Aptamer-targeted inhibition of mTOR in T cells enhances antitumor immunity
Alexey Berezhnoy, Iris Castro, Agata Levay, Thomas R. Malek, Eli Gilboa
Alexey Berezhnoy, Iris Castro, Agata Levay, Thomas R. Malek, Eli Gilboa
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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 2

4-1BB-raptor conjugate decreases mTORC1 activity in activated OT-I cells in mice.

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4-1BB-raptor conjugate decreases mTORC1 activity in activated OT-I cells...
CD45.2 C57BL/6 mice were transferred with congenic CD45.1 OT-I cells, activated in situ with OVA peptide, and treated either with rapamycin or with 4-1BB aptamer-GFP siRNA (4-1BB-GFP) or 4-1BB aptamer-raptor siRNA (4-1BB-raptor) conjugates, as described in Methods. Forty-eight hours after treatment, splenocytes were stained for (A) phosphorylated S6 riboprotein (mTORC1 activity) and CD45.1 (OT-I cells) (n = 6) or (C) CD8, CD45.1, and either phosphorylated S6 (p235) or phosphorylated Akt (p473) (mTORC2 activity) (n = 3). (C) Mice were adoptively transferred with 1 × 106 OT-I cells and 4 × 106 T cell EL4 lymphoma cells and injected 24 hours later with OVA peptide and LPS. EL4 cells can be distinguished from host cells by staining with a Vβ12 TCR chain–specific antibody. Twenty-four hours after peptide injection, splenocytes were isolated and stained for OT-I (CD45.1), EL4 (Vβ12), and 4-1BB. OT-I and EL4 can be readily distinguished by CD45.1 and Vβ12 staining. (D) Four and 24 hours after peptide administration, mice were injected i.p. with 1.5 μg rapamycin or i.v. with 1 nmole 4-1BB-raptor conjugate, or were left untreated (filled gray area). Mice were analyzed for mTORC1 activity by staining for phosphorylated S6 (pS6) in OT-1 (blue) and EL4 (red) cells. (C and D) n = 1, with three time points, one of which is shown. (E) Forty-eight hours after peptide LPS vaccination, splenocytes were isolated from 4-1BB-GFP conjugate (green), 4-1BB-raptor conjugate (red), or from mock-treated mice (gray). CD45.1+CD8+ (OT-I, upper panel) and CD45.1-CD8+ (host CD8+ T cells, lower panel) were sorted to >99.5% purity, and β-actin, Cd8, and raptor mRNA levels were quantified in each population by qRT-PCR.