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"Plasmacytoid Dendritic Cells Both Produce Interferon-f and Respond to Interferon-f Stimulation"

by
Zhiwei Yin
Molecular Pathology & Immunology Program

B.S. 1996, Tianjin Medical University



Thesis Advisor: Patricia Fitzgerald-Bocarsly, Ph.D.

Professor

Department of Pathology & Laboratory Medicine

Monday, May 3, 2010
MSB C-555, 1:30 P.M.


Abstract

PDC are considered to be professional type I interferon (IFN) producing cells in that they produce 10-100-fold more IFN- in response to enveloped virus or synthetic TLR7 and 9 agonists than other cell types. We were interested in determining the ability of human pDC to produce IFN-f in response to enveloped viruses and synthetic Toll-like receptor (TLR) agonists and whether these cells respond to IFN-f treatment. We found that HSV induced a 10-20-fold increase in both IFN-1 and IFN-2 transcripts in pDC compared with the unstimulated controls. We developed an intracellular flow cytometry assay using antibodies to IFN-1 and -f2 to assess the expression of IFN-f protein by pDC. A subset of human pDC were found to express intracellular IFN-f as well as IFN- after stimulation with HSV, influenza virus, Sendai virus, or AT-2 inactivated HIV-1 as well as a synthetic TLR9 agonist, CpG A, (but not CpGB) and the TLR7 agonist imiquimod, although fewer cells responded with IFN-f expression than IFN-. By using ELISA assay, the secretion of IFN- by virus-stimulated pDC was found to be time-dependent and cumulative. In order to understand the mechanism of induction of IFN-܃nin pDC, a variety of approaches were utilized. First, we found that cross-linking of CD4 and BDCA2 molecules on pDC inhibited HSV-induced IFN- production. Similar to the production of IFN- in pDC, the HSV-induced IFN- production by pDC was found to be mediated through TLR9. The HSV-induced IFN- production was virus-proliferation independent. Exogenous IFN- was not sufficient to induce IFN- production by pDC, indicating virus induced IFN- production of pDC results from the direct stimulation of virus instead of indirect action of endogenous IFN-. Unstimulated and stimulated pDC were found to express high levels of IFN-f receptor at both the mRNA and protein levels. Moreover, exogenous IFN-f treatment of pDC (priming) led to upregulation of the intracellular expression of both IFN- and IFN-f. In addition, exogenous IFN-f, like IFN-, was found to inhibit apoptosis of pDC. We conclude that pDC are major producers of IFN-f in response to viral stimulation and also express receptors for this cytokine. Moreover, these receptors are functional in that IFN-f has autocrine effects that strengthen the antiviral effect of the pDC by increasing IFN- and IFN-f production and promoting pDC survival. With a better understanding the production of IFN-f and its effects on pDC, we can focus on the role of pDC and IFN-f in the immune system and a variety of diseases, such as AIDS, cancer, autoimmune diseases with the goal to discover new therapeutic strategies to cure these diseases.


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