RESUMEN
We have previously shown that T cell receptor-activated mouse T helper (Th)1 clones induce the production of interleukin (IL)-12 by splenic antigen-presenting cells (APC). Here, we show that the expression of CD40L by activated T cells is critical for T cell-dependent IL-12 production by mouse macrophages. IL-12 was produced in cultures containing alloreactive Th1 clones stimulated with allogeneic peritoneal macrophages, or in cultures of splenocytes stimulated with anti-CD3. Anti-CD40L monoclonal antibodies (mAb) inhibited the production of IL-12, but not IL-2, in these cultures by approximately 90% and had dramatic inhibitory effects on antigen-dependent proliferation of Th1 clones. In addition, both activated T cells and a Th1 clone derived from CD40L knockout mice failed to induce IL-12 production from splenic APC or peritoneal macrophages. Finally, macrophages cultured in the absence of T cells produced IL-12 upon stimulation with soluble recombinant CD40L in combination with either supernatants from activated Th1 clones or with interferon-gamma and granulocyte/macrophage colony-stimulating factor. Thus, both CD40L-dependent and cytokine-mediated signals from activated T cells are required to induce the production of IL-12 by macrophages. A blockade at the level of IL-12 production may explain, at least in part, the dramatic ability of anti-CD40L mAb to inhibit disease in animal models that are dependent upon the generation of a cell-mediated immune response. Moreover, a defect in T cell-dependent induction of IL-12 may contribute to the immune status of humans that lack functional CD40L.
Asunto(s)
Antígenos CD40/farmacología , Interleucina-12/biosíntesis , Macrófagos Peritoneales/metabolismo , Glicoproteínas de Membrana/farmacología , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Ligando de CD40 , Sistema Libre de Células/inmunología , Células Clonales , Citocinas/farmacología , Interacciones Farmacológicas/inmunología , Femenino , Activación de Linfocitos , Masculino , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Noqueados , Proteínas Recombinantes/farmacología , Linfocitos T/inmunologíaRESUMEN
A ligand was cloned for murine OX40, a member of the TNF receptor family, using a T cell lymphoma cDNA library. The ligand (muOX40L) is a type II membrane protein with significant identity to human gp34 (gp34), a protein whose expression on HTLV-1-infected human leukemic T cells is regulated by the tax gene. The predicted structures of muOX40L and gp34 are similar to, but more compact than, those of other ligands of the TNF family. Mapping of the muOX40L gene revealed tight linkage to gld, the FasL gene, on chromosome 1. gp34 maps to a homologous region in the human genome, 1q25. cDNAs for human OX40 receptor were cloned by cross-hybridization with muOX40, and gp34 was found to bind the expressed human receptor. Lymphoid expression of muOX40L was detected on activated T cells, with higher levels found on CD4+ rather than CD8+ cells. The cell-bound recombinant ligands are biologically active, co-stimulating T cell proliferation and cytokine production. Strong induction of IL-4 secretion by muOX40L suggests that this ligand may play a role in regulating immune responses. In addition, the HTLV-1 regulation of gp34 suggests a possible connection between virally induced pathogenesis and the OX40 system.
Asunto(s)
Glicoproteínas de Membrana , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos de Superficie , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 1 , Clonación Molecular , Citocinas/biosíntesis , Femenino , Regulación de la Expresión Génica , Virus Linfotrópico T Tipo 1 Humano/metabolismo , Humanos , Ligandos , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Ligando OX40 , Receptores OX40 , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Linfocitos T/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genética , Factores de Necrosis TumoralAsunto(s)
Glicoproteínas de Membrana , Receptores del Factor de Necrosis Tumoral/química , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Secuencia de Bases , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Ligando OX40 , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/fisiología , Proteínas Recombinantes de Fusión , Homología de Secuencia , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/química , Factores de Necrosis TumoralRESUMEN
Murine and human CD40 ligand (CD40L) were recently cloned, expressed, and shown to possess potent activity on human and murine B cells, including stimulation of proliferation and Ig secretion in the presence of cytokines. In addition to its action on B lymphocytes, this report demonstrates that CD40L induced both CD4+ and CD8+ T cells isolated from murine lymphoid tissues to proliferate in the presence of submitogenic dosages of Con A, PHA, CD3 mAb, and TCR-alpha beta mAb. The presence of CD40L during suboptimal TCR stimulation resulted in increased expression of the activation Ags IL-2R alpha and CD69 and increased IL-2 production. Taken together, these results show that CD40L is a potent activator of murine T cells and suggest that CD40L is involved in the regulation of T cell function mediated through T:T cell interaction.
