Graduation Year

2019

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Pearlie K. Epling-Burnette, PharmD., Ph.D.

Committee Member

Daniel Abate-Daga, Ph.D.

Committee Member

John L. Cleveland, Ph.D.

Committee Member

Shari A. Pilon-Thomas, Ph.D.

Committee Member

Sheng Wei, M.D.

Committee Member

Daniel D. Billadeau, Ph.D.

Keywords

anti-tumor immunity, cereblon, immunometabolism, polyamines, regulatory T cells

Abstract

The immune system is responsible for surveillance against pathogens and malignancies and adoptive transfer of antigen-specific T is a powerful new cancer treatment option. There is a clinical need to understand more about the normal regulatory processes that prevent immunosurveillance and how to manipulate this for clinical benefit. This project aims to understand T cells from three different angles. First, the biological role of the negative regulator cereblon (CRBN) was investigated in CD8+ T cells and my studies showed that higher levels of specific derivatives downstream of the amino acid glutamine may be beneficial to T cells in the immune microenvironment. Then, the functional role of these specific glutamine metabolites was studied in antigen-specific CD8+ T cells. Finally, suppressive populations of regulatory T cells (Treg cells) were measured in patients at risk for skin cancer. Together, these results expanded our understanding of T cells in various facets of .anti-tumor immunity and immune surveillance.

Immunomodulatory drugs (IMiDs) potentiate T cell effector functions through cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex. However, the physiological roles of CRBN in T cells are unclear. Here, we report CRBN expression is regulated by T cell receptor (TCR) activation and harnesses metabolism of CD8+ T cells. Crbn deficiency in CD8+ T cells augments cellular metabolism manifest by elevated bioenergetics, with supraphysiological levels of polyamines secondary to enhanced glucose and amino acid transport, and increased expression of metabolic enzymes including ornithine decarboxylase. IMiD treatment similarly augments human CD8+ T cell metabolism. Notably, control of metabolism by immunomodulatory drugs and Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, while Crbn deficient T cells exacerbate graft-versus-host disease in vivo, they endow tumor infiltrating lymphocytes with superior anti-tumor reactivity following adoptive transfer, revealing a new targetable pathway for adoptive T cell immunotherapy.

Polyamine biosynthesis is a well-conserved metabolic pathway that is increased upon activation in T cells, though the role that these metabolites serve in activated T cells has not been fully resolved. Novel implications for the role of polyamines in T cell metabolism and asymmetric cell division are presented here. First, it was found that antigen-activated CD8+ T cells prefer glutamine for the carbon source of polyamines over arginine, which can also produce polyamines as a product of the urea cycle. We also report that glutamine-derived polyamines are necessary for increased glycolysis observed after T cell receptor activation and that biosynthetic blockade with pharmacological inhibitors of the rate limiting enzyme ornithine decarboxylase (OCD) increases the spare respiratory capacity of activated CD8+ T cells. Further, polyamines act as a critical functional regulator for eukaryotic translation initiation factor 5A (eIF5a) through a process called hypusination. While hypusinated eIF5a is necessary for normal clonal expansion of antigen-activated CD8+ T cells, it is not involved with the coupled distribution of CD8 and Myc proteins during asymmetric cell division suggesting that the role of polyamines in this process is independent of eIF5a. Together, these findings shed light on how polyamines control normal cellular functions of CD8+ T cells.

Ultraviolet radiation (UVR) causing DNA damage is a well-documented risk factor for non-melanoma skin cancer (NMSC). Although poorly understood, UVR may also indirectly contribute to carcinogenesis by promoting immune evasion. We report the first epidemiological study designed to investigate the association between quantitative measures of UVR, obtained using a spectrophotometer, and circulating T regulatory (Treg) cells. In addition to total Treg cells, the proportion of functionally distinct Treg subsets defined by CD45RA and CD27 phenotypic markers, graded expression of FoxP3 and CD25, and those expressing cutaneous lymphocyte-associated antigen (CLA) and the chemokine receptor CCR4 were enumerated in 350 individuals undergoing routine skin cancer screening exams and determined not to have prevalent skin cancer. No associations were identified for UVR exposure and the overall proportion of circulating Tregs. However, Treg subpopulations with an activation-associated phenotype, CD45RA-/CD27-, and those expressing cutaneous homing receptors were significantly and positively associated with UVR. These subpopulations of Treg cells also differed by age, sex, and race. After stratification by natural skin tone, and adjusting for age and sex, we found that spectrophotometer-based measures of UVR exposure but not self-reported measures of past sun exposure, were positively correlated with the highest levels of these Treg subpopulations, particularly among lighter skinned individuals. Findings from this large epidemiologic study highlight the diversity of human Treg cell subpopulations associated with UVR, and raise questions about the specific coordinated expression of CD45RA, CD27, CCR4, and CLA on Treg cells and the possibility that UVR contributes to NMSC carcinogenesis through Treg-mediated immune evasion.

Here, questions are addressed about fundamental aspects of T cell biology that includes their role in tumor immune surveillance, translational principles for the design and implementation of novel immunotherapies, and a comprehensive analysis of immunometabolism related to human and mouse experimental systems. Through interdisciplinary approaches, some of the questions about immunobiology and immunotherapy of cancer have been successfully addressed.

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