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1.
Nature ; 603(7900): 328-334, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35197632

RESUMO

Effective antitumour immunity depends on the orchestration of potent T cell responses against malignancies1. Regression of human cancers has been induced by immune checkpoint inhibitors, T cell engagers or chimeric antigen receptor T cell therapies2-4. Although CD8 T cells function as key effectors of these responses, the role of CD4 T cells beyond their helper function has not been defined. Here we demonstrate that a trispecific antibody to HER2, CD3 and CD28 stimulates regression of breast cancers in a humanized mouse model through a mechanism involving CD4-dependent inhibition of tumour cell cycle progression. Although CD8 T cells directly mediated tumour lysis in vitro, CD4 T cells exerted antiproliferative effects by blocking cancer cell cycle progression at G1/S. Furthermore, when T cell subsets were adoptively transferred into a humanized breast cancer tumour mouse model, CD4 T cells alone inhibited HER2+ breast cancer growth in vivo. RNA microarray analysis revealed that CD4 T cells markedly decreased tumour cell cycle progression and proliferation, and also increased pro-inflammatory signalling pathways. Collectively, the trispecific antibody to HER2 induced T cell-dependent tumour regression through direct antitumour and indirect pro-inflammatory/immune effects driven by CD4 T cells.


Assuntos
Neoplasias da Mama , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Feminino , Humanos , Camundongos , Receptor ErbB-2/genética
2.
J Immunol ; 211(11): 1656-1668, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37850953

RESUMO

Regulatory T cells (Tregs) are critical mediators of immune tolerance and play a diametric role in cancer and autoimmunity. Tumor-infiltrating Tregs are often associated with poor prognosis in solid tumors because their enrichment in the tumor microenvironment contributes to immunosuppression. Conversely, dysregulation in the Treg compartment can disrupt self-tolerance, leading to autoimmunity. In the present study, we describe what is, to our knowledge, a novel regulator of Tregs, the GTPase activator regulator of G protein 1 (RGS1), demonstrating that RGS1-deficient human Tregs show downregulation of Treg-associated genes and are less immunosuppressive. These RGS1-deficient Tregs exhibit perturbations to the FOXP3-c-MYC transcriptional axis and downstream metabolic and autophagy programs by shifting their energy demands toward glycolysis and rendering them less autophagic. Taken together, RGS1 may serve as an apical node of Treg function by regulating the FOXP3-c-MYC transcriptional axis, thereby providing a therapeutic rationale for targeting RGS1 for treatment of cancer and autoimmune diseases.


Assuntos
Doenças Autoimunes , Neoplasias , Proteínas RGS , Humanos , Linfócitos T Reguladores , Doenças Autoimunes/patologia , Autoimunidade , Neoplasias/patologia , Autofagia/genética , Fatores de Transcrição Forkhead/metabolismo , Microambiente Tumoral , Proteínas RGS/genética , Proteínas RGS/metabolismo
4.
Immunity ; 38(3): 489-501, 2013 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-23521885

RESUMO

The motheaten mouse has long served as a paradigm for complex autoimmune and inflammatory disease. Null mutations in Ptpn6, which encodes the nonreceptor protein-tyrosine phosphatase Shp1, cause the motheaten phenotype. However, Shp1 regulates multiple signaling pathways in different hematopoietic cell types, so the cellular and molecular mechanism of autoimmunity and inflammation in the motheaten mouse has remained unclear. By using floxed Ptpn6 mice, we dissected the contribution of innate immune cells to the motheaten phenotype. Ptpn6 deletion in neutrophils resulted in cutaneous inflammation, but not autoimmunity, providing an animal model of human neutrophilic dermatoses. By contrast, dendritic cell deletion caused severe autoimmunity, without inflammation. Genetic and biochemical analysis showed that inflammation was caused by enhanced neutrophil integrin signaling through Src-family and Syk kinases, whereas autoimmunity resulted from exaggerated MyD88-dependent signaling in dendritic cells. Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease.


