RESUMEN
The 4-1BB glycoprotein is a member of the tumor necrosis factor receptor superfamily and binds to a high-affinity ligand (4-1BBL) expressed on several antigen-presenting cells such as macrophages and activated B cells. Expression of 4-1BB is restricted to primed CD4+ and CD8+ T cells, and 4-1BB signaling either by binding to 4-1BBL or by antibody ligation delivers a dual mitogenic signal for T-cell activation and growth. These observations suggest an important role for 4-1BB in the amplification of T cell-mediated immune responses. We now show that administration of anti-4-1BB monoclonal antibodies can eradicate established large tumors in mice, including the poorly immunogenic Ag104A sarcoma and the highly tumorigenic P815 masto cytoma. The immune response induced by anti-4- 1BB monoclonal antibodies is mediated by both CD8+ and CD4+ T cells and is accompanied by a marked augmentation of tumor-selective cytolytic T-cell activity. Our data suggest that a similar approach may be efficacious for immunotherapy of human cancer.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Sarcoma de Mastocitos/terapia , Receptores de Factor de Crecimiento Nervioso/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Sarcoma Experimental/terapia , Animales , Antígenos CD , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Femenino , Sarcoma de Mastocitos/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos DBA , Trasplante de Neoplasias , Sarcoma Experimental/inmunología , Linfocitos T Citotóxicos/inmunología , Células Tumorales Cultivadas , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
During T cell activation, CD4 is intimately involved in colocalizing the T cell receptor (TCR) with its specific peptide ligand bound to class II molecules of the major histocompatibility complex (MHC). Previously, the COOH-terminal residues, Trp62/63, which flank the immunodominant epitope of hen egg lysozyme (HEL 52-61), were shown to have a profound effect on TCR recognition. CD4 maintains the fidelity of this interaction when short peptides are used. To determine which portion of CD4 was responsible for this effect, a series of CD4 mutants were made and transfected into CD4 loss variants of two HEL 52-61-specific T cell hybridomas. Surprisingly, some CD4 mutants that failed to interact with MHC class II molecules (D2 domain mutant) or with p56kk (cytoplasmic-tailless mutant) restored responsiveness. Nevertheless, a significant reduction in association between cytoplasmic-tailless CD4 and the TCR, as determined by fluorescence resonance energy transfer, was observed. Thus, neither colocalization of CD4 and the TCR nor signal transduction via CD4 was solely responsible for the functional restoration of these T cell hybridomas by wild-type CD4. However, substitution of the two membrane proximal domains of murine CD4 (D3 and D4) with domains from human CD4 or intercellular adhesion molecule 1 not only abrogated its ability to restore function, but also substantially reduced its ability to associate with the TCR. Furthermore, the mouse/human CD4 chimera had a potent dominant negative effect on T cell function in the presence of equimolar concentrations of wild-type CD4. These data suggest that the D3/D4 domains of CD4 may interact directly or indirectly with the TCR-CD3 complex and influence the signal transduction processes. Given the striking structural differences between CD4 and CD8 in this region, these data define a novel and unique function for CD4.
Asunto(s)
Antígenos CD4/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos CD/biosíntesis , Antígenos CD/inmunología , Antígenos CD/metabolismo , Antígenos CD4/biosíntesis , Antígenos CD4/metabolismo , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/inmunología , Molécula 1 de Adhesión Intercelular/metabolismo , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T/biosíntesis , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Tetraciclina/farmacología , TransfecciónRESUMEN
The 4-1BB receptor (CDw137), a member of the tumor necrosis factor receptor superfamily, has been shown to costimulate the activation of T cells. Here we show that anti-mouse 4-1BB monoclonal antibodies (mAbs) inhibit thymus-dependent antibody production by B cells. Injection of anti-4-1BB mAbs into mice being immunized with cellular or soluble protein antigens induced long-term anergy of antigen-specific T cells. The immune response to the type II T cell-independent antigen trinintrophenol-conjugated Ficoll, however, was not suppressed. Inhibition of humoral immunity occurred only when anti-4-1BB mAb was given within 1 wk after immunization. Anti-4-1BB inhibition was observed in mice lacking functional CD8(+) T cells, indicating that CD8(+) T cells were not required