the IL-7R, the phosphorylation of Stat5, its dimerization and relocation to the nucleus, enabling a transcriptional programming that governs commitment, survival and proliferation of lymphoid lineage cells. Miz-1 is critical in this signal transduction pathway, since it controls the expression of Socs1, an inhibitor of JAKs and thus of Stat5 activation and Bcl-2 expression. A lack of Miz-1 blocks IL-7 mediated signaling, which is detrimental for early B- and T-lymphoid development. These functions of Miz-1 during early lymphocyte development are c-Myc-independent. In contrast, when c-Myc is constitutively over-expressed, for instance during c-Myc induced lymphomagenesis, the interaction between Miz-1 and c-Myc becomes important and critical for the initiation and maintenance of c-Myc-dependent lymphoid malignancies.
Assuntos
Linfócitos B/imunologia , Fatores de Transcrição Kruppel-Like/metabolismo , Linfoma/patologia , Linfócitos T/imunologia , Linfócitos B/citologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Biológicos , Proteínas Proto-Oncogênicas c-bcl-6 , Transdução de Sinais , Linfócitos T/citologia , Fatores de Transcrição/metabolismoRESUMO
In T cells, two members of the Dok family, Dok-1 and Dok-2, are predominantly expressed. Recent evidence suggests that they play a negative role in T-cell signaling. In order to define whether Dok proteins regulate T-cell development, we have generated transgenic mice overexpressing Dok-1 in thymocytes and peripheral T cells. We show that overexpression of Dok-1 retards the transition from the CD4(-) CD8(-) to CD4(+) CD8(+) stage. Moreover, there is a specific expansion of PLZF-expressing Vγ1.1(+) Vδ6.3(+) T cells. This subset of γδ T cells acquires innate characteristics including rapid IL-4 production following stimulation and requiring SLAM-associated adaptor protein (SAP) for their development. Moreover, Dok-1 overexpression promotes the generation of an innate-like CD8(+) T-cell population that expresses Eomesodermin. Altogether, these findings identify a novel role for Dok-1 in the regulation of thymic differentiation and in particular, in the development of PLZF(+) γδ T cells.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Células T Matadoras Naturais/metabolismo , Fosfoproteínas/biossíntese , Fosfoproteínas/genética , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/genética , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Animais , Proteínas de Ligação a DNA/metabolismo , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoproteínas/metabolismo , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteínas de Ligação a RNA/metabolismo , Proteína Associada à Molécula de Sinalização da Ativação LinfocitáriaRESUMO
T cells originate from early T lineage precursors that have entered the thymus and differentiate through well-defined steps. Mice deficient for the BTB/POZ domain of zinc finger protein-1 (Miz-1) almost entirely lack early T lineage precursors and have a CD4(-)CD8(-) to CD4(+)CD8(+) block causing a strong reduction in thymic cellularity. Miz-1(ΔPOZ) pro-T cells cannot differentiate in vitro and are unable to relay signals from the interleukin-7R (IL-7R). Both STAT5 phosphorylation and Bcl-2 up-regulation are perturbed. The high expression levels of SOCS1 found in Miz-1(ΔPOZ) cells probably cause these alterations. Moreover, Miz-1 can bind to the SOCS1 promoter, suggesting that Miz-1 deficiency causes a deregulation of SOCS1. Transgenic overexpression of Bcl-2 or inhibition of SOCS1 restored pro-T cell numbers and their ability to differentiate, supporting the hypothesis that Miz-1 is required for the regulation of the IL-7/IL-7R/STAT5/Bcl-2 signaling pathway by monitoring the expression levels of SOCS1.
Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Células Progenitoras Linfoides/fisiologia , Receptores de Interleucina-7/fisiologia , Animais , Diferenciação Celular/imunologia , Linhagem da Célula/imunologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/química , Fatores de Transcrição Kruppel-Like/genética , Células Progenitoras Linfoides/imunologia , Células Progenitoras Linfoides/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estrutura Terciária de Proteína , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Dedos de Zinco/genéticaRESUMO
Miz-1 is a Broad-complex, Tramtrack and Bric-à-brac/pox virus zinc finger domain (BTB/POZ)-containing protein expressed in lymphoid precursors that can activate or repress transcription. We report in this article that mice expressing a nonfunctional Miz-1 protein lacking the BTB/POZ domain (Miz-1(ΔPOZ)) have a severe differentiation block at the pre-T cell "ß-selection" checkpoint, evident by a drastic reduction of CD4(-)CD8(-) double-negative-3 (DN3) and DN4 cell numbers. T cell-specific genes including Rag-1, Rag-2, CD3ε, pTα, and TCRß are expressed in Miz-1-deficient cells and V(D)J recombination is intact, but few DN3/DN4 cells express a surface pre-TCR. Miz-1-deficient DN3 cells are highly apoptotic and do not divide, which is consistent with enhanced expression of p53 target genes such as Cdkn1a, PUMA, and Noxa. However, neither coexpression of the antiapoptotic protein Bcl2 nor the deletion of p21(CIP1) nor the combination of both relieved Miz-1-deficient DN3/DN4 cells from their differentiation block. Only the coexpression of rearranged TCRαß and Bcl2 fully rescued Miz-1-deficient DN3/DN4 cell numbers and enabled them to differentiate into DN4TCRß(+) and double-positive cells. We propose that Miz-1 is a critical factor for the ß-selection checkpoint and is required for both the regulation of p53 target genes and proper expression of the pre-TCR to support the proliferative burst of DN3 cells during T cell development.
Assuntos
Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Proteínas Nucleares/imunologia , Proteínas Inibidoras de STAT Ativados/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Proteína Supressora de Tumor p53/biossíntese , Animais , Ciclo Celular , Separação Celular , Citometria de Fluxo , Expressão Gênica , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas Nucleares/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Inibidoras de STAT Ativados/genética , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/citologia , Linfócitos T/imunologia , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína LigasesRESUMO
Modification of histones is critically involved in regulating chromatin structure and gene expression. The zinc finger protein Gfi1 silences transcription by recruiting a complex of histone modifying enzymes such as LSD-1/CoRest and HDAC-1 to target gene promoters. Here we present evidence that Gfi1 forms a complex with the p150 subunit of the histone chaperone chromatin assembly factor-1 (Caf-1). Gfi1 and p150 interact at endogenous expression levels and co-localize in distinct sub-nuclear structures. We show that p150 enhances Gfi1-mediated transcriptional repression and that it occupies Gfi1 target gene promoters in transfected cells and primary murine T cells only in the presence of Gfi1. Finally, size exclusion chromatography shows a fraction of p150 to coelute with Gfi1, LSD-1 and HDAC-1 and thus provides evidence that p150 is part of the Gfi1 repression complex. Since p150 binds directly to histones H3 and H4, our findings suggest that p150 may link the DNA-bound Gfi1 repressor complex to histones enabling modifications required for transcriptional silencing.
Assuntos
Fator 1 de Modelagem da Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Dedos de Zinco , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Fator 1 de Modelagem da Cromatina/genética , Proteínas de Ligação a DNA/genética , Imunofluorescência , Células HL-60 , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Knockout , Células NIH 3T3 , Regiões Promotoras Genéticas/genética , Ligação Proteica , Interferência de RNA , Timo/citologia , Timo/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The lack of appropriate experimental models often limits our ability to investigate the establishment of infections in specific tissues. To reproduce the structural and spatial organization of vaginal mucosae to study human immunodeficiency virus type-1 (HIV-1) infection, we used the self-assembly technique to bioengineer tridimensional vaginal mucosae using human cells extracted from HIV-1-negative healthy pre- and postmenopausal donors. We produced a stroma, free of exogenous material, that can be adapted to generate near-to-native vaginal tissue with the best complexity obtained with seeded epithelial cells on the organ-specific stroma. The autologous engineered tissues had mechanical properties close to native mucosa and shared similar glycogen production, which declined in reconstructed tissues of the postmenopausal donor. The in vitro-engineered tissues were also rendered immune competent by adding human monocyte-derived macrophages (MDMs) on the epithelium or in the stroma layers. The model was infected with HIV-1, and viral replication and transcytosis were observed when immunocompetent reconstructed vaginal mucosa tissue has incorporated MDMs into the stroma and infected with free HIV-1 green fluorescent protein (GFP) viral particles. These data illustrate a natural permissiveness of immunocompetent untransformed human vaginal mucosae to HIV-1 infection. This model offers a physiological tool to explore viral load, HIV-1 transmission in an environment that may contribute to the virus propagation, and new antiviral treatments in vitro. Impact statement This study introduces an innovative immunocompetent three-dimensional human organ-specific vaginal mucosa free of exogenous material for in vitro modeling of human immunodeficiency virus type-1 (HIV-1) infection. The proposed model is histologically close to native tissue, especially by presenting glycogen accumulation in the epithelium's superficial cells, responsive to estrogen, and able to sustain a monocyte-derived macrophage population infected or not by HIV-1 during â¼2 months.