Asunto(s)
Glicoproteínas de Membrana/inmunología , Linfocitos T/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Ligando de CD40 , Antígenos CD8/metabolismo , Citocinas/biosíntesis , Femenino , Humanos , Interleucinas/farmacología , Ligandos , Activación de Linfocitos , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mitógenos/administración & dosificación , Embarazo , Receptores de Antígenos de Linfocitos T alfa-beta/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Bazo/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
The stimulatory requirements for T cells bearing gamma delta T cell receptors are distinct from those of alpha beta T cells. We have analyzed the ability of the CD40 ligand (CD40L) to activate neonatal thymic gamma delta T cells. CD40L is expressed on activated T cells and has been shown to induce B cell proliferation and Ig secretion as well as monocyte activation. We now demonstrate that, in the presence of an anti-TCR-gamma delta Ab, CD40L is able to induce the proliferation of neonatal thymic gamma delta cells. The presence of CD40L also leads to enhanced expression of a variety of activation-associated Ag including CD25, CD69, CD44, and Ly6C. In addition to proliferation, CD40L induces lectin-mediated cytolytic activity in thymic gamma delta T cells as well as the production of IFN-gamma and TNF-alpha. We were unable to detect IL-2 or IL-4 production in response to CD40L, and Ab-blocking studies indicate that the mechanism of activation appears to involve IL-1 but is independent of IL-2, IL-4, and IL-7. These results suggest that, in addition to its effects on B cells and monocytes, CD40L can costimulate the activation of thymic gamma delta T cells.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Subgrupos de Linfocitos T/inmunología , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Antígenos de Diferenciación de Linfocitos T/metabolismo , Antígeno B7-1/metabolismo , Antígenos CD40 , Citotoxicidad Inmunológica , Técnicas In Vitro , Interferón gamma/biosíntesis , Interleucina-1/antagonistas & inhibidores , Interleucina-2/antagonistas & inhibidores , Interleucina-2/biosíntesis , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T gamma-delta/antagonistas & inhibidores , Transducción de Señal/inmunología , Factor de Necrosis Tumoral alfa/biosíntesis , Regulación hacia ArribaRESUMEN
Biotinylated interleukin-4 (IL-4) was used to examine IL-4 receptor (IL-4R) expression on a range of human B-cell lines by flow cytometry. Using high concentrations of biotinylated IL-4, we have identified a novel low-affinity IL-4 receptor expressed at high levels on pre-B lines. Expression of this low-affinity receptor did not correlate with detected mRNA levels for the previously cloned receptor or with reactivity of two anti-human IL-4R monoclonal antibodies (MoAb). Radiolabeled IL-4 cross-linking studies using pre-B lines showed a doublet of 65 to 75 Kd in contrast to the 110- to 130-Kd molecule detected on cells expressing the cloned IL-4R. A soluble IL-4 binding protein (IL-4bp) was purified from the supernatants of three pre-B lines expressing the low-affinity receptor on their surface. IL-4bp could block both IL-4-mediated CD23 induction on tonsil B cells and IL-4-induced inhibition of proliferation of the pre-B line JM1. Partial N-terminal amino acid sequence was obtained from purified IL-4bp that confirmed this protein to be novel. A 12 amino acid peptide based on the IL-4bp sequence was used to produce a polyclonal antiserum that was reactive with purified IL-4bp, and also bound to the surface of pre-B cells but not to murine CTLL cells transfected with the human IL-4R. Blocking MoAb against the previously characterized high-affinity receptor inhibited IL-4-mediated proliferation of hIL-4R+ CTLL cells but had no effect on IL-4-induced inhibition of JM1 cell proliferation, and only partially inhibited IL-4-mediated CD23 and sIgM induction and proliferation of tonsil B cells. The data presented here provide evidence for a novel cell-surface expressed low-affinity IL-4R that also exists as a biologically active soluble IL-4 binding protein.
