TSC2 S1365A mutation potently regulates CD8+ T cell function and differentiation and improves adoptive cellular cancer therapy
Chirag H. Patel, Yi Dong, Navid Koleini, Xiaoxu Wang, Brittany L. Dunkerly-Eyring, Jiayu Wen, Mark J. Ranek, Laura M. Bartle, Daniel B. Henderson, Jason Sagert, David A. Kass, Jonathan D. Powell
Chirag H. Patel, Yi Dong, Navid Koleini, Xiaoxu Wang, Brittany L. Dunkerly-Eyring, Jiayu Wen, Mark J. Ranek, Laura M. Bartle, Daniel B. Henderson, Jason Sagert, David A. Kass, Jonathan D. Powell
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Research Article Cell biology Immunology

TSC2 S1365A mutation potently regulates CD8+ T cell function and differentiation and improves adoptive cellular cancer therapy

Abstract

MTORC1 integrates signaling from the immune microenvironment to regulate T cell activation, differentiation, and function. TSC2 in the tuberous sclerosis complex tightly regulates mTORC1 activation. CD8+ T cells lacking TSC2 have constitutively enhanced mTORC1 activity and generate robust effector T cells; however, sustained mTORC1 activation prevents generation of long-lived memory CD8+ T cells. Here we show that manipulating TSC2 at Ser1365 potently regulated activated but not basal mTORC1 signaling in CD8+ T cells. Unlike nonstimulated TSC2-KO cells, CD8+ T cells expressing a phosphosilencing mutant TSC2-S1365A (TSC2-SA) retained normal basal mTORC1 activity. PKC and T cell receptor (TCR) stimulation induced TSC2 S1365 phosphorylation, and preventing this with the SA mutation markedly increased mTORC1 activation and T cell effector function. Consequently, SA CD8+ T cells displayed greater effector responses while retaining their capacity to become long-lived memory T cells. SA CD8+ T cells also displayed enhanced effector function under hypoxic and acidic conditions. In murine and human solid-tumor models, SA CD8+ T cells used as adoptive cell therapy displayed greater antitumor immunity than WT CD8+ T cells. These findings reveal an upstream mechanism to regulate mTORC1 activity in T cells. The TSC2-SA mutation enhanced both T cell effector function and long-term persistence/memory formation, supporting an approach to engineer better CAR-T cells for treating cancer.

Authors

Chirag H. Patel, Yi Dong, Navid Koleini, Xiaoxu Wang, Brittany L. Dunkerly-Eyring, Jiayu Wen, Mark J. Ranek, Laura M. Bartle, Daniel B. Henderson, Jason Sagert, David A. Kass, Jonathan D. Powell

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Figure 3

Mutating TSC2 at S1365 augments mTORC1 activity and function in murine CD8+ T cells upon stimulation.

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Mutating TSC2 at S1365 augments mTORC1 activity and function in murine C...
(A) Immunoblot analysis of mTORC1 activity at baseline (time 0) and 0.5 and 2 hours after stimulation with αCD3/αCD28 in CD8+ T cells expressing WT or TSC2 SA/SA. Summary data to the right (n = 4/group); 2-way ANOVA, Sidaks multiple comparisons test: * P < 0.00003; ** P ≤ 0.003 versus Time 0, 120 min; †P = 0.012 versus WT at 120 min). (B) mTORC1 activity assessed by intracellular staining for mTORC1 activity via phospho-S6 levels in naive WT, TSC2 WT, SA/SA, or TSC2–/– CD8+ T cells. Geo-MFI, geometric mean fluorescence intensity. Summary data to right; n = 4, 5, 3 for groups left to right; 1-way ANOVA, ****P = 3 × 10–11, Holm-Šídáks multiple-comparisons test. (C) Example immunoblot for mTORC1 activation (pS6K-1) from similar experiment as in A but with TSC2 WT/SA CD8+ T cells. (D) Naive WT, WT/SA, and SA/SA CD8+ T cells stimulated with αCD3 and αCD28 to activate TCR signaling for 60 minutes and 18 hours and mTORC1 activity assessed by intracellular staining for mTORC1 activity via phospho-S6 levels. (E) WT and SA/SA CD8+ T cells stimulated with αCD3 and αCD28 with or without rapamycin to inhibit mTOR, and cell cycle entry assessed by BrdU+ staining by flow cytometry. n = 4 biologic replicates, 5 control, 2 rapamycin; P value Kruskal Wallis test. (F) Same experiment (without rapamycin) with cell counts measured after 24 and 48 hours. n = 5/group; significance found with Kruskal Wallis test. (G) WT, WT/SA, and SA/SA mutant OTI CD8+ T cells stimulated with OVA I peptide and cell proliferation analyzed by flow cytometry on day 2 and day 3 after activation. (H) CD8+ T cells from WT, WT/SA, and SA/SA genotypes, activated and expanded in IL-2, and then examined for cytokine analysis of IFN-γ, TNF-α, and IL-2 after restimulating with αCD3/αCD28. Data are representative of at least 2 independent experiments.