Understanding and Optimizing Gamma-Delta T cells as a Platform for Cellular Immunotherapies Restricted; Files Only

Abstract

Gamma-delta T cells are a subset of T cells with both adaptive and innate capabilities. Their ability to recognize phosphoantigens via TCR and respond to stress signals via NK receptors, make them a unique platform for cancer immunotherapies. Much work has been done to successfully expand these cells both in vivo and ex vivo. Clinical trials in cancer patients have found gamma-delta T cell products to be safe yet effective only in a small population of patients. Continuing work is being done to optimize gamma-delta T cells so that they successfully home to, infiltrate, and lyse tumors. To do this, it is essential to understand the phenotype of expansion products and how different growth conditions can influence product candidates. An attempt to characterize the phenotypic differences in gamma-delta T cells expanded in OpTmizer versus RPMI+10% FBS is presented here. We found that cells grown in RPMI + 10% FBS for 12 days yielded a higher fold expansion with more NKG2A and CD94, while OpTmizer produced more CD56+ cells. Other NK and T cell markers (NKG2D, NKp46, CD16, CD161, CD137L, and CD69) did not correlate with growth conditions and some key activating receptors were lost by day 12. To bypass donor variability and differences in growth conditions, we sought to optimize an mRNA transfection strategy to express any chemoreceptor. For these studies a DNA backbone was created that could be easily transcribed in vitro into mRNA and electroporated into ex vivo expanded gamma-delta T cells. We used multiple chemoreceptors (XCR1, CXCR4, CCR2, CXCR3, and CCR7) to test our DNA backbone and found that they expressed in HEK 293T cells but were not optimal for expression in gamma-delta T cells. We ultimately hoped to increase CXCR4 expression on gamma-delta T cells and co-express it with SCF CARs to increase homing to the bone marrow. Further work is needed to create an optimal mRNA product for transfection and expression into gamma-delta T cells.

Table of Contents

Introduction

Adaptive functions of Gamma-Delta T cells

Innate Functions of Gamma-Delta T cells

Receptors

CD56

CD16

NKG2D

CD94/NKG2

CD161

CD137/CD137L

Chemoreceptors

XCR1

CXCR4

CCR2

CXCR3

CCR7

Gamma-Delta T Cells as Cancer Immunotherapies

The Plasticity of Gamma-Delta T cells

Rational and Hypothesis

Methods and Materials

Cell Lines and Cell Cultures

Gamma-Delta T Cell Ex Vivo Expansion

Cell Surface Staining and Flow Cytometry

gBlock Construction

mRNA Production

Transfection of mRNA into HEK 293T Cells

Transfection of mRNA into Gamma-Delta T Cells

Results

Discussion

References

About this Master's Thesis

Rights statement

• Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.

School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Cancer Biology
Degree	M.S.
Submission	Master's Thesis
Language	English
Research Field	Health Sciences, Oncology Health Sciences, Immunology Biology, Cell
Keyword	Cancer Immunotherapy Chemokine Receptors mRNA Genetic Engineering Receptors Gamma-Delta T cells
Committee Chair / Thesis Advisor	Trent Spencer, Emory University
Committee Members	Curtis J. Henry, Emory University Sunil Raikar, Emory University

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