RESUMO
The F-BAR domain containing protein CIP4 (Cdc42 interacting protein 4) interacts with Cdc42 and WASP/N-WASP and is thought to participate in the assembly of filamentous actin. CIP4(-/-) mice had normal T- and B-lymphocyte development but impaired T cell-dependent antibody production, IgG antibody affinity maturation, and germinal center (GC) formation, despite an intact CD40L-CD40 axis. CIP4(-/-) mice also had impaired contact hypersensitivity (CHS) to haptens, and their T cells failed to adoptively transfer CHS. Ovalbumin-activated CD4(+) effector T cells from CIP4(-/-)/OT-II mice migrated poorly to antigen-challenged skin. Activated CIP4(-/-) T cells exhibited impaired adhesion and polarization on immobilized VCAM-1 and ICAM-1 and defective arrest and transmigration across murine endothelial cell monolayers under shear flow conditions. These results demonstrate an important role for CIP4 in integrin-dependent T cell-dependent antibody responses and GC formation and in integrin-mediated recruitment of effector T cells to cutaneous sites of antigen-driven immune reactions.
Assuntos
Movimento Celular , Integrinas/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Animais , Linfócitos B/imunologia , Adesão Celular , Polaridade Celular , Dermatite Alérgica de Contato/genética , Dermatite Alérgica de Contato/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Antígenos de Histocompatibilidade Menor , Molécula 1 de Adesão de Célula Vascular/imunologiaRESUMO
Atopic dermatitis (AD) is a common allergic inflammatory skin disease caused by a combination of intense pruritus, scratching, and epicutaneous (e.c.) sensitization with allergens. To explore the roles of IL-21 and IL-21 receptor (IL-21R) in AD, we examined skin lesions from patients with AD and used a mouse model of allergic skin inflammation. IL-21 and IL-21R expression was upregulated in acute skin lesions of AD patients and in mouse skin subjected to tape stripping, a surrogate for scratching. The importance of this finding was highlighted by the fact that both Il21r-/- mice and WT mice treated with soluble IL-21R-IgG2aFc fusion protein failed to develop skin inflammation after e.c. sensitization of tape-stripped skin. Adoptively transferred OVA-specific WT CD4+ T cells accumulated poorly in draining LNs (DLNs) of e.c. sensitized Il21r-/- mice. This was likely caused by both DC-intrinsic and nonintrinsic effects, because trafficking of skin DCs to DLNs was defective in Il21r-/- mice and, to a lesser extent, in WT mice reconstituted with Il21r-/- BM. More insight into this defect was provided by the observation that skin DCs from tape-stripped WT mice, but not Il21r-/- mice, upregulated CCR7 and migrated toward CCR7 ligands. Treatment of epidermal and dermal cells with IL-21 activated MMP2, which has been implicated in trafficking of skin DCs. These results suggest an important role for IL-21R in the mobilization of skin DCs to DLNs and the subsequent allergic response to e.c. introduced antigen.
Assuntos
Dermatite Atópica , Imunização , Inflamação/imunologia , Receptores de Interleucina-21/imunologia , Pele , Transferência Adotiva , Animais , Células Cultivadas , Dermatite Atópica/imunologia , Dermatite Atópica/patologia , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Humanos , Inflamação/induzido quimicamente , Inflamação/patologia , Interleucinas/genética , Interleucinas/imunologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ovalbumina/imunologia , Ovalbumina/farmacologia , Receptores CCR7/genética , Receptores CCR7/imunologia , Receptores de Interleucina-21/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Pele/imunologia , Pele/patologia , Baço/citologia , Baço/imunologiaRESUMO
The Wiskott-Aldrich syndrome (WAS) interacting protein (WIP) stabilizes the WAS protein (WASP), the product of the gene mutated in WAS. WIP-deficient T cells have low WASP levels, limiting the usefulness of WIP KO mice in defining the role of WIP in T cell function. To define this role, we compared WIP/WASP double KO (DKO) mice to WASP KO mice on DO11.10 background. T cell development was normal in both strains, but peripheral T cell numbers were significantly decreased in DKO mice. WASP KO T cells proliferated and secreted IL-2 normally in response to OVA peptide (OVAp). In contrast, T cells from DKO mice proliferated poorly in response to OVAp in vitro, and cutaneous hapten hypersensitivity was deficient in these mice. DKO T cells up-regulated CD25 expression and secreted normal amounts of IL-2 after antigen stimulation, but had defective response to IL-2, evidenced by failure to further up-regulate CD25 expression, phosphorylate STAT5, and induce expression of STAT5-dependent genes. DKO, but not WASP KO, T cells had a disrupted subcortical actin cytoskeleton and impaired actin polymerization after T cell antigen receptor (TCR) ligation. These results indicate that WIP is essential for IL-2 signaling and responsiveness in T cells, possibly because of its critical role in TCR-triggered actin cytoskeletal reorganization.
