Anti-CD83 Monoclonal antibody, clone IC26f [PE]

The cytoplasmic nucleotide oligomerization domain 2 (NOD2) receptor recognizes the bacterial cell wall component muramyl dipeptide (MDP). NOD2 ligation initiates the nuclear factor kappa B and the mitogen-activated protein kinase cascades. However, administering MDP alone is insufficient to elicit strong cytokine responses in various immune cells, including dendritic cells (DCs). Because the simultaneous presence of various microbial products and cytokines in inflamed tissues modulates DC function, we initiated this study to examine how interferon gamma (IFN gamma), a central modulator of inflammation, affects the NOD2-mediated signaling pathway in human conventional DCs (cDCs). Synergistic stimulation of DCs with MDP and IFN gamma increased the expression of CD40, CD80, CD83, CD86, and human leukocyte antigen DQ proteins and significantly elevated the production of pro-inflammatory cytokines IL-1 beta, IL-6, IL-12, and tumour necrosis factor (TNF), as well as anti-inflammatory cytokine IL-10. Furthermore, the simultaneous presence ofMDP and IFN gamma was necessary to decrease IkB alpha protein levels. By investigating various mechanisms implicated in MDPand IFN gamma-mediated signaling pathways, we revealed that the increased production of pro-inflammatory cytokines is highly dependent on the X-linked inhibitor of apoptosis protein (XIAP) but not on cellular IAP1 and IAP2. We also found that the NOD2 signaling pathway is regulated by the mammalian target of rapamycin (mTOR) but is not affected by phosphatidylinositol-3 kinase or signal transducer and activator of transcription 1 inhibition. Our results demonstrate, for the first time, that IFN gamma positively affects NOD2-mediated signaling in human cDCs, in a manner considerably dependent on XIAP and partially dependent on mTOR.

Dendritic cells (DCs) as professional antigen presenting cells, are important in the initiation of the primary immune response. The present study compared the morphology, phenotypes and function between monocyte-derived human DCs produced from a conventional culturing system containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 (IL-4-DC) and DCs generated by the stimulation of GM-CSF and interferon (IFN)-alpha (IFN-DC). When compared with IL-4-DC in morphology, IFN-DC contained more organelles, including endoplasmic reticulum and myelin figures, whereas mature (m)IL-4-DC contained more vacuoles in the cells. The spikes of IFN-DC were shorter and thicker. The expression of phenotypes between immature IFN-DC and IL-4-DC were diverse. Following maturation with tumor necrosis factor-alpha, IFN-DC and IL-4-DC upregulated the expression of cluster of differentiation (CD) 11c and CD83. Conversely, immature IFN-DC and IL-4-DC secreted few inflammatory cytokines including interleukin (IL)-18, IL-23, IL-12p70, IL-1 beta and anti-inflammatory IL-10. Following maturation, large amounts of the cytokines were secreted by these two DCs and mIFN-DC secreted more cytokines compared with mIL-4-DC in general. Furthermore, immature IFN-DC and IL-4-DC loaded with cytomegalovirus (CMV)-pp65 protein were unable to induce the priming of T cells, as evaluated by the intracellular staining with IFN-gamma. Notably, mature DCs exhibited the ability to present CMV-pp65 protein and activate T cells. The mIFN-DC activated a greater proportion of autologous CD4(+) T cells (0.91 vs. 0.31%, P<0.001) and CD8(+) T cells (0.90 vs. 0.48%, P<0.001) to secret IFN-gamma compared with mIL-4-DC. The results suggested that the morphology, phenotypes and cytokine secretion of IFN-DC and IL-4-DC were diverse. The mIFN-DC were more effective in priming and cross-priming T cells when compared with IL-4-DC.