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 3

NAC establishes a gene expression signature of long-lived memory T cells.

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NAC establishes a gene expression signature of long-lived memory T cells...
(A) CD8+ Tn cells (n = 6) were activated in the absence (CTRL) or presence of NAC, as in Figure 2E, and their transcriptome was analyzed by RNAseq. Volcano plot representation of differentially expressed genes (DEGs) between NAC-treated and CTRL CD8+ T cells with a log2 fold change >1 and adjusted P < 0.05 (q value). (B) From left to right (top): relative expression level of TFs or activation/exhaustion related genes in CTRL versus NAC-treated samples; from left to right (bottom): relative expression level of effector function or homing/adhesion-related genes in CTRL versus NAC-treated samples. (C and D) Validation of selected RNAseq transcripts, as determined by qPCR (C; n = 3–5 HD in n = 2–3 exp.) or by FACS (D; similar data were obtained from n = 3 more HD). In D, a representative staining in PBMCs is depicted as an additional control. (E) Transcripts per million (TPM) (mean ± SEM) of HNRNPLL gene, as obtained from RNAseq data, as in A. *P < 0.05, paired Student’s t test. (F) Gene set enrichment analysis (GSEA) of mTOR signaling pathway (c2.cp.kegg.v6.1) between CTRL and NAC-treated CD8+ T cells from data as in A. (G) CD8+ Tn cells were activated in the absence (CTRL) or presence of NAC as in Figure 2E and then washed and restimulated with anti-CD3/28 for 30 minutes or left unstimulated. The bar graph shows the (mean ± SEM) frequencies of Ser240/244 pS6+ T cells (n = 6 HD from n = 2 exp.). Statistical analyses were performed with parametric paired Student’s t test (in C for LEF1 and TCF7 expression and E and G). In C, significance of PRDM1 expression was evaluated with nonparametric Wilcoxon test. *P < 0.05, **P < 0.01.