RESUMO
The magnitude and quality of the germinal center (GC) response decline with age, resulting in poor vaccine-induced immunity in older individuals. A functional GC requires the co-ordination of multiple cell types across time and space, in particular across its two functionally distinct compartments: the light and dark zones. In aged mice, there is CXCR4-mediated mislocalization of T follicular helper (TFH) cells to the dark zone and a compressed network of follicular dendritic cells (FDCs) in the light zone. Here we show that TFH cell localization is critical for the quality of the antibody response and for the expansion of the FDC network upon immunization. The smaller GC and compressed FDC network in aged mice were corrected by provision of TFH cells that colocalize with FDCs using CXCR5. This demonstrates that the age-dependent defects in the GC response are reversible and shows that TFH cells support stromal cell responses to vaccines.
Assuntos
Linfócitos T Auxiliares-Indutores , Vacinas , Animais , Camundongos , Linfócitos B , Células T Auxiliares Foliculares , Centro Germinativo , EnvelhecimentoRESUMO
Ageing profoundly changes our immune system and is thought to be a driving factor in the morbidity and mortality associated with infectious disease in older people. We have previously shown that the impaired immunity to vaccination that occurs in aged individuals is partly attributed to the effect of age on T follicular helper (Tfh) cell formation. In this study, we examined how age intrinsically affects Tfh cell formation in both mice and humans. We show increased formation of Tfh precursors (pre-Tfh) but no associated increase in germinal centre (GC)-Tfh cells in aged mice, suggesting age-driven promotion of only early Tfh cell differentiation. Mechanistically, we show that ageing alters TCR signalling which drives expression of the Notch-associated transcription factor, RBPJ. Genetic or chemical modulation of RBPJ or Notch rescues this age-associated early Tfh cell differentiation, and increased intrinsic Notch activity recapitulates this phenomenon in younger mice. Our data offer mechanistic insight into the age-induced changes in T-cell activation that affects the differentiation and ultimately the function of effector T cells.
Assuntos
Diferenciação Celular/imunologia , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Receptores CXCR5/metabolismo , Células T Auxiliares Foliculares/imunologia , Envelhecimento , Animais , Feminino , Humanos , CamundongosRESUMO
The GTPases of the immunity-associated proteins (GIMAP) GTPases are a family of proteins expressed strongly in the adaptive immune system. We have previously reported that in human cells one member of this family, GIMAP6, interacts with the ATG8 family member GABARAPL2, and is recruited to autophagosomes upon starvation, suggesting a role for GIMAP6 in the autophagic process. To study this possibility and the function of GIMAP6 in the immune system, we have established a mouse line in which the Gimap6 gene can be inactivated by Cre-mediated recombination. In mice bred to carry the CD2Cre transgene such that the Gimap6 gene was deleted within the T and B cell lineages there was a 50-70% reduction in peripheral CD4+ and CD8+ T cells. Analysis of splenocyte-derived proteins from these mice indicated increased levels of MAP1LC3B, particularly the lipidated LC3-II form, and S405-phosphorylation of SQSTM1. Electron microscopic measurements of Gimap6-/- CD4+ T cells indicated an increased mitochondrial/cytoplasmic volume ratio and increased numbers of autophagosomes. These results are consistent with autophagic disruption in the cells. However, Gimap6-/- T cells were largely normal in character, could be effectively activated in vitro and supported T cell-dependent antibody production. Treatment in vitro of CD4+ splenocytes from GIMAP6fl/flERT2Cre mice with 4-hydroxytamoxifen resulted in the disappearance of GIMAP6 within five days. In parallel, increased phosphorylation of SQSTM1 and TBK1 was observed. These results indicate a requirement for GIMAP6 in the maintenance of a normal peripheral adaptive immune system and a significant role for the protein in normal autophagic processes. Moreover, as GIMAP6 is expressed in a cell-selective manner, this indicates the potential existence of a cell-restricted mode of autophagic regulation.
Assuntos
Imunidade Adaptativa/genética , Autofagossomos/imunologia , Autofagia/imunologia , Linfócitos T CD4-Positivos/imunologia , GTP Fosfo-Hidrolases/fisiologia , Animais , Linhagem Celular , GTP Fosfo-Hidrolases/genética , Células HEK293 , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/fisiologia , Fosforilação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Sequestossoma-1/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologiaRESUMO
T follicular helper (Tfh) cells are a distinct type of CD4+ T cell specialized in providing help to B cells during the germinal centre (GC) reaction. As such, they are critical determinants of the quality of an antibody response following antigen challenge. Excessive production of Tfh cells can result in autoimmunity whereas too few can result in inadequate protection from infection. Hence, their differentiation and maintenance must be tightly regulated to ensure appropriate but limited help to B cells. Unlike the majority of other CD4+ T-cell subsets, Tfh cell differentiation occurs in three phases defined by their anatomical location. During each phase of differentiation the emerging Tfh cells express distinct patterns of co-receptors, which work together with the T-cell receptor (TCR) to drive Tfh differentiation. These signals provided by both TCR and co-receptors during Tfh differentiation alter proliferation, survival, metabolism, cytokine production and transcription factor expression. This review will discuss how engagement of TCR and co-receptors work together to shape the formation and function of Tfh cells.