for the induction of anergy. Analysis of the requirements for the anti-4-1BB-mediated inhibition of humoral immunity revealed that suppression could not be adoptively transferred with T cells from anti-4-1BB-treated mice. Transfer of BALB/c splenic T cells from sheep red blood cell (SRBC)-immunized and anti-4-1BB-treated mice together with normal BALB/c B cells into C.B-17 severe combined immunodeficient mice failed to generate an anti-SRBC response. However, B cells from the SRBC-immunized, anti-4-1BB-treated BALB/c mice, together with normal naive T cells, exhibited a normal humoral immune response against SRBC after transfer, demonstrating that SRBC-specific B cells were left unaffected by anti-4-1BB mAbs.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Formación de Anticuerpos , Tolerancia Inmunológica , Receptores de Factor de Crecimiento Nervioso/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T/fisiología , Animales , Anticuerpos Monoclonales/farmacocinética , Antígenos CD , Eritrocitos/inmunología , Femenino , Ficoll/análogos & derivados , Ficoll/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratas , Trinitrobencenos/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
Three human lymphocyte antigens recognized by monoclonal antibodies OKIa1, OKT9, and OKT10 were found to be minimally represented on resting peripheral T cells (all three) and thymocytes (OKIa1 and OKT9). These antigens, which are present on "activated" T cells, were promptly displayed on "resting" T cells or thymocytes following cross-linking of surface-bound monoclonal antibody by horse alpha-mouse IgG. These experiments suggested that membrane perturbations may induce the expression of certain antigens that are normally present in an unexpressed form in resting cells.
Asunto(s)
Antígenos de Superficie/inmunología , Membrana Celular/inmunología , Linfocitos T/inmunología , Anticuerpos Monoclonales , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoglobulina GRESUMEN
The 4-1BB receptor is an inducible type I membrane protein and member of the tumor necrosis factor receptor (TNFR) superfamily that is rapidly expressed on the surface of CD4+ and CD8+ T cells after antigen- or mitogen-induced activation. Cross-linking of 4-1BB and the T cell receptor (TCR) on activated T cells has been shown to deliver a costimulatory signal to T cells. Here, we expand upon previously published studies by demonstrating that CD8+ T cells when compared with CD4+ T cells are preferentially responsive to both early activation events and proliferative signals provided via the TCR and 4-1BB. In comparison, CD28-mediated costimulatory signals appear to function in a reciprocal manner to those induced through 4-1BB costimulation. In vivo examination of the effects of anti-4-1BB monoclonal antibodies (mAbs) on antigen-induced T cell activation have shown that the administration of epitope-specific anti-4-1BB mAbs amplified the generation of H-2d-specific cytotoxic T cells in a murine model of acute graft versus host disease (GVHD) and enhanced the rapidity of cardiac allograft or skin transplant rejection in mice. Cytokine analysis of in vitro activated CD4+ and CD8+ T cells revealed that anti-4-1BB costimulation markedly enhanced interferon-gamma production by CD8+ T cells and that anti-4-1BB mediated proliferation of CD8+ T cells appears to be IL-2 independent. The results of these studies suggest that regulatory signals delivered by the 4-1BB receptor play an important role in the regulation of cytotoxic T cells in cellular immune responses to antigen.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunidad Celular , Activación de Linfocitos/inmunología , Receptores de Factor de Crecimiento Nervioso/inmunología , Receptores del Factor de Necrosis Tumoral/inmunología , Animales , Antígenos CD , Linfocitos T CD8-positivos/citología , División Celular/inmunología , Ratones , Ratones Endogámicos BALB C , Receptores de Factor de Crecimiento Nervioso/biosíntesis , Receptores del Factor de Necrosis Tumoral/biosíntesis , Transducción de Señal/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
