CD1- and CD1+ porcine blood dendritic cells are enriched for the orthologues of the two major mammalian conventional subsets

Abstract

Conventional dendritic cells (cDC) are professional antigen-presenting cells that induce immune activation or tolerance. Two functionally specialised populations, termed cDC1 and cDC2, have been described in humans, mice, ruminants and recently in pigs. Pigs are an important biomedical model species and a key source of animal protein; therefore further understanding of their immune system will help underpin the development of disease prevention strategies. To characterise cDC populations in porcine blood, DC were enriched from PBMC by CD14 depletion and CD172a enrichment then stained with lineage mAbs (Lin; CD3, CD8α, CD14 and CD21) and mAbs specific for CD172a, CD1 and CD4. Two distinct porcine cDC subpopulations were FACSorted CD1^- cDC (Lin^-CD172⁺ CD1^-CD4^-) and CD1⁺ cDC (Lin^-CD172a⁺ CD1⁺ CD4^-), and characterised by phenotypic and functional analyses. CD1⁺ cDC were distinct from CD1^- cDC, expressing higher levels of CD172a, MHC class II and CD11b. Following TLR stimulation, CD1⁺ cDC produced IL-8 and IL-10 while CD1^- cDC secreted IFN-α, IL-12 and TNF-α. CD1^- cDC were superior in stimulating allogeneic T cell responses and in cross-presenting viral antigens to CD8 T cells. Comparison of transcriptional profiles further suggested that the CD1^- and CD1⁺ populations were enriched for the orthologues of cDC1 and cDC2 subsets respectively.

Figure 1. Sorting strategy for the isolation of porcine blood DC populations.

Blood DC, enriched by magnetic depletion of CD14⁺ cells and selection of CD172a⁺ cells, were stained with mAbs to CD172a and lineage markers (lin; CD3, CD8α, CD21) (A). Large (gate 1) CD172a⁺ lin⁻ (gate 2) blood DC subsets were then sorted on expression CD1 and CD4: CD4⁻CD1⁻ cDC (gate 3), CD4⁻CD1⁺ cDC (gate 4) and CD1⁻CD4⁺ pDC (gate 5). Sorted blood DC subsets showed >95% purity when assessed by flow cytometry (B).

Figure 2. Sorted porcine blood DC populations and monocytes express distinct cell surface phenotypes.

Freshly isolated monocytes, CD1⁻ cDC, CD1⁺ cDC and pDC were stained with a panel of DC markers (black histograms) and corresponding host/isotype matched control antibodies (grey histograms) and analysed by flow cytometry. Representative data is shown from 1 of 3 individual pigs analysed.

Figure 3. Assessment of the effect of cell culture on the phenotype of sorted porcine blood DC populations and monocytes.

Expression of MHC class II DR and CD80/86 by monocytes, CD1⁻ cDC, CD1⁺ cDC and pDC upon isolation (fresh) or following an 18 h culture (cultured) was assessed by flow cytometry. MHC class II DR and CD80/86 staining (black histograms) was compared against the corresponding isotype control antibody (grey histograms) and representative data is shown from 1 of 3 individual pigs analysed.

Figure 4. Differential responses of sorted porcine blood DC populations and monocytes to PAMP stimulation.

Monocytes, CD1⁻ cDC, CD1⁺ cDC and pDC were cultured in the presence of poly(I:C), LPS, CpG-ODN 2007 or in medium alone for 18 h. Cytokine content of cell-free culture supernatants were then assessed by multiplex (TNF-α, IFN-γ, IFN-α, IL-10 and IL-8) or singleplex (IL-12) ELISAs and data presented as the mean cytokine concentration of triplicate pooled supernatants from 3 pigs ± SEM.

Figure 5. Assessment of the antigen uptake, processing and presentation capabilities of porcine blood DC populations and monocytes.

The ability of blood DC populations and monocytes to endocytose soluble and phagocytose particulate antigen was examined using Alexa Fluor-647^®-conjugated ovalbumin either in soluble form or encapsulated in PLGA nanoparticles. Antigen uptake was determined after 1 h by flow cytometry. Antigen uptake was measured by mean fluorescence intensity measurements and data presented are the mean 4 °C corrected antigen uptake at 37 °C for triplicate cultures from 1/3 representative experiments (A). Stimulation of antigen-specific CD4⁺ and CD8⁺ T cell IFN-γ responses by sorted blood DC populations and monocytes pulsed with a 28mer synthetic peptide carrying defined CD4 and CD8 T cell epitopes (long peptide) or inactivated PRRSV was assessed by flow cytometry. The unstimulated or, in the case of PRRSV, the mock-virus stimulated corrected mean % IFN-γ⁺ live, singlet CD8 (CD4⁻CD8α^high; left y-axis) and memory CD4 (CD4⁺ CD8α^low; right y-axis) T cells for triplicate cultures from 1 of 2 experiments are presented (B). Allogeneic T cell stimulatory capacity of monocytes and DC populations was assessed in a mixed-leukocyte reaction and lymphoproliferation assessed by ³H-thymidine incorporation. The data are presented as the mean incorporated counts per minute (cpm) of triplicate cultures ± SEM from 1 of 2 experiments (C). For all plots error bars represent SEM. Values were compared using a two-tailed un-paired t-test and significance indicated by ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 6. Gene expression profiling of porcine blood DC and monocytes.

Principle component analysis (PCA) analysis of the isolated cell populations Monocytes (black), pDC (red), CD1⁺ cDC (blue) and CD1⁻ cDC (green) clusters showing two principle components representing 65.6% of total gene variation. Data for sorted blood DCs subsets and monocytes from three pigs are presented.