Assuntos
Diferenciação Celular , Centro Germinativo/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Formação de Anticorpos , Antígenos/imunologia , Centro Germinativo/citologia , Centro Germinativo/metabolismo , Humanos , Fenótipo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
An effective immune system depends upon the survival of mature T cells in the periphery. Members of the GIMAP family of GTPases have been proposed to regulate this homeostasis, supported by the paucity of peripheral T cells in rodents deficient for either GIMAP1 or GIMAP5. It is unclear whether this lack of T cells is a consequence of an ontological defect, causing the thymus to generate and export T cells incapable of surviving in the periphery, or whether (alternatively or additionally) mature T cells intrinsically require GIMAP1 for survival. Using the ERT2 Cre+ transgene, we conditionally deleted Gimap1 in C57BL/6 mice and demonstrate that GIMAP1 is intrinsically required for the survival of mature T cells in the periphery. We show that, in contrast to GIMAP5, this requirement is independent of the T-cells' activation status. We investigated the nature of the survival defect in GIMAP1-deficient CD4+ T cells and show that the death occurring after GIMAP1 ablation is accompanied by mitochondrial depolarization and activation of the extrinsic apoptotic pathway. This study shows that GIMAP1 is critical for maintaining the peripheral T-cell pool in mice and offers a potent target for the treatment of T-cell-mediated diseases.
Assuntos
Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores Etários , Alelos , Animais , Apoptose/genética , Apoptose/imunologia , Caspases/metabolismo , Sobrevivência Celular/genética , Proteínas de Ligação ao GTP/deficiência , Deleção de Genes , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Proteínas de Membrana/deficiência , Camundongos , Camundongos Transgênicos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
An effective immune system depends upon regulation of lymphocyte function and homeostasis. In recent years, members of the GTPases of the immunity associated protein (GIMAP) family were proposed to regulate T cell homeostasis. In contrast, little is known about their function and mode of action in B cells. We used a combination of transgenic mice and in vivo and in vitro techniques to conditionally and electively ablate GIMAP1 in resting and activated peripheral B cells. Our data suggest that GIMAP1 is absolutely essential for the survival of peripheral B cells, irrespective of their activation state. Together with recent data showing increased expression of GIMAP1 in B cell lymphomas, our work points to the possible potential of GIMAP1 as a target for manipulation in a variety of B cell-mediated diseases.
Assuntos
Linfócitos B/imunologia , Proteínas de Ligação ao GTP/imunologia , Ativação Linfocitária/imunologia , Proteínas de Membrana/imunologia , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Células Cultivadas , Proteínas de Ligação ao GTP/genética , Centro Germinativo/imunologia , Linfoma de Células B/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: GTPases of the immunity-associated protein family (GIMAPs) are predominantly expressed in mature lymphocytes. Studies of rodents deficient in GIMAP1, GIMAP4, or GIMAP5 have demonstrated that these GTPases regulate lymphocyte survival. In contrast to the other family members, GIMAP8 contains three potential GTP-binding domains (G-domains), a highly unusual feature suggesting a novel function for this protein. To examine a role for GIMAP8 in lymphocyte biology we examined GIMAP8 expression during lymphocyte development. We also generated a mouse deficient in GIMAP8 and examined lymphocyte development and function. PRINCIPAL FINDINGS: We show that GIMAP8 is expressed in the very early and late stages of T cell development in the thymus, at late stages during B cell development, and peripheral T and B cells. We find no defects in T or B lymphocyte development in the absence of GIMAP8. A marginal decrease in the number of recirculating bone marrow B cells suggests that GIMAP8 is important for the survival of mature B cells within the bone marrow niche. We also show that deletion of GIMAP8 results in a delay in apoptotic death of mature T cell in vitro in response to dexamethasone or γ-irradiation. However, despite these findings we find that GIMAP8-deficient mice mount normal primary and secondary responses to a T cell dependent antigen. CONCLUSIONS: Despite its unique structure, GIMAP8 is not required for lymphocyte development but appears to have a minor role in maintaining recirculating B cells in the bone marrow niche and a role in regulating apoptosis of mature T cells.