Assuntos
Autoimunidade/imunologia , Células Dendríticas/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Animais , Autoimunidade/genética , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Calgranulina A/genética , Calgranulina A/imunologia , Calgranulina A/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Células Dendríticas/metabolismo , Derme/imunologia , Derme/metabolismo , Derme/patologia , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Inflamação/genética , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Neutrófilos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Quinase Syk , Quinases da Família src/imunologia , Quinases da Família src/metabolismo
5.
Proc Natl Acad Sci U S A ; 111(44): 15741-5, 2014 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-25331893

RESUMO

There are many transmembrane receptor-like proteins whose ligands have not been identified. A strategy for finding ligands when little is known about their tissue source is to screen each extracellular protein individually expressed in an array format by using a sensitive functional readout. Taking this approach, we have screened a large collection (3,191 proteins) of extracellular proteins for their ability to activate signaling of an orphan receptor, leukocyte tyrosine kinase (LTK). Only two related secreted factors, FAM150A and FAM150B (family with sequence similarity 150 member A and member B), stimulated LTK phosphorylation. FAM150A binds LTK extracellular domain with high affinity (K(D) = 28 pM). FAM150A stimulates LTK phosphorylation in a ligand-dependent manner. This strategy provides an efficient approach for identifying functional ligands for other orphan receptors.


Assuntos
Citocinas/metabolismo , Proteoma/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Citocinas/genética , Feminino , Células HEK293 , Humanos , Masculino , Fosforilação/fisiologia , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína , Proteoma/genética , Proteômica , Receptores Proteína Tirosina Quinases/genética
6.
J Cell Sci ; 125(Pt 22): 5535-45, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-22976304

RESUMO

Macrophages migrate to sites of insult during normal inflammatory responses. Integrins guide such migration, but the transmission of signals from integrins into the requisite cytoskeletal changes is poorly understood. We have discovered that the hematopoietic adaptor protein Skap2 is necessary for macrophage migration, chemotaxis, global actin reorganization and local actin reorganization upon integrin engagement. Binding of phosphatidylinositol [3,4,5]-triphosphate to the Skap2 pleckstrin-homology (PH) domain, which relieves its conformational auto-inhibition, is critical for this integrin-driven cytoskeletal response. Skap2 enables integrin-induced tyrosyl phosphorylation of Src-family kinases (SFKs), Adap, and Sirpα, establishing their roles as signaling partners in this process. Furthermore, macrophages lacking functional Sirpα unexpectedly have impaired local integrin-induced responses identical to those of Skap2(-/-) macrophages, and Skap2 requires Sirpα for its recruitment to engaged integrins and for coordinating downstream actin rearrangement. By revealing the positive-regulatory role of Sirpα in a Skap2-mediated mechanism connecting integrin engagement with cytoskeletal rearrangement, these data demonstrate that Sirpα is not exclusively immunoinhibitory, and illuminate previously unexplained observations implicating Skap2 and Sirpα in mouse models of inflammatory disease.


Assuntos
Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/metabolismo , Receptores Imunológicos/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Bovinos , Quimiotaxia/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Células HEK293 , Humanos , Integrinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Modelos Biológicos , Polimerização/efeitos dos fármacos , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos
7.
J Immunol ; 185(6): 3256-67, 2010 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-20696858

RESUMO

During responses against viruses and malignancies, naive CD8 T lymphocytes expand to form both short-lived effector cells and a population containing cells with the potential to be long-lived and participate in memory responses (memory precursor effector cells). The strength of antigenic, costimulatory, and cytokine signals during responses impacts the magnitude and type of CD8 populations formed. In vitro studies have revealed that the tyrosine phosphatase Src homology region 2 domain-containing phosphatase-1 (SHP-1) regulates signal transduction from receptors on T cells including the TCR, helping set the activation threshold, and therefore may shape responses of mature CD8 T cells in vivo. Analysis of CD8 T cells from motheaten mice, which are globally deficient in SHP-1, proved problematic due to cell-extrinsic effects of SHP-1 deficiency in non-T cells on CD8 T cells. Therefore, a conditional knockout of SHP-1 in mature single-positive T cells was developed to analyze cell-intrinsic consequences of complete and partial SHP-1 deficiency on CD8 T cell responses to acute viral infection. The results demonstrated that SHP-1 has disparate effects on subpopulations of responding cells, limiting the magnitude and quality of primary and secondary responses by reducing the number of short-lived effector cells generated without affecting the size of the memory precursor effector cell pool that leads to formation of long-term memory.


Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Inibidores do Crescimento/fisiologia , Memória Imunológica , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Cricetinae , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Inibidores do Crescimento/deficiência , Inibidores do Crescimento/genética , Memória Imunológica/genética , Depleção Linfocítica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Células-Tronco/citologia , Células-Tronco/imunologia , Células-Tronco/metabolismo , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo
8.
J Immunother Cancer ; 10(9)2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36096532

RESUMO

BACKGROUND: Cancer immunotherapy has revolutionized cancer treatment. However, considering the limited success of immunotherapy to only some cancer types and patient cohorts, there is an unmet need for developing new treatments that will result in higher response rates in patients with cancer. Immunoglobulin-like transcript 2 (ILT2), a LILRB family member, is an inhibitory receptor expressed on a variety of immune cells including T cells, natural killer (NK) cells and different myeloid cells. In the tumor microenvironment, binding of class I MHC (in particular HLA-G) to ILT2 on immune cells mediates a strong inhibitory effect, which manifests in inhibition of antitumor cytotoxicity of T and NK cells, and prevention of phagocytosis of the tumor cells by macrophages. METHODS: We describe here the development and characteristics of BND-22, a novel, humanized monoclonal antibody that selectively binds to ILT2 and blocks its interaction with classical MHC I and HLA-G. BND-22 was evaluated for its binding and blocking characteristics as well as its ability to increase the antitumor activity of macrophages, T cells and NK cells in various in vitro, ex vivo and in vivo systems. RESULTS: Collectively, our data suggest that BND-22 enhances activity of both innate and adaptive immune cells, thus generating robust and comprehensive antitumor immunity. In humanized mice models, blocking ILT2 with BND-22 decreased the growth of human tumors, hindered metastatic spread to the lungs, and prolonged survival of the tumor-bearing mice. In addition, BND-22 improved the antitumor immune response of approved therapies such as anti-PD-1 or anti-EGFR antibodies. CONCLUSIONS: BND-22 is a first-in-human ILT2 blocking antibody which has demonstrated efficient antitumor activity in various preclinical models as well as a favorable safety profile. Clinical evaluation of BND-22 as a monotherapy or in combination with other therapeutics is under way in patients with cancer. TRIAL REGISTRATION NUMBER: NCT04717375.


Assuntos
Antígenos HLA-G , Neoplasias , Animais , Antígenos HLA-G/metabolismo , Humanos , Imunoterapia , Células Matadoras Naturais , Camundongos , Neoplasias/tratamento farmacológico , Linfócitos T , Microambiente Tumoral
9.
Nat Med ; 10(8): 849-57, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15273746

RESUMO

Noonan syndrome is a common human autosomal dominant birth defect, characterized by short stature, facial abnormalities, heart defects and possibly increased risk of leukemia. Mutations of Ptpn11 (also known as Shp2), which encodes the protein-tyrosine phosphatase Shp2, occur in approximately 50% of individuals with Noonan syndrome, but their molecular, cellular and developmental effects, and the relationship between Noonan syndrome and leukemia, are unclear. We generated mice expressing the Noonan syndrome-associated mutant D61G. When homozygous, the D61G mutant is embryonic lethal, whereas heterozygotes have decreased viability. Surviving Ptpn11(D61G/+) embryos ( approximately 50%) have short stature, craniofacial abnormalities similar to those in Noonan syndrome, and myeloproliferative disease. Severely affected Ptpn11(D61G/+) embryos ( approximately 50%) have multiple cardiac defects similar to those in mice lacking the Ras-GAP protein neurofibromin. Their endocardial cushions have increased Erk activation, but Erk hyperactivation is cell and pathway specific. Our results clarify the relationship between Noonan syndrome and leukemia and show that a single Ptpn11 gain-of-function mutation evokes all major features of Noonan syndrome by acting on multiple developmental lineages in a gene dosage-dependent and pathway-selective manner.


