Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

John L. Cleveland, Ph.D.

Committee Member

George Blanck, Ph.D.

Committee Member

Niketa Patel, Ph.D.

Committee Member

Shari Pilon-Thomas, Ph.D.

Committee Member

Joshua Gamsby, Ph.D.

Committee Member

Susan Gilmour, Ph.D.

Keywords

Immunometabolism, Polyamines, Hypusination, CD8+ T cells

Abstract

T cells are critical effectors of the immune response against pathogens and malignancies and, given the recent success of T-cell immunotherapies in cancer, there is now an urgent need to enhance the efficacy and durability of T-cell immune responses to improve anti-tumor immunity. Formation of memory T cells is a critical step of a successful immune response as it ensures maintenance of long-term immunity and protection from re-exposure to foreign antigens. Notably, metabolism regulates the fate and function of T cells and is a powerful tool to modulate T-cell activity and memory differentiation. Accordingly, metabolic interventions to augment T-cell memory differentiation and function in adoptive T cells therapies, including tumor infiltrating lymphocytes (TILs) and chimeric antigen receptor (CAR) T-cell therapies, are attractive strategies to enhance anti-tumor immunity and achieve durable clinical responses in cancer patients.

The amino acid glutamine is an important substrate for effector T cell proliferation and cytokine production. However, the glutamine metabolic network appears to play complex roles in the fate and function of different T cell subsets. We showed in Elmarsafawi et al. that the polyamines, putrescine, spermidine and spermine are derived from glutamine in CD8+ T cells. Glutamine derived polyamines play an important role in activation induced growth and proliferation in T cells. Further, the polyamine spermidine is involved in a post-translational modification known as hypusination, which uniquely occurs in eukaryotic translation factor 5A (eIF5A) and its ortholog eIF5A2, where it augments peptide bond formation as well as nascent peptide release.

Other have shown that the polyamine-hypsuine axis controls CD4+ T cell lineage fidelity, yet the roles of polyamines in controlling CD8+ T cell fate and function are unclear. This project has provided new insights into the role of the glutamine-polyamine-hypsuine axis in the development of memory CD8+ T cells and tested if targeting this pathway represents an effective therapeutic strategy to enhance adoptive T cell therapy responses. First, we have shown that modulating glutamine levels or catabolism controls the expression of CD69, a T-cell activation and tissue resident memory T (TRM) cell marker, in CD8+ T cells. Secondly, using pharmacologic approaches, we have shown that the polyamine-hypsuine circuit controls TRM cell development, fate, and function both ex vivo and in vivo in the bone marrow, an important TRM niche. Furthermore, these findings were validated by genetic studies which showed that T cell-specific deletion of ornithine decarboxylase (Odc), or deoxyhypusine synthase (Dhps), which are essential enzymes that direct polyamine biosynthesis or hypusination, respectively, reprograms CD8+ T cells into TRM-like cells having superior mitochondria and enhanced cytotoxic function that are manifest by increased production of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and granzyme Finally, our preliminary studies focused on defining the mechanisms by which the polyamine-hypusine circuit controls TRM cell fate and functions suggest that this control is epigenetic in nature, at both the level of the genome and histone modifications.

Most importantly, our studies establish the exciting translational potential of our findings to cellular immunotherapies, specifically therapies that are based upon TIL and CAR-T cell therapies. Specifically, we have shown that inhibiting the polyamine-hypusine circuits endows (a) human CD8+ T cells from peripheral blood and sarcoma TIL and (b) human CD8+ CAR-T cells with superior TRM phenotypes that include enhanced polyfunctionality as evidenced by increased production of IFN-gamma and TNF-alpha, as well as of the cytotoxicity-associated molecules granzyme B and perforin.

In conclusion the doctoral dissertation studies presented herein have established critical roles of the glutamine-polyamine-hypusine circuit in controlling CD8+ T cell fate and function in both human and mouse experimental systems, and they provide evidence supporting the notion that targeting this pathway can benefit immunotherapies, including TILs or CAR-T cells. They also have shed light on therapeutic design considerations for implementing polyamine-hypusine targeting therapies in the clinic. Firstly, we have shown that the polyamine-hypusine axis could be targeted pharmacologically or genetically and thus the effects of genetic versus pharmacologic manipulations of this pathway in T cells should be assessed for implementation in adoptive T cell therapies. Secondly, our studies suggest that interventions targeting ODC should be complemented by therapy that can overcome compensatory effects of polyamine uptake to maximize therapeutic efficacy.

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