The human immunodeficiency virus (HIV) binds to CD4-positive cells through interaction of its envelope glycoprotein (gp120) with the CD4 molecule. CD4 is a prominent immunoregulatory molecule, and chronic exposure to antibody against CD4 (anti-CD4) has been shown to cause immunodeficiency in mice. T cell-dependent in vitro immune responses can also be inhibited by anti-CD4. Experimental findings reported here indicate that CD4-bound gp120 attracts gp120-specific antibodies derived from the blood of HIV-seropositive individuals to form a trimolecular complex with itself and CD4. Thus targeted to CD4, the gp120-specific antibody functions as an antibody to CD4; it cross-links and modulates the CD4 molecules and suppresses the activation of T cells as measured by mobilization of intracellular calcium (Ca2i+). The synergism between gp120 and anti-gp120 in blocking T cell activation occurs at low concentrations of both components. Neither gp120 nor anti-gp120 inhibits T cell activation by itself in the concentrations tested.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Linfocitos T CD4-Positivos/inmunología , Anticuerpos Anti-VIH/inmunología , Antígenos VIH/inmunología , VIH/inmunología , Proteínas de los Retroviridae/inmunología , Reacciones Antígeno-Anticuerpo , Calcio/fisiología , Relación Dosis-Respuesta Inmunológica , Proteína gp120 de Envoltorio del VIH , Humanos , Recubrimiento Inmunológico , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
A single-chain Fv (sFv) was expressed from the variable regions of the CD40-specific mAb G28-5. The molecule bound CD40 with a high affinity (2.2 nM) and was a monomer in solution. Surprisingly, G28-5 sFv was a potent CD40 agonist that rapidly crosslinked CD40 on the cell surface but did not crosslink CD40-Ig in solution. G28-5 sFv was a more potent agonist than G28-5 IgG and was able to stimulate CD40 responses by B cells and monocytes. G28-5 IgG partially blocked, whereas G28-5 sFv augmented CD40 responses during stimulation with natural ligand (gp39-CD8 fusion protein). These results indicate that the functional activity of ligands built from the binding site of G28-5 is highly dependent upon the size and physical properties of the molecule both in solution and on the cell surfaces.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Antígenos CD40/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Células Cultivadas , Clonación Molecular , Endotelio/citología , Expresión Génica , Humanos , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Monocitos/inmunología , FN-kappa B/inmunologíaRESUMEN
A method for antigen-specific generation of antibody-forming B cells in cultures of human peripheral blood mononuclear (PBM) cells based on the Mishell-Dutton system has recently been established in this laboratory. Comparing PBM cell cultures from healthy donors and from patients with advanced cancer we found the latter to be unresponsive in our assay. Passage of PBM cell suspension over Sephadex G-10 columns restored the response of patient PBM cells to normal levels. The cell population trapped on the column can be recovered and its inhibitory potential demonstrated by its graded addition to cells eluted from the column. The cell responsible for inhibition is sensitive to treatment with OKT8 antibody and complement, indicating its T cell nature. Passage of PBM cells from healthy individuals did not alter antibody responses substantially but made the activation requirements less stringent.
Asunto(s)
Linfocitos B/citología , Separación Celular/métodos , Linfocitos T Reguladores , Animales , Células Productoras de Anticuerpos/citología , Células Productoras de Anticuerpos/inmunología , Linfocitos B/inmunología , Cromatografía en Gel/métodos , Técnica de Placa Hemolítica , Humanos , Melanoma/inmunología , Fenotipo , Linfocitos T Reguladores/inmunologíaRESUMEN
BACKGROUND: 4-1BB (CD137) is a T cell costimulatory molecule that promotes T cell activation. In this study, we investigated the role of 4-1BB costimulation in allogeneic T cell responses. METHODS: Vascularized heart transplantation, allogeneic mixed leukocyte reaction (MLR), and graft versus host disease models were used to examine 4-1BB and 4-1BBL expression. In addition, agonistic anti-4-1BB antibodies were used in MLR to functionally analyze T cell responses. RESULTS: Using a heart transplant model, we found that 4-1BB and 4-1BBL transcripts were both expressed in rejecting cardiac grafts. In the allogeneic MLR, 4-1BB was expressed on both activated CD4 and CD8 T cells and 4-1BB was expressed on T cells after multiple cell divisions in vivo. Functionally, 4-1BB was a potent stimulator of proliferation, cytokine secretion, and CD25 expression by CD8 T cells, but 4-1BB signals had a weak effect on the proliferation of CD4 T cells. Because 4-1BB promoted the secretion of IL-2 and the expression of CD25 on CD8 T cells, we investigated whether IL-2 was the only factor whereby 4-1BB signals induced CD8 T cell proliferation. Although IL-2 was required for optimal CD8 T cell proliferation, 4-1BB also costimulated CD8 T cell proliferation independently of IL-2. CONCLUSIONS: This study demonstrates that 4-1BB is expressed on activated, maximally divided T cells and shows that 4-1BB promotes CD8 T cell proliferation by enhancing signals through the IL-2 receptor and by other mechanisms independent of the IL-2 pathway.
