RESUMEN
TWEAK is a member of the TNF ligand family that induces angiogenesis in vivo. We report cloning of a receptor for TWEAK (TweakR) from a human umbilical vein endothelial cell (HUVEC) library. The mature form of TweakR has only one hundred and two amino acids and six cysteine residues in its extracellular region. Five different assays demonstrate TWEAK-TweakR binding, and the interaction affinity constant (Kd) is within a physiologically relevant range of 2.3 +/- 0.1 nM. The TweakR cytoplasmic domain binds TRAFs 1, 2, and 3. Cross-linking of TweakR induces HUVEC growth, and mRNA levels are upregulated in vitro by a variety of agents and in vivo following arterial injury. Soluble TweakR inhibits endothelial cell migration in vitro and corneal angiogenesis in vivo.
Asunto(s)
Endotelio Vascular/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Clonación Molecular , Citocina TWEAK , Humanos , Ligandos , Datos de Secuencia Molecular , Neovascularización Fisiológica , Ratas , Alineación de Secuencia , Receptor de TWEAK , Factores de Necrosis TumoralRESUMEN
CD40 is a member of the tumor necrosis factor receptor family and was first identified with a monoclonal antibody raised against bladder carcinoma. Recombinant human CD40L has been shown previously to have a direct antitumor effect on an ovarian cancer cell line and ovarian carcinoma cells isolated from ascites fluid. We show here that rhuCD40L inhibits the growth of several ovarian adenocarcinomas derived from surgical specimens and grown as xenografts in severe combined immunodeficient mice. Two 14-day treatment cycles were more effective than one. This effect is apparently not mediated by natural killer cells, because blocking natural killer cell activity by antiasialo GM-1 did not diminish this effect. In addition to suppression of tumor growth, treatment with rhuCD40L resulted in an increased expression of FasL, an increase in apoptosis, and histological changes including increased fibrosis and areas of tumor destruction. Using this model, we examined the efficacy of rhuCD40L in combination with chemotherapeutic agents. The antitumor effect of rhuCD40L in combination with 4 mg/kg cisplatin (CDDP) was increased over the effect of CDDP alone. Furthermore, rhuCD40L increased the efficacy of a suboptimal dose of CDDP (2mg/kg) such that it matched that of high-dose CDDP alone. These data suggest a role for rhuCD40L therapy in combination with platinum based regimens for primary treatment of epithelial ovarian tumors.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ligando de CD40/farmacología , Cisplatino/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Animales , Cisplatino/administración & dosificación , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Humanos , Ratones , Ratones SCID , Proteínas Recombinantes/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
New members of the extended MHC class I-like family were identified based on their ability to bind human cytomegalovirus glycoprotein UL16 and/or their mutual homology. Soluble UL16 binding proteins (ULBP) competed with each other for binding to NK cells. Treatment of human and mouse NK cells with ULBP led to increased production of cytokines/chemokines, proliferation, cytotoxic activity and up-regulation of activation-associated surface molecules. The presence of ULBP during the stimulation phase of the CTL assay caused increased cytotoxic activity. Addition of soluble recombinant UL16 protein inhibited the biological activities mediated by ULBP, suggesting the existence of a novel mechanism utilized by CMV to evade elimination by the host immune system.
Asunto(s)
Proteínas Portadoras/metabolismo , Citomegalovirus/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Proteínas del Envoltorio Viral/metabolismo , Animales , Unión Competitiva , Proteínas Portadoras/antagonistas & inhibidores , División Celular/efectos de los fármacos , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Citomegalovirus/inmunología , Citotoxicidad Inmunológica , Citometría de Flujo , Proteínas Ligadas a GPI , Humanos , Inmunoglobulina G/inmunología , Péptidos y Proteínas de Señalización Intercelular , Interleucina-15/inmunología , Péptidos y Proteínas de Señalización Intracelular , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Activación de Linfocitos/efectos de los fármacos , Recuento de Linfocitos , Proteínas de la Membrana , Ratones , Ratones SCID , Unión Proteica , Solubilidad , Bazo/citología , Linfocitos T Citotóxicos/inmunología , Proteínas del Envoltorio Viral/farmacologíaRESUMEN
The human cytomegalovirus glycoprotein, UL16, binds to two members of a novel family of molecules, the ULBPs, and to the MHC class I homolog, MICB. The ULBPs are GPI-linked glycoproteins belonging to the extended MHC class I family but are only distantly related to MICB. The ULBP and MICB molecules are ligands for the activating receptor, NKG2D/DAP10, and this interaction is blocked by a soluble form of UL16. The ULBPs stimulate cytokine and chemokine production from NK cells, and expression of ULBPs in NK cell-resistant target cells confers susceptibility to NK cell cytotoxicity. Masking of NK cell recognition of ULBP or MIC antigens by UL16 provides a potential mechanism by which human cytomegalovirus-infected cells might evade attack by the immune system.
