RESUMO
T helper 17 (Th17) cells are pathogenic in many inflammatory diseases, but also support the integrity of the intestinal barrier in a non-inflammatory manner. It is unclear what distinguishes inflammatory Th17 cells elicited by pathogens and tissue-resident homeostatic Th17 cells elicited by commensals. Here, we compared the characteristics of Th17 cells differentiating in response to commensal bacteria (SFB) to those differentiating in response to a pathogen (Citrobacter rodentium). Homeostatic Th17 cells exhibited little plasticity towards expression of inflammatory cytokines, were characterized by a metabolism typical of quiescent or memory T cells, and did not participate in inflammatory processes. In contrast, infection-induced Th17 cells showed extensive plasticity towards pro-inflammatory cytokines, disseminated widely into the periphery, and engaged aerobic glycolysis in addition to oxidative phosphorylation typical for inflammatory effector cells. These findings will help ensure that future therapies directed against inflammatory Th17 cells do not inadvertently damage the resident gut population.
Assuntos
Citrobacter rodentium/imunologia , Infecções por Enterobacteriaceae/imunologia , Microbioma Gastrointestinal/imunologia , Intestinos/imunologia , Células Th17/imunologia , Animais , Plasticidade Celular , Células Cultivadas , Citocinas/metabolismo , Glicólise , Homeostase , Memória Imunológica , Inflamação , Camundongos , Camundongos TransgênicosRESUMO
Interleukin 9 (IL-9) is a cytokine linked to lung inflammation, but its cellular origin and function remain unclear. Here we describe a reporter mouse strain designed to map the fate of cells that have activated IL-9. We found that during papain-induced lung inflammation, IL-9 production was largely restricted to innate lymphoid cells (ILCs). IL-9 production by ILCs depended on IL-2 from adaptive immune cells and was rapidly lost in favor of other cytokines, such as IL-13 and IL-5. Blockade of IL-9 production via neutralizing antibodies resulted in much lower expression of IL-13 and IL-5, which suggested that ILCs provide the missing link between the well-established functions of IL-9 in the regulation of type 2 helper T cell cytokines and responses.
Assuntos
Citocinas/metabolismo , Interleucina-9/metabolismo , Linfócitos/metabolismo , Pneumonia/imunologia , Células Th2/imunologia , Animais , Anticorpos Bloqueadores/administração & dosagem , Células Cultivadas , Citocinas/imunologia , Genes Reporter/genética , Imunidade Inata , Interleucina-9/genética , Interleucina-9/imunologia , Pulmão , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/imunologia , Linfócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Papaína/administração & dosagem , Comunicação Parácrina , Pneumonia/induzido quimicamente , Células Th2/efeitos dos fármacosRESUMO
Here we describe a reporter mouse strain designed to map the fate of cells that have activated interleukin 17A (IL-17A). We found that IL-17-producing helper T cells (T(H)17 cells) had distinct plasticity in different inflammatory settings. Chronic inflammatory conditions in experimental autoimmune encephalomyelitis (EAE) caused a switch to alternative cytokines in T(H)17 cells, whereas acute cutaneous infection with Candida albicans did not result in the deviation of T(H)17 cells to the production of alternative cytokines, although IL-17A production was shut off in the course of the infection. During the development of EAE, interferon-γ (IFN-γ) and other proinflammatory cytokines in the spinal cord were produced almost exclusively by cells that had produced IL-17 before their conversion by IL-23 ('ex-T(H)17 cells'). Thus, this model allows the actual functional fate of effector T cells to be related to T(H)17 developmental origin regardless of IL-17 expression.
