Plasmacytoid Dendritic Cells Migrate in Afferent Skin Lymph

Abstract

Conventional dendritic cells enter lymph nodes by migrating from peripheral tissues via the lymphatic route, whereas plasmacytoid dendritic cells (pDC), also called IFN-producing cells (IPC), are described to gain nodes from blood via the high endothelial venules. We demonstrate here that IPC/pDC migrate in the afferent lymph of two large mammals. In sheep, injection of type A CpG oligodinucleotide (ODN) induced lymph cells to produce type I IFN. Furthermore, low-density lymph cells collected at steady state produced type I IFN after stimulation with type A CpG ODN and enveloped viruses. Sheep lymph IPC were found within a minor BnegCD11cneg subset expressing CD45RB. They presented a plasmacytoid morphology, expressed high levels of TLR-7, TLR-9, and IFN regulatory factor 7 mRNA, induced IFN-γ production in allogeneic CD4pos T cells, and differentiated into dendritic cell-like cells under viral stimulation, thus fulfilling criteria of bona fide pDC. In mini-pig, a CD4posSIRPpos subset in afferent lymph cells, corresponding to pDC homologs, produced type I IFN after type A CpG-ODN triggering. Thus, pDC can link innate and acquired immunity by migrating from tissue to draining node via lymph, similarly to conventional dendritic cells.

Bluetongue Virus Targets Conventional Dendritic Cells in Skin Lymph

“…CD11c, which is not expressed by pDCs (37), was chosen to sort the lymph cDCs and proceed to BTV RNA detection. As controls, CD4 ϩ T cells, which represent the major subset in afferent lymph (60% of TL cells [39]) were sorted from the same samples (Fig.…”

“…Lymph cell subsets were enriched by either positive immunomagnetic cell sorting (Myltenyi Biotech) or negative immunomagnetic cell sorting (Dynal). For the positive selection of CD4 ϩ T cells or cDCs, the anti-CD4 (ST4) or anti-CD11c (mix of OM1 and BAQ153) monoclonal antibodies (MAbs) were used as previously described (37). In some experiments involving "untouched" cDCs, the cells were enriched by negative sorting using a mixture of anti-CD4 (ST4), -CD8 (CACT80C), -␥/␦ T cells (86D), -B cells (DU2-104), and -CD14 (CAM36), followed by a mix of magnetic Dynabeads (Dynal) coated with human MAb anti-mouse immunoglobulin G (IgG) and rat MAb anti-mouse IgM, according to the manufacturer's recommendations.…”

“…Uninfected cDCs were used as controls for basal autologous cDC-induced proliferation. After 3 days in culture, the cell supernatants were collected for the detection of gamma interferon (IFN-␥) and IL-10, using a ruminant-specific enzyme-linked immunosorbent assay (37). After the 5-day cultures were complete, the cells were labeled for CD4 (GC50A1) and CD8 (7C2) detection as described above, and the CD4 ϩ and CD8 ϩ cells that had divided were analyzed by FACS.…”

“…Lymph cells (5 ϫ 10 6 ) were treated with primary antibodies (2 g/ml) in FACS medium (RPMI 1640 containing 4% horse serum) for 30 min at 4°C, washed twice, and further incubated with FITC, phycoerythrin, cyanine 5, or tricolor-conjugated purified goat IgG directed to specific murine isotypes (Caltag Laboratories) at a 1:200 final dilution. In some instances, double labeling involving two primary IgG1 were conducted as previously described (37). For the intracellular detection of NS2, labeled cells were treated with the Fix and Perm kit (Caltag Laboratories), following the manufacturer's instructions, and then intracellularly labeled with rabbit serum directed against NS2 (1:500; raised in our lab by immunizing rabbits with purified recombinant NS2).…”

“…Labeling of lymph cells was conducted with mouse MAb antiruminant determinants that have been described previously (15,37) …”