Asunto(s)
Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Citomegalovirus/inmunología , Citomegalovirus/metabolismo , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/metabolismo , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/genética , Línea Celular , Citomegalovirus/patogenicidad , Citotoxicidad Inmunológica , Cartilla de ADN/genética , Proteínas Ligadas a GPI , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular , Ligandos , Proteínas de la Membrana , Datos de Secuencia Molecular , Subfamilia K de Receptores Similares a Lectina de Células NK , Receptores de Células Asesinas Naturales , Homología de Secuencia de AminoácidoRESUMEN
CD7 is a 40-kDa protein found primarily on T, NK, and pre-B cells; the function of the CD7 protein in the immune system is largely unknown. The K12 (SECTM1) protein was originally identified by its location just upstream of the CD7 locus. The K12 gene encodes a transmembrane protein of unknown function. In order to clone a K12-binding protein, we generated a soluble version of the human K12 protein by fusing its extracellular domain to the Fc portion of human IgG(1). Flow cytometry experiments showed that the K12-Fc fusion protein bound at high levels to both human T and NK cells. Precipitation experiments using K12-Fc on (35)S-radiolabeled NK cells lysates indicated that the K12 cognate was an approximately 40-kDa protein. A human peripheral blood T cell cDNA expression library was screened with the K12-Fc protein, and two independent, positive cDNA clones were identified and sequenced. Both cDNAs encoded the same protein, which was CD7. Thus, K12 and CD7 are cognate proteins that are located next to each other on human chromosome 17q25. Additionally, we have cloned the gene encoding the mouse homologue of K12, shown that it maps near the mouse CD7 gene on chromosome 11, and established that the mouse K12 protein binds to mouse, but not human, CD7. Mouse K12-Fc inhibited in a dose-dependent manner concanavalin A-induced proliferation, but not anti-TcRalpha/beta induced proliferation, of mouse lymph node T cells. Human K12-Fc stimulated the up-regulation of CD25, CD54, and CD69 on human NK cells in vitro.
Asunto(s)
Antígenos CD7/genética , Cromosomas Humanos Par 17 , Proteínas de la Membrana/genética , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD7/metabolismo , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/análisis , ADN Complementario/genética , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Daily treatment of mice with fms-like tyrosine kinase 3 ligand (Flt3L) leads to a significant increase in the number of dendritic cells and induces antitumor immunity. Here, we show that Flt3L and CD40 ligand (CD40L) synergize in the generation of immune responses against two poorly immunogenic tumors, leading to complete tumor rejection in a high proportion of mice. Rechallenge of the Flt3L + CD40L-treated mice with the immunizing tumor resulted in complete inhibition of tumor growth, indicating that these animals had developed long-lasting antitumor immunity. In addition, we demonstrate that endogenous CD40L plays a critical role in antitumor immunity, since blockade of CD40-CD40L interactions in vivo prevents the generation of antitumor immunity in therapeutic and vaccination protocols. Dendritic cells generated in mice treated with Flt3L alone or in combination with CD40L were equally potent in stimulating allogeneic T cells and expressed similar levels of MHC class II, CD80, and CD86. However, mice treated with Flt3L + CD40L had significantly more dendritic cells than mice treated with either of the cytokines alone, suggesting that CD40L promotes the proliferation and/or survival of dendritic cells generated by Flt3L treatment. Dendritic cells generated in this manner are likely to be involved in the priming of antitumor immune responses.