Assuntos
Inflamação , Interleucina-17/imunologia , Linfócitos T/imunologia , Animais , Encefalomielite Autoimune Experimental/imunologia , Citometria de Fluxo , Genes Reporter , Interferon gama/imunologia , Interleucina-17/genética , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
The aryl hydrocarbon receptor (AhR), a transcription factor known for mediating xenobiotic toxicity, is expressed in B cells, which are known targets for environmental pollutants. However, it is unclear what the physiological functions of AhR in B cells are. We show here that expression of Ahr in B cells is up-regulated upon B-cell receptor (BCR) engagement and IL-4 treatment. Addition of a natural ligand of AhR, FICZ, induces AhR translocation to the nucleus and transcription of the AhR target gene Cyp1a1, showing that the AhR pathway is functional in B cells. AhR-deficient (Ahr-/-) B cells proliferate less than AhR-sufficient (Ahr+/+) cells following in vitro BCR stimulation and in vivo adoptive transfer models confirmed that Ahr-/- B cells are outcompeted by Ahr+/+ cells. Transcriptome comparison of AhR-deficient and AhR-sufficient B cells identified cyclin O (Ccno), a direct target of AhR, as a top candidate affected by AhR deficiency.
Assuntos
Linfócitos B/fisiologia , Proliferação de Células , Receptores de Hidrocarboneto Arílico/metabolismo , Ciclinas/metabolismo , Citocromo P-450 CYP1A1/biossíntese , Perfilação da Expressão Gênica , Interleucina-4/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Hidrocarboneto Arílico/deficiência , Transcrição GênicaRESUMO
Chronic viral infections of the hematopoietic system are associated with bone marrow dysfunction, to which both virus-mediated and immune-mediated effects may contribute. Using unresolving noncytopathic Friend virus (FV) infection in mice, we showed that unregulated CD4(+) T cell response to FV caused IFN-gamma-mediated bone marrow pathology and anemia. Importantly, bone marrow pathology was triggered by relative insufficiency in regulatory T (Treg) cells and was prevented by added Treg cells, which suppressed the local IFN-gamma production by FV-specific CD4(+) T cells. We further showed that the T cell receptor (TCR) repertoire of transgenic Treg cells expressing the beta chain of an FV-specific TCR was virtually devoid of FV-specific clones. Moreover, anemia induction by virus-specific CD4(+) T cells was efficiently suppressed by virus-nonspecific Treg cells. Thus, sufficient numbers of polyclonal Treg cells may provide substantial protection against bone marrow pathology in chronic viral infections.
Assuntos
Anemia/imunologia , Medula Óssea/imunologia , Medula Óssea/fisiopatologia , Linfócitos T CD4-Positivos/imunologia , Vírus da Leucemia Murina de Friend/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Infecções por Retroviridae/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Anemia/metabolismo , Anemia/virologia , Animais , Medula Óssea/patologia , Linfócitos T CD4-Positivos/metabolismo , Doença Crônica , Vírus da Leucemia Murina de Friend/patogenicidade , Técnicas de Silenciamento de Genes , Interferon gama/biossíntese , Interferon gama/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/metabolismo , Infecções por Retroviridae/patologia , Infecções por Retroviridae/fisiopatologia , Infecções por Retroviridae/virologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
IL-22 is a cytokine that regulates tissue homeostasis at barrier surfaces. A variety of IL-22-producing cell types is known, but identification on the single-cell level remains difficult. Therefore, we generated a fate reporter mouse that would allow the identification of IL-22-producing cells and their fate mapping in vivo. To trace IL-22-expressing cells, a sequence encoding Cre recombinase was cloned into the Il22 locus, and IL22(Cre) mice were crossed with reporter mice expressing enhanced yellow fluorescence protein (eYFP) under control of the endogenous Rosa26 promoter. In IL22(Cre)R26R(eYFP) mice, the fluorescent reporter permanently labels cells that have switched on Il22 expression, irrespective of cytokine production. Despite a degree of underreporting, eYFP expression was detectable in nonimmune mice and restricted to group 3 innate lymphoid cells (ILC3) in the gut and γδ T cells in skin or lung. Upon skin challenge with imiquimod, eYFP(+) γδ and CD4 T cells expanded in the skin. Infection with Citrobacter rodentium initially was controlled by ILC3, followed by expansion of eYFP(+) CD4 T cells, which were induced in innate lymphoid follicles in the colon. No eYFP expression was detected in small intestinal Th17 cells, and they did not expand in the immune response. Colonic eYFP(+) CD4 T cells exhibited plasticity during infection with expression of additional cytokines, in contrast to ILC3, which remained largely stable. Single-cell quantitative PCR analysis of eYFP(+) CD4 T cells confirmed their heterogeneity, suggesting that IL-22 expression is not confined to particular subsets or a dedicated Th22 subset.
