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"BDCA3/CD141/Thrombomodulin
A novel surface marker for characterizing a highly functional subtype of human blood plasmacytoid dendritic cells"

by
Tiffany Shih
Infection, Immunity & Inflammation track, Multidisciplinary PhD Program
M.S., 2011, Rutgers, State University of NJXNJMS (Formerly UMDNJ), Newark, NJ
B.A., 2010, University of California Berkeley, Berkeley, California


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

Wednesday, June 15, 2016
10:30 A.M., MSB C-555


Abstract

Dendritic cells (DCs) are a heterogeneous population of immune cells that regulate both innate and adaptive immunity. Human blood DCs are broadly characterized as pDC (BDCA2+), mDC1 (BDCA1+), and mDC2 (BDCA3+(CD141)). While much work has focused on the functional role of CD141hi mDCs, we investigated the maturation potential and functional significance of a novel subtype of classically defined pDCs (BDCA2+) that express intermediate levels of BDCA3. Using flow cytometry, we identified four human blood DC subsets: BDCA2+/3-, BDCA2+/3int, BDCA2-/3int, and BDCA2-/3hi. Upon stimulation with TLR9 agonists CpG-A or HSV-1, the BDCA2+/3int DCs had the highest frequency of IFN-+ and TNF-+ production, while mDC2 did not respond. Also, both pDC populations were the highest IFN-f producers in response to HSV-1. Virus-stimulated BDCA2+/3int DCs became more activated and mature with higher expression of co-stimulatory and migratory receptors than the BDCA2+/3-cells. IRF7, the master regulator of IFN production in pDCs, was constitutively expressed by both pDC subsets but not BDCA2- DCs. The BDCA2+/3int DCs were more potent than BDCA2+/3neg cells at the preferential acquisition of cellular material from HSV-infected Raji cells. These data indicate that BDCA2+/3int DCs exhibit major classical pDC functions but are phenotypically and functionally distinct from BDCA2-/3int and BDCA2-/3hi DCs. At both the RNA transcript and surface protein level, we found that BDCA3 is an inducible marker after treatment with IL-3, a survival factor commonly used for culturing pDC in vitro. BDCA3 is inducible on sort-purified BDCA2+/3neg DC by HSV-1 and IL-3 treatment, but BDCA2+/3int pDC fractions do not interconvert to BDCA2+/3neg pDCs. Analysis of morphology by AMNIS ImageStream showed no difference in circularity and size between the BDCA2+/3int and BDCA2+/3neg DCs, but mDC2 were larger and less circular than the pDC subsets. Both BDCA2+/3neg and BDCA2+/3int pDC fractions were detectable by multi-parameter flow cytometry in human bone marrow, and we observed that BDCA2+/3int and BDCA2+/3neg pDCs, BDCA2neg/3int DC and BDCA2neg/3hi subsets expressed CD34 at intermediate levels. Cross-linking BDCA3 surface receptors with microbeads inhibited the production of IFN- by the total population of BDCA2+CD123+ pDC from PBMC. Together, these results describe a novel, high functioning BDCA3+ pDC subset and demonstrated that BDCA3 is not a marker unique to mDC, and is highly inducible by 10-fold on pDC in a non-TLR dependent manner.


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