Asunto(s)
Células Dendríticas/citología , Células Dendríticas/inmunología , Glicoproteínas de Membrana/administración & dosificación , Glicoproteínas de Membrana/fisiología , Proteínas de la Membrana/administración & dosificación , Proteínas de la Membrana/fisiología , Sarcoma Experimental/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/fisiología , Animales , Antígenos CD40/fisiología , Ligando de CD40 , Recuento de Células , División Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/metabolismo , Sinergismo Farmacológico , Femenino , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Inyecciones Subcutáneas , Interleucina-12/biosíntesis , Interleucina-12/genética , Ligandos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Trasplante de Neoplasias , ARN Mensajero/biosíntesis , Sarcoma Experimental/genética , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Regulación hacia Arriba/inmunologíaRESUMEN
CD40 is present on B cells, monocytes, dendritic cells, and endothelial cells, as well as a variety of neoplastic cell types, including carcinomas. CD40 stimulation by an antibody has previously been demonstrated to induce activation-induced cell death in aggressive histology human B-cell lymphoma cell lines. Therefore, we wanted to assess the effects of a recombinant soluble human CD40 ligand (srhCD40L) on human breast carcinoma cell lines. Human breast carcinoma cell lines were examined for CD40 expression by flow cytometry. CD40 expression could be detected on several human breast cancer cell lines and this could be augmented with interferon-gamma. The cell lines were then incubated with a srhCD40L to assess effects on in vitro growth. srhCD40L significantly inhibited the proliferation of the CD40(+) human breast cancer cell lines. This inhibition could also be augmented with interferon-gamma. Viability was also affected and this was shown to be due to increased apoptosis of the cell lines in response to the ligand. Treatment of tumor-bearing mice was then performed to assess the in vivo efficacy of the ligand. Treatment of tumor-bearing SCID mice with the ligand resulted in significant increases in survival. Thus, CD40 stimulation by its ligand directly inhibits human breast carcinoma cells in vitro and in vivo. These results suggest that srhCD40L may be of clinical use to inhibit human breast carcinoma growth.
Asunto(s)
Neoplasias de la Mama/patología , Antígenos CD40/fisiología , Glicoproteínas de Membrana/uso terapéutico , Animales , Antígenos CD/genética , Antígenos CD/fisiología , Apoptosis , Neoplasias de la Mama/inmunología , Antígenos CD40/genética , Ligando de CD40 , Carcinoma Ductal de Mama/patología , División Celular , Supervivencia Celular , Femenino , Citometría de Flujo , Humanos , Ligandos , Glicoproteínas de Membrana/toxicidad , Ratones , Ratones SCID , Proteínas Recombinantes/uso terapéutico , Proteínas Recombinantes/toxicidad , Trasplante Heterólogo , Células Tumorales CultivadasAsunto(s)
Células Dendríticas/inmunología , Supervivencia de Injerto/inmunología , Trasplante de Corazón/inmunología , Interleucina-17/inmunología , Receptores de Interleucina/uso terapéutico , Linfocitos T/inmunología , Animales , Células de la Médula Ósea/inmunología , Células Cultivadas , Interleucina-17/antagonistas & inhibidores , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos , Receptores de Interleucina/inmunología , Receptores de Interleucina/metabolismo , Receptores de Interleucina-17 , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Linfocitos T/efectos de los fármacos , Trasplante HomólogoRESUMEN
Eight different CD40 mAb shared with soluble trimeric CD40 ligand (sCD40LT) the capacity to rescue germinal center (GC) B cells from spontaneous apoptosis and to suppress antigen receptor-driven apoptosis in group I Burkitt's lymphoma cells. Three mAb (G28-5, M2 and M3) mimicked sCD40LT in its ability to promote strong homotypic adhesion in resting B cells, whereas others (EA5, BL-OGY/C4 and 5C3) failed to stimulate strong clustering. Binding studies revealed that only those mAb that promoted strong B cell clustering bound at, or near to, the CD40L binding site. While all eight mAb and sCD40LT were capable of synergizing with IL-4 or phorbol ester for promoting DNA synthesis in resting B cells, co-stimulus-independent activation of the cells into cycle through CD40 related directly to the extent of receptor cross-linking. Thus, mAb which bound outside the CD40L binding site synergized with sCD40LT for promoting DNA synthesis; maximal levels of stimulation were achieved by presenting any of the mAb on CD32 transfectants in the absence of sCD40LT or by cross-linking bound sCD40LT with a second antibody. Monomeric sCD40L, which was able to promote rescue of GC B cells from apoptosis, was unable to drive resting B cells into cycle. These studies demonstrate that CD40-dependent rescue of human B cells from apoptosis requires minimal cross-linking and is essentially epitope independent, whereas the requirements for promoting cell cycle progression and homotypic adhesion are more stringent. Possible mechanisms underlying these differences and their physiological significance are discussed.
