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1.
Crit Rev Immunol ; 33(3): 219-43, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23756245

RESUMEN

Caspase recruitment domain-containing membrane-associated guanylate kinase protein-1 (CARMA1), a member of the membrane associated guanylate kinase (MAGUK) family of kinases, is essential for T lymphocyte activation and proliferation via T-cell receptor (TCR) mediated NF-κB activation. Recent studies suggest a broader role for CARMA1 regulating other T-cell functions as well as a role in non-TCR-mediated signaling pathways important for lymphocyte development and functions. In addition, CARMA1 has been shown to be an important component in the pathogenesis of several human diseases. Thus, comprehensively defining its mechanisms of action and regulation could reveal novel therapeutic targets for T-cell-mediated diseases and lymphoproliferative disorders.


Asunto(s)
Proteínas Adaptadoras de Señalización CARD/inmunología , Guanilato Ciclasa/inmunología , Linfocitos T/inmunología , Animales , Proteínas Adaptadoras de Señalización CARD/química , Guanilato Ciclasa/química , Humanos , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal
2.
J Immunol ; 189(6): 2879-89, 2012 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-22875804

RESUMEN

Influenza is a major cause of morbidity and mortality in the United States. Studies have shown that excessive T cell activity can mediate pneumonitis in the setting of influenza infection, and data from the 2009 H1N1 pandemic indicate that critical illness and respiratory failure postinfection were associated with greater infiltration of the lungs with CD8+ T cells. T cell Ig and mucin domain 3 (Tim3) is a negative regulator of Th1/Tc1-type immune responses. Activation of Tim3 on effector T cells has been shown to downregulate proliferation, cell-mediated cytotoxicity, and IFN-γ production, as well as induce apoptosis. In this article, we demonstrate that deletion of the terminal cytoplasmic domain of the Tim3 gene potentiates its ability to downregulate Tc1 inflammation, and that this enhanced Tim3 activity is associated with decreased phosphorylation of the TCR-CD3ζ-chain. We then show that mice with this Tim3 mutation infected with influenza are protected from morbidity and mortality without impairment in viral clearance or functional heterotypic immunity. This protection is associated with decreased CD8+ T cell proliferation and decreased production of inflammatory cytokines, including IFN-γ. Furthermore, the Tim3 mutation was protective against mortality in a CD8+ T cell-specific model of pneumonitis. These data suggest that Tim3 could be targeted to prevent immunopathology during influenza infection and demonstrate a potentially novel signaling mechanism used by Tim3 to downregulate the Tc1 response.


Asunto(s)
Infecciones por Orthomyxoviridae/inmunología , Receptores Virales/metabolismo , Regulación hacia Arriba/inmunología , Animales , Complejo CD3/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Citotoxicidad Inmunológica/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Receptor 2 Celular del Virus de la Hepatitis A , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/mortalidad , Fosforilación/genética , Fosforilación/inmunología , Receptores Virales/genética , Receptores Virales/fisiología , Eliminación de Secuencia/genética , Eliminación de Secuencia/inmunología , Transducción de Señal/genética , Transducción de Señal/inmunología , Análisis de Supervivencia , Regulación hacia Arriba/genética
3.
J Immunol ; 187(12): 6197-207, 2011 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-22075698

RESUMEN

CARMA1 is a lymphocyte-specific scaffold protein necessary for T cell activation. Deletion of CARMA1 prevents the development of allergic airway inflammation in a mouse model of asthma due to a defect in naive T cell activation. However, it is unknown if CARMA1 is important for effector and memory T cell responses after the initial establishment of inflammation, findings that would be more relevant to asthma therapies targeted to CARMA1. In the current study, we sought to elucidate the role of CARMA1 in T cells that have been previously activated. Using mice in which floxed CARMA1 exons can be selectively deleted in T cells by OX40-driven Cre recombinase (OX40(+/Cre)CARMA1(F/F)), we report that CD4(+) T cells from these mice have impaired T cell reactivation responses and NF-κB signaling in vitro. Furthermore, in an in vivo recall model of allergic airway inflammation that is dependent on memory T cell function, OX40(+/Cre)CARMA1(F/F) mice have attenuated eosinophilic airway inflammation, T cell activation, and Th2 cytokine production. Using MHC class II tetramers, we demonstrate that the development and maintenance of Ag-specific memory T cells is not affected in OX40(+/Cre)CARMA1(F/F) mice. In addition, adoptive transfer of Th2-polarized OX40(+/Cre)CARMA1(F/F) Ag-specific CD4(+) T cells into wild-type mice induces markedly less airway inflammation in response to Ag challenge than transfer of wild-type Th2 cells. These data demonstrate a novel role for CARMA1 in effector and memory T cell responses and suggest that therapeutic strategies targeting CARMA1 could help treat chronic inflammatory disorders such as asthma.


