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
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.
KeywordsAdoptive T-cell therapy Cell death N-acetyl-cysteine Akt Foxo1 PD1
Activation-induced cell death
Rapid expansion protocol
Tyrosinase related protein 1
The retroviral plasmid DNA encoding the MSGV-1 TRP-1 TCR was a kind gift from N. Restifo to C. Paulos.
MJS and CV-J conceived and designed the study. CV-J directed the project and edited the final draft of the manuscript. MJS carried out all experiments except for the aspects described for co-authors below and drafted the manuscript. GS was responsible for transduction and REP of human T cells. MMW transduced murine T cells. EG-M was responsible for the biostatistical analysis of animal experiments. GS, MMW, and EG-M drafted the corresponding method sections. CMP provided oversight for murine T-cell transduction and contributed towards the design of the study. MIN was responsible for direction and oversight of the clinical trial and contributed towards the design of the study. All authors read and approved the final manuscript.
This work was supported by the National Institutes of Health Grant P01CA154778. The Cell Evaluation and Therapy Shared Resource of the Hollings Cancer Center, Medical University of South Carolina was in part supported by the National Institutes of Health Grant P30 CA138313.
Compliance with ethical standards
Conflict of interest
All authors declare that they have no conflict of interest.
Ethical approval and ethical standards
Apheresis and cells from melanoma patients were obtained as part of clinical trial that is registered with clinicaltrials.gov NCT01586403 and was approved by the Institutional Review Board at Loyola Medical University Center (LU 203732). All procedures performed in studies using human participants were in accordance with the ethical standards of the institutional and/or national research committee, and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All animal experiments were performed with approval by the Institutional Animal Care and Use Committee at the Medical University of South Carolina to ensure that ethical regulatory and policy mandates governing the use of animals in research are met (Animal Welfare Assurance #A3428-01). All methods were performed in accordance with the relevant guidelines and regulations.
Informed consent was obtained from all individual participants included in the study.
TRP-1 TCR transgenic mice, originally obtained from Dr. Nicolas Restifo, were provided by Dr. Chrystal Paulos. Recipient C57BL6 mice were purchased from Jackson Laboratories.
Cell line authentication
The tumor-cell lines in this study are used to determine T-cell reactivity and specificity, so the critical feature is their HLA and antigen expression. Cells are, therefore, routinely checked to ensure their antigen (tyrosinase) and expression HLA (HLA-A2 for MEL624) is correct.
- 3.Johnson LA, June CH (2016) Driving gene-engineered T cell immunotherapy of cancer. Cell Res 38–58Google Scholar
- 15.Moore T, Wagner CR, Scurti GM et al (2017) Clinical and immunologic evaluation of three metastatic melanoma patients treated with autologous melanoma-reactive TCR-transduced T cells. Cancer Immunol Immunother. https://doi.org/10.1007/s00262-017-2073-0