Asunto(s)
Apoptosis , Linfocitos B/inmunología , Antígenos CD40/inmunología , Centro Germinal/inmunología , Activación de Linfocitos , Linfocitos B/citología , Unión Competitiva , Ligando de CD40 , Adhesión Celular , Ciclo Celular , Mapeo Epitopo , Epítopos , Centro Germinal/citología , Humanos , Recubrimiento Inmunológico , Glicoproteínas de MembranaRESUMEN
Antibodies to CD40 have been demonstrated to promote B-cell growth and differentiation in vitro. In order to determine if CD40 stimulation could promote antigen-specific human immunoglobulin (Ig) production in vivo, we examined the effects of anti-human CD40 MoAb in an in vivo system where human peripheral blood lymphocytes (huPBL) were engrafted into mice with severe combined immune deficiency (SCID). The huPBL-SCID mice were then given various doses of diphtheria-tetanus toxoid (DT) vaccine and were examined for the presence of human DT-specific antibodies by ELISA. Surprisingly, treatment with anti-CD40 significantly lowered background DT responses versus untreated chimeras in unimmunized huPBL-SCID mice. However, after immunization, huPBL-SCID mice treated with anti-CD40 MoAb responded to a significantly greater extent in response to the vaccine compared with control huPBL-SCID mice, although total Ig levels were sometimes lower in anti-CD40-treated mice. The predominant Ig isotype induced after immunization was IgG. Thus, CD40 stimulation promotes human secondary IgG responses in huPBL-SCID mice. These data demonstrate that CD40 stimulation is capable of promoting antigen-specific human B-cell responses in vivo.
Asunto(s)
Antígenos CD40/farmacología , Quimera/inmunología , Inmunoglobulina G/biosíntesis , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Antígenos CD40/inmunología , Toxoide Diftérico/inmunología , Toxoide Diftérico/farmacología , Vacuna contra Difteria y Tétanos , Ensayo de Inmunoadsorción Enzimática , Epítopos , Gangliósido G(M1)/inmunología , Gangliósido G(M1)/farmacología , Humanos , Inmunización Secundaria , Inmunoglobulina G/inmunología , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones , Ratones SCID , Toxoide Tetánico/inmunología , Toxoide Tetánico/farmacología , Vacunas Combinadas/inmunología , Vacunas Combinadas/farmacologíaRESUMEN
IL-17 is a T cell-derived cytokine that stimulates stromal cells and macrophages to secrete proinflammatory cytokines. We hypothesized that IL-17 might play a role in alloimmune responses, and that interference with its activity might suppress allograft rejection. IL-17R:Fc or control IgG was added at the start of mouse MLR or was administered i.p. (100-500 microg/day) for different durations post-transplant to murine recipients of MHC-mismatched cardiac allografts. IL-17R:Fc (50-200 microg/ml) markedly inhibited T cell proliferation in vitro and significantly prolonged nonvascularized cardiac allograft median survival time from 13 to 20 days (100 microg/day; days 0 and 1) or to 19 days (100-300 microg/day; days 0-4). Survival of vascularized grafts was also extended significantly from 10.5 to 19 days by IL-17R:Fc (500 microg/day; days 0-6). To address a possible mechanism by which IL-17 may promote alloreactivity, we examined the influence of IL-17 on the differentiation and function of bone marrow-derived cells propagated in granulocyte-macrophage CSF with or without IL-4 to promote dendritic cell (DC) growth. A minor proportion of CD11c+ DC expressed the IL-17R. IL-17 promoted the maturation of DC progenitors, as evidenced by increased cell surface expression of CD11c, costimulatory molecules (CD40, CD80, CD86), and MHC class II Ag, and allostimulatory capacity. IL-17 had a lesser effect on the phenotype and function of more fully differentiated myeloid DC. These findings suggest a role for IL-17 in allogeneic T cell proliferation that may be mediated in part via a maturation-inducing effect on DC. IL-17 appears to be a novel target for therapeutic intervention in allograft rejection.