Assuntos
Proteínas de Transporte/fisiologia , Interleucina-2/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Actinas/metabolismo , Actinas/ultraestrutura , Animais , Antígenos/imunologia , Proteínas de Transporte/genética , Proliferação de Células , Proteínas do Citoesqueleto , Citoesqueleto/imunologia , Citoesqueleto/ultraestrutura , Interleucina-2/farmacologia , Camundongos , Camundongos Knockout , Receptores de Interleucina-2/biossíntese , Linfócitos T/efeitos dos fármacos , Timo/crescimento & desenvolvimento , Timo/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/fisiologiaRESUMO
BACKGROUND: The Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are caused by mutations in WAS, which encodes for WAS protein (WASP). The WASP-interacting protein (WIP) stabilizes WASP, as evidenced by severely decreased WASP levels in T cells from WIP-deficient mice. The majority of missense mutations in patients with WAS/XLT are located in the WIP-binding domain of WASP and might result in dissociation of the WASP-WIP complex and WASP degradation. OBJECTIVE: To restore WASP levels and correct T-cell function in WAS/XLT patients with mutations in the WIP-binding domain of WASP. METHODS: WIP, and a WIP-derived 41-amino acid-long peptide, which interacts with WASP and was designated nanoWIP (nWIP), were fused to enhanced green fluorescent protein and introduced by electroporation into EBV-transformed B cells, and by retroviral transduction into purified blood T cells from patients with WAS. WASP levels were measured by intracellular fluorescence-activated cell sorting staining. The actin cytoskeleton was visualized by intracellular phalloidin staining. RESULTS: Introduction of WIP and nWIP restored WASP levels to normal in EBV-transformed B-cell lines from XLT patients with missense mutations in the WIP-binding domain of WASP and residual WASP levels, and corrected the defective spreading and pseudopodia formation of their T cells in response to immobilized anti-CD3. CONCLUSION: A WASP-binding WIP-derived peptide stabilizes WASP in cells from XLT patients with missense mutations that disrupt WIP binding, and corrects their T-cell actin cytoskeleton defect. This may provide a novel therapeutic strategy for these patients.
Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfócitos T/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Linfócitos B/metabolismo , Western Blotting , Separação Celular , Citoesqueleto/patologia , Eletroporação , Citometria de Fluxo , Humanos , Imunoprecipitação , Células Jurkat , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Mutação de Sentido Incorreto , Peptídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/patologia , Transdução Genética , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
The tyrosine kinase JAK3 plays a well-established role during normal lymphocyte development and is constitutively phosphorylated in several lymphoid malignancies. However, its contribution to lymphomagenesis remains elusive. In this study, we used the newly identified activating JAK3A572V mutation to elucidate the effect of constitutive JAK3 signaling on murine lymphopoiesis. In a bone marrow transplantation model, JAK3A572V induces an aggressive, fatal, and transplantable lymphoproliferative disorder characterized by the expansion of CD8(+)TCRalphabeta(+)CD44(+)CD122(+)Ly-6C(+) T cells that closely resemble an effector/memory T-cell subtype. Compared with wild-type counterparts, these cells show increased proliferative capacities in response to polyclonal stimulation, enhanced survival rates with elevated expression of Bcl-2, and increased production of interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), correlating with enhanced cytotoxic abilities against allogeneic target cells. Of interest, the JAK3A572V disease is epidermotropic and produces intraepidermal microabscesses. Taken together, these clinical features are reminiscent of those observed in an uncommon but aggressive subset of CD8(+) human cutaneous T-cell lymphomas (CTCLs). However, we also observed a CD4(+) CTCL-like phenotype when cells are transplanted in an MHC-I-deficient background. These data demonstrate that constitutive JAK3 activation disrupts T-cell homeostasis and induces lymphoproliferative diseases in mice.
Assuntos
Linfócitos T CD8-Positivos/patologia , Janus Quinase 3/fisiologia , Linfopoese/fisiologia , Transtornos Linfoproliferativos/etiologia , Mutação Puntual , Proteínas Recombinantes de Fusão/fisiologia , Subpopulações de Linfócitos T/patologia , Animais , Antígenos Ly/análise , Transplante de Medula Óssea , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/química , Indução Enzimática , Humanos , Receptores de Hialuronatos/análise , Subunidade beta de Receptor de Interleucina-2/análise , Janus Quinase 3/biossíntese , Janus Quinase 3/genética , Linfoma Cutâneo de Células T/patologia , Linfopoese/genética , Transtornos Linfoproliferativos/enzimologia , Transtornos Linfoproliferativos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Quimera por Radiação , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Pele/patologia , Subpopulações de Linfócitos T/químicaRESUMO
Genetic defects in the transcription factor forkhead box protein P3 (Foxp3) cause immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX). IPEX is thought to be due to a defect in naturally arising CD4+ Tregs. In this issue of the JCI, Bacchetta and colleagues demonstrate that patients with IPEX and missense mutations in Foxp3 provide insight into the role of various domains of Foxp3 in the development and function of Tregs (see the related article beginning on page 1713).