Asunto(s)
Asma/inmunología , Proteínas Adaptadoras de Señalización CARD/fisiología , Células Th2/inmunología , Enfermedad Aguda , Traslado Adoptivo , Animales , Asma/genética , Asma/patología , Proteínas Adaptadoras de Señalización CARD/deficiencia , Proteínas Adaptadoras de Señalización CARD/genética , Polaridad Celular/genética , Polaridad Celular/inmunología , Células Cultivadas , Enfermedad Crónica , Modelos Animales de Enfermedad , Memoria Inmunológica/genética , Memoria Inmunológica/inmunología , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/fisiología , Integrasas/genética , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/fisiología , Receptores OX40/biosíntesis , Receptores OX40/genética , Hipersensibilidad Respiratoria/genética , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/prevención & control , Células Th2/citología , Células Th2/trasplante
4.
Immunology ; 136(3): 352-60, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22486311

RESUMEN

Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non-overlapping terminal and internal epitopes of the F. tularensis LPS O-polysaccharide (OAg). In the current study, we used F. tularensis LPS oligosaccharides of defined OAg repeat length as molecular rulers in competition ELISA to demonstrate that the epitope targeted by the terminal OAg-binding mAb FB11 is contained within one tetrasaccharide repeat whereas the epitope targeted by the internal OAg-binding mAb Ab52 spans two tetrasaccharide repeats. Both mAbs conferred survival to BALB/c mice infected intranasally with the F. tularensis type B live vaccine strain and prolonged survival of BALB/c mice infected intranasally with the highly virulent F. tularensis type A strain SchuS4. The protective effects correlated with reduced bacterial burden in mAb-treated infected mice. These results indicate that an oligosaccharide with two OAg tetrasaccharide repeats covers both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularaemia. Furthermore, the FB11 and Ab52 mAbs could serve as reporters to monitor the response of vaccine recipients to protective B-cell epitopes of F. tularensis OAg.


Asunto(s)
Epítopos de Linfocito B/química , Francisella tularensis/inmunología , Antígenos O/química , Infecciones del Sistema Respiratorio/inmunología , Infecciones del Sistema Respiratorio/prevención & control , Tularemia/inmunología , Tularemia/prevención & control , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Monoclonales , Carga Bacteriana , Vacunas Bacterianas/inmunología , Modelos Animales de Enfermedad , Femenino , Francisella tularensis/patogenicidad , Ratones , Ratones Endogámicos BALB C , Oligosacáridos/química , Oligosacáridos/inmunología , Infecciones del Sistema Respiratorio/microbiología , Tularemia/microbiología
5.
Immunol Lett ; 114(1): 16-22, 2007 Nov 30.
Artículo en Inglés | MEDLINE | ID: mdl-17920694

RESUMEN

Athymic nude mice bearing subcutaneous tumor xenografts of the human anti-colorectal cancer cell line SW480 were used as a preclinical model to explore anti-tumor immunotherapies. Intratumor or systemic treatment of the mice with murine anti-SW480 serum, recombinant anti-SW480 polyclonal antibodies, or the anti-colorectal cancer monoclonal antibody CO17-1A, caused retardation or regression of SW480 tumor xenografts. Interestingly, when mice that had regressed their tumors were re-challenged with SW480 cells, these mice regressed the new tumors without further antibody treatment. Adoptive transfer of spleen cells from mice that had regressed their tumors conferred anti-tumor immunity to naïve nude mice. Pilot experiments suggest that the transferred anti-tumor immunity is mediated by T cells of both gammadelta and alphabeta lineages. These results demonstrate that passive anti-tumor immunotherapy can elicit active immunity and support a role for extra-thymic gammadelta and alphabeta T cells in tumor rejection. Implications for potential immunotherapies include injection of tumor nodules in cancer patients with anti-tumor antibodies to induce anti-tumor T cell immunity.


