The use of N-acetyl cysteine (NAC) during ex vivo expansion of TCR-engineered human T cells for adoptive cell transfer provides protection against activation-induced cell death, increases granzyme B production, and enhances antitumor efficacy in vitro. Although NAC did not drive cells to a central memory phenotype, it did reduce the expansion-induced upregulation of CD57 (senescence) and PD-1. Mechanistically, NAC likely maintains activity of the P13K/Akt pathway, causing degradation of Foxo1 and thus reduced expression of EOMES and PD-1.

Therapeutic outcomes for adoptive cell transfer (ACT) therapy are constrained by the quality of the infused T cells. The rapid expansion necessary to obtain large numbers of cells results in a more terminally differentiated phenotype with decreased durability and functionality. N-acetyl cysteine (NAC) protects against activation-induced cell death (AICD) and improves anti-tumor efficacy of Pmel-1 T cells in vivo. Here, we show that these benefits of NAC can be extended to engineered T cells and significantly increases T-cell survival within the tumor microenvironment. The addition of NAC to the expansion protocol of human TIL13838I TCR-transduced T cells that are under evaluation in a Phase I clinical trial, demonstrated that findings in murine cells extend to human cells. Expansion of TIL13838I TCR-transduced T cells in NAC also increased their ability to kill target cells in vitro. Interestingly, NAC did not affect memory subsets, but diminished up-regulation of senescence (CD57) and exhaustion (PD-1) markers and significantly decreased expression of the transcription factors EOMES and Foxo1. Pharmacological inhibition of the PI3K/Akt pathway ablates the decrease in Foxo1 induced by NAC treatment of activated T cells. This suggests a model in which NAC through PI3K/Akt activation suppresses Foxo1 expression, thereby impacting its transcriptional targets EOMES, PD-1, and granzyme B. Taken together, our results indicate that NAC exerts pleiotropic effects that impact the quality of TCR-transduced T cells and suggest that the addition of NAC to current clinical protocols should be considered.

Author Info: (1) Department of Microbiology and Immunology, Medical University of South Carolina, MSC 250504, 173 Ashley Avenue, Charleston, SC, 29425, USA. (2) Department of Surgery, Loyola

Author Info: (1) Department of Microbiology and Immunology, Medical University of South Carolina, MSC 250504, 173 Ashley Avenue, Charleston, SC, 29425, USA. (2) Department of Surgery, Loyola University, Maywood, IL, USA. (3) Department of Microbiology and Immunology, Medical University of South Carolina, MSC 250504, 173 Ashley Avenue, Charleston, SC, 29425, USA. (4) Department of Public Health Sciences, Medical University of South Carolina, Charleston, SC, USA. (5) Department of Microbiology and Immunology, Medical University of South Carolina, MSC 250504, 173 Ashley Avenue, Charleston, SC, 29425, USA. (6) Department of Surgery, Loyola University, Maywood, IL, USA. (7) Department of Microbiology and Immunology, Medical University of South Carolina, MSC 250504, 173 Ashley Avenue, Charleston, SC, 29425, USA. johnsocv@musc.edu.

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