Asunto(s)
Células Dendríticas/patología , Rechazo de Injerto/inmunología , Rechazo de Injerto/patología , Interleucina-17/fisiología , Células Madre/patología , Adyuvantes Inmunológicos/fisiología , Animales , Diferenciación Celular/inmunología , Coristoma/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Oído Externo/irrigación sanguínea , Trasplante de Corazón/inmunología , Trasplante de Corazón/patología , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Inmunofenotipificación , Inmunosupresores/farmacología , Integrina alfaXbeta2/biosíntesis , Interleucina-17/metabolismo , Activación de Linfocitos/genética , Prueba de Cultivo Mixto de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Receptores de Interleucina/biosíntesis , Receptores de Interleucina/genética , Receptores de Interleucina-17 , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Células Madre/inmunología , Células Madre/metabolismo , Linfocitos T/inmunología , Regulación hacia Arriba/inmunologíaRESUMEN
Recent progress in the understanding of immune function indicates that the interaction of CD40L with its receptor, CD40, plays a pivotal role in both humoral immunity and cell-mediated defense against pathogens. Functional studies of this interaction on both dendritic cells and malignant cells have demonstrated that CD40L also plays an important role in immune surveillance and anti-tumor immunity. CD40L exists in nature predominantly as a membrane-anchored molecule. To develop CD40L as a potential therapeutic, it is important to optimize soluble forms of this molecule that could be used in a clinical setting. Several reports have shown that soluble forms of CD40L, like CD40 antibodies, are biologically active. In the present report we demonstrate that the incorporation of an isoleucine zipper trimerization motif significantly enhances the biological activity of soluble CD40L.
Asunto(s)
Isoleucina/química , Leucina Zippers , Glicoproteínas de Membrana/metabolismo , Animales , Biopolímeros , Antígenos CD40/metabolismo , Ligando de CD40 , Células CHO , Rastreo Diferencial de Calorimetría , Cricetinae , Electroforesis en Gel de Poliacrilamida , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/aislamiento & purificación , Conformación Proteica , TermodinámicaRESUMEN
The tumor necrosis factor family molecule Ox40-ligand (Ox40L) has been identified as a potential costimulatory molecule and also has been implicated in T cell homing and B cell activation. To ascertain the essential functions of Ox40L, we generated and characterized Ox40L-deficient mice. Mice lacking Ox40L exhibit an impaired contact hypersensitivity response, a dendritic cell-dependent T cell-mediated response, due to defects in T cell priming and cytokine production. In contrast, Ox40L-deficient mice do not have defects in T cell homing or humoral immune responses. In vitro, Ox40L-deficient dendritic cells are defective in costimulating T cell cytokine production. Thus, Ox40L has a critical costimulatory function in vitro and in vivo for dendritic cell:T cell interactions.
Asunto(s)
Células Dendríticas/inmunología , Glicoproteínas de Membrana , Receptores del Factor de Necrosis Tumoral/inmunología , Linfocitos T/inmunología , Células 3T3 , Animales , Antígenos T-Independientes/inmunología , Dermatitis por Contacto/inmunología , Haptenos/inmunología , Hemocianinas/inmunología , Hipersensibilidad Tardía/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ligando OX40 , Ovalbúmina/inmunología , Receptores del Factor de Necrosis Tumoral/genética , Factores de Necrosis TumoralRESUMEN
Posttransplant infection associated with host immune deficiency is the major cause of nonrelapse mortality of human bone marrow transplant recipients. In a new murine model of posttransplant infection, allogeneic bone marrow transplant recipients were infected with herpes simplex virus-1 (HSV-1) via intraperitoneal inoculation 12 weeks after transplantation. Allogeneic transplant recipients with graft-versus-host disease (GVHD) had significantly increased mortality from HSV-1 encephalitis, with deficiencies of both specific anti-HSV-1 antibody and total serum IgG2a. GVHD mice displayed a Th2 cytokine profile (increased interleukin-4 [IL-4] and decreased interferon-gamma) and decreased lipopolysaccharide (LPS) responses, suggesting that both T-cell and B-cell defects contributed to the impaired production of antibody. Because passive transfer of hyperimmune serum protected mice from HSV-1 infection, we hypothesized that CD40 ligand (CD40L), which induces B-cell maturation, would protect mice from HSV-1 infection. CD40L-treated GVHD mice showed elevated IgG2a levels and increased survival compared with vehicle-treated transplant recipients.