Assuntos
Dosagem de Genes , Mutação/genética , Síndrome de Noonan/genética , Proteínas Tirosina Fosfatases/genética , Animais , Linhagem da Célula/genética , Anormalidades Craniofaciais/genética , Modelos Animais de Doenças , Comunicação Atrioventricular/genética , Comunicação Atrioventricular/metabolismo , Citometria de Fluxo , Genes Dominantes , Cardiopatias Congênitas/genética , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Mutantes , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transtornos Mieloproliferativos/genética , Testes de Precipitina , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/metabolismo
10.
Oncoimmunology ; 9(1): 1811605, 2020 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-33224628

RESUMO

TGFß is a pleiotropic cytokine that may have both tumor inhibiting and tumor promoting properties, depending on tissue and cellular context. Emerging data support a role for TGFß in suppression of antitumor immunity. Here we show that SAR439459, a pan-TGFß neutralizing antibody, inhibits all active isoforms of human and murine TGFß, blocks TGFß-mediated pSMAD signaling, and TGFß-mediated suppression of T cells and NK cells. In vitro, SAR439459 synergized with anti-PD1 to enhance T cell responsiveness. In syngeneic tumor models, SAR439459 treatment impaired tumor growth, while the combination of SAR439459 with anti-PD-1 resulted in complete tumor regression and a prolonged antitumor immunity. Mechanistically, we found that TGFß inhibition with PD-1 blockade augmented intratumoral CD8+ T cell proliferation, reduced exhaustion, evoked proinflammatory cytokines, and promoted tumor-specific CD8+ T cell responses. Together, these data support the hypothesis that TGFß neutralization using SAR439459 synergizes with PD-1 blockade to promote antitumor immunity and formed the basis for the ongoing clinical investigation of SAR439459 in patients with cancer (NCT03192345).


Assuntos
Terapia de Imunossupressão , Receptor de Morte Celular Programada 1 , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Humanos , Tolerância Imunológica , Camundongos
11.
Trends Biochem Sci ; 28(6): 284-93, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12826400

RESUMO

Src homology-2 (SH2) domain-containing phosphatases (Shps) are a small, highly conserved subfamily of protein-tyrosine phosphatases, members of which are present in both vertebrates and invertebrates. The mechanism of regulation of Shps by ligand binding is now well understood. Much is also known about the normal signaling pathways regulated by each Shp and the consequences of Shp deficiency. Recent studies have identified mutations in human Shp2 as the cause of the inherited disorder Noonan syndrome. Shp2 mutations might also contribute to the pathogenesis of some leukemias. In addition, Shp2 might be a key virulence determinant for the important human pathogen Helicobacter pylori. Despite these efforts, however, the key targets of each Shp have remained elusive. Identifying these substrates remains a major challenge for future research.


Assuntos
Proteínas Tirosina Fosfatases , Transdução de Sinais , Tirosina/metabolismo , Domínios de Homologia de src , Animais , Sítios de Ligação , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Leucemia/metabolismo , Mutação/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Síndrome
12.
J Exp Med ; 210(7): 1419-31, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23797092

