Generation of T cell lines expressing wild type and mutant CD3ε FRET sensors and characterization of CD3ε regulation by plasma membrane binding

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CHOI-UNDERGRADUATERESEARCHOPTIONTHESIS-2021.pdf (4.32 MB)
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Choi, Yujin
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Wallace H. Coulter Department of Biomedical Engineering
The joint Georgia Tech and Emory department was established in 1997
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Undergraduate Scholarship
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http://hdl.handle.net/1853/66279
Abstract
T cells are a crucial part of our adaptive immune response by protecting our system against pathogens and cancer. They recognize specific foreign peptides on the surface of antigen presenting cells (APC) through T cell receptors (TCR) placed on the surface of T cells. Peptides bound to major histocompatibility complexes (pMHC) form bonds with these TCR and allow them to recognize antigen fragments, eventually activating the T cells. In their resting state, the CD3ε and ζ tails are bound to the inner leaflet of the plasma membrane (PM) by ionic interactions, which prevents phosphorylation. TCR-pMHC bond formation allows for CD3 tails’ dissociation from the PM, as observed through Fluorescence Resonance Energy Transfer (FRET). This study aims to create a new, improved FRET pair between CD3ε and PM for wild-type CD3ε as well as a panel of mutated CD3ε which target key steps in the chain of events following TCR-pMHC bond formation until ITAM phosphorylation by Lck, with the hope of elucidating the mechanism of TCR triggering and signal transduction from the extracellular binding site to the intracellular tyrosines. FRET pair for the wild-type CD3ε was created through lentiviral transfection of Jurkat cells through spinfection, followed by single cell sorting conducted with Fluorescence-activated cell sorting (FACS) Aria Fusion machine. Transduced cells expressing Green Fluorescent Protein (GFP) were selected through gating and then diluted into ~0.5 cells/well to be seeded onto a 96 well plate. Top 3 cells were selected for GFP strength and proliferated, thus creating stable cell lines for the wild-type CD3ε FRET pair. CD3ε mutation constructs of the K76T, ITAM, BRS, and RK motifs were then created using polymerase chain reaction (PCR) to insert the mutations in pLenti virus (pLv) and pTwist plasmids. The formation of colonies after transformation and the sequencing results indicate that the mutations were successfully and correctly inserted in the plasmids for each type of mutations: K76T, BRS, and ITAM. Since these plasmids are confirmed for their integrity through sequencing, they will be used to create stable cell lines expressing mutated CD3ε for the new FRET pair. The stable cell lines will be produced through transfection of HEK 293T cells, followed by transduction of Jurkat cells using virus harvested post-transfection. These stable cell lines expressing mutated CD3ε will then be used to explore the mechanism underpinning peptide strength-dependent CD3ε-PM release and its role in antigen recognition in the future.
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2021-12
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Undergraduate Thesis
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