Asunto(s)
Trasplante de Médula Ósea/efectos adversos , Herpes Simple/prevención & control , Herpesvirus Humano 1 , Glicoproteínas de Membrana/uso terapéutico , Animales , Ligando de CD40 , Femenino , Herpes Simple/etiología , Herpes Simple/mortalidad , Humanos , Terapia de Inmunosupresión/efectos adversos , Ratones , Ratones Endogámicos CBA , Proteínas Recombinantes/uso terapéutico , Trasplante HomólogoRESUMEN
Resting B (rB) cells are known to be incompetent APCs in vitro, which alone can induce specific unresponsiveness to single minor histocompatibility (miH) Ags and, when combined with CD40 pathway blockade, can induce hyporesponsiveness to MHC molecules in vivo. Here we show that anti-CD40 ligand (CD40L) mAb does not prevent the expression of B7-2 on allogeneic rB cells in vivo but did prolong donor-specific cardiac allograft survival. Moreover, pretreatment with professional APCs combined with anti-CD40L mAb induced hyporesponsiveness to alloantigens in vivo. rB cells from CD40 knockout mice were unable to induce unresponsiveness, while graft prolongation was achieved in CD40L knockout recipients pretreated with wild-type rB cells. These data suggest that CD40-CD40L interactions in the recipient play a critical role in the induction of hyporesponsiveness to alloantigens in vivo and that the effect of the CD40 pathway may be independent of its effect on the B7 costimulatory pathway.
Asunto(s)
Antígenos CD40/fisiología , Tolerancia Inmunológica/inmunología , Inmunoconjugados , Isoantígenos/inmunología , Abatacept , Adyuvantes Inmunológicos , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacología , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/trasplante , Antígenos CD , Antígenos de Diferenciación/farmacología , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/trasplante , Antígenos CD40/genética , Antígenos CD40/inmunología , Ligando de CD40 , Antígeno CTLA-4 , Supervivencia de Injerto/genética , Supervivencia de Injerto/inmunología , Trasplante de Corazón/inmunología , Humanos , Inmunofenotipificación , Inyecciones Intravenosas , Interfase/inmunología , Isoantígenos/administración & dosificación , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
CD40 is a member of the TNF receptor family that was initially described on the surface of B cells. Recently, CD40 has also been described on mesenchymal cells, such as endothelial cells and fibroblasts, where engagement by its ligand CD40 ligand can lead to up-regulation of costimulatory and cell adhesion molecules, as well as secretion of proinflammatory cytokines. Since airway inflammation potentially involves cell-cell interactions of T cells and eosinophils (which express CD40 ligand) with airway smooth muscle (ASM) cells, we postulated that ASM may express CD40 and that engagement of ASM CD40 may modulate smooth muscle cell function. We demonstrate that CD40 is expressed on cultured human ASM and that expression can be increased by treatment with TNF-alpha or IFN-gamma. Cross-linking CD40 on ASM resulted in enhanced IL-6 secretion and an increase in intracellular calcium concentrations, which were dependent on calcium influx. We show that CD40-mediated signaling events include protein tyrosine phosphorylation and activation of NF-kappaB. Pretreatment of ASM with the tyrosine kinase inhibitors genistein or herbimycin inhibited the rapid mobilization of calcium induced via CD40, suggesting that calcium mobilization was coupled to activation of protein tyrosine kinases. In addition, inhibition of calcium influx inhibited both CD40-mediated NF-kappaB activation and enhancement of IL-6 secretion. These results delineate a potentially important CD40-mediated signal-transduction pathway in ASM, involving protein tyrosine kinase-dependent calcium mobilization, NF-kappaB activation, and IL-6 production. Together, these results suggest a mechanism whereby T cell/smooth muscle cell interactions may potentiate airway inflammation.