Assuntos
GTP Fosfo-Hidrolases/deficiência , Animais , Apoptose , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The generation of high-affinity Abs is essential for immunity and requires collaboration between B and T cells within germinal centers (GCs). By using novel mouse models with a conditional deletion of the p110δ catalytic subunit of the PI3K pathway, we established that p110δ is required in T cells, but not in B cells, for the GC reaction. We found the formation of T follicular helper (T(FH)) cells to be critically dependent on p110δ in T cells. Furthermore, by deleting phosphatase and tensin homolog deleted on chromosome 10, which opposes p110δ in activated T cells, we found a positive correlation between increased numbers of T(FH) cells and GC B cells. These results are consistent with the hypothesis that T cell help is the limiting factor in the GC reaction. P110δ was not required for the expression of B cell lymphoma 6, the downregulation of CCR7, or T cell entry into primary follicles. Instead, p110δ was the critical catalytic subunit for ICOS downstream signaling and the production of key T(FH) cytokines and effector molecules. Our findings support a model in which the magnitude of the GC reaction is controlled by the activity of the PI3K pathway in T(FH) cells.
Assuntos
Formação de Anticorpos/imunologia , Centro Germinativo/imunologia , Ativação Linfocitária/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Linfócitos T Auxiliares-Indutores/enzimologia , Transferência Adotiva , Animais , Linfócitos B/imunologia , Western Blotting , Separação Celular , Classe I de Fosfatidilinositol 3-Quinases , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Centro Germinativo/enzimologia , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Fosfatidilinositol 3-Quinases/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
The guanosine triphosphatases (GTPases) of the immunity-associated protein (GIMAP) family of putative GTPases has been implicated in the regulation of T-lymphocyte development and survival. A mouse conditional knockout allele was generated for the immune GTPase gene GIMAP1. Homozygous loss of this allele under the influence of the lymphoid-expressed hCD2-iCre recombinase transgene led to severe (> 85%) deficiency of mature T lymphocytes and, unexpectedly, of mature B lymphocytes. By contrast there was little effect of GIMAP1 deletion on immature lymphocytes in either B or T lineages, although in vitro studies showed a shortening of the survival time of both immature and mature CD4(+) single-positive thymocytes. These findings show a vital requirement for GIMAP1 in mature lymphocyte development/survival and draw attention to the nonredundant roles of members of the GIMAP GTPase family in these processes.
Assuntos
Linfócitos B/citologia , Diferenciação Celular/imunologia , GTP Fosfo-Hidrolases/metabolismo , Linfócitos T/citologia , Animais , Western Blotting , Separação Celular , Sobrevivência Celular , Citometria de Fluxo , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase , Transdução de Sinais/imunologiaRESUMO
RhoG is a Rho family small GTPase implicated in cytoskeletal regulation, acting either upstream of or in parallel to Rac1. The precise function(s) of RhoG in vivo has not yet been defined. We have identified a novel role for RhoG in signaling the neutrophil respiratory burst stimulated by G protein-coupled receptor agonists. Bone marrow-derived neutrophils from RhoG knockout (RhoG(-/-)) mice exhibited a marked impairment of oxidant generation in response to C5a or fMLP, but normal responses to PMA or opsonized zymosan and normal bacterial killing. Activation of Rac1 and Rac2 by fMLP was diminished in RhoG(-/-) neutrophils only at very early (5 s) time points (by 25 and 32%, respectively), whereas chemotaxis in response to soluble agonists was unaffected by lack of RhoG. Additionally, fMLP-stimulated phosphorylation of protein kinase B and p38MAPK, activation of phospholipase D, and calcium fluxes were equivalent in wild-type and RhoG(-/-) neutrophils. Our results define RhoG as a critical component of G protein-coupled receptor-stimulated signaling cascades in murine neutrophils, acting either via a subset of total cellular Rac relevant to oxidase activation and/or by a novel and as yet undefined interaction with the neutrophil NADPH oxidase.
Assuntos
NADPH Oxidases/metabolismo , Neutrófilos/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Cálcio/metabolismo , Catecolaminas/farmacologia , Células Cultivadas , Quimiotaxia de Leucócito , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Humanos , Imidazolinas/farmacologia , Camundongos , Camundongos Knockout , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Fosfolipase D/metabolismo , Transdução de Sinais , Solubilidade , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/agonistas , Proteínas rho de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Rac GTPases regulate cytoskeletal structure, gene expression, and reactive oxygen species (ROS) production. Rac2-deficient neutrophils cannot chemotax, produce ROS, or degranulate upon G protein-coupled receptor (GPCR) activation. Deficiency in PI3Kgamma, an upstream regulator of Rac, causes a similar phenotype. P-Rex1, a guanine-nucleotide exchange factor (GEF) for Rac, is believed to link GPCRs and PI3Kgamma to Rac-dependent neutrophil responses. We have investigated the functional importance of P-Rex1 by generating a P-Rex1(-/-) mouse. P-Rex1(-/-) mice are viable and healthy, with apparently normal leukocyte development, but with mild neutrophilia. In neutrophils from P-Rex1(-/-) mice, GPCR-dependent Rac2 activation is impaired, whereas Rac1 activation is less compromised. GPCR-dependent ROS formation is absent in lipopolysaccharide (LPS)-primed P-Rex1(-/-) neutrophils, but less affected in unprimed or TNFalpha-primed cells. Recruitment of P-Rex1(-/-) neutrophils to inflammatory sites is impaired. Surprisingly, chemotaxis of isolated neutrophils is only slightly reduced, with a mild defect in cell speed, but normal polarization and directionality. Secretion of azurophil granules is unaffected. In conclusion, P-Rex1 is an important regulator of neutrophil function by mediating a subset of Rac-dependent neutrophil responses. However, P-Rex1 is not an essential regulator of neutrophil chemotaxis and degranulation.