Assuntos
Infecções por HIV , HIV-1 , Feminino , Hormônios , Humanos , Mucosa , VaginaRESUMO
T(h) cells have long been divided into two subsets, T(h)1 and T(h)2; however, recently, T(h)17 and inducible regulatory T (iTreg) cells were identified as new T(h) cell subsets. Although T(h)1- and T(h)2-polarizing cytokines have been shown to suppress T(h)17 and iTreg development, transcriptional regulation of T(h)17 and iTreg differentiation by cytokines remains to be clarified. In this study, we found that expression of the growth factor independent 1 (Gfi1) gene, which has been implicated in T(h)2 development, was repressed in T(h)17 and iTreg cells compared with T(h)1 and T(h)2 lineages. Gfi1 expression was enhanced by the IFN-gamma/STAT1 and IL-4/STAT6 pathways, whereas it was repressed by the transforming growth factor-beta1 stimulation at the promoter level. Over-expression of Gfi1 strongly reduced IL-17A transcription in the EL4 T cell line, as well as in primary T cells. This was due to the blockade of recruitment of retinoid-related orphan receptor gammat to the IL-17A promoter. In contrast, IL-17A expression was significantly enhanced in Gfi1-deficient T cells under T(h)17-promoting differentiation conditions as compared with wild-type T cells. In contrast, the impacts of Gfi1 in iTregs were not as strong as in T(h)17 cells. Taken together, these data strongly suggest that Gfi1 is a negative regulator of T(h)17 differentiation, which represents a novel mechanism for the regulation of T(h)17 development by cytokines.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Interleucina-17/imunologia , Receptores do Ácido Retinoico/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Regulação para Baixo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-4/imunologia , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares , RNA Mensageiro/metabolismo , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT6/imunologia , Fator de Transcrição STAT6/metabolismo , Linfócitos T Reguladores/imunologia , Fatores de Transcrição/genética , Regulação para CimaRESUMO
Tissue engineering offers novel therapies for vaginal reconstruction in patients with congenital vaginal agenesis such as Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. This study aims to reconstruct a prevascularized tissue-engineered model of human vaginal mucosa (HVM) using the self-assembly approach, free of exogenous materials. In this study, a new cell culture method was used to enhance microcapillary network formation while maintaining sufficient biomechanical properties for surgical manipulation. Human vaginal fibroblasts were coseeded with human umbilical vein endothelial cells (HUVECs). Transduction of HUVEC with a vector that allows the expression of both green fluorescent protein (GFP) and luciferase allowed the monitoring of the formation of a microvascular network in vitro and the assessment of the viability and stability of HUVEC in vivo. Two reconstructed vaginal mucosa grafts, a prevascularized, and a nonvascularized control were implanted subcutaneously on the back of 12 female nude mice and monitored for up to 21 days. Prevascularized grafts demonstrated signs of earlier vascularization compared with controls. However, there were no differences in graft survival outcomes in both groups. The finding of mouse red blood cells within GFP-positive capillaries 1 week after implantation demonstrates the capacity of the reconstructed capillary-like network to connect to the host circulation and sustain blood perfusion in vivo. Furthermore, sites of inosculation between GFP-positive HUVEC and mouse endothelial cells were observed within prevascularized grafts. Our results demonstrate that the addition of endothelial cells using a hybrid approach of self-assembly and reseeding generates a mature capillary-like network that has the potential to become functional in vivo, offering an optimized prevascularized HVM model for further translational research. Impact statement This study introduces a prevascularized tissue-engineered model of human vaginal mucosa (HVM), which is adapted for surgical applications. The prevascularization of tissue-engineered grafts aims to enhance graft survival and is an interesting feature for sexual function. Various scaffold-free cell culture methods were tested to reconstruct a mature microcapillary network within HVM grafts while meeting biomechanical needs for surgery. Moreover, this animal study assesses the vascular functionality of prevascularized grafts in vivo, serving as a proof of concept for further translational applications. This research underlines the continuous efforts to optimize current models to closely mimic native tissues and further improve surgical outcomes.