RESUMO

The protein-tyrosine phosphatase Shp1 is expressed ubiquitously in hematopoietic cells and is generally viewed as a negative regulatory molecule. Mutations in Ptpn6, which encodes Shp1, result in widespread inflammation and premature death, known as the motheaten (me) phenotype. Previous studies identified Shp1 as a negative regulator of TCR signaling, but the severe systemic inflammation in me mice may have confounded our understanding of Shp1 function in T cell biology. To define the T cell­intrinsic role of Shp1, we characterized mice with a T cell­specific Shp1 deletion (Shp1fl/fl CD4-cre). Surprisingly, thymocyte selection and peripheral TCR sensitivity were unaltered in the absence of Shp1. Instead, Shp1(fl/fl) CD4-cre mice had increased frequencies of memory phenotype T cells that expressed elevated levels of CD44. Activation of Shp1-deficient CD4⁺ T cells also resulted in skewing to the Th2 lineage and increased IL-4 production. After IL-4 stimulation of Shp1- deficient T cells, Stat 6 activation was sustained, leading to enhanced Th2 skewing. Accordingly, we observed elevated serum IgE in the steady state. Blocking or genetic deletion of IL-4 in the absence of Shp1 resulted in a marked reduction of the CD44hi population. Therefore, Shp1 is an essential negative regulator of IL-4 signaling in T lymphocytes.


Assuntos
Homeostase/imunologia , Interleucina-4/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Transdução de Sinais/imunologia , Linfócitos T/enzimologia , Linfócitos T/imunologia , Animais , Memória Imunológica , Interleucina-4/deficiência , Interleucina-4/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Células Th2/enzimologia , Células Th2/imunologia
13.
Diabetes ; 61(8): 1949-58, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22698917

RESUMO

The protein-tyrosine phosphatase Shp1 negatively regulates insulin action on glucose homeostasis in liver and muscle, but its potential role in obesity-linked insulin resistance has not been examined. To investigate the role of Shp1 in hepatic insulin resistance, we generated hepatocyte-specific Shp1 knockout mice (Ptpn6(H-KO)), which were subjected to extensive metabolic monitoring throughout an 8-week standard chow diet (SD) or high-fat diet (HFD) feeding. We report for the first time that Shp1 expression is upregulated in metabolic tissues of HFD-fed obese mice. When compared with their Shp1-expressing Ptpn6(f/f) littermates, Ptpn6(H-KO) mice exhibited significantly lowered fasting glycemia and heightened hepatic insulin sensitivity. After HFD feeding, Ptpn6(H-KO) mice developed comparable levels of obesity as Ptpn6(f/f) mice, but they were remarkably protected from liver insulin resistance, as revealed by euglycemic clamps and hepatic insulin signaling determinations. Although Ptpn6(H-KO) mice still acquired diet-induced peripheral insulin resistance, they were less hyperinsulinemic during a glucose tolerance test because of reduced insulin secretion. Ptpn6(H-KO) mice also exhibited increased insulin clearance in line with enhanced CC1 tyrosine phosphorylation in liver. These results show that hepatocyte Shp1 plays a critical role in the development of hepatic insulin resistance and represents a novel therapeutic target for obesity-linked diabetes.


Assuntos
Resistência à Insulina/fisiologia , Fígado/metabolismo , Obesidade/fisiopatologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Animais , Glicemia/metabolismo , Dieta Hiperlipídica , Deleção de Genes , Insulina/metabolismo , Camundongos , Obesidade/metabolismo
14.
Invest Ophthalmol Vis Sci ; 53(10): 6628-37, 2012 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-22930722

RESUMO

PURPOSE: To determine if the progression of age-related macular degeneration (AMD) is associated with complement activation in the eye. METHODS: Immunohistochemistry and ELISAs were used to determine the distribution, concentration, and activation of the alternative pathway complement proteases factor B (FB) and factor D (FD) and the central complement protein C3 in genotyped human postmortem donor eyes graded as having no or minimal drusen (category 1; controls), large drusen (category 3), and large drusen with advanced AMD (category 4). RESULTS: C3, FB, and FD were present in vitreous and Bruch's membrane choroid (BM/C) interface of the macula of eyes in all tested AMD severity categories (n = 100). C3, FB, and FD were predominantly located to the choroidal vasculature and Bruch's membrane and, together with the serum proteins transferrin and albumin, elevated in BM/C extracts of category 4 eyes (n = 23) compared with category 1 eyes (n = 24). A significant increase in FB activation was found only in vitreous of category 4 eyes (n = 23) compared with category 1 eyes (n = 25). Genetic variants of complement factor H (CFH), C3, C2, and FB associated with increased risk of AMD were correlated with alternative pathway complement activation in vitreous, but not with complement proteins in BM/C protein extracts. CONCLUSIONS: Increased activation of the alternative complement pathway in vitreous was controlled by disease stage and genetic variation in the complement pathway, supporting a role for complement activation in AMD disease pathogenesis.