Asunto(s)
Linfocitos B/química , Interleucina-4/metabolismo , Receptores Mitogénicos/química , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Reactivos de Enlaces Cruzados , Expresión Génica , Humanos , Datos de Secuencia Molecular , ARN Mensajero/genética , Receptores de Interleucina-4 , Receptores Mitogénicos/inmunología , SolubilidadRESUMEN
Signaling through the cell surface molecule, CD40, is known to play an important role in the proliferation and differentiation of B lymphocytes. Using the thymoma cell line EL4, we recently identified and cloned a cDNA encoding a murine ligand for the CD40 molecule (mCD40-L) and showed that it has biological activity in vitro. A cDNA encoding a human homologue of the mCD40-L was isolated using crosshybridization techniques from an activated peripheral blood T cell library. The predicted amino acid sequence indicates that this human ligand for CD40 (hCD40-L) is a 261 amino acid type II membrane protein that exhibits 78% amino acid identity with its murine counterpart. Northern blot and FACS analyses suggest that the hCD40-L is restricted in its expression to T lymphocytes, and that it is most abundant on the CD4+ T cell subpopulation. Cells transfected with hCD40-L caused the proliferation of human tonsil B cells in the absence of costimuli and, in the presence of interleukin 4, induced immunoglobulin E secretion from purified human B cells. A comparison of the efficacy of the hCD40-L and mCD40-L in these assays is presented.
Asunto(s)
Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos B/fisiología , Linfocitos B/inmunología , Inmunoglobulina E/metabolismo , Activación de Linfocitos , Glicoproteínas de Membrana/fisiología , Secuencia de Aminoácidos , Animales , Antígenos CD/metabolismo , Secuencia de Bases , Antígenos CD40 , Ligando de CD40 , Células Cultivadas , Clonación Molecular , Humanos , Inmunoglobulina E/biosíntesis , Interleucina-4/farmacología , Cinética , Linfoma , Glicoproteínas de Membrana/genética , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/farmacología , Homología de Secuencia de Aminoácido , Linfocitos T/inmunología , Células Tumorales CultivadasRESUMEN
We have identified the murine thymoma line EL4 as a source of biologically active CD40 ligand. Using a biotin-labeled soluble CD40.Fc fusion protein, consisting of the extracellular domain of human CD40 and the Fc region of human IgG1, EL4 cells were subjected to repeated flow cytometric cell sorting to select for cells with enhanced biotinylated CD40.Fc binding. After nine rounds of sorting, the number of CD40.Fc binding sites/cell had risen from 450 on the unsorted parental EL4 cells to 15,000 on EL40.9 cells (EL4 cells sorted with biotinylated CD40.Fc for nine rounds). Scatchard analysis of radiolabeled CD40.Fc binding revealed that the surface-expressed CD40 ligand on parental EL4 and EL40.9 cells bound its receptor with a single class of high-affinity sites (Kd = 0.5 nM). Supernatant (SN) from the sorted EL40.9 cells was found to contain human and murine B cell stimulatory activity which could be removed by preclearing with immobilized CD40.Fc, confirming the presence of soluble CD40 ligand in the preparations. EL40.9 supernatant enhanced soluble CD23 (sCD23) release and induced IgE secretion from interleukin 4-stimulated human B cells. In addition, EL40.9 SN contained proliferative activity for anti-IgM-activated murine B cells which could be removed by treatment with immobilized CD40.Fc. However, the same SN had no demonstrable activity on the proliferation of human B cells. The results presented here describe, for the first time, a source of membrane-bound and soluble CD40 ligand. The soluble form of this murine ligand has activity on murine and human B cells and induces some of the functional responses predicted for the ligand based on the action of stimulatory antibodies directed against the human CD40 surface molecule.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Animales , Antígenos de Diferenciación de Linfocitos B/biosíntesis , Linfocitos B/inmunología , Antígenos CD40 , Citometría de Flujo , Humanos , Inmunoglobulina E/metabolismo , Interleucina-4/farmacología , Ligandos , Activación de Linfocitos , Ratones , Receptores Fc/biosíntesis , Receptores de IgERESUMEN