Assuntos
Homeostase , Infecções/metabolismo , Interleucinas/biossíntese , Animais , Citrobacter rodentium/imunologia , Análise por Conglomerados , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Infecções por Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Ordem dos Genes , Marcação de Genes , Genes Reporter , Loci Gênicos , Homozigoto , Infecções/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Interleucinas/genética , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Interleucina 22RESUMO
The human CD2 (hCD2) locus control region (LCR) inserted in the mouse CD8 gene complex activates expression of the CD8 genes in T cell subsets in which the CD8 locus is normally silenced (e.g., CD4(+) single-positive T cells). In this article, we show that, in conditional mCD8/hCD2-LCR (CD8/LCR) knock-in mice, the continuous presence of the hCD2-LCR is required for this effect. Deletion of the inserted hCD2-LCR in a developmental stage and cell lineage-specific manner revealed that the temporary presence of the LCR during early development does not permanently alter the expression pattern of the CD8 genes. As a result, cells that have been affected by the insertion of the LCR can convert to their destined phenotype once the LCR is removed. DNaseI hypersensitive sites 1 and 2 of the hCD2-LCR influence the expression of the CD8 genes in a similar manner as does the full LCR, whereas insertion of hypersensitive site 3 alone of the LCR does not result in a changed expression pattern. This analysis revealed a dynamic interaction between the hCD2-LCR and the endogenous regulatory elements of the CD8 genes.
Assuntos
Antígenos CD2/genética , Antígenos CD8/genética , Regulação da Expressão Gênica/imunologia , Região de Controle de Locus Gênico/genética , Linfopoese/genética , Linfócitos T/citologia , Animais , Southern Blotting , Antígenos CD2/imunologia , Antígenos CD8/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Separação Celular , Citometria de Fluxo , Expressão Gênica , Técnicas de Introdução de Genes , Humanos , Região de Controle de Locus Gênico/imunologia , Camundongos , Linfócitos T/imunologiaRESUMO
To study the influence of a locus control region (LCR) on the expression of a highly characterized, developmentally regulated locus, we have targeted the hCD2-LCR as a single copy into the endogenous mouse CD8 gene complex. Two knock-in mouse lines that differ in the integration site of the hCD2-LCR within the mCD8 gene complex were generated, and the influence on expression of the CD8 coreceptor was assessed. In these mice the normal developmental silencing of the CD8 genes in the CD4 lineage is deregulated, and the mice develop CD4(+) cells that also express the CD8 genes. This is accompanied by the physical maintenance of the CD8 genes within an extended loop away from their subchromosomal territory. Further analysis of these mice revealed unexpected fluid chromatin dynamics, whereby the LCR can be initially dominant over the endogenous CD8 gene-repressive regulatory processes present in CD4(+) cells but is continuously contested by them, resulting in the eventual inactivation of the inserted LCR, probably as a result of multiple rounds of replication.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígenos CD8/genética , Linhagem da Célula/genética , Replicação do DNA , Região de Controle de Locus Gênico , Animais , Antígenos CD2/genética , Linfócitos T CD4-Positivos/citologia , Linhagem Celular , Núcleo Celular/genética , Técnicas de Introdução de Genes , Linfopenia/imunologia , CamundongosRESUMO
Cryptosporidium is a leading cause of diarrheal-related deaths in children, especially in resource-poor settings. It also targets the immunocompromised, chronically infecting people living with HIV and primary immunodeficiencies. There is no vaccine or effective treatment. Although it is known from human cases and animal models that CD4+ T cells play a role in curbing Cryptosporidium, the role of CD8+ T cells remains to be defined. Using a Cryptosporidium tyzzeri mouse model, we show that gut-resident CD8+ intraepithelial lymphocytes (IELs) confer resistance to parasite growth. CD8+ IELs express and depend on the ligand-dependent transcription factor aryl hydrocarbon receptor (AHR). AHR deficiency reduces CD8+ IELs, decreases their cytotoxicity, and worsens infection. Transfer of CD8+ IELs rescues severely immunodeficient mice from death following Cryptosporidium challenge. Finally, dietary supplementation of the AHR pro-ligand indole-3-carbinol in newborn mice promotes resistance to infection. Therefore, common dietary metabolites augment the host immune response to cryptosporidiosis, protecting against disease.
