RESUMEN
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
G28-5 sFv-PE40 is a single-chain immunotoxin that is cytotoxic toward malignant B cells expressing CD40. Human monocytes, which also express cell surface CD40, were found to be insensitive to the immunotoxin. Activation of the monocytic cell line THP-1, or freshly isolated peripheral blood monocytes with IFN-gamma, but not IL-3, GM-CSF, IL-6, or TNF-alpha, greatly sensitized the cells toward G28-5 sFv-PE40, lowering the EC50 value from > 10 micrograms/ml to 80 ng/ml. This sensitization could not be explained simply by the two- to threefold increase in cell surface CD40 expression induced by IFN-gamma since TNF-alpha or the combination of TNF-alpha and IL-6 gave similar increases in CD40 expression but did not sensitize the cells to the immunotoxin. Internalization of G28-5 sFv-PE40 after IFN-gamma activation was also increased threefold, reflective of the increase in CD40 expression. IFN-gamma-treated but not -untreated THP-1 cells produced IL-6 and TNF-alpha following incubation with G28-5 sFv-PE40, indicating an association between CD40 signaling, which induces cytokine production, and sensitivity to the immunotoxin. HUVECs also express CD40 but were found to be insensitive to the anti-CD40 immunotoxin. A combination of IFN-gamma and TNF-alpha, but neither cytokine alone, sensitized the endothelial cells to G28-5 sFv-PE40. These data show that activation with cytokines can sensitize monocytes and endothelial cells to an immunotoxin targeted to CD40, most likely by altering the trafficking and/or processing of the immunotoxin after receptor-mediated internalization.
Asunto(s)
ADP Ribosa Transferasas , Toxinas Bacterianas , Antígenos CD40/inmunología , Antígenos CD40/farmacología , Exotoxinas/farmacología , Inmunotoxinas/farmacología , Interferón gamma/farmacología , Monocitos/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de Virulencia , Antígenos CD40/biosíntesis , Células Cultivadas , Resistencia a Medicamentos , Endocitosis , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Interleucina-6/farmacología , Leucemia Monocítica Aguda/patología , Monocitos/inmunología , Monocitos/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/inmunología , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales , Exotoxina A de Pseudomonas aeruginosaRESUMEN
CD40 is a glycoprotein of about 50 kDa that plays a crucial role in B cell growth and differentiation. It is found on the surface of B cells, follicular dendritic cells, monocytes, and some endothelial, epithelial, and carcinoma cells. Engagement of CD40 with anti-CD40 mAbs, gp39 expressed on the cell surface or soluble forms of gp39, primes B cells to efficiently respond to subsequent stimulatory signals leading to B cell proliferation, differentiation, and isotype switching. Peripheral monocytes also express CD40 on the cell surface and expression in increased following treatment with IFN-gamma. Using a soluble murine CD8/human gp39 fusion protein (sgp39) we have found that CD40 plays a crucial role in the regulation of monocyte function. Stimulation of human peripheral monocytes with sgp39 induced homotypic aggregation and significantly increased the expression of several cell-surface proteins including CD54, MHC class II, CD86, and CD40. Soluble gp39 also dramatically enhanced monocyte survival, preventing the onset of apoptosis that normally occurs upon withdrawal of serum. Finally, in the absence of any costimulatory molecules, sgp39 stimulated monocytes to produce TNF-alpha, IL-1 beta, IL-6, and IL-8. These results suggest that ligation of CD40 on human monocytes induces phenotypic changes that would be expected to influence T cell activation by the monocyte and also to enhance or prolong inflammatory responses.
Asunto(s)
Antígenos CD40/inmunología , Inflamación/inmunología , Glicoproteínas de Membrana/inmunología , Monocitos/inmunología , Animales , Antígenos CD/biosíntesis , Ligando de CD40 , Agregación Celular/efectos de los fármacos , Células Cultivadas , Citocinas/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Humanos , Glicoproteínas de Membrana/farmacología , Ratones , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
The effect of interferon-gamma (IFN-gamma) on the ability of human monocytic cells to process exogenous (major histocompability complex class II) antigens was investigated. The processing (i.e. protein degradation) of antigens that were internalized via Fc gamma receptor (Fc gamma R) was followed for various times after treatment of cells with IFN-gamma. THP-1 cells that had been treated with IFN-gamma for 4 h degraded antigen, internalized as an immune complex, at an enhanced rate. After 24 h of IFN-gamma treatment the rate of processing was similar to untreated cells. Unexpectedly, in cells which had been treated for 48-72 h there was a significant decrease in the rate of processing of the exogenous antigen. These effects were not due to changes in the rate of internalization of immune complex. The inhibition of the rate of processing was independent of the type of antigen, was dependent on the dose of IFN-gamma, and also occurred with normal human peripheral monocytes. Analysis of the degraded peptides by high-pressure liquid chromatography indicated that some of the peptides generated in the IFN-gamma-treated cells were both quantitatively and qualitatively different from the peptides generated in untreated cells. These data suggest that IFN-gamma modulates the way in which antigens, internalized through Fc receptors as immune complexes, are processed. Additionally, the results imply that decreases in the rate of antigen processing may lead to more efficient antigen presentation.