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 4

CD8+ T cells expressing TSC2 SA mutation have greater effector function while still preserving memory formation and recall capacity.

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CD8+ T cells expressing TSC2 SA mutation have greater effector function ...
WT and mutant TSC2 SA/SA transgenic CD8+ T cells were coadoptively transferred (1:1) into naive WT hosts followed with acute pathogen infection. (A) Flow cytometry plot of transferred OTI CD8+ T cells (top) and summary data (bottom) showing percent of WT versus SA/SA genotype from donor population 8 days after exposure to LM-OVA in spleen (n = 6) and blood (n = 17). *P < 0.05, ****P < 0.0001 paired 2-tailed t test. (B–D) Phenotypic and functional analysis of donor WT and mutant TSC2 SA/SA CD8+ T cells during acute infection from A. * P < 0.05, n = 6. (E) Triple coadoptive transfer of WT, WT/SA, or SA/SA TSC2 OTI CD8+ T cells into mice then infected with LM-OVA with relative number of cells in each group identified 1 week later. n = 9/group; *P < 0.05, ****P < 0.0001, 1-way ANOVA, Sidak’s multiple-comparison test. (F) Percent present of donor WT versus mutant TSC2 SA/SA CD8+ T cells 44 days after injection (blood, memory phase). n = 9; **P = 0.004, Wilcoxon 2-tailed signed-rank test. (G) Naive mice received equal cotransfer of memory OTI CD8+ WT or SA expressing T cells for recall with LM-OVA. At day 90, the percent of memory WT and mutant TSC2SA OTI CD8+ T cells in spleen (top, combined n = 3) and characterized based on KLRG1 (–) and CD62L (+) for memory phenotype (lower panels). These sorted memory CD8+ T cells were then injected in equal numbers (1:1) to naive WT recipients (top, right) who were subsequently infected with LM-OVA to assess memory recall ability. (H) After 5-days after infection, graphical summary of donor WT and SA/SA CD8+ T cells in spleen. **P = 0.003 Wilcoxon, n = 16. Data are representative of at least 3 independent experiments except E, with 2.