Date of Award

2017

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

College

College of Graduate Studies

First Advisor

Christina Voelkel-Johnson

Second Advisor

Craig C. Beeson

Third Advisor

Shikhar Mehrotra

Fourth Advisor

Michael Nishimura

Fifth Advisor

Chrystal Paulos

Abstract

The adoptive transfer of autologous melanoma antigen-specific T cells has demonstrated a remarkable improvement in clinical outcomes for patients with late- stage metastatic melanoma. However, the majority of patients do not achieve a durable response. To achieve a sufficient quantity of cells for transfer, T cells undergo a rapid expansion protocol which makes them more susceptible to activation-induced cell death (AICD). As the persistence of transferred T cells is necessary for optimal patient response, limitation of persistence via AICD is likely a constraint on clinical efficacy. The accumulation of oxidative stress caused by TCR restimulation has previously been demonstrated to be necessary for the onset of AICD. The data contained within in this thesis reveal that accumulation of ROS escalates into the incursion of the appearance of γH2AX foci, which are indicative of DNA damage, and activation of the DNA damage response pathway characterized by autophosphorylation of ATM on Ser1981 and ATM mediated phosphorylation of the tumor suppressor p53 on Ser15. Treatment with the glutathione pro-drug N-acetyl cysteine (NAC) significantly reduced the upregulation of γH2AX and subsequent ATM activation and cell death. Additionally, both murine Pmel-1 T cells and TIL1383I TCR transduced therapeutic human T cells exhibited less susceptibility to the upregulation of γH2AX and onset of AICD when NAC was added to the medium during extended culture. Both Pmel-1 T cells and TRP-1 TCR transduced murine splenocytes cultured in NAC prior to adoptive transfer into B16F10 challenged mice exhibited enhanced control of tumor burden and survival of recipient mice. TIL1383I TCR transduced T cells cultured in NAC demonstrated reduced expression of the exhaustion and senescence markers PD-1 and CD57 as well as the exhaustion associated transcription factors EOMES and Foxo1. Taken together, the results contained in this thesis demonstrate the addition of NAC to the rapid expansion of therapeutic T cells bolsters the overall fitness and anti-melanoma functionality of the cells and could potentially improve the quality and therapeutic efficacy of adoptive T cell therapeutics infused into patients.

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