Route of Administration of the TLR9 Agonist CpG Critically Determines the Efficacy of Cancer Immunotherapy in Mice

Contains fulltext : 81648.pdf (publisher's version ) (Open Access)BACKGROUND: The TLR9 agonist CpG is increasingly applied in preclinical and clinical studies as a therapeutic modality to enhance tumor immunity. The clinical application of CpG appears, however, less successful than would be predicted from animal studies. One reason might be the different administration routes applied in most mouse studies and clinical trials. We studied whether the efficacy of CpG as an adjuvant in cancer immunotherapy is dependent on the route of CpG administration, in particular when the tumor is destructed in situ. METHODOLOGY/PRINCIPAL FINDINGS: In situ tumor destruction techniques are minimally invasive therapeutic alternatives for the treatment of (nonresectable) solid tumors. In contrast to surgical resection, tumor destruction leads to the induction of weak but tumor-specific immunity that can be enhanced by coapplication of CpG. As in situ tumor destruction by cryosurgery creates an instant local release of antigens, we applied this model to study the efficacy of CpG to enhance antitumor immunity when administrated via different routes: peritumoral, intravenous, and subcutaneous but distant from the tumor. We show that peritumoral administration is superior in the activation of dendritic cells, induction of tumor-specific CTL, and long-lasting tumor protection. Although the intravenous and subcutaneous (at distant site) exposures are commonly used in clinical trials, they only provided partial protection or even failed to enhance antitumor responses as induced by cryosurgery alone. CONCLUSIONS/SIGNIFICANCE: CpG administration greatly enhances the efficacy of in situ tumor destruction techniques, provided that CpG is administered in close proximity of the released antigens. Hence, this study helps to provide directions to fully benefit from CpG as immune stimulant in a clinical setting

Route of CpG administration affects T cell activation.

<p>(<b>A</b>) Percentages of IFN-γ⁺ T cells within the CD4⁺ or CD8⁺ populations after cryo ablation +/− CpG. B16-OVA tumor-bearing mice were treated with cryo ablation combined with CpG administration via the indicated routes. Seven days after ablation, spleen, tumor-draining lymph nodes and non-draining lymph nodes were isolated. Cells were stimulated with PMA/ionomycin and stained for CD8, CD4 and IFN-γ. * indicate significant differences (p<0.05) between the indicated CpG-treated group and the Naïve and Cryo group. (<b>B</b>) IFN-γ production after cryo ablation +/− CpG. Cells were stimulated with OVA for 96 hours and IFN-γ was measured in supernatant by ELISA. The mean levels of the Cryo+CpG i.v. groups in both organs are significantly different from all other groups (n = 6/group, 2 experiments).</p

Timing of i.v. CpG injections determines antitumor immunity.

<p>Established B16OVA tumors were treated with cryo ablation alone or in combination with i.v. CpG administration concurrent with ablation, or 1 day before or after ablation. Forty days later, naïve and treated tumor-free mice (7–10 mice per group) received a s.c. re-challenge with tumor cells (25.000–50.000 B16OVA cells) on the contra-lateral flank and survival was monitored. Representative of 2 experiments is shown.</p

Quantity and quality of lymph node DC.

<p>(<b>A</b>) Absolute numbers of CD11c⁺ cells in the draining lymph nodes 2 days after ablation. Tumor-bearing mice were left untreated (NT) or were subjected to cryo ablation (cryo). In addition, some groups of mice received CpG via the indicated routes. Brackets indicate significant differences (p<0.05) between indicated groups (n = 4–6/group, 2 similar experiments). (<b>B</b>) Uptake of antigen after cryo ablation. OVA-Alexa488 was injected in B16OVA tumors (6–9 mm) just prior to cryo ablation to monitor the fate of antigen after in situ tumor destruction. Mice were additionally treated with CpG via the indicated routes. Two days after ablation, the uptake of antigen was analyzed in CD11c⁺ cells enriched from pools of tumor-draining lymph nodes. Numbers in the panels indicate the percentage of OVA⁺ of all CD11c⁺ cells. Data from one representative mouse is shown per group. (<b>C</b>) Quantitative analyses of replicates of experiments as shown in (B) (5 mice/group, representative of 2 experiments). (<b>D</b>) Uptake of antigen and CpG by CD11c⁺ cells monitored after p.t. and i.v. CpG-Cy5 administration and intra-tumoral injection of OVA-Alexa488. Data from one representative mouse is shown (n = 4/group). (<b>E</b>) The expression of CD80 on CD11c⁺ antigen-loaded cells. * indicates significantly different (p<0.05) from all other groups (n = 5/group, 2 similar experiments).</p

Route of CpG administration determines efficacy of antitumor immunity.

<p>(<b>A</b>) Kaplan-Meier survival curves of naïve mice versus mice that have been treated with cryo ablation alone, or in combination with concurrent CpG administration via the indicated routes. Established B16OVA tumors on the right femur were treated with cryo ablation alone or in combination with CpG administrations via different routes: p.t., i.v., or s.c. contra lateral of the tumor. Forty days later, naïve and tumor-free mice (8–13 mice per group) received a s.c. re-challenge with tumor cells (25.000 B16OVA cells). (<b>B</b>) Tumor size determined every 2–3 days after treatment. Data is representative of two independent experiments.</p

Route of CpG administration determines induction of tumor-specific CTL.

<p>(<b>A</b>) Representative dot plots of CD8⁺OVA-Kb tetramer⁺ cells of individual mice. Mice bearing B16OVA tumors (6–9 mm) were subjected to cryo ablation alone or received additional CpG injections via the indicated routes. Ten days after tumor destruction, cells from spleen and tumor draining lymph nodes were isolated and re-stimulated with IFN-γ-treated γ-irradiated B16OVA tumor cells. Cultures were cleaned by a Ficoll step after 3–4 days of culture and at day 8–9 cells were analyzed for the presence of tumor-specific CTL using APC-labeled OVA-K_b tetramers. Cells were gated on CD8⁺ T cells. Data from one representative mouse per group is shown. (<b>B</b>) Quantitative analyses of collective data as shown in (A) (mean levels of 2 separate experiments (4–6 mice per group/experiment)). * indicates significant differences (p<0.05) of the indicated group compared to all other groups.</p

Broad defects in the energy metabolism of leukocytes underlie immunoparalysis in sepsis

The acute phase of sepsis is characterized by a strong inflammatory reaction. At later stages in some patients, immunoparalysis may be encountered, which is associated with a poor outcome. By transcriptional and metabolic profiling of human patients with sepsis, we found that a shift from oxidative phosphorylation to aerobic glycolysis was an important component of initial activation of host defense. Blocking metabolic pathways with metformin diminished cytokine production and increased mortality in systemic fungal infection in mice. In contrast, in leukocytes rendered tolerant by exposure to lipopolysaccharide or after isolation from patients with sepsis and immunoparalysis, a generalized metabolic defect at the level of both glycolysis and oxidative metabolism was apparent, which was restored after recovery of the patients. Finally, the immunometabolic defects in humans were partially restored by therapy with recombinant interferon-γ, which suggested that metabolic processes might represent a therapeutic target in sepsis