Asunto(s)
Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/uso terapéutico , Inmunoterapia Activa , Inmunoterapia Adoptiva , Neoplasias Experimentales/terapia , Animales , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/inmunología , Trasplante Heterólogo
6.
Immunol Lett ; 112(2): 92-103, 2007 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-17764754

RESUMEN

Tularemia is caused by the Gram-negative facultative intracellular bacterium Francisella tularensis, which has been classified as a category A select agent-a likely bioweapon. The high virulence of F. tularensis and the threat of engineered antibiotic resistant variants warrant the development of new therapies to combat this disease. We have characterized 14 anti-Francisella hybridoma antibodies derived from mice infected with F. tularensis live vaccine strain (LVS) for potential use as immunotherapy of tularemia. All 14 antibodies cross-reacted with virulent F. tularensis type A clinical isolates, 8 bound to a purified preparation of LVS LPS, and 6 bound to five protein antigens, identified by proteome microarray analysis. An IgG2a antibody, reactive with the LPS preparation, conferred full protection when administered either systemically or intranasally to BALB/c mice post challenge with a lethal dose of intranasal LVS; three other antibodies prolonged survival. These anti-Francisella hybridoma antibodies could be converted to chimeric versions with mouse V regions and human C regions to serve as components of a recombinant polyclonal antibody for clinical testing as immunotherapy of tularemia. The current study is the first to employ proteome microarrays to identify the target antigens of anti-Francisella monoclonal antibodies and the first to demonstrate the systemic and intranasal efficacy of monoclonal antibodies for post-exposure treatment of respiratory tularemia.


Asunto(s)
Anticuerpos Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Hibridomas/inmunología , Tularemia/inmunología , Tularemia/terapia , Administración Intranasal , Traslado Adoptivo , Animales , Anticuerpos Antibacterianos/clasificación , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/clasificación , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Vacunas Bacterianas/administración & dosificación , Línea Celular Tumoral , Reacciones Cruzadas , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Francisella tularensis/inmunología , Francisella tularensis/patogenicidad , Humanos , Hibridomas/microbiología , Inmunización/métodos , Lipopolisacáridos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Análisis por Matrices de Proteínas , Tularemia/microbiología
7.
Elife ; 42015 May 22.
Artículo en Inglés | MEDLINE | ID: mdl-25998054

RESUMEN

The balance between Th17 and T regulatory (Treg) cells critically modulates immune homeostasis, with an inadequate Treg response contributing to inflammatory disease. Using an unbiased chemical biology approach, we identified a novel role for the dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A in regulating this balance. Inhibition of DYRK1A enhances Treg differentiation and impairs Th17 differentiation without affecting known pathways of Treg/Th17 differentiation. Thus, DYRK1A represents a novel mechanistic node at the branch point between commitment to either Treg or Th17 lineages. Importantly, both Treg cells generated using the DYRK1A inhibitor harmine and direct administration of harmine itself potently attenuate inflammation in multiple experimental models of systemic autoimmunity and mucosal inflammation. Our results identify DYRK1A as a physiologically relevant regulator of Treg cell differentiation and suggest a broader role for other DYRK family members in immune homeostasis. These results are discussed in the context of human diseases associated with dysregulated DYRK activity.


Asunto(s)
Diferenciación Celular/inmunología , Homeostasis/inmunología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T Reguladores/metabolismo , Células Th17/metabolismo , Animales , Técnicas de Cultivo de Célula , Harmina/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Quinasas DyrK
8.
Monoclon Antib Immunodiagn Immunother ; 33(4): 235-45, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-25171003

