TSC2 S1365A mutation potently regulates CD8+ T cell function and differentiation and improves adoptive cellular cancer therapy
Chirag H. Patel, … , David A. Kass, Jonathan D. Powell
Chirag H. Patel, … , David A. Kass, Jonathan D. Powell
Published October 3, 2023
Citation Information: JCI Insight. 2023;8(21):e167829. https://doi.org/10.1172/jci.insight.167829.
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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.