Graduation Term

2023

Degree Name

Master of Science (MS)

Department

School of Biological Sciences

Committee Chair

Laura A Vogel

Committee Member

Rachel M Bowden

Abstract

B cells are crucial components of the immune system during an infection that produce antibodies. It is not known how B cells function or if different cell subsets exist in reptiles. Understanding how B cells function in reptiles can help protect endangered species by clarifying how their immune system may be different from other vertebrates but may also allow for the development of new human medicines. First, we aimed to identify turtle leukocytes based on their morphological characteristics. Blood smears were made and photos were taken on a light microscope equipped with a digital camera. Using this method, we were able to identify turtle erythrocytes, thrombocytes, heterophils, eosinophils, basophils, monocytes, and lymphocytes. Additionally, since many B and T cell molecules are highly conserved, we hypothesized that antibodies used to detect B and T cells in human and mice would cross-react with turtle cells. We stained turtle B and T cells with monoclonal antibodies, known to bind human and mouse B and T cells, conjugated to different fluorochromes to determine if they would also bind turtle B and T cells. We found that turtle B cells were largely not cross-reactive with many of the monoclonal antibodies we used; however, a different population of turtle leukocytes was anti-CD45R positive. Turtle T cells were able to be identified with anti-CD3 monoclonal antibody. Although many of the proteins are highly conserved, we were unable to identify a monoclonal antibody that cross reacted and could be used for live T cell identification. We have previously found that reptile B cells had a special function that most human B cells do not – they could engulf and destroy invaders using phagocytosis. We hypothesized that reptilian B cells differentiate during infection to either phagocytosis or antibody secretion depending on the size of the target particle. Particles that were too large to be engulfed would prompt the B cell to secrete antibodies while smaller invaders would be destroyed by phagocytosis. To test this hypothesis, we collected blood samples from wild red-eared slider turtles. Lymphocytes were incubated with fluorescent polystyrene beads (1 μm or 6 μm) to simulate invading pathogens. We used flow cytometry to determine if a B cell had captured any beads (or how many). We added an additional fluorescent dye to identify B cells from other white blood cell types. The results of our phagocytic assay showed that turtle B cells were able to engulf multiple 1 μm beads as predicted but also were able to engulf one or two 6 μm beads. We also performed ELISpot assays to examine antibody secretion. Turtle leukocytes were incubated for several days, with some samples receiving an additional infection signal, lipopolysaccharide (LPS). We found that turtle B cells could be detected with this method and that the cells incubated with LPS created more spots, indicating that they secreted more antibodies. These results further our understanding of how B cells function in reptiles.

Access Type

Thesis-Open Access

DOI

https://doi.org/10.30707/ETD2023.20231004061828389763.999978

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