In Vitro Expansion of Murine γδ T Cells and Transfection of Human γδ T cells for CAR T Cell Therapy Público

Abstract

CAR T cell therapy has emerged as one of the most promising ways to treat cancer and is currently being studied as a potential treatment for AML (Leick et al. 2022). In particular, γδ T cells have special promise due to their ability to kill cancer in an off-the-shelf manner without inducing graft-versus-host-disease (GVHD). We want to develop a syngeneic mouse model to study whether murine γδ T cells from the bone marrow preferentially home to the bone marrow. Our study focused on developing a protocol for expanding murine γδ T cells. In this study, we tested the ability of CD3/CD28 beads, zoledronate, lipopolysaccharides (LPS), and Concanavalin A (ConA) to activate murine γδ T cells. Our findings have shown that of our tested activators, ConA provides the greatest percentage of murine γδ T cells, but is lacking in its expansion ability. We have also found that when using ConA, by the end of the expansion, there is a greater number of murine γδ T cells from the spleen rather than from the bone marrow. In addition, we optimized a protocol for transfecting human γδ T cells with an SCF-CAR through electroporation. This was done by collecting blood samples from human donors and isolating the PBMCs, performing an αβ T cell depletion on day 6, and cryopreserving them on day 14. When required for experiments, they were thawed and electroporated. The next day, flow cytometry analysis was done to determine the success of the electroporation conditions, where we found the Biorad 4 mm cuvettes electroporated for 2 ms at 500 volts were considered to be the optimal condition. This work has the ability to provide tools that will help us learn more about the biology of murine and human γδ T cells.

Table of Contents

Introduction.......................................................................................................................................................................1

Methods.............................................................................................................................................................................6

Results..............................................................................................................................................................................18

Discussion.........................................................................................................................................................................23

References.........................................................................................................................................................................27

Tables and Figures

Figure 1.............................................................................................................................................................................31

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Table 1..............................................................................................................................................................................37

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Table 2...............................................................................................................................................................................54

Figure 23............................................................................................................................................................................55

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Health Sciences, Immunology Biology, Cell Biology, General
Palabra Clave	murine γδ T cells electroporation CAR T cell therapy γδ T cells
Committee Chair / Thesis Advisor	Spencer, H. Trent, Emory University
Committee Members	Kissick, Haydn, Emory University Comstra, Skye, Emory University

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