Asunto(s)
Receptores de Factor de Crecimiento Nervioso/fisiología , Receptores del Factor de Necrosis Tumoral/fisiología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos CD , Citocinas/biosíntesis , Trasplante de Corazón/inmunología , Interleucina-2/fisiología , Activación de Linfocitos , Ratones , Ratones Endogámicos , Receptores de Interleucina-2/biosíntesis , Trasplante Homólogo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
Several studies have suggested that prostaglandin E2 (PGE2) might influence the phagocytic activity of macrophage cells. The present study was designed to examine the in vivo effects of PGE2, the prostaglandin synthesis inhibitor meclofenamate, the prostaglandin precursor arachidonic acid, and the biologically inactive fatty acid 11,14,17-eicosatrienoic acid on phagocytosis by peritoneal macrophage cells in the rat. Following 3 days of treatment with either agent, fluorescent methacrylate microbeads were injected intraperitoneally into all rats. Peritoneal exudates were harvested after administration of the microbeads and the percent phagocytosis determined in macrophage cells using a fluorescence-activated cell sorter (FACS II). The administration of PGE2 was associated with a significant decrease in the percentage of peritoneal macrophages ingesting the fluorescent methacrylate microbeads. In contrast, treatment with arachidonic acid or 11,14,17-eicosatrienoic acid significantly enhanced the percentage of phagocytic macrophage cells. A significant increase in the number of macrophages undergoing phagocytosis of the methacrylate microbeads was also observed in rats treated with meclofenamate. This later observation, taken together with the inhibitory effect induced by PGE2 on macrophage phagocytosis, points to a potential modulator role of PGE2 on the phagocytic activity of macrophages. These data also suggest that arachidonic acid might influence macrophage phagocytosis by a mechanism independent of PGE2.
Asunto(s)
Ácidos Araquidónicos/farmacología , Macrófagos/fisiología , Fagocitosis/efectos de los fármacos , Prostaglandinas E/farmacología , Animales , Ácido Araquidónico , Dinoprostona , Fluoresceínas , Macrófagos/efectos de los fármacos , Macrófagos/ultraestructura , Masculino , Microscopía Electrónica , Ácidos Polimetacrílicos , Ratas , Ratas EndogámicasRESUMEN
Cell surface proteins of the tumor necrosis factor (TNF) family of receptors have been intimately involved in inducing T cell death. A feature of these family members that is less well studied is their ability to rescue T cells from apoptosis. One such member is 4-1BB; an activation induced surface receptor on CD4 and CD8 T cells. This study demonstrates that the costimulatory effects of 4-1BB, which was found to enhance clonal expansion, required cross-linking of the receptor. The survival of the activated CD8 T cells following expansion was not associated with an increase in Bcl-2 expression. Provided that 4-1BB signaling was present, the amplification of activated CD8 T cell growth in vivo was independent of CD28 ligation. In vivo clonal expansion of activated CD4 T cells, however, was not as responsive to 4-1BB cross-linking. Moreover, 4-1BB-induced expansion was comparable to that mediated by LPS which can incite multiple costimulatory signals. Furthermore, LPS-mediated growth and survival of superantigen (SAg) stimulated T cells appeared to be partially dependent on interactions between 4-1BB and 4-1BB ligand (4-1BBL).
