The Immunostimulatory Activity of Unmethylated and Methylated CpG Oligodeoxynucleotide Is Dependent on Their Ability To Colocalize with TLR9 in Late Endosomes

Jong, Susan de; Basha, Genc; Wilson, Kaley D.; Kazem, Mikameh; Cullis, Pieter R.; Jefferies, Wilf; Tam, Ying K.

doi:10.4049/jimmunol.0802442

Cited by 36 publications

(22 citation statements)

References 26 publications

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“…Accordingly, nanoparticles have been employed to augment CpG uptake and function (32–37). Lipid nanoparticles, for example, can not only improve CpG uptake and immunopotency (32), but as demonstrated recently, are critical in sequestering TLR9 migration from endoplasmic reticulum to the late endosomal compartment where it can interact with CpG (35). Although we did not correlate subcellular colocalization of CNT-CpG with TLR9 in this study, rapid CNT-CpG sequestration into cytoplasmic compartments seen here suggests that CNTs were responsible for augmenting CpG prostimulatory function by facilitating its uptake.…”

Section: Discussionmentioning

confidence: 99%

Carbon Nanotubes Enhance CpG Uptake and Potentiate Antiglioma Immunity

Zhao

Alizadeh

Zhang

et al. 2011

Clinical Cancer Research

154

122

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Purpose: Stimulation of toll-like receptor-9 (TLR9) by CpG oligodeoxynucleotides (CpG) has been shown to counteract the immunosuppressive microenvironment and to inhibit tumor growth in glioma models. Because TLR9 is located intracellularly, we hypothesized that methods that enhance its internalization may also potentiate its immunostimulatory response. The goal of this study was to evaluate carbon nanotubes (CNT) as a CpG delivery vehicle in brain tumor models.Experimental Design: Functionalized single-walled CNTs were conjugated with CpG (CNT-CpG) and evaluated in vitro and in mice bearing intracranial GL261 gliomas. Flow cytometry was used to assess CNTCpG uptake and antiglioma immune response. Tumor growth was measured by bioluminescent imaging, histology, and animal survival.Results: CNT-CpG was nontoxic and enhanced CpG uptake both in vitro and intracranial gliomas. CNTmediated CpG delivery also potentiated proinflammatory cytokine production by primary monocytes. Interestingly, a single intracranial injection of low-dose CNT-CpG (but not free CpG or blank CNT) eradicated intracranial GL261 gliomas in half of tumor-bearing mice. Moreover, surviving animals exhibited durable tumor-free remission (>3 months), and were protected from intracranial tumor rechallenge, demonstrating induction of long-term antitumor immunity.Conclusions: These findings suggest that CNTs can potentiate CpG immunopotency by enhancing its delivery into tumor-associated inflammatory cells.

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Section: Discussionmentioning

confidence: 99%

Carbon Nanotubes Enhance CpG Uptake and Potentiate Antiglioma Immunity

Zhao

Alizadeh

Zhang

et al. 2011

Clinical Cancer Research

154

122

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“…Spleens were harvested, minced in small pieces, and digested in 1 mg/ml collagenase D (Roche) and CD11b + and CD11c + cells were isolated ex vivo using magnetic beads as described previously. 50 Cell isolates were aliquoted, and GAPDH and α-Tubulin protein expression was assessed by flow cytometry as described. Data were acquired using LSRII flow cytometer after gating 10,000 events from the F4-80 + /CD11b + for MΦ or CD11c high for DCs and analyzed by FlowJo software.…”

Section: Methodsmentioning

confidence: 99%

Influence of Cationic Lipid Composition on Gene Silencing Properties of Lipid Nanoparticle Formulations of siRNA in Antigen-Presenting Cells

et al. 2011

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Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.

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“…Others propose that the binding is independent of the CpG-motif base composition and that mtDNA phosphodiester 2'-deoxyribose backbone is responsible for activation (153). Furthermore, there have been reports suggesting that the preceding stages of endosomal compartmentalization of TLR9 is essential for the receptor to discriminate between endogenous and pathogenic DNA, and subsequent ligand binding to TLR9 (147,153,154). …”

Section: Tlr9 Can Be Activated By Mtdnamentioning

confidence: 99%