Assuntos
Mucosa/irrigação sanguínea , Mucosa/citologia , Engenharia Tecidual/métodos , Vagina/irrigação sanguínea , Vagina/citologia , Animais , Técnicas de Cultura de Células , Feminino , Humanos , Camundongos , Camundongos Nus , Alicerces Teciduais/químicaRESUMO
MHC-independent αßTCRs (TCRs) recognize conformational epitopes on native self-proteins and arise in mice lacking both MHC and CD4/CD8 coreceptor proteins. Although naturally generated in the thymus, these TCRs resemble re-engineered therapeutic chimeric antigen receptor (CAR) T cells in their specificity for MHC-independent ligands. Here we identify naturally arising MHC-independent TCRs reactive to three native self-proteins (CD48, CD102, and CD155) involved in cell adhesion. We report that naturally arising MHC-independent TCRs require high affinity TCR-ligand engagements in the thymus to signal positive selection and that high affinity positive selection generates a peripheral TCR repertoire with limited diversity and increased self-reactivity. We conclude that the affinity of TCR-ligand engagements required to signal positive selection in the thymus inversely determines the diversity and self-tolerance of the mature TCR repertoire that is selected.
Assuntos
Seleção Clonal Mediada por Antígeno , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Tolerância a Antígenos Próprios/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/fisiologia , Animais , Antígenos CD/metabolismo , Antígenos CD8/imunologia , Moléculas de Adesão Celular/metabolismo , Ligantes , Antígeno-1 Associado à Função Linfocitária/metabolismo , Complexo Principal de Histocompatibilidade/genética , Camundongos , Camundongos Knockout , Ligação Proteica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores Virais/imunologiaAssuntos
Antígenos CD4/imunologia , Antígenos CD8/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Animais , Apresentação de Antígeno , Seleção Clonal Mediada por Antígeno/genética , Humanos , Complexo Principal de Histocompatibilidade/fisiologia , Camundongos , Timócitos/citologia , Timócitos/imunologiaRESUMO
The αß T cell receptor (TCR) repertoire on mature T cells is selected in the thymus, but the basis for thymic selection of MHC-restricted TCRs from a randomly generated pre-selection repertoire is not known. Here we perform comparative repertoire sequence analyses of pre-selection and post-selection TCR from multiple MHC-sufficient and MHC-deficient mouse strains, and find that MHC-restricted and MHC-independent TCRs are primarily distinguished by features in their non-germline CDR3 regions, with many pre-selection CDR3 sequences not compatible with MHC-binding. Thymic selection of MHC-independent TCR is largely unconstrained, but the selection of MHC-specific TCR is restricted by both CDR3 length and specific amino acid usage. MHC-restriction disfavors TCR with CDR3 longer than 13 amino acids, limits positively charged and hydrophobic amino acids in CDR3ß, and clonally deletes TCRs with cysteines in their CDR3 peptide-binding regions. Together, these MHC-imposed structural constraints form the basis to shape VDJ recombination sequences into MHC-restricted repertoires.
Assuntos
Regiões Determinantes de Complementaridade/química , Regiões Determinantes de Complementaridade/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Timo/imunologia , Sequência de Aminoácidos , Animais , Regiões Determinantes de Complementaridade/genética , Ativação Linfocitária , Complexo Principal de Histocompatibilidade/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/genética , Análise de Sequência de Proteína , Linfócitos T/imunologia , Linfócitos T/metabolismo , Recombinação V(D)JRESUMO
Twenty years ago, Dr. François A. Auger, the founder of the Laboratory of Experimental Organogenesis (LOEX), introduced the self-assembly technique. This innovative technique relies on the ability of dermal fibroblasts to produce and assemble their own extracellular matrix, differing from all other tissue-engineering techniques that use preformed synthetic scaffolds. Nevertheless, the use of the self-assembly technique was limited for a long time due to its main drawbacks: time and cost. Recent scientific breakthroughs have addressed these limitations. New protocol modifications that aim at increasing the rate of extracellular matrix formation have been proposed to reduce the production costs and laboratory handling time of engineered tissues. Moreover, the introduction of vascularization strategies in vitro permits the formation of capillary-like networks within reconstructed tissues. These optimization strategies enable the large-scale production of inexpensive native-like substitutes using the self-assembly technique. These substitutes can be used to reconstruct three-dimensional models free of exogenous materials for clinical and fundamental applications.
