Aptamer-targeted inhibition of mTOR in T cells enhances antitumor immunity
Alexey Berezhnoy, … , Thomas R. Malek, Eli Gilboa
Alexey Berezhnoy, … , Thomas R. Malek, Eli Gilboa
Published December 2, 2013
Citation Information: J Clin Invest. 2014;124(1):188-197. https://doi.org/10.1172/JCI69856.
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Research Article Oncology

Aptamer-targeted inhibition of mTOR in T cells enhances antitumor immunity

Abstract

Recent studies have underscored the importance of memory T cells in mediating protective immunity against pathogens and cancer. Pharmacological inhibition of regulators that mediate T cell differentiation promotes the differentiation of activated CD8+ T cells into memory cells. Nonetheless, pharmacological agents have broad targets and can induce undesirable immunosuppressive effects. Here, we tested the hypothesis that aptamer-targeted siRNA inhibition of mTOR complex 1 (mTORC1) function in CD8+ T cells can enhance their differentiation into memory T cells and potentiate antitumor immunity more effectively than the pharmacologic inhibitor rapamycin. To specifically target activated cells, we conjugated an siRNA targeting the mTORC1 component raptor to an aptamer that binds 4-1BB, a costimulatory molecule that is expressed on CD8+ T cells following TCR stimulation. We found that systemic administration of the 4-1BB aptamer-raptor siRNA to mice downregulated mTORC1 activity in the majority of CD8+ T cells, leading to the generation of a potent memory response that exhibited cytotoxic effector functions and enhanced vaccine-induced protective immunity in tumor-bearing mice. In contrast, while treatment with the general mTORC1 inhibitor rapamycin also enhanced antigen-activated CD8+ T cell persistence, the cytotoxic effector functions of the reactivated memory cells were reduced and the alloreactivity of DCs was diminished. Consistent with the immunological findings, mice treated with rapamycin, but not with 4-1BB aptamer-raptor siRNA, failed to reject a subsequent tumor challenge.

Authors

Alexey Berezhnoy, Iris Castro, Agata Levay, Thomas R. Malek, Eli Gilboa

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Figure 4

4-1BB-raptor–generated memory OT-I cells exhibit proliferative and cytotoxic effector functions.

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4-1BB-raptor–generated memory OT-I cells exhibit proliferative and cytot...
CD45.2 C57BL/6 mice were transferred with CD45.1 OT-I cells, activated with OVA peptide, and treated either with rapamycin or with 4-1BB-GFP or 4-1BB-raptor conjugates. (A) On day 30, mice were revaccinated with OVA peptide, and 3 days later the number of CD8+CD45.1 OT-I cells in the spleen was determined (n = 6). (B) On day 30, splenocytes were isolated, 5 × 104 CD8+CD45.1 OT-I cells were mixed with a 1:1 mixture of 5 × 106 CFSEhi OVA and CFSElo influenza NP peptide–pulsed splenocytes, then injected into naive mice. Lysis of OVA targets was determined, calculated as the percentage of reduced CFSEhi cells relative to CFSElo cells (n = 8). (C) OT-I–bearing mice were vaccinated with OVA peptide and LPS and treated with rapamycin or administered 4-1BB-GFP or 4-1BB-raptor conjugates, as described in Methods. Fourteen days after peptide administration, mice were injected again with peptide and LPS and sacrificed 72 hours later, and splenocytes were isolated (Experiment 1), or mice were sacrificed after 30 days, but not injected again with peptide and LPS (Experiment 2). Either total splenocytes or purified CD8 cells containing 104 OT-I cells were used as effectors against 51Cr-labeled OVA peptide–loaded EL4 targets at a 1:10 effector/target ratio, and specific lysis was determined as described in Methods. Statistical analysis for 4-1BB-raptor versus rapamycin; total splenocytes, P = 0.028; purified CD8 cells, P = 0.785 (n = 2).