Asunto(s)
Antígenos CD40/fisiología , Músculo Liso/inmunología , Transducción de Señal/inmunología , Tráquea/inmunología , Antígenos CD40/biosíntesis , Antígenos CD40/metabolismo , Ligando de CD40 , Calcio/metabolismo , Calcio/fisiología , Células Cultivadas , Espacio Extracelular/metabolismo , Espacio Extracelular/fisiología , Humanos , Interferón gamma/farmacología , Interleucina-6/biosíntesis , Interleucina-6/metabolismo , Líquido Intracelular/metabolismo , Ligandos , Glicoproteínas de Membrana/fisiología , Músculo Liso/citología , Músculo Liso/enzimología , Músculo Liso/metabolismo , FN-kappa B/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Tráquea/citología , Tráquea/enzimología , Tráquea/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
It has been proposed that the induction of cellular immunity and resistance to intracellular pathogens is dependent upon CD40 ligand (CD40L). In the present study we show that this proposal is not ubiquitously supported. Mice genetically deficient in CD40L (CD40LKO) were resistant to i.v. infection with Mycobacterium tuberculosis when assessed by survival and bacteriologic burden in the spleen, liver, and lungs. Infected CD40LKO mice developed granulomas that lacked epithelioid cells and were less numerous and markedly smaller than those observed in control mice. Upon stimulation with purified protein derivative of M. tuberculosis, CD4+ T cells from infected CD40LKO mice proliferated and produced high levels of IFN-gamma but not IL-4. Finally, spleen cells from CD40LKO mice stimulated with M. tuberculosis produced IL-12, TNF, and nitric oxide levels comparable to those produced by control cells. In contrast to original proposals, these data clearly show that protective Thl immunity can be achieved against intracellular pathogens (e.g., Mycobacterium) independently of CD40L.
Asunto(s)
Antígenos CD40/fisiología , Glicoproteínas de Membrana/fisiología , Mycobacterium tuberculosis/inmunología , Tuberculosis/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/sangre , Antígenos CD40/genética , Ligando de CD40 , Inmunidad Celular/genética , Inmunidad Innata/genética , Interleucina-12/biosíntesis , Ligandos , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tuberculosis/genética , Tuberculosis/microbiología , Tuberculosis/patología , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
We examined T-cell proliferation in five patients with X-linked hyper-IgM syndrome (XHIM), using a panel of antigens and lectins. All patients had impaired antigen-induced proliferation, whereas their lectin responses were normal. Thus, in addition to severely depressed antibody responses, patients with XHIM have a defect in antigen-specific T-cell proliferation, which may explain their susceptibility to pathogens such as Pneumocystis carinii.
Asunto(s)
Antígenos/inmunología , Ligamiento Genético , Hipergammaglobulinemia/inmunología , Inmunoglobulina M , Síndromes de Inmunodeficiencia/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Cromosoma X , Antígenos Fúngicos , Antígenos CD40/genética , Candida/inmunología , Concanavalina A , Criptosporidiosis/inmunología , Toxoide Diftérico , Susceptibilidad a Enfermedades/inmunología , Humanos , Hipergammaglobulinemia/genética , Síndromes de Inmunodeficiencia/genética , Lectinas , Ligandos , Masculino , Fitohemaglutininas , Neumonía por Pneumocystis/inmunología , Mitógenos de Phytolacca americana , Toxoide TetánicoRESUMEN
CD40 is a molecule present on multiple cell types including B lymphocyte lineage cells. CD40 has been shown to play an important role in B cell differentiation and activation in vitro, although little is known concerning the effects of CD40 stimulation in vivo. We therefore examined the effects of CD40 stimulation in mice using a syngeneic bone marrow transplantation (BMT) model in an effort to augment B cell recovery after high dose therapy with hematopoietic reconstitution. After the BMT, mice were treated with or without 2-6 microg of a soluble recombinant murine CD40 ligand (srmCD40L) given intraperitoneally twice a week. A significant increase in B cell progenitors (B220+/ surface IgM-) was observed in the bone marrow of mice receiving the srmCD40L. The treated recipients also demonstrated improved B-cell function with increases in total serum immunoglobulin and increased splenic mitogen responsiveness to LPS being noted. Additionally, srmCD40L treatment promoted secondary lymphoid organ repopulation, accelerating germinal center formation in the lymph nodes. Total B cell numbers in the periphery were not significantly affected even with continuous srmCD40L administration. Lymphocytes obtained from mice treated with the ligand also had increases in T cell mitogen and anti-CD3 mAb responsiveness and acquired the capability to produce IL-4. Surprisingly, treatment with srmCD40L also produced hematopoietic effects in mice, resulting in an increase of BM and splenic hematopoietic progenitor cells in the mice after BMT. Treatment with srmCD40L significantly increased granulocyte and platelet recovery in the peripheral blood. Incubation of BMC with srmCD40L in vitro also resulted in increased progenitor proliferation, demonstrating that the hematopoietic effects of the ligand may be direct. Thus, stimulation of CD40 by its ligand may be beneficial in accelerating both immune and hematopoietic recovery in the setting of bone marrow transplantation.