Assuntos
Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , História do Século XX , História do Século XXI , Humanos , Engenharia Tecidual/históriaRESUMO
Many diseases necessitate the substitution of vaginal tissues. Current replacement therapies are associated with many complications. In this study, we aimed to create bioengineered neovaginas with the self-assembly technique using autologous vaginal epithelial (VE) and vaginal stromal (VS) cells without the use of exogenous materials and to document the survival and incorporation of these grafts into the tissues of nude female mice. Epithelial and stromal cells were isolated from vaginal biopsies. Stromal cells were driven to form collagen sheets, 3 of which were superimposed to form vaginal stromas. VE cells were seeded on top of these stromas and allowed to mature in an air-liquid interface. The vaginal equivalents were implanted subcutaneously in female nude mice, which were sacrificed after 1 and 2 weeks after surgery. The in vitro and animal-retrieved equivalents were assessed using histologic, functional, and mechanical evaluations. Vaginal equivalents could be handled easily. VE cells formed a well-differentiated epithelial layer with a continuous basement membrane. The equivalent matrix was composed of collagen I and III and elastin. The epithelium, basement membrane, and stroma were comparable to those of native vaginal tissues. The implanted equivalents formed mature vaginal epithelium and matrix that were integrated into the mice tissues. Using the self-assembly technique, in vitro vaginal tissues were created with many functional and biological similarities to native vagina without any foreign material. They formed functional vaginal tissues after in vivo animal implantation. It is appropriate for vaginal substitution and disease modeling for infectious studies, vaginal applicants, and drug testing.
Assuntos
Imageamento Tridimensional , Engenharia Tecidual/métodos , Vagina/fisiologia , Adulto , Animais , Biomarcadores/metabolismo , Separação Celular , Feminino , Imunofluorescência , Sobrevivência de Enxerto , Humanos , Implantes Experimentais , Camundongos Nus , Fenótipo , Reprodutibilidade dos Testes , Transplante Autólogo , Vagina/ultraestrutura , Adulto JovemRESUMO
Cells obtained from a patient's biopsy have to be expanded after extraction to produce autologous tissues, but standard cell culture conditions often limit their growth or lifespan and could induce early and inadequate cell differentiation. Moreover, it has previously been reported that the air-liquid interface, that induces maturation of the urothelium, stimulated inadequate differentiation associated with aberrant keratin-14 expression. The aim of this study was to test the benefits of hypoxia during expansion of urothelial cells and maturation of the bladder epithelium in the context of tissue engineering. Bladder mucosa substitutes were reconstructed using the self-assembly method with urothelial cells (UCs) expanded in normoxia or hypoxia. Hypoxia improved UCs expansion until passage P7, whereas normoxic conditions limited the use of UCs to passage P4. Maturation of the urothelium was also compared in normoxic vs. hypoxic conditions. Using laminin V, p63, Ki-67, keratin-5 and -14, Claudin-4 and zonula occludens protein-1, we show a better organization of the basal UC layer in hypoxia despite a thinner intermediate layer. Finally, barrier function was assessed by permeation tests. Cell culture in hypoxia allowed the generation of bioengineered urological tissue closer to native bladder characteristics, which represents a promising avenue to circumvent the lack of adequate tissues for reconstructive surgery. Copyright © 2017 John Wiley & Sons, Ltd.
