Purpose/Objective: B7h, expressed by several cell types, binds Inducible co-stimulator (ICOS) expressed by activated T cells. This work investigated the effect of B7h triggering by ICOS on human monocyte-derived dendritic cells (DC). Materials and methods: Monocytes were induced to differentiate to immature DC (iDC) by a 5-day culture with GM-CSF and IL-4. They were then induced to mature for 2 days with LPS in the absence (mDC) and the presence (mDCICOS) of ICOS-Fc, a recombinant form of ICOS fused to the human or mouse IgG1 Fc; the mutant F119SICOSFc, which doesn’t bind B7h, and CTLA-4-Fc were used as controls. Supernatants were then collected for cytokine analysis by ELISA. In the antigen presentation assay, keyhole limpet hemocyanin (KLH) was added to DC with during the maturation step in the presence of the different ICOS-constructs; these DC were then cocultured with autologous T cells, and IFN-gamma production from CD4+ and CD8+ T cells was assessed by ELISpot. In the adhesion assay, endothelial cells (EC) were treated or not with the different ICOS-Fc constructs and incubated with iDC or mDC; adherent cells were then counted by the Image Pro Plus Software. In the migration assay, iDC or mDC were plated onto the Boyden chamber in the presence of appropriate chemoattractants and the different ICOS constructs. Cells on the bottom of the filter were stained with crystal-violet and counted. Results: Results showed that mDCICOS produced higher amounts of IL-23 and IL-10, and lower amounts of TNF-a and IL-6 than mDC. Moreover upon pulsing with KLH, mDCICOS were better stimulators of IFN-gamma production in CD8+ T cells than mDC, whereas no difference was found in CD4+ T cells. Finally, ICOS-Fc inhibited adhesion of both iDC and mDC to EC, and their migratory activity. Conclusions: These data suggests that ‘reverse’ signaling mediated by B7h modulates DC maturation by influencing cytokine secretion and potentiating antigen cross-presentation. Moreover, it modulates DC adhesiveness and migration and might thus influence their recruitment into tissues
Triggering of B7h by the inducible costimulator modulates maturation and migration of monocyte-derived dendritic cells
OCCHIPINTI, SERGIO;DIANZANI, Chiara;GIOVARELLI, Mirella;
2012-01-01
Abstract
Purpose/Objective: B7h, expressed by several cell types, binds Inducible co-stimulator (ICOS) expressed by activated T cells. This work investigated the effect of B7h triggering by ICOS on human monocyte-derived dendritic cells (DC). Materials and methods: Monocytes were induced to differentiate to immature DC (iDC) by a 5-day culture with GM-CSF and IL-4. They were then induced to mature for 2 days with LPS in the absence (mDC) and the presence (mDCICOS) of ICOS-Fc, a recombinant form of ICOS fused to the human or mouse IgG1 Fc; the mutant F119SICOSFc, which doesn’t bind B7h, and CTLA-4-Fc were used as controls. Supernatants were then collected for cytokine analysis by ELISA. In the antigen presentation assay, keyhole limpet hemocyanin (KLH) was added to DC with during the maturation step in the presence of the different ICOS-constructs; these DC were then cocultured with autologous T cells, and IFN-gamma production from CD4+ and CD8+ T cells was assessed by ELISpot. In the adhesion assay, endothelial cells (EC) were treated or not with the different ICOS-Fc constructs and incubated with iDC or mDC; adherent cells were then counted by the Image Pro Plus Software. In the migration assay, iDC or mDC were plated onto the Boyden chamber in the presence of appropriate chemoattractants and the different ICOS constructs. Cells on the bottom of the filter were stained with crystal-violet and counted. Results: Results showed that mDCICOS produced higher amounts of IL-23 and IL-10, and lower amounts of TNF-a and IL-6 than mDC. Moreover upon pulsing with KLH, mDCICOS were better stimulators of IFN-gamma production in CD8+ T cells than mDC, whereas no difference was found in CD4+ T cells. Finally, ICOS-Fc inhibited adhesion of both iDC and mDC to EC, and their migratory activity. Conclusions: These data suggests that ‘reverse’ signaling mediated by B7h modulates DC maturation by influencing cytokine secretion and potentiating antigen cross-presentation. Moreover, it modulates DC adhesiveness and migration and might thus influence their recruitment into tissues
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