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Title: Phenotypic, ultra-structural and functional characterization of bovine peripheral blood dendritic cell subsets

Author
item SEI, JANET - University Of Vermont
item OCHOA, AMANDA - University Of Vermont
item Bishop, Elizabeth
item BARLOW, JOHN - University Of Vermont
item Golde, William

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/2014
Publication Date: 10/8/2014
Citation: Sei, J.J., Ochoa, A.S., Bishop, E.A., Barlow, J.W., Golde, W.T. 2014. Phenotypic, ultra-structural and functional characterization of bovine peripheral blood dendritic cell subsets. PLoS One. 9(10):e109273. DOI: 10.1371/journal.pone.0109273.

Interpretive Summary: Control of livestock infectious diseases like foot-and-mouth disease virus (FMDV), is best achieved by vaccination against infection. The induction of an adaptive immune response is the critical feature of vaccines. This response is initiated by cells in the blood and lymph nodes termed dendritic cells (DC). Vaccines are formulated with pathogen-derived antigens to drive pathogen specific immunity and are most effective when they contain adjuvants. We now know that a primary role of the adjuvant is to nonspecifically activate dendritic cells. Thus, in designing and testing vaccines, understanding the biology of DCs is very important. FMDV outbreaks are economically devastating in endemic countries causing significant losses in livestock production. For FMDV free countries like the US, the threat of an introduction of the virus is very real and if it happened, would cost the US economy billions of dollars while the outbreak is controlled. Because this virus is so contagious for cattle and swine, vaccines have been selected that very quick to induce protective immunity. The vaccine formulations that have fast onset of immunity turn out to have a composition, both the killed virus and the adjuvant additives, that are very efficient at stimulating DCs. As we develop new and improved vaccine for FMDV for the needs of today’s livestock producers, understanding DC function is critical to design of better vaccines. A lot of work has been done in human DC biology and the genetic control of DC function has been intensely studied in mice. The biology of cattle DCs is much less understood. In this manuscript we characterize circulating DC populations by phenotype, function and morphology using techniques to isolate DC subsets away from other cells in the blood. We demonstrate 3 distinct populations of DCs with unique function and phenotype. We also show that similar populations exist in the lymphoid tissues. Homologous populations have been described in humans and swine. These data provide valuable information for the development and refinement of cattle vaccine formulations.

Technical Abstract: Dendritic cells (DC) are multifunctional cells that bridge the gap between innate and adaptive immune systems. In bovine, significant information is lacking on the precise identity and role of peripheral blood DC subsets. In this study, we identify and characterize bovine peripheral blood DC subsets directly ex vivo, without further in vitro manipulation. Multi-color flow cytometric analysis revealed that three DC subsets could be identified. Bovine plasmacytoid DC were phenotypically identified by a unique pattern of cell surface protein expression including CD4, exhibited an extensive endoplasmic reticulum and Golgi apparatus, efficiently internalized and degraded exogenous antigen, and were the only peripheral blood cells specialized in the production of type I IFN following activation with Toll-like receptor (TLR) agonists. Conventional DC were identified by using different patterns of cell surface protein expression including CD11c, MHC class II, and CD80, among others, displayed extensive dendritic protrusions on their plasma membrane, expressed very high levels of MHC class II and co-stimulatory molecules, were highly efficient in the internalization and degradation of exogenous antigen, and readily produced detectable levels of TNF-alpha in response to TLR activation. Our investigations also revealed a third novel DC subset that may be a precursor of conventional DC that were MHC class IIpositive and CD11cnegative. These cells exhibited a smooth plasma membrane with a rounded nucleus, produced TNF-alpha in response to TLR-activation (albeit lower than CD11c+ DC), and were the least efficient in internalization/degradation of exogenous antigen. These studies define three bovine blood DC subsets with distinct phenotypic and functional characteristics that can be analyzed during immune responses to pathogens and vaccinations of cattle.