Assuntos
Fatores de Transcrição Forkhead/imunologia , Doenças Genéticas Ligadas ao Cromossomo X/imunologia , Poliendocrinopatias Autoimunes/imunologia , Enteropatias Perdedoras de Proteínas/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Fatores de Transcrição Forkhead/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Humanos , Camundongos , Mutação de Sentido Incorreto , Fenótipo , Poliendocrinopatias Autoimunes/genética , Enteropatias Perdedoras de Proteínas/genéticaRESUMO
The adapter 3BP2 is involved in leukocyte signaling downstream Src/Syk-kinases coupled immunoreceptors. Here, we show that 3BP2 directly interacts with the endocytic scaffold protein CIN85 and the actin-binding protein HIP-55. 3BP2 co-localized with CIN85 and HIP-55 in T cell rafts and at the T cell/APC synapse, an active zone of receptors and proteins recycling. A binding region of CIN85 SH3 domains on 3BP2 was mapped to a PVPTPR motif in the first proline-rich region of 3BP2, whereas the C-terminal SH3 domain of HIP-55 bound a more distal proline-rich domain of 3BP2. Together, our data suggest an unexpected role of 3BP2 in endocytic and cytoskeletal regulation through its interaction with CIN85 and HIP-55.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Citoesqueleto/metabolismo , Endocitose , Humanos , Técnicas In Vitro , Células Jurkat , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Modelos Biológicos , Prolina/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Linfócitos T/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Domínios de Homologia de src/genéticaRESUMO
Patients with ectodermal dysplasia with immunodeficiency (ED-ID) caused by mutations in the inhibitor of NF-κB α (IκBα) are susceptible to severe recurrent infections, despite normal T and B cell numbers and intact in vitro lymphocyte function. Moreover, the outcome of hematopoietic stem cell transplantation (HSCT) in these patients is poor despite good engraftment. Mice heterozygous for the IκBα S32I mutation found in patients exhibited typical features of ED-ID. Strikingly, the mice lacked lymph nodes, Peyer's patches, splenic marginal zones, and follicular dendritic cells and failed to develop contact hypersensitivity (CHS) or form germinal centers (GCs), all features not previously recognized in patients and typical of defective noncanonical NF-κB signaling. Lymphotoxin ß receptor (LTßR)-driven induction of chemokines and adhesion molecules mediated by both canonical and noncanonical NF-κB pathways was impaired, and levels of p100 were markedly diminished in the mutant. IκBα mutant â Rag2(-/-), but not WTâIκBα mutant, bone marrow chimeras formed proper lymphoid organs and developed CHS and GCs. Defective architectural cell function explains the immunodeficiency and poor outcome of HSCT in patients with IκBα deficiency and suggests that correction of this niche is critical for reconstituting their immune function.
Assuntos
Heterozigoto , Proteínas I-kappa B/genética , Tecido Linfoide/embriologia , Tecido Linfoide/imunologia , Mutação , Organogênese/genética , Organogênese/imunologia , Animais , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Códon , Dermatite de Contato/genética , Dermatite de Contato/imunologia , Dermatite de Contato/metabolismo , Modelos Animais de Doenças , Hipersensibilidade Tardia/genética , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Tardia/metabolismo , Proteínas I-kappa B/metabolismo , Síndromes de Imunodeficiência/etiologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Transgênicos , Inibidor de NF-kappaB alfa , Fosforilação , Proteólise , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptores Toll-Like/metabolismo , Fatores de Necrose Tumoral/metabolismoRESUMO
BACKGROUND: The chemokine CCL25, and its receptor CCR9, constitute a unique chemokine/receptor pair, which regulates trafficking of T lymphocytes to the small intestine under physiological conditions and is an attractive target for small bowel Crohn's disease drug development. We have previously shown that CCL25/CCR9 interactions regulate the recovery from acute dextran sulfate sodium-induced colonic inflammation. In this study, we explored whether these interactions also regulate chronic colitis development in 2 independent murine models of experimental colitis. METHODS: Histological flow cytometry and qPCR analyses were performed to evaluate the role of CL25 and CCR9 in chronic colonic inflammation induced by serial exposures to dextran sulfate sodium salts or by adoptive transfer of CD45RB(hi) CD4(+) T cell into lymphopenic mice devoid of CCL25/CCR9 interactions. RESULTS: Chronic dextran sulfate sodium exposure results in exacerbated colitis in mice deficient for either CCR9 or CCL25 when compared with wild-type control mice. Although CCR9-deficient T cells traffic to the colon and induce severe colitis similar to wild-type T cells in the CD45RB transfer model, naive wild-type T cells induce more severe disease in recipient animals devoid of CCL25 expression. CONCLUSIONS: CCL25/CCR9 interactions are required for modulating protection against large intestinal inflammation in 2 models of chronic colitis. These data may have implications for the potential effects of disrupting CCL25/CCR9 interactions in humans in the setting of intestinal disorders including inflammatory bowel disease.