Assuntos
Antígenos de Diferenciação/metabolismo , Diferenciação Celular , Proliferação de Células , Células Epiteliais/metabolismo , Urotélio/metabolismo , Hipóxia Celular , Células Cultivadas , Células Epiteliais/citologia , Humanos , Urotélio/citologiaRESUMO
Myc-interacting zinc finger protein 1 (Miz-1) is a BTB/POZ domain transcription factor that regulates complex processes such as proliferation and apoptosis. Constitutively Miz-1-deficient animals arrest embryonic development at E14.5 due to severe anemia and fetal liver cells lacking Miz-1 show a high cell death rate and a significant reduction of mature Ter119(+)ckit(-) or Ter119(+)CD71(-/low) cells. Consistently, the numbers of BFU-Es and CFU-Es were severely reduced in colony forming assays. Mice with conditional Miz-1 alleles deleted around E14.5 were born at expected ratios, but had reduced numbers of erythrocytes, and showed an increase in reticulocytes and Macro-RBCs in the peripheral blood. When challenged with the hemolytic agent phenylhydrazine (PHZ), Miz-1 deficient mice responded with a severe anemia after 4 days of treatment, but showed a delay in the recovery from this anemia with regard to RBC counts, hematocrit and hemoglobin levels compared to controls. In addition, an accumulation of immature CD71(+)Ter119(+) cells occurred in the bone marrow and spleen of mice lacking a functional Miz-1. We conclude from our studies that Miz-1 is important for erythroid differentiation and development. Moreover, Miz-1 is necessary to maintain a peripheral red blood cell homeostasis in particular in response to hemolysis after oxidative stress.
RESUMO
The transcriptional repressor Gfi1 is a nuclear zinc-finger protein that is expressed in T cell precursors in the thymus, but is down-regulated in mature, resting T cells. Gfi1 expression rises transiently to levels seen in thymocytes upon antigenic activation. We show here that lack of Gfi1 causes delayed cell cycle entry and apoptosis after antigenic stimulation in both mature CD4+ and CD8+ T cells ex vivo. DNA micro-array analysis demonstrated that this correlated with an up-regulation of the death receptor CD95, the proapoptotic factors Bad and Apaf1 and the cell cycle inhibitor p21, and a down-regulation of Bcl-2 expression in Gfi1-/- T cells. Surprisingly, while Gfi1-deficient CD4+ T cells showed the same defective behavior in vivo, Gfi1-deficient CD8+ T cells showed no aberration in vivo and were fully able to mount an anti-viral immune response. This indicates that Gfi1 exerts different functions in CD4+ and CD8+ T cells very likely by maintaining different genetic programs in both cell types, and appears to be essential for the CD4 helper T cell immune response but dispensable for the function of cytotoxic CD8+ T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Animais , Antígenos/imunologia , Apoptose/genética , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Ciclo Celular/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Ativação Linfocitária/genética , Linfopenia/genética , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Receptores de Interleucina-7/biossíntese , Receptores de Interleucina-7/genética , Transdução de Sinais/fisiologia , Vírus Formadores de Foco no Baço/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genéticaRESUMO
The human-specific p35 isoform of the invariant chain (Ii) includes an R-X-R endoplasmic reticulum (ER) retention motif that is inactivated upon HLA-DR binding. Although the masking is assumed to involve the cytoplasmic tails of class II molecules, the mechanism underlying this function remains to be investigated. Moreover, in light of the polymorphic nature of the class II cytosolic tails, little is known about the capacity of various isotypes or alleles to overcome the retention signal of Iip35. To gain further insights into these issues, we first addressed the proposed role of the HLA-DR cytoplasmic tails. As shown by flow cytometry, the presence of Iip35 in transfected HeLa cells prevented surface expression of HLA-DR molecules lacking their cytoplasmic tails (DRalphaTM/betaTM). These truncated class II molecules and Iip35 accumulated in the ER, and co-localized with calnexin, as determined by confocal microscopy. Sensitivity of DRalphaTM/betaTM to endoglycosidase H treatment confirmed that these molecules do not reach the trans-Golgi network when associated with Iip35. Further characterization revealed that the beta chain cytosolic tail is critical for efficient ER egress of class II/Iip35 complexes. Interestingly, our results clearly demonstrate for the first time that DP and DQ isotypes can also overcome the retention motif of Iip35 through a mechanism involving their very distinctive polymorphic beta chain cytoplasmic tails. Altogether, these results further dissect the masking of di-basic retention signals, and emphasize the interplay between class II molecules and Ii for the transport of the complex to the endocytic pathway.