N-acetyl cysteine protects anti-melanoma cytotoxic T cells from exhaustion induced by rapid expansion via the downmodulation of Foxo1 in an Akt-dependent manner

Cancer Immunol Immunother. 2018 Apr;67(4):691-702. doi: 10.1007/s00262-018-2120-5. Epub 2018 Feb 2.


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.

Keywords: Adoptive T-cell therapy; Akt; Cell death; Foxo1; N-acetyl-cysteine; PD1.

MeSH terms

  • Acetylcysteine / pharmacology*
  • Animals
  • Cells, Cultured
  • Forkhead Box Protein O1 / metabolism*
  • Free Radical Scavengers / pharmacology
  • Gene Expression Regulation, Neoplastic / drug effects*
  • Humans
  • Immunotherapy, Adoptive*
  • Lymphocyte Activation
  • Melanoma / drug therapy*
  • Melanoma / immunology
  • Melanoma / metabolism
  • Mice
  • Phosphatidylinositol 3-Kinases / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Signal Transduction
  • T-Lymphocytes, Cytotoxic / immunology*


  • FOXO1 protein, human
  • Forkhead Box Protein O1
  • Free Radical Scavengers
  • Phosphatidylinositol 3-Kinases
  • Proto-Oncogene Proteins c-akt
  • Acetylcysteine