The CD40 surface molecule is a 277-amino-acid glycoprotein expressed on B lymphocytes, epithelial cells and some carcinoma cell lines. Monoclonal antibodies against CD40 mediate a variety of effects on B lymphocytes, including induction of intercellular adhesion, short- and long-term proliferation, differentiation and enhanced tyrosine phosphorylation of proteins. In addition, germinal centre centrocytes are prevented from undergoing apoptosis by activation through CD40 and receptor for antigen. These data indicate that CD40 could be a receptor for an unknown ligand with important functions in B-cell development and activation. This hypothesis is strengthened by the homology of the extracellular region of the CD40 molecule with a family of cell-surface glycoproteins that includes the receptors for nerve growth factor and tumour necrosis factor. Here we report the cloning of a ligand for CD40 that is expressed on the cell surface of activated T cells and mediates B-cell proliferation in the absence of co-stimulus, as well as IgE production in the presence of interleukin-4.
Asunto(s)
Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos B/metabolismo , Glicoproteínas de Membrana/genética , Secuencia de Aminoácidos , Animales , Linfocitos B/metabolismo , Secuencia de Bases , Antígenos CD40 , Ligando de CD40 , Humanos , Inmunoglobulina E/metabolismo , Ligandos , Glicoproteínas de Membrana/fisiología , Ratones , Datos de Secuencia Molecular , Unión Proteica , Linfocitos T/metabolismo , Células Tumorales CultivadasRESUMEN
Although numerous in vitro studies have demonstrated that cytokines are involved in the generation of alloreactive effector cells, the role of these regulatory substances in vivo is less well defined. We have recently cloned and expressed cDNAs encoding both membrane bound and soluble forms of the murine IL-4R. The effects of murine rIL-4 and a recombinant, soluble, extracellular portion of the murine IL-4R soluble(s) IL-4R on the generation of alloresponsiveness in vivo were evaluated by measuring the lymphoproliferative response to a localized injection of allogeneic cells and the survival of cardiac allografts. Administration of IL-4 to BALB/c mice resulted in a slight augmentation of the anti-C57BL/6 lymphoproliferative response compared to that occurring in control, mouse serum albumin-(MSA) treated mice. In contrast, the sIL-4R suppressed this response to allogeneic cells in a dose-dependent manner, with a dose of 50 micrograms/kg/day causing nearly complete inhibition of the response as compared to the response observed in controls. The inhibitory effect of sIL-4R was reversed by simultaneous administration of IL-4. A neutralizing antibody against IL-4 (11B11) and another against the IL-4R (M1) were also effective inhibitors of the response when given at 100- to 1000-fold higher concentrations than the amount of sIL-4R required to inhibit the response. In cardiac allograft experiments, BALB/c mice were engrafted with newborn C57BL/6 hearts in the ear pinnae and treated with sIL-4R (50 micrograms/kg) or MSA. Such allografts survived an average of 4 days longer in sIL-4R-treated mice than in MSA-treated controls. In conclusion, neutralization of IL-4 inhibits alloresponsiveness in vivo. These results confirm a regulatory role for this pleiotropic cytokine in allograft rejection and suggest a therapeutic value for IL-4 antagonists alone or in combination with other immunosuppressive regimens.