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ligando 4-1BB , Animales , Antígenos CD , Antígenos CD28/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Diferenciación Celular , Células Clonales , Femenino , Lipopolisacáridos/metabolismo , Lipopolisacáridos/farmacología , Activación de Linfocitos , Ratones , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Superantígenos/metabolismo , Superantígenos/farmacología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis TumoralAsunto(s)
Formación de Anticuerpos , Antígenos de Diferenciación/farmacología , Inmunoconjugados , Inmunosupresores/farmacología , Linfocitos T/inmunología , Abatacept , Análisis de Varianza , Animales , Anticuerpos Monoclonales/farmacología , Antígenos CD , Antígeno CTLA-4 , Hemocianinas/inmunología , Humanos , Inmunoglobulina G/farmacología , Terapia de Inmunosupresión , Macaca fascicularis , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
BACKGROUND: Allergic asthma is a T-helper type 2 (Th2) cell-mediated chronic disease that is characterized by airway hyperreactivity (AHR) and chronic eosinophilic airway inflammation. Several studies suggest co-stimulatory molecules like CD137 as potential targets for therapeutic interventions in allergic airway disease. Recently, we could show in a murine asthma model that administration of an agonistic antibody against the receptor of the co-stimulatory molecule CD137 prevented and even reversed an already-established asthma phenotype. OBJECTIVE: The purpose of this study was to analyse the effect of stimulation of the CD137 ligand by a monoclonal antibody (CD137L mAb). METHODS: To induce an asthma-like phenotype, BALB/c mice were sensitized to ovalbumin (OVA), followed by an intrapulmonary allergen challenge. Anti-CD137L or control mAb were applied 1 day before OVA immunization or after the asthma phenotype was already established. RESULTS: Stimulation of the CD137L instead of the receptor by CD137L mAb prevents the development of an asthma-like phenotype but does not reverse established disease. While the receptor-mediated effect is partly mediated by anergy of CD4(+) T cells and partly by induction of IFN-gamma-producing CD8(+) T cells, the effect of the CD137L mAb is completely dependent on IFN-gamma-producing CD8(+) T cells: blockade of IFN-gamma and depletion of CD8(+) T cells fully abrogated the observed protective effect. In vitro experiments showed that the anti-CD137L mAb ligates directly to CD8(+) T cells and induces the generation of IFN-gamma by this cell population. CONCLUSION: Our results demonstrate that anti-CD137L mAb prevents disease development via IFN-gamma-producing CD8(+) T cells but is inferior to stimulation of the receptor that reverses established disease by a mechanism including CD4(+) T cell anergy.
Asunto(s)
Ligando 4-1BB/inmunología , Anticuerpos Monoclonales/administración & dosificación , Asma/inmunología , Linfocitos T CD8-positivos/inmunología , Interferón gamma/biosíntesis , Animales , Asma/prevención & control , Modelos Animales de Enfermedad , Femenino , Inmunidad Celular , Ratones , Ratones Endogámicos BALB C , Células Th2/inmunologíaRESUMEN
Activation of peripheral blood T cells, and the leukemic T cell line Jurkat, as measured by mobilization of intracellular calcium, by an anti-TCR antibody is blocked by mAb (T191) to the leukocyte common Ag (CD45). T191 also blocked down-regulation of the CD3-TCR complex induced by an anti-CD3 mAb. Vanadate, a phosphotyrosine phosphatase inhibitor, partially blocks the effect of T191 and restored mobilization of intracellular calcium. Assays of the immunoprecipitates of T191 and CD45 from both Jurkat-BM1 and peripheral T cells showed that the immune complexes had intrinsic phosphatase activity. A parallel immunoprecipitate using a mAb (4-10) against HLA class I showed no such activity. Further analysis of the T191 immunocomplex revealed activity against phosphotyrosine, p-nitrophenylphosphate, and [32P-poly-glu-tyr, but not against phosphoserine. Phosphatase activity was inhibited by Vanadate, but not by Zn2+ or F-. These results show that CD45 is a phosphotyrosine phosphatase, and strongly suggest that tyrosine phosphorylation/dephosphorylation is critically involved in activation of T cells through the TCR-CD3 complex.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Diferenciación/metabolismo , Reactivos de Enlaces Cruzados , Antígenos de Histocompatibilidad/metabolismo , Activación de Linfocitos , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Anticuerpos Monoclonales/fisiología , Antígenos de Diferenciación/inmunología , Unión Competitiva , Complejo CD3 , Línea Celular , Antígenos de Histocompatibilidad/inmunología , Humanos , Antígenos Comunes de Leucocito , Activación de Linfocitos/efectos de los fármacos , Pruebas de Precipitina , Proteínas Tirosina Quinasas/inmunología , Linfocitos T/enzimología , Linfocitos T/metabolismo , Vanadatos/farmacologíaRESUMEN
Activation of human PBL T cells with solid phase anti-CD3 mAb or during the course of an MLR response gives rise to the association of CD4 or CD8 molecules with the protein tyrosine phosphatase, CD45, on the cell surface. This paired association of cell-surface molecules occurs late in the activation cycle and appears to be dependent upon Ti-CD3-mediated signaling because mitogen-driven activation does not induce formation of the complex. Maximal association occurred 72 to 96 h after exposure to anti-CD3 mAb on both CD4+ and CD8+ T cells. In contrast, association between CD8 and CD45 during an MLR response did not occur until day 6 of a MLR whereas CD4-CD45 association was detected by 72 h of culture. The kinetics of association between CD4 or CD8 and CD45 was measured by fluorescence resonance energy transfer and confirmed by immunoprecipitation of dithiobis succinimidylpropionate or disuccinimidyl suberate cross-linked 125I-labeled resting or activated T cells. The molecules that co-precipitated with either CD4 or CD8 and had an apparent kDa of 180 to 205 could be immunodepleted with anti-CD45 mAb. Furthermore, CD4 or CD8 immunoprecipitates from 96-h activated T cells contained significant levels of protein tyrosine phosphatase activity whereas corresponding immunoprecipitates from resting or recently activated T cells showed little protein tyrosine phosphatase activity. This association may allow CD45 to engage and dephosphorylate lck or another CD4- or CD8-associated substrate in order to reset the receptor complex to receive a new set of stimuli. Our observations suggest that synergistic signaling provided as a consequence of CD4 or CD8 association with the TCR after antigenic stimulation may develop on a different temporal scale than that observed after soluble anti-CD4+ anti-CD3 heteroconjugate antibody cross-linking.
