Date of Award

Summer 7-28-2023

Embargo Period

7-28-2028

Document Type

Dissertation - MUSC Only

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Chrystal M. Paulos

Second Advisor

Jessica E. Thaxton

Third Advisor

Gregory B. Lesinski

Fourth Advisor

Haydn T. Kissick

Fifth Advisor

Mark P. Rubinstein

Sixth Advisor

Shikhar Mehrotra

Abstract

Half of melanoma patients experience objective responses when treated with adoptive T cell transfer (ACT) therapy. Despite clinical success, there remain barriers in sustained tumor immunity in some patients. Positive therapeutic outcomes with ACT therapy in patients with melanoma are correlated with infusion of less differentiated T cell products. T cell bioenergetic fitness is tightly associated with ‘stemness’ and is denoted by maintenance of mitochondrial mass and respiration capacity in the tumor. Furthermore, PI3Kδ signaling has been associated with T cell differentiation and loss of mitochondrial fidelity. We hypothesized that PI3Kδ inhibition would generate stem-like memory T cells (TSCM) that provide protection against melanoma by enhancing mitochondrial bioenergetics. To test this idea, we expanded melanoma specific CD8+ T cells in the presence of increasing concentrations of Idelalisib, a PI3Kδ specific inhibitor, and infused them into melanoma bearing mice to test antitumor activity. In vitro we tested T cell stemness by flow cytometry and RNA sequencing. We next assessed mitochondrial qualities such as mass, membrane potential, reactive oxygen species, and respiratory capacity. Mitochondrial transcription was measured via mitochondrial mRNA relative quantification and protein levels of electron transport chain proteins. We found that Idelalisib treatment enriches stemness features in T cells that have potent antitumor activity in melanoma. The adoptively transferred T cells transcriptionally resembled TSCM cells. Metrics of improved bioenergetic fitness were elevated in a dose dependent manner. Moreover, mitochondrially encoded electron transport chain gene expression was selectively enhanced at the RNA and protein level. RNA-seq identified REXO2 as a differentially expressed gene, a novel regulator of mitochondrial transcription. Ablation of REXO2 using CRISPR-Cas9 vastly impaired the antitumor activity of stem-like memory T cells. Furthermore, we found Tcf-1 and Lef-1 functioned as transcriptional regulators of REXO2. Our findings indicate that long-lasting tumor immunity of adoptively stem-like memory T cells can be induced in vitro by blocking PI3Kδ. We also discovered a unique role for REXO2 in mitochondrial transcription and TSCM mediated tumor immunity. These findings suggest that modulating mitochondrial transcription is a potential target to bolster the activity of tumor specific T cells and is independent of their stemness qualities.

Rights

Copyright is held by the author. All rights reserved.

Download

Available for download on Friday, July 28, 2028

Share

COinS