Assuntos
Criptosporidiose , Cryptosporidium , Criança , Humanos , Camundongos , Animais , Criptosporidiose/parasitologia , Linfócitos T CD8-Positivos , Ligantes , DietaRESUMO
The aryl hydrocarbon receptor (AHR) is an environmental sensor that integrates microbial and dietary cues to influence physiological processes within the intestinal microenvironment, protecting against colitis and colitis-associated colorectal cancer development. Rapid tissue regeneration upon injury is important for the reinstatement of barrier integrity and its dysregulation promotes malignant transformation. Here we show that AHR is important for the termination of the regenerative response and the reacquisition of mature epithelial cell identity post injury in vivo and in organoid cultures in vitro. Using an integrative multi-omics approach in colon organoids, we show that AHR is required for timely termination of the regenerative response through direct regulation of transcription factors involved in epithelial cell differentiation as well as restriction of chromatin accessibility to regeneration-associated Yap/Tead transcriptional targets. Safeguarding a regulated regenerative response places AHR at a pivotal position in the delicate balance between controlled regeneration and malignant transformation.
Assuntos
Mucosa Intestinal , Receptores de Hidrocarboneto Arílico , Colo/patologia , Mucosa Intestinal/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Transdução de Sinais , Células-Tronco/metabolismoRESUMO
The CD8alphabeta heterodimer is integral to the selection of the class I-restricted lineage in the thymus; however, the contribution of the CD8beta chain to coreceptor function is poorly understood. To understand whether the CD8beta membrane proximal stalk region played a role in coreceptor function, we substituted it with the corresponding sequence from the CD8alpha polypeptide and expressed the hybrid molecule in transgenic mice in place of endogenous CD8beta. Although the stalk-swapped CD8beta was expressed on the cell surface as a disulfide-bonded heterodimer at equivalent levels of expression to an endogenous CD8beta molecule, it failed to restore selection of CD8(+) class I MHC-restricted T cells and it altered the response of peripheral T cells. Thus, the stalk region of the CD8beta polypeptide has an essential role in ensuring functionality of the CD8alphabeta heterodimer and its replacement compromises the interaction of CD8 with peptide-MHC complexes.