Asunto(s)
Antígenos CD/metabolismo , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Antígenos de Histocompatibilidad/metabolismo , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/fisiología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Complejo CD3 , Humanos , Técnicas In Vitro , Antígenos Comunes de Leucocito , Prueba de Cultivo Mixto de Linfocitos , Sustancias Macromoleculares , Fitohemaglutininas/inmunología , Proteínas Tirosina Fosfatasas/metabolismo , Agregación de Receptores , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de SeñalRESUMEN
After recognition of Ag/MHC and ligation of a costimulatory molecule, resting T cells will clonally expand and then delete to very low levels. Previously, it was shown that deletion can be prevented by coinjection of cytokines or proinflammatory agents such as adjuvants. Here, we demonstrate that ligation of 4-1BB blocks deletion of superantigen-activated T cells in the absence of adjuvant or additional cytokine treatment. Nearly 10 times as many staphylococcal enterotoxin A-specific T cells were detected in the spleens of mice injected 21 days previously with staphylococcal enterotoxin A and an agonist anti-4-1BB Ab compared with mice given staphylococcal enterotoxin A and a control IgG. Even though both CD4- and CD8-activated T cells expressed 4-1BB, a higher proportion of CD8 T cells were rescued compared CD4 T cells. These data suggest that although 4-1BB provides costimulation, it may also promote long-term T cell survival.
Asunto(s)
Linfocitos T CD8-positivos/citología , Factor de Necrosis Tumoral alfa/fisiología , Ligando 4-1BB , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Muerte Celular/inmunología , Supervivencia Celular/inmunología , Células Clonales , Enterotoxinas/farmacología , Femenino , Ligandos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos A , Ratones Endogámicos C57BL , Staphylococcus aureus/inmunología , Superantígenos/farmacología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Hybridomas secreting monoclonal antibodies against an apparent strain-specific cell surface antigen of Neisseria gonorrhoeae were produced. Spleen cells from BALB/c mice immunized with whole gonococci were fused with mouse myeloma cell line Sp2/0, and hybrid cells were selected in culture. One hybridoma that secreted antibodies reactive with the immunizing strain was cloned by limiting dilution to obtain cell lines secreting monoclonal antibodies. These antibodies reacted with purified outer membranes from the immunizing strain as well as with whole gonococci. Binding of antibodies to whole gonococci was highly strain specific, with most gonococcal strains showing less than 1% of the binding with the immunizing strain. Antibodies did not bind to the other Neisseria species tested. Binding of monoclonal antibodies to whole gonococci of the immunizing strain was not dependent on state of piliation. The extent of antibody binding did vary in different colonial variants of the immunizing strain. Antibody bound to cells from colonies that were transparent or of intermediate opacity, but did not bind to cells from deeply opaque colony variants.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos/inmunología , Neisseria gonorrhoeae/inmunología , Animales , Anticuerpos Monoclonales , Antígenos Bacterianos , Antígenos de Superficie , Reacciones Cruzadas , Epítopos , Ratones , Neisseria gonorrhoeae/clasificación , Neisseria gonorrhoeae/citología , Especificidad de la EspecieRESUMEN
The binding and subsequent cross-linking of murine IgG2a or human IgG to the Fc gamma R on the monocytic cell line THP-1 induced a rapid, dose-dependent increase in tyrosine phosphorylation of several proteins (a doublet centered around 110 kDa, and bands at 80, 60, and 52 kDa) and smaller increases in other proteins. This phosphorylation was accompanied by an increase in intracellular free Ca2+. The signaling required the cross-linking of the IgG, either through a biotin-avidin complex or with a F(ab')2 second antibody. Cross-linking of an F(ab')2 fragment of mAb 32.2 to Fc gamma RI (CD64) or an Fab fragment of mAb