Assuntos
Complemento C3/metabolismo , Fator B do Complemento/metabolismo , Fator D do Complemento/metabolismo , Via Alternativa do Complemento/genética , Degeneração Macular/genética , Corpo Vítreo/metabolismo , Idoso , Idoso de 80 Anos ou mais , Lâmina Basilar da Corioide/metabolismo , Corioide/metabolismo , Ativação do Complemento , Complemento C3/genética , Fator B do Complemento/genética , Fator D do Complemento/genética , Fator H do Complemento/genética , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Genótipo , Humanos , Imuno-Histoquímica , Degeneração Macular/metabolismo , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Doadores de Tecidos
15.
PLoS One ; 6(4): e19233, 2011 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-21559430

RESUMO

BACKGROUND: MMTV-Cre mouse lines have played important roles in our understanding about the functions of numerous genes in mouse mammary epithelial cells during mammary gland development and tumorigenesis. However, numerous studies have not included MMTV-Cre mice as controls, and many investigators have not indicated which of the different MMTV-Cre founder lines were used in their studies. Here, we describe a lactation defect that severely limits the use of one of the most commonly used MMTV-Cre founder lines. METHODOLOGY/PRINCIPAL FINDINGS: To explore the role of protein tyrosine phosphatase Shp1 in mammary gland development, mice bearing the floxed Shp1 gene were crossed with MMTV-Cre mice and mammary gland development was examined by histological and biochemical techniques, while lactation competency was assessed by monitoring pup growth. Surprisingly, both the Shp1fl/+;MMTV-Cre and MMTV-Cre female mice displayed a severe lactation defect when compared to the Shp1 fl/+ control mice. Histological and biochemical analyses reveal that female mice expressing the MMTV-Cre transgene, either alone or in combination with floxed genes, exhibit defects in lobuloalveolar expansion, presence of large cytoplasmic lipid droplets in luminal alveolar epithelial cells postpartum, and precocious induction of involution. Using a PCR-based genotyping method, the three different founder lines can be distinguished, and we determined that the MMTV-Cre line A, the most widely used MMTV-Cre founder line, exhibits a profound lactation defect that limits its use in studies on mammary gland development. CONCLUSIONS/SIGNIFICANCE: The identification of a lactation defect in the MMTV-Cre line A mice indicates that investigators must use MMTV-Cre alone mice as control in studies that utilize Cre recombinase to excise genes of interest from mammary epithelial cells. Our results also suggest that previous results obtained in studies using the MMTV-Cre line A line should be re-evaluated if the controls did not include mice expressing only Cre recombinase.


Assuntos
Lactação/genética , Glândulas Mamárias Animais/metabolismo , Vírus do Tumor Mamário do Camundongo/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Animais , Apoptose , Feminino , Integrases/metabolismo , Glândulas Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase/métodos , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT5/genética
16.
Annu Rev Immunol ; 25: 473-523, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17291189

RESUMO

Tyrosyl phosphorylation plays a critical role in multiple signaling pathways regulating innate and acquired immunity. Although tyrosyl phosphorylation is a reversible process, we know much more about the functions of protein-tyrosine kinases (PTKs) than about protein-tyrosine phosphatases (PTPs). Genome sequencing efforts have revealed a large and diverse superfamily of PTPs, which can be subdivided into receptor-like (RPTPs) and nonreceptor (NRPTPs). The role of the RPTP CD45 in immune cell signaling is well known, but those of most other PTPs remain poorly understood. Here, we review the mechanism of action, regulation, and physiological functions of NRPTPs in immune cell signaling. Such an analysis indicates that PTPs are as important as PTKs in regulating the immune system.