RESUMEN

The O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis (Ft), which is both a capsular polysaccharide and a component of lipopolysaccharide, is comprised of tetrasaccharide repeats and induces antibodies mainly against repeating internal epitopes. We previously reported on several BALB/c mouse monoclonal antibodies (MAbs) that bind to internal Ft OAg epitopes and are protective in mouse models of respiratory tularemia. We now characterize three new internal Ft OAg IgG2a MAbs, N203, N77, and N24, with 10- to 100-fold lower binding potency than previously characterized internal-OAg IgG2a MAbs, despite sharing one or more variable region germline genes with some of them. In a mouse model of respiratory tularemia with the highly virulent Ft type A strain SchuS4, the three new MAbs reduced blood bacterial burden with potencies that mirror their antigen-binding strength; the best binder of the new MAbs, N203, prolonged survival in a dose-dependent manner, but was at least 10-fold less potent than the best previously characterized IgG2a MAb, Ab52. X-ray crystallographic studies of N203 Fab showed a flexible binding site in the form of a partitioned groove, which cannot provide as many contacts to OAg as does the Ab52 binding site. These results reveal structural features of antibodies at the low end of reactivity with multi-repeat microbial carbohydrates and demonstrate that such antibodies still have substantial protective effects against infection.


Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales de Origen Murino/inmunología , Francisella tularensis/genética , Antígenos O/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Monoclonales de Origen Murino/genética , Secuencia de Bases , Cristalografía por Rayos X , Francisella tularensis/inmunología , Inmunoensayo , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Análisis de Secuencia de ADN
9.
PLoS One ; 9(6): e99847, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24968190

RESUMEN

The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA. In a mouse model of respiratory tularemia with the highly pathogenic Ft type A strain SchuS4, the Ab64 and N200 IgG2a mAbs, which block each other's binding to and are sensitive to the same two point mutations in FtGroEL, reduced bacterial burden indicating that they target protective GroEL B-cell epitopes. The Ab64 and N200 epitopes, as well as those of three other mAbs with different crossblocking profiles, Ab53, N3, and N30, were mapped by hydrogen/deuterium exchange-mass spectrometry (DXMS) and visualized on a homology model of FtGroEL. This model was further supported by its experimentally-validated computational docking to the X-ray crystal structures of Ab64 and Ab53 Fabs. The structural analysis and DXMS profiles of the Ab64 and N200 mAbs suggest that their protective effects may be due to induction or stabilization of a conformational change in FtGroEL.


Asunto(s)
Linfocitos B/inmunología , Proteínas Bacterianas/inmunología , Chaperonina 60/inmunología , Francisella tularensis/inmunología , Tularemia/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/clasificación , Anticuerpos Monoclonales/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión de Anticuerpos , Chaperonina 60/química , Chaperonina 60/genética , Epítopos/genética , Epítopos/inmunología , Ratones , Datos de Secuencia Molecular , Mutación Puntual , Unión Proteica
10.
Hybridoma (Larchmt) ; 30(1): 19-28, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21466282

RESUMEN

The lipopolysaccharide (LPS) of Francisella tularensis (Ft), the Gram negative bacterium that causes tularemia, has been shown to be a main protective antigen in mice and humans; we have previously demonstrated that murine anti-Ft LPS IgG2a monoclonal antibodies (MAbs) can protect mice against otherwise lethal intranasal infection with the Ft live vaccine strain (LVS). Here we show that four IgG2a anti-LPS MAbs are specific for the O-polysaccharide (O-antigen [OAg]) of Ft LPS. But whereas three of the MAbs bind to immunodominant repeating internal epitopes, one binds to a unique terminal epitope of Ft OAg. This was deduced from its even binding to both long and short chains of the LPS ladder in Western blots, its rapid decrease in ELISA binding to decreasing solid-phase LPS concentrations, its inability to compete for LPS binding with a representative of the other three MAbs, and its inability to immunoprecipitate OAg despite its superior agglutination titer. Biacore analysis showed the end-binding MAb to have higher bivalent avidity for Ft OAg than the internal-binding MAbs and provided an immunogenicity explanation for the predominance of internal-binding anti-Ft OAg MAbs. These findings demonstrate that non-overlapping epitopes can be targeted by antibodies to Ft OAg, which may inform the design of vaccines and immunotherapies against tularemia.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Francisella tularensis/inmunología , Inmunoglobulina G/inmunología , Lipopolisacáridos/inmunología , Antígenos O/inmunología , Anticuerpos Monoclonales/genética , Unión Competitiva/inmunología , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Epítopos/genética , Inmunoglobulina G/genética , Inmunoprecipitación , Antígenos O/genética
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