IL-12-Impaired and IL-12-Secreting Dendritic Cells Produce IL-23 upon CD154 Restimulation

Abstract

Experimental studies in monkeys on the basis of ex vivo-generated, reinjected dendritic cells (DCs) allow investigations of primate DC biology in vivo. To study in vitro and in vivo properties of DCs with a reduced capacity to produce IL-12, we adapted findings obtained in vitro with human cells to the rhesus macaque model. Following exposure of immature monocyte-derived monkey DCs to the immunomodulating synthetic polypeptide glatiramer acetate (GA) and to dibutyryl-cAMP (d-cAMP; i.e., a cAMP enhancer that activates DCs but inhibits the induction of Th1 immune responses), the resulting DCs displayed a mature phenotype with enhanced Ag-specific T cell stimulatory function, notably also for memory Th1 cells. Phosphorylation of p38 MAPK was not induced in GA/d-cAMP-activated DCs. Accordingly, these cells secreted significantly less IL-12p40 (p ≤ 0.001) than did cytokine-activated cells. However, upon restimulation with rhesus macaque CD154, GA/d-cAMP-activated DCs produced IL-12p40/IL-23. Additionally, DCs activated by proinflammatory cytokines following protocols for the generation of cells used in clinical studies secreted significantly more IL-23 upon CD154 restimulation than following prior activation. Two days after intradermal injection, GA/d-cAMP-activated fluorescence-labeled DCs were detected in the T cell areas of draining lymph nodes. When similarly injected, GA/d-cAMP as well as cytokine-activated protein-loaded DCs induced comparable Th immune responses characterized by secretion of IFN-γ, TNF, and IL-17, and transiently expanded FOXP3+ regulatory T cells. Reactivation of primate DCs through CD154 considerably influences their immmunostimulatory properties. This may have a substantial impact on the development of innovative vaccine approaches.

Protumor vs Antitumor Functions of IL-17

“…In addition, IL-23 overexpression is likely to increase systemic IL-23 levels, leading to the growth and survival of CD8 ϩ memory T cells. IL-23 can be induced in Propionibacterium acnes-conditioned dendritic cells upon re-stimulation with CD154 (71). In a model of the P. acnes infection, CD40-deficient animals had impaired IL-17 but not IFN-␥ response.…”

Comparative Analysis of the Interaction of Helicobacter pylori with Human Dendritic Cells, Macrophages, and Monocytes

“…Immature monocyte-derived DCs were generated by culturing 1 ϫ 10 6 monocytes/ml in complete RPMI 1640 medium containing 2 mM L-glutamine (Merck, Darmstadt, Germany, or Gibco) and 10 mM HEPES (Gibco) supplemented with 10% FCS, and penicillin (100 U/ml)-streptomycin (100 g/ml) (Sigma) as well as 1,000 U/ml of granulocyte-macrophage colony-stimulating factor ([GM-CSF] Bayer HealthCare Pharmaceuticals, VA) and 100 U/ml recombinant IL-4 (R&D Systems, MN) (41). A total of 3 ϫ 10 6 cells per well were cultivated in six-well culture plates (TPP, Trasadingen, Switzerland, or Nunc, Roskilde, Denmark).…”

Nocardia farcinica Activates Human Dendritic Cells and Induces Secretion of Interleukin-23 (IL-23) Rather than IL-12p70

“…Human T cells were enriched from CD14-depleted PBMCs by HLA-DR-negative magnetic bead selection (33). A total of 1 ϫ 10 8 cells/ml were incubated with FITC-conjugated monoclonal antibodies (MAbs; 1:50 dilution) against human HLA-DR (BD Pharmingen, Heidelberg, Germany), the MAbs were washed out, and cells were incubated with anti-FITC antibody-conjugated magnetic beads (Miltenyi Biotec).…”

“…Penicillin (100 U/ml)-streptomycin (100 g/ml) was added in experiments without viable bacteria (see below). The endocytic functions of DCs were studied by using 10 g/ml dextran-fluorescein isothiocyanate (dextran-FITC) (Molecular Probes/Invitrogen) as described previously (33,40).…”

“…Buffy coats (German Red Cross, Berlin, Germany) or peripheral blood from healthy volunteers were used to isolate peripheral blood mononuclear cells (PBMCs). Monocyte-derived DCs were generated from magnetically separated CD14 ϩ monocytes (Miltenyi Biotec, Bergisch-Gladbach, Germany) as previously described (33). Briefly, monocytes were cultured in medium as described above, with supplementation of the medium with 1,000 U/ml human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) (sargramostim; Leukine, Berlex, Richmond, CA) and 100 U/ml human rIL-4 (R&D Systems, Wiesbaden-Nordenstadt, Germany), at 3 ϫ 10 6 cells/well in 6-well cell culture dishes (Nunc, Roskilde, Denmark) for 6 days, and fresh medium and cytokines were added every other day.…”