Assuntos
Antígenos CD8/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Sequência de Aminoácidos , Animais , Antígenos CD8/biossíntese , Antígenos CD8/genética , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Dimerização , Deleção de Genes , Fusão Gênica/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Transgenes/imunologiaRESUMO
The AHR is an environmental sensor and transcription factor activated by a variety of man-made and natural ligands, which has recently emerged as a critical regulator of homeostasis at barrier organs such as the skin. Activation of the AHR pathway downmodulates skin inflammatory responses in animal models and psoriasis clinical samples. In this study, we identify CYP1A1 enzymatic activity as a critical regulator of beneficial AHR signaling in the context of skin inflammation. Mice constitutively expressing Cyp1a1 displayed increased CYP1A1 enzymatic activity in the skin, which resulted in exacerbated immune cell activation and skin pathology, mirroring that observed in Ahr-deficient mice. Inhibition of CYP1A1 enzymatic activity ameliorated the skin immunopathology by restoring beneficial AHR signaling. Importantly, patients with psoriasis displayed reduced activation of the AHR pathway and increased CYP1A1 enzymatic activity compared with healthy donors, suggesting that dysregulation of the AHR/CYP1A1 axis may play a role in inflammatory skin disease. Thus, modulation of CYP1A1 activity may represent a promising alternative strategy to harness the anti-inflammatory effect exerted by activation of the AHR pathway in the skin.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Psoríase/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Estudos de Casos e Controles , Citocromo P-450 CYP1A1/antagonistas & inibidores , Citocromo P-450 CYP1A1/genética , Modelos Animais de Doenças , Feminino , Voluntários Saudáveis , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Psoríase/genética , Psoríase/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Pele/imunologia , Pele/patologia , Adulto JovemRESUMO
L-selectin mediates rolling of lymphocytes in high endothelial venules (HEVs) of peripheral lymph nodes (PLNs). Cross-linking of L-selectin causes proteolytic shedding of its ectodomain, the physiological significance of which is unknown. To determine whether L-selectin shedding regulates lymphocyte migration, a mutant form that resists shedding (LdDeltaP-selectin) was engineered. Transgenic mice expressing either LDeltaP or wild-type (WT) L-selectin on T cells were crossed with L-selectin knockout (KO) mice. The cellularity and subset composition of secondary lymphoid organs did not differ between LDeltaP and WT mice, however, they were different from C57BL/6. Plasma levels of soluble L-selectin in LDeltaP mice were reduced to <5% of WT and C57BL/6 mice. The rolling properties of T lymphocytes from LDeltaP and WT mice on immobilized L-selectin ligands were similar. Furthermore, similar numbers of LDeltaP and WT T lymphocytes were recruited from the bloodstream into PLNs in mice, although LDeltaP T cells transmigrated HEVs more slowly. WT, but not LDeltaP-selectin, underwent rapid, metalloproteinase-dependent shedding after TCR engagement, and LDeltaP T cells retained the capacity to enter PLNs from the bloodstream. These results suggest that the ability to shed L-selectin is not required for T cell recirculation and homing to PLNs. However, L-selectin shedding from antigen-activated T cells prevents reentry into PLNs.
Assuntos
Antígenos/imunologia , Movimento Celular/imunologia , Selectina L/imunologia , Linfócitos T/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Selectina L/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Sequences proximal to transgene integration sites are able to deregulate transgene expression resulting in complex position effect phenotypes. In addition, transgenes integrated as repeated arrays are susceptible to repeat-induced gene silencing. Using a Cre recombinase-based system we have addressed the influence of transgene copy number (CN) on expression of hCD2 transgenes. CN reduction resulted in a decrease, increase or no effect on variegation depending upon the site of integration. This finding argues that repeat-induced gene silencing is not the principle cause of hCD2 transgene variegation. These results also suggest that having more transgene copies can be beneficial at some integration sites. The transgenic lines examined in this report also exhibited a form of imprinting, which was manifested by decreased levels of expression and increased levels of variegation, upon maternal transmission; and this correlated with DNA hypermethylation and a reduction in epigenetic chromatin modifications normally associated with active genes.