IV.3 to Fc gamma RII (CDw32) gave similar results to those observed with intact murine IgG2a or human IgG. Cross-linking of a F(ab')2 fragment of mAb 3G8 to Fc gamma RIII (CD16) had very little effect. Increases in both tyrosine phosphorylation and intracellular free Ca2+ were significantly reduced in a dose-dependent manner upon treatment of THP-1 cells with the tyrosine kinase inhibitors herbimycin-A, genistein, or erbstatin. Additionally, there was a marked inhibition of both Ca2+ mobilization and tyrosine phosphorylation when a F(ab')2 fragment of a mAb (T191) to the protein tyrosine phosphatase CD45, was co-cross-linked with either Hu-IgG, Mu-IgG2a, F(ab')2 anti-Fc gamma RI, or Fab anti-Fc gamma RII. Taken together these results suggest that signaling through Fc gamma RI (CD64) and Fc gamma RII (CDw32) in the monocytic leukemia cell line THP-1 gives rise to rapid tyrosine phosphorylation of several proteins followed by an increase in intracellular calcium. In addition, CD45 is able to inhibit the intracellular signaling when it is brought into close proximity to the Fc gamma R. This suggests that this transmembrane tyrosine phosphatase may regulate the stimulation of the cells through the Fc gamma R.
Asunto(s)
Calcio/metabolismo , Antígenos Comunes de Leucocito/fisiología , Receptores de IgG/fisiología , Tirosina/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Benzoquinonas , Línea Celular , Humanos , Fragmentos Fab de Inmunoglobulinas/fisiología , Inmunoglobulina G/fisiología , Lactamas Macrocíclicas , Ratones , Fosforilación , Quinonas/farmacología , Rifabutina/análogos & derivadosRESUMEN
Aggregation (e.g., through cross-linkage) of cell surface molecules is in various biologic systems a necessary event in cellular activation. Examining the Ia molecule on B cells we found that aggregation is a function of the surface Ag mobility; the higher the fraction of immobile molecules on the plane of the membrane, the better Ia forms aggregates and patches. We identify two factors that control Ia mobility and aggregability. One factor is the M1s-1a gene product; the other factor is an Ia-reactive immunogenic peptide. Both factors increase Ia aggregability and reduce the MHC Ag mobility.
Asunto(s)
Linfocitos B/fisiología , Antígenos de Histocompatibilidad Clase II/inmunología , Sitios Menores de Histocompatibilidad , Péptidos/inmunología , Animales , Membrana Celular/fisiología , Citoesqueleto/fisiología , Antígenos de Histocompatibilidad Clase I/fisiología , Fragmentos Fc de Inmunoglobulinas/inmunología , Fluidez de la Membrana , Ratones , Ratones Endogámicos , Ovalbúmina/inmunología , Agregación de Receptores , Receptores Inmunológicos/fisiologíaRESUMEN
By employing flow cytometric analysis and fluorescence resonance energy transfer (FRET), we examined the physical relationship between the T-cell receptor-CD3 complex (Ti-CD3) and the CD4 molecule on helper T cells. Through the use of an L3T4-negative murine T-cell hybridoma infectant expressing the human CD4 gene and having antigen specificity for HLA-DR, we show that binding of the Ti-CD3 complex with an anti-CD3 monoclonal antibody induces its redistribution proximal to cell-surface CD4. FRET efficiency was 9.4% on cells labeled with rhodaminated anti-CD3 and fluoresceinated anti-CD4. FRET was found to be temperature dependent, since similarly treated cells held at 4 degrees C displayed a FRET efficiency of less than 1%. Energy transfer was evident within 3 min after warming cells to 37 degrees C. Energy transfer was not detected between Ti-CD3 and the abundantly expressed leukocyte common antigen (CD45). Of greater significance was our observation that hybridomas infected with a truncated CD4 gene lacking the cytoplasmic domain failed to transfer energy despite the fact that CD4 was expressed on the cell surface at levels equivalent to or greater than the wild type. These studies suggest that after crosslinking of the Ti-CD3 on CD4+ T cells, a physical association occurs between the antigen receptor complex and CD4 and that the association is dependent upon the presence of the cytoplasmic domain of CD4.