We utilized the cell-specific surface expression of BDCA-4 antigens on pDCs to reproducibly obtain these populations at greater than 95% purity using MACS followed by FACS (Figure C). FACS sorting for BDCA-4+ cells not co-expressing the B cell marker CD20 or the T cell marker, CD3, eliminated contaminating lymphocytes retained following the first isolation (Figures A and B).
Figure 1 Separation of BDCA-4 subpopulation by MACS and FACS. Representative Flow Cytometry plot evaluating the purity of BDCA-4 populations from Ficol-separated PBMCs obtained from normal healthy donors. Leukocytes stained with BDCA-4-PE and the lymphocyte markers (more ...)
Gene arrays to evaluate the gene expression in DC’s during maturation in vitro
have revealed upregulation of genes involved in cell adhesion and motility, immune response, growth control and lipid metabolism affecting cell growth and differentiation, signal transduction, ion channel activities and membrane function [18
]. More recently Lindstedt et al. published the gene family clustering of human blood and tonsillar DC subsets from healthy children undergoing tonsillectomy using Affymetrix gene arrays [21
]. We chose the Affymetrix system to assess gene profiles of pure populations of unstimulated de novo
BDCA-4 cells obtained from consented healthy adult donors.
While gene arrays of DC’s have been compared to several immune cells, including T-lymphocytes, B-lymphocytes and macrophages [18
], we compared the genetic profiles of four separate isolations of BDCA-4+
cells from healthy donors to CD14+
cells devoid of BDCA-4 cells as this cell population is commonly used as DC precursors in cancer vaccine trials. Out of over 22,000 genes assessed between both populations of cells, 448 genes including the BDCA-4 gene were upregulated at least three-fold compared to those expressed in CD14+ cells (see GSE11943 for gene array datasets). While the majority of the genes encoded proteins of unknown function, the remaining upregulated genes involved several biologic processes including the immune response, cell proliferation, development, protein amino acid phosphorylation, metabolism, the ubquitin cycle, cell cycle, transcription and signal transduction pathways (Figure A). Nineteen of these genes were involved in the immune and inflammatory response (Figure B). Interestingly, the Granzyme B gene was overexpressed over 200-fold in all four BDCA-4 cell samples with RT PCR confirming an 18,000 to 25,000 fold upregulated expression of this gene relative to CD14 cells (Figure B). Granzyme-B is expressed by innate and adaptive immune effector cells and plays key roles in the destruction of tumor cells and cells infected with intracellular pathogens such as viruses [22
]. Rissoan et al. identified high levels of mRNA expression in both resting and activated pDC obtained from cord blood and tonsillar tissue [23
] and this enzyme is upregulated by a pDC leukemia [24
]. Our results reinforce the recently published data reported by Tel J et al. which indicates that this DC cell subtype can be skewed as a killer DC subset when exposed to viral vaccines [25
]. The gene array data demonstrating overexpression of Granzyme B at baseline suggests a genetic potential for effector function by pDC in vivo
Figure 2 Summary of BDCA-4 dendritic cell genes upregulated (A) and genes involved in the immune response. (A) Biologic processes of genes upregulated at least three fold using the OntoExpress software as discussed in the materials and methods section. (B) List (more ...)
Our data demonstrate unique immune-related genes expressed in adult peripheral blood plasmacytoid dendritic cell subtypes relative to autologous CD14 monocytes and complement the previously reported differential gene expression of DC subsets obtained from the peripheral blood and tonsils of children [21
Interestingly, we found a slightly different pDC gene expression profile compared to that reported by Lindstedt et al. While our results also demonstrate several immune-related genes upregulated in this subset including IL18R1, IL3RA (CD123) and TLR-7, additional upregulated genes not reported by Lindstedt et al. included the leukocyte-associated immunoglobulin like receptor, IL-18 receptor accessory protein, and lymphotoxin beta. One limitation of our study was that with the human HU133A chip we could not assess the previously demonstrated upregulated genes CXCR3, CLECSF7 and TLR-9 genes [21
There are a number of reasons to explain why our results differ from those previously published. First, we used the more stringent cutoff of greater than or equal to three-fold up- or down-regulation in our analyses to further eliminate potential gene signal noise while Lindstedt et al. used ≥ two-fold [21
]. Also, the pDC used in Lindstedt’s study were obtained from children who underwent tonsillectomies [21
] while our pDC came from the peripheral blood of healthy adults. Aging can affect both the phenotype and the function of human monocytes and leukocytes [26
]. Additionally, cord blood-derived monocytes and adult monocytes have been demonstrated to have differential gene profiles at rest and when stimulated with LPS [27
]. Therefore it is conceivable that the unstimulated pDC from our older adult donors would express different levels of immune-related genes when compared to the same subtypes obtained from children undergoing tonsillectomies.
In summary, we have utilized FACS and MACS to successfully isolate highly purified populations of pDC from healthy donors and present the genetic signatures of these cells. We have identified several upregulated genes in pDC when compared to the CD14+ monocytes with the gene encoding the effector enzyme, Granzyme B, being the most upregulated in our isolated pDC, suggesting an effector function of these cells in the peripheral blood of healthy adults. The genetic profiles from the pDC in our studies differed from those published by Lindstedt et al. This may be due to a combination of setting different cutoff points for considering significant up- and down-regulation of gene expression as well as using cells obtained from older donors. Our results yield further information regarding unstimulated pDCs de novo providing an additional foundation for developing human DC tumor vaccines.