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"PHENOTYPIC AND FUNCTIONAL CHARACTERIZATION OF THE CD2+ AND CD2- SUBSETS OF HUMAN PLASMACYTOID DENDRITIC CELLS"

by
David Mwangi
Molecular Pathology and Immunology Program
M.S. 2005, Kean University
B.S. 1994, Egerton University


Thesis Advisor: Patricia Fitzgerald-Bocarsly, Ph.D.
Professor
Department of Pathology and Laboratory Medicine

Friday, February 22, 2013
1.00 P.M., MSB Room C-555


Abstract

pDC secrete type I and III interferons and pro-inflammatory cytokines upon viral stimulation through activation of endosomal TLR7 or TLR9. In this study, we have identified a stable subset of human pDC that expresses CD2. In vitro, no intraconversion of the subsets was observed. We hypothesized that the CD2+ and CD2- pDC may be functionally distinct populations that may differentially modulate the immune response. To address this hypothesis, functional and phenotypic analyses of the pDC subsets were performed.
To evaluate the cytokine secretion profiles of the pDC subsets, PBMC were stimulated with viruses and cytokine expression was measured by intracellular flow cytometry and ELISA assay. These cytokine production studies demonstrated that while at 6 h, more CD2- pDC than CD2+ pDC expressed IFN-уn in response to HSV stimulation, the CD2+ pDC subset had more sustained production of IFN- and IFN-f when compared to the CD2- pDC. In addition, there were more IL-6-expressing cells in the CD2- population following 6 hr of viral stimulation while CD2+ pDC had a higher percentage of TNF--expressing pDC at this time-point.
pDC express CD4, CCR5, and CXCR4, the primary receptor and co-receptors, respectively, for the HIV envelope protein gp120. In vitro, pDC can be infected with R5 and X4 strains of HIV-1. A preferential loss of circulating CD2- pDC has been reported in HIV-infected individuals, but the functional significance of this observation is not known. To begin to address this issue, we compared the expression of the HIV receptor/co-receptors on pDC subsets. pDC were stimulated with viruses and TLR-7/9 agonists and the surface expression of CD4, CCR5 and CXCR4 measured by flow cytometry. The CD2+ and CD2- pDC were found to express comparable levels of CD4. However, CD2+ pDC expressed higher levels of CXCR4 but not CCR5 as compared to the CD2- pDC. This suggests that HIV may have differential tropism for the CD2+ and CD2- pDC.
It has been reported that pDC isolated from lymph nodes show a higher expression of CD2 when compared to peripheral blood. In addition, pDC express high constitutive levels of CD62L and need to express CCR7 in order to migrate. To evaluate the expression profile of migration-related molecules on pDC, we stimulated pDC with the viruses HSV-1, HIVMN-AT2, Flu and the non-viral TLR-7/9 agonists CPGA and CPGB. The expression of migration molecules was measured using flow cytometry. Our data show that, upon activation, the CD2+ subset exhibits a higher expression of CD62L and CCR7, which are molecules particularly involved in pDC migration. Consistent with this observation, we observed that CD2+ pDC exhibited greater migration in response to the CCR7 ligand, CCL19, than did the CD2- pDC.
Upon stimulation with the viruses HSV-1, AT2-inactivated HIVMN, Flu and the cytokine IFN-, CD2+ pDC expressed higher levels of activation markers CD40, CD83, and HLA-DR and the co-stimulatory molecules CD80 and CD86. Subsequent experiments demonstrated that blocking CD2 on pDC inhibits pDC nibbling of HSV-infected MCF7 breast cancer cells. Furthermore, the CD2+ subset had a significantly higher expression of the C-type lectins mannose receptor and Dectin-1. These findings suggest that CD2+ pDC have the potential to be more efficient at antigen presentation following viral stimulation.
Overall our data shows, for the first time, that CD2+ and CD2- populations represent phenotypically and functionally distinct subsets of pDC. Since it has been reported that CD2- pDC are preferentially lost in HIV-infected individuals, future studies will address the significance of our findings with respect to the role of pDC in HIV-1 infection.


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