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"PLASMACYTOID DENDRITIC CELL INTERACTIONS WITH VIRUS AND VIRUS-INFECTED CELLS: FOCUS ON HSV-INFECTED MDDC AND HIV-INFECTED T CELLS"

by
Evan S. Jacobs
Pathology and Laboratory Medicine
B.S. 2002, University of Maryland at College Park



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

Professor

Dept. of Pathology and Laboratory Medicine

Friday, March 26, 2010
2:00 p.m., MSB B-610


Abstract

Plasmacytoid dendritic cells (pDC) serve as a link between the innate and adaptive immune systems. pDC are lin-, CD123+, BDCA2/4+ cells and are well-known for their ability to produce large quantities of interferon- (IFN-) in response to viruses and TLR-7 and -9 stimulation. Additionally, they express the chemokine receptors CCR5 and CXCR4, as well as the CD4 receptor, all of which are important in HIV infection. In this study, we examined the interaction between pDC and HSV-infected monocyte-derived dendritic cells (MDDC) to demonstrate whether pDC work intimately with myeloid DC (mDC) in order to direct an efficient immune response. The membrane dye PKH-67 along with GFP-HSV construct were utilized in concert with a new technology, imaging flow cytometry (AMNIS ImageStream®), to analyze how pDC are able to interact with HSV-infected vs. uninfected MDDC. Using imaging flow cytometry we confirmed an intimate preferential interaction between pDC and HSV-infected vs. uninfected MDDC where pDC conjugated with and took up both membrane and cytoplasmic material preferentially from HSV-infected MDDC a process termed “nibbling”, which resulted in IFN- production. These results indicate that pDC can receive interferogenic signals from a heterogeneous population of cells including mDC in order to initiate and direct the immune response.
We followed up these studies with a similar analysis of how pDC interact with HIV-infected cells. Using both the H9 cell line chronically-infected with HTLVIIIB as well as acutely-infected CD4+ T cells infected with wither CCR5-tropic and CXCR4 tropic viruses, we demonstrated a similar interaction where pDC are capable of conjugating with and internalizing membrane from HIV-infected cells. More importantly, we demonstrated that in the process of nibbling, the majority of these conjugations actually end up in large single cell, multi-nuclear syncytia (fusions) between the pDC and the HIV-infected cell. We show that these fusions occur with both CCR5-tropic and CXCR4-tropic viruses, including CCR5-tropic viruses that have not been associated with syncytia formation. We also show that this occurs in both autologous and heterologous cultures of pDC and infected CD4+ T cells. These data provide a potential mechanism for the depletion of pDC that is observed in HIV infection.
Kinetics of fusions were studied over 20 hour co-cultures, where the percentage of pDC: HIV-infected cell fusions increased along with a concurrent loss of pDC events in culture. We also demonstrate that the pDC: T cell fusion was dependent on the gp120: CD4 interaction, gp120: co-receptor interaction, as well as the HIV fusion intermediate, 6-bundle helix, and that blockade of these structures lead to a significant reduction in fusion. We also demonstrate that formation of these fusions correlated with a loss of pDC IFN- production, which was rescued along with nibbling of material from the HIV-infected cells by treatment of co-culutres with the CXCR4 antagonist, AMD3100, or the fusion inhibitor, T20.
We subsequently examined the consequences of pDC interaction with non-cell associated virus, and how various treatments to block the CD4 receptor, CXCR4 or CCR5 receptors, fusion, or virus replication affected pDC function. Surface and intracellular flow cytometry were utilized to determine the levels of CD40, CCR7, CD83, and CD86 expression, which are markers of pDC maturation and for production of IFN-. Our results indicate differences in the interaction of pDC with live forms of HIV vs. inactivated HIV. Additionally we show that there are differences in the pDC maturation phenotype when stimulated with CXCR4-tropic viruses or CCR5-tropic viruses. Finally, using a cohort of HIV-infected individuals grouped into ages between 18-39 (6 donors) and 50-65 (9 donors) along with age-matched controls, we compared the phenotype and function of HIV-infected younger vs older individuals. Our data indicates that pDC numbers and function in older vs younger HIV infected individuals were similar to each other and similar to healthy older individuals. Additionally, we observed an increased level of pDC activation (indicated by CD40 expression) on HIV aging vs older individuals. These data are consistent with the hypothesis that the immune system of an HIV-infected individual is more similar in activation and function to that of a much older but relatively healthy individual Together, this dissertation describes a novel interaction between pDC and virus and virus infected cells and how these processes are subverted in HIV infection.


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