Assuntos
Antígenos CD2/genética , Inativação Gênica , Impressão Genômica , Transgenes , Animais , Antígenos CD2/metabolismo , Imunoprecipitação da Cromatina , Metilação de DNA , Citometria de Fluxo , Dosagem de Genes , Humanos , Região de Controle de Locus Gênico , Camundongos , Camundongos Transgênicos , Linfócitos T/imunologiaRESUMO
The transcriptional program of early embryonic development is tightly regulated by a set of well-defined transcription factors that suppress premature expression of differentiation genes and sustain the pluripotent identity. It is generally accepted that this program can be perturbed by environmental factors such as chemical pollutants; however, the precise molecular mechanisms remain unknown. The aryl hydrocarbon receptor (AHR) is a widely expressed nuclear receptor that senses environmental stimuli and modulates target gene expression. Here, we have investigated the AHR interactome in embryonic stem cells by mass spectrometry and show that ectopic activation of AHR during early differentiation disrupts the differentiation program via the chromatin remodeling complex NuRD (nucleosome remodeling and deacetylation). The activated AHR/NuRD complex altered the expression of differentiation-specific genes that control the first two developmental decisions without affecting the pluripotency program. These findings identify a mechanism that allows environmental stimuli to disrupt embryonic development through AHR signaling.
Assuntos
Diferenciação Celular , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Mapas de Interação de Proteínas , Receptores de Hidrocarboneto Arílico/química , Receptores de Hidrocarboneto Arílico/genéticaRESUMO
To investigate the temporal regulation of the commitment of immature thymocytes to either the CD4(+) or the CD8(+) lineage in the thymus, we developed a transgenic mouse that expressed a tetracycline-inducible gene encoding the tyrosine kinase zeta chain-associated protein kinase of 70 kD (Zap70), which restored development in Zap70(-/-) thymocytes arrested at the preselection, CD4(+)CD8(+) double-positive (DP) stage. After induction of the expression of Zap70 and the production of Zap70 protein, CD4(+) single-positive (SP) cells that expressed Zbtb7b (which encodes the CD4(+) T cell-associated transcription factor ThPOK) became abundant within 30 hours, whereas CD8(+) SP cells were not detectable until day 4. We found that mature CD4(+) and CD8(+) cells arose from phenotypically distinct subsets of DP thymocytes that developed with different kinetics and contrasting sensitivities to stimulation of the T cell antigen receptor (TCR). In wild-type mice, expression of endogenous Zap70 progressively increased during maturation of the DP subsets, and the abundance of Zap70 protein determined the sensitivity of the cells to stimulation of the TCR. This temporal gradient in the amount of Zap70 protein enabled the selection of CD4(+) and CD8(+) repertoires in separate temporal windows and at different TCR signaling thresholds, thereby facilitating discrimination of distinct positive selection signals in these lineages.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proteína-Tirosina Quinase ZAP-70/genética , Proteína-Tirosina Quinase ZAP-70/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Antígenos CD5/metabolismo , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Cinética , Camundongos , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de SinaisRESUMO
The role for IL-7R expression in the differentiation of effector T cells into resting memory remains controversial. Here, using a conditional IL-7R transgenic model, we were able to test directly whether CD8 effector T cells require IL-7R expression for their differentiation into resting memory cells. In the absence of IL-7R expression, effector cells transferred into "full" hosts underwent a protracted and unremitting contraction compared with IL-7R-expressing control cells and were unable to develop into long-term resting memory cells. Surprisingly, when the same effector cells were transferred into empty T-cell-deficient hosts, they could generate long-lived fully functional resting memory cells independently of IL-7R expression. Formation of these latter cells was found to be dependent on IL-15, because the same IL-7R-deficient effector cells were rapidly lost from IL-15-deficient hosts, having a half-life of less than 40 hours. Therefore, our data suggest that, under physiological conditions, both IL-7 and IL-15 synergize to promote the formation of memory cells directly by limiting the contraction of effectors that occurs following an immune response and that reexpression of IL-7R is a key checkpoint in the regulation of this process.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Receptores de Interleucina-7/metabolismo , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/citologia , Interleucina-15/deficiência , Interleucina-15/genética , Interleucina-15/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Receptores de Interleucina-7/deficiência , Receptores de Interleucina-7/genéticaRESUMO
It has been shown previously that a human CD2 (hCD2) disabled locus control region (LCR) transgene is unable to establish an open chromatin configuration in all the T cells, and this leads to position effect variegation of the transgene. In this study we show that thymus-specific overexpression of human high mobility group box transcription factor 1 (HBP1), a transcription factor that binds a specific sequence within the hCD2 LCR, affects thymus cellularity as well as the number of CD8(+) thymocytes in two independent transgenic mouse lines and increases the proportion of T cells that fully activate the transgenic locus in hCD2 variegating mice in a sequence-specific dependent manner. This finding suggests that overexpression of HBP1 can affect lineage commitment and can relieve the suppressive influence of heterochromatin, allowing thymocytes to express the variegating target locus more efficiently. These effects could be the result of direct HBP1 action on LCR activity. Alternatively, the extra HBP1 molecules may sequester repressive elements away from the LCR, thus allowing transcription permissive states to form on the transgene locus.