Assuntos
Genoma Humano/imunologia , Imunidade Inata , Família Multigênica/imunologia , Proteínas Tirosina Fosfatases/imunologia , Transdução de Sinais/imunologia , Animais , Genoma Humano/genética , Humanos , Família Multigênica/genética , Proteínas Tirosina Fosfatases/genética , Transdução de Sinais/genética
17.
Immunity ; 27(1): 35-48, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17600736

RESUMO

Spontaneous loss-of-function mutations in the protein-tyrosine phosphatase Shp1 cause the motheaten phenotype, characterized by widespread inflammation and autoimmunity. Because Shp1 is expressed in all hematopoietic cells, it has been unclear which aspects of the motheaten phenotypes are primary effects of Shp1 deficiency. We generated mice (Ptpn6(f/f);CD19-cre) that delete Shp1 specifically in B cells. Analysis of these mice indicates that the increase in B-1a cells in motheaten mice is a cell-autonomous consequence of Shp1 deficiency. Shp1-deficient B-1a cells could be derived from adult bone marrow and had N-nucleotide additions, consistent with an adult origin. Shp1 deficiency altered calcium response evoked by B cell antigen receptors and impaired CD40-evoked proliferation. Young Ptpn6(f/f);CD19-cre mice exhibited elevated serum immunoglobulins and impaired antibody responses to immunization, whereas older Ptpn6(f/f);CD19-cre mice developed systemic autoimmunity, characterized by DNA antibodies and immune complex glomerulonephritis. Thus, Shp1 deficiency in B cells alone perturbs B cell development and causes autoimmune disease.


Assuntos
Doenças Autoimunes/enzimologia , Doenças Autoimunes/genética , Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/imunologia , Diferenciação Celular/imunologia , Deleção de Genes , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Animais , Complexo Antígeno-Anticorpo/metabolismo , Doenças Autoimunes/patologia , Subpopulações de Linfócitos B/citologia , Diferenciação Celular/genética , Células Cultivadas , Glomerulonefrite/enzimologia , Glomerulonefrite/genética , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência
18.
Immunity ; 25(5): 769-81, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17081782

RESUMO

T cell activation through the T cell receptor (TCR) is subsequently modified by secondary signals that are either stimulatory or inhibitory. We show that CEACAM1 adhesion molecule isoforms containing a long cytoplasmic domain inhibited multiple T cell functions as a consequence of TCR ligation. Overexpression of CEACAM1 resulted in decreased proliferation, allogeneic reactivity, and cytokine production in vitro and delayed type hypersensitivity and inflammatory bowel disease in mouse models in vivo. Conditioned deletion of CEACAM1 in T cells caused increased TCR-CD3 complex signaling. This T cell regulation was dependent upon the presence of immunoreceptor tyrosine-based inhibition motifs (ITIM) within the cytoplasmic domain of CEACAM1 and the Src homology 2 domain-containing protein tyrosine-phosphatase 1 (SHP1) in the T cell. Thus, CEACAM1 overexpression or deletion in T cells resulted in T cell inhibition or activation, respectively, revealing a role for CEACAM1 as a class of inhibitory receptors potentially amenable to therapeutic manipulation.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígeno Carcinoembrionário/imunologia , Ativação Linfocitária/imunologia , Isoformas de Proteínas/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Transferência Adotiva , Motivos de Aminoácidos , Animais , Linfócitos T CD4-Positivos/metabolismo , Antígeno Carcinoembrionário/química , Colite/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Immunoblotting , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Isoformas de Proteínas/química , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transfecção
19.
Proc Natl Acad Sci U S A ; 102(29): 10238-43, 2005 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-16006513

