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 4

NAC induces metabolic reprogramming in generated CD8+ Tscm cells.

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NAC induces metabolic reprogramming in generated CD8+ Tscm cells. (A) Ge...
(A) Gene set enrichment analysis (GSEA) of glycolysis signaling pathway (c2.cp.reactome.v6.1) between CTRL and NAC-treated CD8+ T cells activated for 8 days, as in Figure 2A, and as assessed by RNAseq, as in Figure 3A (n = 6). (B) Transcripts per million (TPM) (mean ± SEM) (n = 6) of differentially expressed glycolytic genes assessed by RNAseq, as in Figure 3A. (C) Representative FACS histograms of 2-NBDG (fluorescent glucose analog) and Bodipy FL C16 (fluorescent fatty acid synthetic precursor) uptake by CTRL and NAC-treated CD8+ T cells, activated for 8 days, as in Figure 2A. (D and E) Cumulative data (mean ± SEM; n = 9 HD, n = 5 exp.) of 2-NBDG (D) and Bodipy FL C16 (E) uptake, as in C. (F) CPT1a TPM (mean ± SEM) and (G) relative gene expression level of selected lipases in CTRL and NAC-treated CD8+ T cells, activated for 8 days, as in Figure 2E, and as assessed by RNAseq, as in Figure 3A (n = 6). (H) Representative CCR7 and CD45RO expression, as detected by FACS, in human circulating CD8+ Tn cells activated with anti-CD3/28, IL-2, and IL-12 in the presence of NAC, orlistat, or a combination of both. Numbers in the plots indicate the frequency of cells identified by the gates. (I) Percentage (mean ± SEM) of cytokine production in response to PMA/ionomycin stimulation by cells cultured, as in H (n = 5 HD in n = 2 exp.). Statistical analyses for B and D–F were performed with parametric paired Student’s t test and for I with nonparametric Wilcoxon test. *P < 0.05, ****P < 0.0001.