Genetic profiles of plasmacytoid (BDCA-4 expressing) DC subtypes-clues to DC subtype function in vivo

Abstract

Among the dendritic cell (DC) subsets, plasmacytoid DC's (pDC) are thought to be important in the generation of both antiviral and antitumor responses. While pDC may be useful in developing dendritic cell-based tumor vaccines, the low frequency of these cells in the peripheral blood has hampered attempts to understand their biology. To provide better insight into the biology of pDC, we isolated these unperturbed cells from the peripheral blood of healthy donors in order to further characterize their gene expression. Using gene array technology we compared the genetic profiles of these cells to those of CD14+ monocytes isolated from the same donors and found several immune related genes upregulated in this cell population. This is the first description, to our knowledge, of gene expression in this subset of DCs obtained from the peripheral blood of adult human donors without exposure in vitro to cytokine or growth factors. Understanding the natural genetic profiles of this dendritic cell subtype as well as others such as the BDCA-1 expressing myeloid DCs may enable us to manipulate these cells ex-vivo to generate enhanced DC-based tumor vaccines inducing more robust antitumor responses.

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 CD3-FITC and CD20-FITC and evaluated by flow cytometry: Prior to sorting (A), after Magnetic Bead Sorting (MACS) (B), and after MACS followed by Fluorescence-Activated Cell Sorting (FACS) (C). R2 indicates the left upper quadrant sorting gate, R3 indicates gate for lymphocytes removed by FACS.

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 of specific genes upregulated in BDCA-4 cells relative to CD14+ cells. ** Genes confirmed to be upregulated using RT-PCR as discussed in the materials and methods section. Numbers in parentheses indicate two separate values of fold upregulation of the selected upregulated genes confirmed by RT-PCR.