Assuntos
Quimiocinas CC/metabolismo , Colite/imunologia , Colite/fisiopatologia , Imunidade Inata/fisiologia , Receptores CCR/metabolismo , Animais , Quimiocinas CC/imunologia , Doença Crônica , Citoproteção , Modelos Animais de Doenças , Feminino , Citometria de Fluxo , Imunidade Inata/imunologia , Masculino , Camundongos , Distribuição Aleatória , Receptores CCR/imunologia , Valores de Referência , Sensibilidade e EspecificidadeRESUMO
Engagement of the B-cell antigen receptor (BCR) activates kinases of the Src and Syk families and signaling complexes assembled by adaptor proteins, which dictate B-cell fate and function. The adaptor 3BP2/SH3BP2, an Abl Src homology domain 3 (SH3)-binding and Syk-kinases interacting protein, exhibits positive regulatory roles in T, natural killer (NK), and basophilic cells. However, its involvement in BCR signaling is completely unknown. Here we show that 3BP2 is tyrosine phosphorylated following BCR aggregation on B lymphoma cells, and that 3BP2 is a substrate for Syk and Fyn, but not Btk. To further explore the function of 3BP2 in B cells, we screened a yeast 2-hybrid B-lymphocyte library and found 3BP2 as a binding partner of Vav proteins. The interaction between 3BP2 and Vav proteins involved both constitutive and inducible mechanisms. 3BP2 also interacted with other components of the BCR signaling pathway, including Syk and phospholipase C gamma (PLC-gamma). Furthermore, overexpression and RNAi blocking experiments showed that 3BP2 regulated BCR-mediated activation of nuclear factor of activated T cells (NFATs). Finally, evidence was provided that 3BP2 functionally cooperates with Vav proteins and Rho GTPases to activate NFATs. Our results show that 3BP2 may regulate BCR-mediated gene activation through Vav proteins.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de Transcrição/metabolismo , Sítios de Ligação , Proteínas de Ciclo Celular , Clonagem Molecular , Regulação da Expressão Gênica , Genes Reporter , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Humanos , Células Jurkat , Fatores de Transcrição NFATC , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-vav , Receptores de Antígenos de Linfócitos B/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Transdução de Sinais , Ativação Transcricional , Transfecção , Domínios de Homologia de srcRESUMO
Actin cytoskeleton dynamics critically regulate T cell activation. We found that the cytoplasmic adaptor HIP-55, a Src/Syk-kinases substrate and member of the drebrin/Abp1 family of actin-binding proteins, localized to the T cell-antigen-presenting cell (APC) contact site in an antigen-dependent manner. Using green fluorescent protein fusion proteins, both Src homology 3 (SH3) and actin binding domains were found necessary for recruitment at the T cell-APC interface. HIP-55 was not implicated in conjugate formation and actin polymerization but regulated distal signaling events through binding and activation of hematopoietic progenitor kinase 1 (HPK1), a germinal center kinase (GCK) family kinase involved in negative signaling in T cells. Using RNA interference and overexpression experiments, the HIP-55-HPK1 complex was found to negatively regulate nuclear factor of activated T cell (NFAT) activation by the T cell antigen receptor. Moreover, we show that HIP-55, which partly co-localized with early endocytic compartments, promoted both basal and ligand-dependent T cell receptor (TCR) down-modulation, resulting in a decreased TCR expression. SH3 and actin-depolymerizing factor homology domains were required for this function. As controls, the expression of CD28 and the glycosylphosphatidylinositol-linked protein CD59 was not affected by HIP-55 overexpression. These results suggest that, in addition to binding to HPK1, HIP-55 might negatively regulate TCR signaling through down-regulation of TCR expression. Our findings show that HIP-55 is a key novel component of the immunological synapse that modulates T cell activation by connecting actin cytoskeleton and TCRs to gene activation and endocytic processes.