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

Saponin-based adjuvants induce cross-presentation in dendritic cells by intracellular lipid body formation

Saponin-based adjuvants (SBAs) are being used in animal and human (cancer) vaccines, as they induce protective cellular immunity. Their adjuvant potency is a factor of inflammasome activation and enhanced antigen cross-presentation by dendritic cells (DCs), but how antigen cross-presentation is induced is not clear. Here we show that SBAs uniquely induce intracellular lipid bodies (LBs) in the CD11b+ DC subset in vitro and in vivo. Using genetic and pharmacological interference in models for vaccination and in situ tumour ablation, we demonstrate that LB induction is causally related to the saponin- dependent increase in cross-presentation and T-cell activation. These findings link adjuvant activity to LB formation, aid the application of SBAs as a cancer vaccine component, and will stimulate development of new adjuvants enhancing T-cell-mediated immunity

NDUFS4 deletion triggers loss of NDUFA12 in Ndufs4−/− mice and Leigh syndrome patients: A stabilizing role for NDUFAF2

Mutations in NDUFS4, which encodes an accessory subunit of mitochondrial oxidative phosphorylation (OXPHOS) complex I (CI), induce Leigh syndrome (LS). LS is a poorly understood pediatric disorder featuring brain-specific anomalies and early death. To study the LS pathomechanism, we here compared OXPHOS proteomes between various Ndufs4−/− mouse tissues. Ndufs4−/− animals displayed significantly lower CI subunit levels in brain/diaphragm relative to other tissues (liver/heart/kidney/skeletal muscle), whereas other OXPHOS subunit levels were not reduced. Absence of NDUFS4 induced near complete absence of the NDUFA12 accessory subunit, a 50% reduction in other CI subunit levels, and an increase in specific CI assembly factors. Among the latter, NDUFAF2 was most highly increased. Regarding NDUFS4, NDUFA12 and NDUFAF2, identical results were obtained in Ndufs4−/− mouse embryonic fibroblasts (MEFs) and NDUFS4-mutated LS patient cells. Ndufs4−/− MEFs contained active CI in situ but blue-native-PAGE highlighted that NDUFAF2 attached to an inactive CI subcomplex (CI-830) and inactive assemblies of higher MW. In NDUFA12-mutated LS patient cells, NDUFA12 absence did not reduce NDUFS4 levels but triggered NDUFAF2 association to active CI. BN-PAGE revealed no such association in LS patient fibroblasts with mutations in other CI subunit-encoding genes where NDUFAF2 was attached to CI-830 (NDUFS1, NDUFV1 mutation) or not detected (NDUFS7 mutation). Supported by enzymological and CI in silico structural analysis, we conclude that absence of NDUFS4 induces near complete absence of NDUFA12 but not vice versa, and that NDUFAF2 stabilizes active CI in Ndufs4−/− mice and LS patient cells, perhaps in concert with mitochondrial inner membrane lipids

Tumor ablation plus co-administration of CpG and saponin adjuvants affects IL-1 production and multifunctional T cell numbers in tumor draining lymph nodes

Background Tumor ablation techniques, like cryoablation, are successfully used in the clinic to treat tumors. The tumor debris remaining in situ after ablation is a major antigen depot, including neoantigens, which are presented by dendritic cells (DCs) in the draining lymph nodes to induce tumor-specific CD8+ T cells. We have previously shown that co-administration of adjuvants is essential to evoke strong in vivo antitumor immunity and the induction of long-term memory. However, which adjuvants most effectively combine with in situ tumor ablation remains unclear.Methods and results Here, we show that simultaneous administration of cytidyl guanosyl (CpG) with saponin-based adjuvants following cryoablation affects multifunctional T-cell numbers and interleukin (IL)-1 induced polymorphonuclear neutrophil recruitment in the tumor draining lymph nodes, relative to either adjuvant alone. The combination of CpG and saponin-based adjuvants induces potent DC maturation (mainly CpG-mediated), antigen cross-presentation (mainly saponin-based adjuvant mediated), while excretion of IL-1β by DCs in vitro depends on the presence of both adjuvants. Most strikingly, CpG/saponin-based adjuvant exposed DCs potentiate antigen-specific T-cell proliferation resulting in multipotent T cells with increased capacity to produce interferon (IFN)γ, IL-2 and tumor necrosis factor-α in vitro. Also in vivo the CpG/saponin-based adjuvant combination plus cryoablation increased the numbers of tumor-specific CD8+ T cells showing enhanced IFNγ production as compared with single adjuvant treatments.Conclusions Collectively, these data indicate that co-injection of CpG with saponin-based adjuvants after cryoablation induces an increased amount of tumor-specific multifunctional T cells. The combination of saponin-based adjuvants with toll-like receptor 9 adjuvant CpG in a cryoablative setting therefore represents a promising in situ vaccination strategy

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

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

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