RESUMO

Mice with heterozygous deletion of the PTEN tumor suppressor gene develop a range of epithelial neoplasia as well as lymphoid hyperplasia. Previous studies suggest that PTEN suppresses tumor formation by acting as a phosphoinositide phosphatase to limit signaling by phosphoinositide 3-kinase (PI3K). Here, we examined the effect of deleting various regulatory subunits of PI3K (p85alpha and p85beta) on epithelial neoplasia and lymphoid hyperplasia in PTEN+/- mice. Interestingly, we found the loss of one p85alpha allele with or without the loss of p85beta led to increased incidence of intestinal polyps. Signaling downstream of PI3K was enhanced in the PTEN+/-p85alpha+/-p85beta-/- polyps, as judged by an increased fraction of both cells with cytoplasmic staining of the transcription factor FKHR and cells with positive staining for the proliferation marker Ki-67. In contrast, the incidence of prostate intraepithelial neoplasia was not significantly altered in PTEN+/- mice heterozygous for p85alpha or null for p85beta, whereas the fraction of proliferating cells in prostate intraepithelial neoplasia was reduced in mice lacking p85beta. Finally, there was no significant change in T lymphocyte hyperplasia in the PTEN+/- mice with various p85 deletions, although anti-CD3-stimulated AKT activation was somewhat reduced in the p85alpha+/- background. These results indicate that decreasing the levels of different p85 regulatory subunits can result in enhanced PI3K signaling in some tissues and decreased PI3K signaling in others, supporting the model that, although p85 proteins are essential for class I(A) PI3K signaling, they can function as inhibitors of PI3K signaling in some tissues and thus suppress tumor formation.


Assuntos
Hiperplasia do Linfonodo Gigante/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Transdução de Sinais/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Western Blotting , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead , Heterozigoto , Imuno-Histoquímica , Pólipos Intestinais/metabolismo , Antígeno Ki-67 , Masculino , Camundongos , PTEN Fosfo-Hidrolase , Sequências Reguladoras de Ácido Nucleico/genética , Linfócitos T/metabolismo , Fatores de Transcrição
20.
J Immunol ; 175(5): 3235-43, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16116214

RESUMO

Cytotoxic lymphocytes express a large family of granule serine proteases, including one member, granzyme (Grz)M, with a unique protease activity, restricted expression, and distinct gene locus. Although a number of Grzs, including GrzM, have been shown to mediate target cell apoptosis in the presence of perforin, the biological activity of Grz has been restricted to control of a number of viral pathogens, including two natural mouse pathogens, ectromelia, and murine CMV (MCMV). In this article, we describe the first reported gene targeting of GrzM in mice. GrzM-deficient mice display normal NK cell/T cell development and homeostasis and intact NK cell-mediated cytotoxicity of tumor targets as measured by membrane damage and DNA fragmentation. GrzM-deficient mice demonstrated increased susceptibility to MCMV infection typified by the presence of more viral inclusions and transiently higher viral burden in the visceral organs of GrzM-deficient mice compared with wild-type (WT) mice. The cytotoxicity of NK cells from MCMV-infected GrzM-deficient mice remained unchanged and, like WT control mice, GrzM-deficient mice eventually effectively cleared MCMV infection from the visceral organs. In contrast, GrzM-deficient mice were as resistant as WT control mice to mouse pox ectromelia infection, as well as challenge with a number of NK cell-sensitive tumors. These data confirm a role for GrzM in the host response to MCMV infection, but suggest that GrzM is not critical for NK cell-mediated cytotoxicity.


Assuntos
Ectromelia Infecciosa/imunologia , Infecções por Herpesviridae/imunologia , Muromegalovirus , Serina Endopeptidases/fisiologia , Animais , Citotoxicidade Imunológica , Granzimas , Infecções por Herpesviridae/patologia , Homeostase , Células Matadoras Naturais/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Muromegalovirus/imunologia , Neoplasias Experimentais/imunologia , Linfócitos T/fisiologia
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