Assuntos
Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/fisiologia , Timo/citologia , Timo/imunologia , Transgenes , Animais , Antígenos CD2/biossíntese , Antígenos CD2/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Citometria de Fluxo , Inibidores do Crescimento/genética , Inibidores do Crescimento/fisiologia , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/imunologia , Timo/enzimologiaRESUMO
Ikaros family members are important regulatory factors in lymphocyte development. Here we show that Ikaros may play an important role in CD4 versus CD8 lineage commitment decisions by demonstrating: (1) that it binds to regulatory elements in the endogenous CD8alpha locus in vivo using thymocyte chromatin immunoprecipitations, (2) that Ikaros suppresses position effect variegation of transgenes driven by CD8 regulatory elements, and (3) that mice with reduced levels of Ikaros and Aiolos show an apparent increase in CD4 populations with immature phenotype, i.e., cells that failed to activate the CD8alpha gene locus. We propose that Ikaros family members function as activators of the CD8alpha gene locus and that their associated activities are critical for appropriate chromatin remodeling transitions during thymocyte differentiation and lineage commitment.
Assuntos
Antígenos CD8/genética , Proteínas de Ligação a DNA , Linfócitos/imunologia , Família Multigênica , Linfócitos T/imunologia , Fatores de Transcrição/genética , Animais , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Mapeamento Cromossômico , Regulação da Expressão Gênica/imunologia , Humanos , Fator de Transcrição Ikaros , Linfonodos/imunologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Fenótipo , Reação em Cadeia da Polimerase , Sequências Reguladoras de Ácido Nucleico , Timo/imunologia , Fatores de Transcrição/deficiência , Dedos de ZincoRESUMO
To generate an experimental model for exploring the function, expression pattern, and developmental regulation of human Ig-like activating and inhibitory receptors, we have generated transgenic mice using two human genomic clones: 52N12 (a 150-Kb clone encompassing the leukocyte Ig-like receptor (LILR)B1 (ILT2), LILRB4 (ILT3), and LILRA1 (LIR6) genes) and 1060P11 (a 160-Kb clone that contains ten killer cell Ig-like receptor (KIR) genes). Both the KIR and LILR families are encoded within the leukocyte receptor complex, and are involved in immune modulation. We have also produced a novel mAb to LILRA1 to facilitate expression studies. The LILR transgenes were expressed in a similar, but not identical, pattern to that observed in humans: LILRB1 was expressed in B cells, most NK cells, and a small number of T cells; LILRB4 was expressed in a B cell subset; and LILRA1 was found on a ring of cells surrounding B cell areas on spleen sections, consistent with other data showing monocyte/macrophage expression. KIR transgenic mice showed KIR2DL2 expression on a subset of NK cells and T cells, similar to the pattern seen in humans, and expression of KIR2DL4, KIR3DS1, and KIR2DL5 by splenic NK cells. These observations indicate that linked regulatory elements within the genomic clones are sufficient to allow appropriate expression of KIRs in mice, and illustrate that the presence of the natural ligands for these receptors, in the form of human MHC class I proteins, is not necessary for the expression of the KIRs observed in these mice.