A Postmigrational Switch among Skin-Derived Dendritic Cells to a Macrophage-Like Phenotype Is Predetermined by the Intracutaneous Cytokine Balance

Abstract

Migration of dendritic cells (DC) to secondary lymphoid organs under proinflammatory conditions coincides with their maturation and acquisition of T cell stimulatory abilities. In contrast, impaired activation of DC, e.g., in tumor-conditioned environments, may hamper their activation and possibly their subsequent migration to lymph nodes, leading to either immunological tolerance or ignorance, respectively. In this study, the influence of cytokines in the peripheral skin microenvironment on the activation state of migrating cutaneous DC was assessed using an ex vivo human skin explant model. We observed a phenotypic shift from mature CD83+ DC to immature CD14+ macrophage-like cells within 7 days subsequent to migration from unconditioned skin. These macrophage-like cells displayed a poor T cell stimulatory ability and lacked expression of CCR7, thus precluding their migration to paracortical T cell areas in the lymph nodes. The balance of suppressive and stimulatory cytokines during the initiation of migration decided the postmigrational fate of DC with IL-10 accelerating and GM-CSF and IL-4 preventing the phenotypic switch, which proved irreversible once established. These observations indicate that, in immunosuppressed environments, a postmigrational DC-to-macrophage shift may hinder T cell activation, but also that it may be prevented by prior conditioning of the tissue microenvironment by GM-CSF and/or IL-4.

Intradermal Delivery of TLR Agonists in a Human Explant Skin Model: Preferential Activation of Migratory Dendritic Cells by Polyribosinic-Polyribocytidylic Acid and Peptidoglycans

“…2, the employed gating strategy is presented. Of note, LC and CD1a + DDCs are mature subsets with a high T cell stimulatory potential, whereas the CD14 + DDCs are immature cells with macrophage-like features and a poor capacity for T cell stimulation (Table I) (12). Beside these three major subsets, also smaller CD1a + CD14 + and CD1a 2 CD14 2 populations (percentages varying per donor and ranging between 5 and 25%) were discernable (Supplemental Fig.…”

“…We previously identified several DC subsets among DCs that had migrated from human skin explants based on multicolor flow cytometry (12). An overview of the phenotype of the three major discernable subsets is presented in Table I, and in Supplemental Fig.…”

“…The cells were subsequently analyzed using a FACSCalibur and Cellquest-Pro FACS analysis software (BD Biosciences). As described, DCs were gated by their high forward and side scatter properties (12). To determine p-ERK expression in the DC subsets, skin-emigrated DCs were stained by FITC-and PerCP-Cy5-labeled mAbs to CD1a and CD14, respectively (both from BD Biosciences), washed, fixed, permeabilized, and stained by a PE-labeled mAb to ERK1/2 (pT202/pY204) using a Phosflow Starter Kit according to the manufacturers' instructions (BD Biosciences) with one notable modification (i.e., overnight permeabilization at 220˚C).…”

“…Moreover, alternative intracellular microbial PRRs, such as nucleotide-binding oligomerization domain (NOD)-like receptors (NOD1 and NOD2) or RIG-I-like helicases (MDA5 and RIG-I), can be expressed by DCs and accessory cells and share ligands with TLRs (7). Therefore, a human skin-explant model was used to explore the in vivo potential of TLR agonists to stimulate LCs and DDCs in their natural complex tissue environment (12). By intradermal delivery of TLR ligands (TLR-Ls), intradermal vaccination with TLR-based adjuvants was thus mimicked ex vivo.…”

Allogeneic Mature Human Dendritic Cells Generate Superior Alloreactive Regulatory T Cells in the Presence of IL-15

“…Skin-derived DCs were allowed to migrate out of skin biopsies during 48 h and subsequently harvested as previously described by de Gruijl et al (35). Numbers of skin-derived DCs varied from 0.5 to 1.5 3 10 4 /explant (data from three to five independent experiments).…”