Assuntos
Degranulação Celular/fisiologia , Quimiotaxia/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Actinas/metabolismo , Animais , Clonagem Molecular , Ativação Enzimática/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos , Camundongos Knockout , Neuropeptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP , Proteína RAC2 de Ligação ao GTPRESUMO
Virus-encoded immune evasion mechanisms provide information on viral pathogenesis and offer a unique opportunity to identify new strategies of immune modulation. Secreted proteins that bind a broad range of chemokines have been identified in recent years in poxviruses and herpesviruses. We discuss the properties of these viral chemokine inhibitors and their potential as new therapeutics to treat human inflammatory diseases.
Assuntos
Quimiocinas/metabolismo , Proteínas Virais/metabolismo , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Quimiocinas/antagonistas & inibidores , Humanos , Ligação Proteica , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/química , Viroses/metabolismo , Viroses/patologia , Vírus/metabolismoRESUMO
The role of PI3K activity in T lymphocyte development is obscure because mice deficient in single PI3K catalytic subunits either die before birth (p110alpha-/- and p110beta-/-) or lack a significant T cell developmental phenotype (p110gamma-/- and p110delta-/-). We have generated mice deficient in both p110gamma and p110delta and show that p110gamma/delta-/- mice have a profound block in T cell development that occurs at the beta-selection checkpoint. We show that pre-TCR-induced signaling is significantly reduced in p110gamma/delta-/- thymocytes and that this results in a concomitant lack of proliferative expansion and increased apoptosis. The survival defect in p110gamma/delta-/- thymocytes is associated with increased levels of the pro-apoptotic molecule Bcl2 interacting mediator of cell death. This work demonstrates that PI3K activity is critical for T cell development and depends on the combined function of p110gamma and p110delta.
Assuntos
Fosfatidilinositol 3-Quinases/fisiologia , Linfócitos T/fisiologia , Animais , Domínio Catalítico , Camundongos , Fosfatidilinositol 3-Quinases/química , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de SinaisRESUMO
Chemokines are small glycosaminoglycan (GAG) binding proteins that direct the migration of leukocytes by signaling through G protein coupled receptors (GPCR). Many viruses encode proteins that disrupt chemokine responses. The murine gammaherpesvirus-68 gene M3 encodes a chemokine binding protein (vCKBP-3), which has no sequence similarity to chemokine receptors. Initial characterization of vCKBP-3 showed that it inhibits receptor binding and chemokine-induced calcium influx. The structural requirements for the chemokines CXCL8 and CCL2 to bind to vCKBP-3 have been determined. Both chemokines bind to vCKBP-3 via their N-loop, a site that can participate in GAG binding for some chemokines. We have investigated the effect of vCKBP-3 on the interaction of chemokines with GAGs. We found that vCKBP-3 can prevent a range of chemokines from binding to GAGs. Moreover, we also found that vCKBP-3 can displace chemokines from a heparin-coated surface. Together, these data imply that vCKBP-3 can inhibit chemokine activity at two distinct levels. First, it inhibits chemokines from binding to their GPCR. Second, it inhibits their GAG binding and disrupts pre-formed chemokine gradients. This dual ability of vCKBP-3 makes it a more effective inhibitor of chemokine activity.
Assuntos
Quimiocinas/metabolismo , Gammaherpesvirinae/fisiologia , Heparina/metabolismo , Proteínas Virais/farmacologia , Animais , Sítios de Ligação , Ligação Competitiva , Células CHO , Quimiocinas/química , Cricetinae , Cricetulus , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/farmacologiaRESUMO
Viruses encode proteins that disrupt chemokine responses. The murine gammaherpesvirus 68 gene M3 encodes a chemokine binding protein (vCKBP-3) which has no sequence similarity to chemokine receptors but inhibits chemokine receptor binding and activity. We have used a panel of CXCL8 analogs to identify the structural requirements for CXCL8 to bind to vCKBP-3 in a scintillation proximity assay. Our data suggest that vCKBP-3 acts by mimicking the binding of chemokine receptors to CXCL8.