Date on Master's Thesis/Doctoral Dissertation

8-2023

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Microbiology and Immunology

Degree Program

Microbiology and Immunology, PhD

Committee Chair

Mitchell, Thomas

Committee Co-Chair (if applicable)

Egilmez, Nejat

Committee Member

Egilmez, Nejat

Committee Member

Kosiewicz, Michele

Committee Member

Smith, Melissa

Committee Member

Watson, Corey

Author's Keywords

Iso-Seq; isoform; FcMR; CXCR5; reference transcriptome; isoform sequencing

Abstract

A thorough understanding of receptor regulation is imperative to predict expression in varying contexts of disease or treatment. Lymphocyte surface receptors are often used as biomarkers and drug targets, making them particularly important for study. For receptors of debated functionality, such as the Fc receptor for IgM (FcMR), understanding regulation can also help to predict expression in vivo to supplement hypotheses of biological roles. Various mechanisms exist for altering receptor surface expression, including direct feedback mechanisms such as ligand-induced endocytosis and broader mechanisms such as transcriptional and translational control. In this dissertation, we explore selected underappreciated mechanisms of lymphocyte receptor regulation. Specifically, we investigate the effects of cell culture conditions on FcMR availability and the potential for global regulation of lymphocyte receptors via isoform variation. FcMR is a constitutively expressed Fc receptor on human T cells, though its function there remains debated. It was previously thought that FcMR was kept low in circulation by FcMR-IgM complex internalization. However, we found that FcMR expression was independent of IgM levels in culture and was higher on direct ex vivo stained lymphocytes than in processed PBMC. Instead, increasing cell culture density inhibited FcMR expression in an apparent cell-contact mediated mechanism, suggesting higher circulating expression of FcMR than previously appreciated and a primary role for FcMR in cell-scarce environments. When next attempting to investigate the potential for isoform-based regulation of FcMR in lymphocytes, we found no applicable isoform-level references. We thus decided to fill this gap using Pacific Biosciences Isoform Sequencing (Iso-Seq) and developed the first Iso-Seq reference transcriptomes of human lymphocytes and activated CD4 T cells. In these references, we discovered many potentially novel transcripts, including end-variant transcripts that only differed from annotated counterparts on their 5’ or 3’ end. Using plasmids designed to express novel CXCR5 end-variant isoforms in a HEK293T cell system, we further validated the potential for novel 5’ end-variants to affect both mRNA stability and protein expression. The studies presented here provide valuable contributions to the understanding of lymphocyte receptor regulation by positing novel regulatory mechanisms that lay the groundwork for many future studies.

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