Enhancing Ex Vivo Expanded γδ T Cell Function by Manipulating Media and Cytokine Induced Memory Phenotypes Open Access

Abstract

Adoptive cell therapy (ACT), particularly through chimeric antigen receptor (CAR) T cell  therapy, is a groundbreaking advancement in oncology primarily utilizing αβ T cells. However, due to limitations such as manufacturing complexity and risk of graft-versus-host disease (GVHD), γδ T cells have emerged as promising ACT candidates due to their MHC-independent cytotoxic capabilities and ability to be used in an “off-the-shelf" manner. This study aimed to direct ex-vivo expanded γδ T cells toward enhanced memory phenotypes by manipulating culture conditions and cytokine exposure. Four distinct culture media were utilized—OpTmizer™, TexMACS™, RPMI 1640, and Lonza TheraPEAK® T-VIVO®—combined with varying concentrations of cytokines, including IL-15 and transforming growth factor-beta (TGF-β) alongside IL-2 and zoledronic acid. γδ T cells were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) and expanded under controlled conditions over a period of 12 days. Expansion efficiency and phenotype expression were assessed through flow cytometry. OpTmizer™ media consistently yielded the highest fold change γδ T cell expansions compared to other media. CXCR4, implicated in cell trafficking to the bone marrow, was not significantly induced by cytokine treatments. The addition of TGF-β did substantially promote a central memory phenotype, reducing effector memory and terminal effector subsets. Moreover, IL-15 combined with TGF-β increased naïve-like γδ T cell populations. Cytokine treatments also influenced cytotoxic markers, with TGF-β consistently suppressing CD56 and CD16 expression. However, IL-15 partially rescued CD56 expression when combined with TGF-β. Both these treatments imply increased persistence in an in-vivo scenario. This research highlights the importance of optimizing γδ T cell expansion protocols through strategic cytokine and media combinations, effectively guiding cell phenotype towards memory subsets which are favorable for adoptive cell therapies. These findings advance the potential of γδ T cells as universally applicable, "off-the-shelf" treatments, able to provide effective, highly persistent, easily deployable therapeutic options with minimized risks of GVHD.

Table of Contents

INTRODUCTION.......................................................................................................................... 1

METHODS.................................................................................................................................. 11

Media Preparation................................................................................................................... 11

Cytokine Preparation............................................................................................................... 11

Isolating γδ T cells from Blood................................................................................................. 12

γδ T Cell Culture..................................................................................................................... 14

Freezing Down γδ T Cells......................................................................................................... 14

Flow Cytometry....................................................................................................................... 15

RESULTS.................................................................................................................................... 17

Expansion Efficiency and Cell Yield........................................................................................... 17

CXCR4 Expression Discrepancy................................................................................................. 22

Effects of Medium and IL-15 and TGF-β on γδ T cell CD56/CD16 Expression................................ 25

Effects of Medium and IL-15 and TGF-β on γδ T cell Phenotype.................................................. 28

DISCUSSION.............................................................................................................................. 32

REFERENCES............................................................................................................................. 39

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Cell Biology, General
Keyword	effector memory γδ T cell expansion γδ T cells T cell central memory γδ T cell phenotype
Committee Chair / Thesis Advisor	Dr. H. Trent Spencer, Emory University
Committee Members	Dr. Arri Eisen, Emory University Dr. Veronika Zarnitsyna, Emory University

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