Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity
Karolina Pilipow, Eloise Scamardella, Simone Puccio, Sanjivan Gautam, Federica De Paoli, Emilia M.C. Mazza, Gabriele De Simone, Sara Polletti, Marta Buccilli, Veronica Zanon, Pietro Di Lucia, Matteo Iannacone, Luca Gattinoni, Enrico Lugli
Karolina Pilipow, Eloise Scamardella, Simone Puccio, Sanjivan Gautam, Federica De Paoli, Emilia M.C. Mazza, Gabriele De Simone, Sara Polletti, Marta Buccilli, Veronica Zanon, Pietro Di Lucia, Matteo Iannacone, Luca Gattinoni, Enrico Lugli
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Research Article Immunology

Antioxidant metabolism regulates CD8+ T memory stem cell formation and antitumor immunity

Abstract

Adoptive T cell transfer (ACT) immunotherapy benefits from early differentiated stem cell memory T (Tscm) cells capable of persisting in the long term and generating potent antitumor effectors. Due to their paucity ex vivo, Tscm cells can be derived from naive precursors, but the molecular signals at the basis of Tscm cell generation are ill-defined. We found that less differentiated human circulating CD8+ T cells display substantial antioxidant capacity ex vivo compared with more differentiated central and effector memory T cells. Limiting ROS metabolism with antioxidants during naive T cell activation hindered terminal differentiation, while allowing expansion and generation of Tscm cells. N-acetylcysteine (NAC), the most effective molecule in this regard, induced transcriptional and metabolic programs characteristic of self-renewing memory T cells. Upon ACT, NAC-generated Tscm cells established long-term memory in vivo and exerted more potent antitumor immunity in a xenogeneic model when redirected with CD19-specific CAR, highlighting the translational relevance of NAC as a simple and inexpensive method to improve ACT.

Authors

Karolina Pilipow, Eloise Scamardella, Simone Puccio, Sanjivan Gautam, Federica De Paoli, Emilia M.C. Mazza, Gabriele De Simone, Sara Polletti, Marta Buccilli, Veronica Zanon, Pietro Di Lucia, Matteo Iannacone, Luca Gattinoni, Enrico Lugli

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Figure 2

Modulating ROS levels regulates effector and memory CD8+ T cell differentiation in vitro.

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Modulating ROS levels regulates effector and memory CD8+ T cell differen...
(A) Representative FACS analysis of CCR7 and CD45RO expression in circulating CD8+ Tn cells activated with anti-CD3/28, IL-2, and IL-12 in the presence of N-acetylcysteine (NAC), reduced glutathione (GSH), vitamin C (vitC), or apocynin (Apo) for 8 days. Treatments were supplemented daily. Additional DMSO control for Apo is shown. Similar data were obtained from n = 8 HD in n = 4 experiments (exp.) (NAC) and n = 3 HD in n = 1 exp. (GSH, vitC, Apo). (B) Representative histogram of CFSE dilution of cells cultured, as in A. NS, CFSE-stained, nonproliferating control cells. (C) Fold change (mean ± SEM) in cell counts compared with baseline (n = 11 HD, n = 6 exp.) and (D) MFI (mean ± SEM) of CellROX, MitoSOX, and TMRM (n = 9 HD, n = 4 exp.) indicative of total ROS levels, O2•–, and ΔΨm, respectively, in CTRL and NAC-treated Tn cells at day 8 of culture, treated as in A. NAC was replaced every 3 days. (E) Representative CCR7 and CD45RO expression, as detected by FACS, of Tn cells activated as in A. NAC and menadione (MD) were replaced every 3 days. (F) FACS analysis of CD45RA, CD27, CD95, and CXCR3 by cells cultured, as in E. PBMCs from a HD are depicted as additional staining control. (G) Proportion (mean ± SEM) of CD8+ T cells with the Tscm, Tcm, Tem, and Tte phenotypes (gated as in Methods) after culture in the indicated conditions (CTRL, n = 13; NAC, n = 13, MD, n = 5 from n = 7 [NAC] and n = 2 [MD] exp.). (H) Percentage (mean ± SEM) of cytokine production in response to PMA/ionomycin stimulation by cells cultured, as in G (CTRL and NAC, n = 18; MD, n = 5 from n = 9 [NAC] and n = 2 [MD] exp.). (I) Pies depicting combinations of cytokine production obtained after stimulation, as in H. In all figures showing FACS dot plots, numbers indicate the percentage of cells identified by the gate. Statistical analyses were performed with nonparametric paired Wilcoxon (C, D, G, and H) and permutation (I) tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.