Table 1.
Antibody Panel for Surface Staining of Bovine Peripheral Blood DC subsets.
Table 2.
Antibody Panel for Intracellular Cytokine Staining.
Figure 1.
Phenotypic characterization of peripheral blood DC subsets.
7-color flow cytometric analysis of bovine PBMC to identify DC. Doublets (B) and dead cells (C) were excluded from the total PBMC population. Using lineage specific antibodies (anti-CD3, anti-CD14, anti-IgM, and anti-CD11b), T cells, monocytes, B cells and NK cells were excluded (D). Lineage negative cells were then gated to identify MHC class II+ and CD4+ cells (E), and the MHC class II+ cells were assessed for CD11c expression (F). Surface expression of DEC205, CD172a, and CD16 by CD4+ DC (G), CD11c+ DC (H), and CD11c− DC (I). Size (FSC) and complexity (SSC) of DC subsets was also assessed by back-gating on the cell of interest. Data are representative of four independent experiments in six different animals. Numbers on plots represent average percentage of cells expressing the surface molecules in six cattle, and error bars represent standard error.
Figure 2.
FACS purification of peripheral blood DC subsets.
Schematic diagram of the DC isolation protocol from PBMC. Following density centrifugation, PBMC were subjected to immuno-magnetic depletion of lineage positive cells, to enrich DC (A). To exclude remnant lineage positive cells present in the enriched DC population, a 5-color sort was performed using a BD FACS Aria II, according to the gating strategy shown in Figure 1A – F. Three major DC subsets that are MHC class II−/CD4+ (C) MHC class II+/CD11c+ (D), and MHC class II+/CD11c− (E). Numbers on plots represent percentage of cells. Data are representative of four independent experiments.
Figure 3.
Transmission electron microscopy of peripheral blood DC subsets.
TEM analyses of freshly isolated DC subsets and cytokine-stimulated CD11c− DC. CD4+ DC display a plasmacytoid phenotype, which includes a prominent ER and dendritic protrusions on the plasma membrane (A). CD11c+ DC exhibit multi-lobulated nucleus, ruffled cell membrane, and do not display the prominent ER (B). CD11c− DC have a smooth plasma membrane and a rounded nucleus (C). Three-day GM-CSF and IL-4-stimulated CD11c− DC display dendritic projections and multi-lobulated nucleus (D). Bars denote 500 nm.
Figure 4.
Expression levels of MHC class II and co-stimulatory molecules by un-stimulated and TLR-activated peripheral blood DC subsets.
Comparison of expression of MHC class II and CD80 by un-stimulated, FACS purified CD4+ DC, CD11c+ DC, and CD11c− DC (A). These DC subsets were stimulated with TLR-agonists or media control for 12 hours and subjected to immuno-staining and flow cytometric analysis. The expression of MHC class II (B) and CD80 (C) by individual DC subsets is shown following TLR-activation. Data shown is one of two experiments with virtually identical results, expressed as mean fluorescence intensity (MFI).
Figure 5.
Pro-inflammatory cytokine production by peripheral blood DC subsets.
PBMC were stimulated for 5 hours with TLR-agonists, and simultaneously treated with Brefeldin A. Cells were immuno-stained with antibodies against surface markers as demonstrated in Figure 1, then intracellularly stained to detect TNF-alpha production. Numbers in plots represent percentage of CD4+ DC (A), CD11c+ DC (B), and CD11c− DC (C) producing TNF-alpha. Plots are representative of one animal. Graphs (D) show TNF-alpha production by DC subsets in 4 animals. Data are representative of two independent experiments. Error bars represent standard deviation.
Figure 6.
Type I IFN production by FACS purified peripheral blood DC subsets.
FACS purified DC subsets and whole PBMC were stimulated with R848 or media control for 20 hours. The supernatant was assessed for the presence of type I IFN by using a Mx-CAT reporter assay. Briefly, in the presence of type I IFN, the type I IFN-inducible Mx promoter would drive the transcription of chloramphenicol acetyltransferase (CAT). CAT protein levels are then detected by an ELISA assay. CAT expression indicated by absorbance at 405 nm (A), and calculated type I IFN levels (B) are shown.
Figure 7.
Real-time PCR quantification of TLR expression by peripheral blood DC subsets.
FACS purified CD4+ DC (A), CD11c+ DC (B), and CD11c− DC (C) were assessed for the expression of TLR3, TLR7, TLR8, and TLR9. Quantification of TLR expression was normalized to expression of GAPDH expression by DC subsets. Data are representative of two independent experiments.
Figure 8.
Internalization and degradation of exogenous antigen by peripheral blood DC subsets.
PBMC were incubated with self-quench fluorescent DQ-OVA for 1.5 hours at 4°C and 37°C. Cells were immuno-stained with surface antibodies to identify DC subsets as outlined in Figure 1. Dot plots show fluorescence of BODIPY that represents cleavage of DQ-OVA by CD4+ DC (A), CD11c+ DC (B), and CD11c− DC (C) from one animal. Calculation of DQ-OVA degradation efficiency by 3 different cattle was performed by subtracting BODIPY fluorescence of 4°C from 37°C (D). Error bars represent standard deviation.
Figure 9.
Phenotypic characterization of DC subsets in secondary lymphoid organs.
Single cell suspensions from retro-pharyngeal, sub-mandibular, pre-scapular, popliteal lymph nodes, and a spleen were prepared, and 7-color flow cytometric analysis performed to identify DC. Doublets, dead cells, and lineage positive cells (T cells, monocytes, B cells and NK cells) were excluded as described in Figure 1. Lineage negative cells were then gated to identify MHC class II+ and CD4+ cells (A), and the MHC class II+ CD4− cells were assessed for CD11c expression (B). Surface expression of DEC205 and CD172a by CD4+ MHC class II− DC (C), MHC class II+/CD4−/CD11c+ DC (D), and MHC class II+/CD4−/CD11c− DC (E) were assessed. Numbers on plots represent percentage of cells expressing the surface markers shown.