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CpG-ODN Facilitates Effective Intratracheal Immunization and Recall of Memory against Neoantigen-Expressing Alveolar Cells.

ABSTRACT: Intrapulmonary immune reactions are impaired by the tolerogenic environment of the lung. This is manifested by the absence of effective endogenous T cell responses upon neoantigen expression. This tolerance is considered to contribute to lung cancer and inefficient immune therapeutic interventions. To investigate the mechanisms contributing to lung tolerance and to overcome these restrictions, we developed a transgenic mouse model with induction of a neoantigen (OVA) exclusively in alveolar type II epithelial cells. This model is characterized by the absence of functional endogenous T cell responses upon OVA neoantigen induction. Standard DNA and protein vaccination protocols resulted in the accumulation of high numbers of antigen-specific CD8 T cells in the lung. However, clearance of antigen-expressing cells was not achieved. To overcome this tolerance, we induced inflammatory conditions by coapplication of the TLR ligands LPS and CpG-ODN during intrapulmonary vaccinations. Both ligands induced high numbers of neoantigen-specific T cells in the lung. However, only coapplication of CpG-ODN was sufficient to establish functional cytotoxic responses resulting in the elimination of neoantigen presenting target cells. Remarkably, CpG-ODN was also crucial for functional memory responses upon re-induction of the neoantigen. The results highlight the need of TLR9 co-stimulation for overcoming tolerization, which might be a key factor for therapeutic interventions.

SUBMITTER: Riehn M

PROVIDER: S-EPMC5630691 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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CpG-ODN Facilitates Effective Intratracheal Immunization and Recall of Memory against Neoantigen-Expressing Alveolar Cells.

Riehn Mathias M Cebula Marcin M Hauser Hansjörg H Wirth Dagmar D

Frontiers in immunology 20170929

Intrapulmonary immune reactions are impaired by the tolerogenic environment of the lung. This is manifested by the absence of effective endogenous T cell responses upon neoantigen expression. This tolerance is considered to contribute to lung cancer and inefficient immune therapeutic interventions. To investigate the mechanisms contributing to lung tolerance and to overcome these restrictions, we developed a transgenic mouse model with induction of a neoantigen (OVA) exclusively in alveolar type ...[more]

PMID: 29038654

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Project description:BackgroundCytosine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN) has been used successfully to induce immune responses against viral and intracellular organisms in mammals. The main objective of this study was to test the effect of CpG-ODN on antigen presenting cells of young foals.MethodsPeripheral blood monocytes of foals (n = 7) were isolated in the first day of life and monthly thereafter up to 3 months of life. Adult horse (n = 7) monocytes were isolated and tested once for comparison. Isolated monocytes were stimulated with IL-4 and GM-CSF (to obtain dendritic cells, DC) or not stimulated (to obtain macrophages). Macrophages and DCs were stimulated for 14-16 hours with either CpG-ODN, LPS or not stimulated. The stimulated and non-stimulated cells were tested for cell surface markers (CD86 and MHC class II) using flow cytometry, mRNA expression of cytokines (IL-12, IFNalpha, IL-10) and TLR-9 using real time quantitative RT-PCR, and for the activation of the transcription factor NF-kappaB p65 using a chemiluminescence assay.ResultsThe median fluorescence of the MHC class II molecule in non-stimulated foal macrophages and DCs at birth were 12.5 times and 11.2 times inferior, respectively, than adult horse cells (p = 0.009). That difference subsided at 3 months of life (p = 0.3). The expression of the CD86 co-stimulatory molecule was comparable in adult horse and foal macrophages and DCs, independent of treatment. CpG-ODN stimulation induced IL-12p40 (53 times) and IFNalpha (23 times) mRNA expression in CpG-ODN-treated adult horse DCs (p = 0.078), but not macrophages, in comparison to non-stimulated cells. In contrast, foal APCs did not respond to CpG-ODN stimulation with increased cytokine mRNA expression up to 3 months of age. TLR-9 mRNA expression and NF-kB activation (NF-kB p65) in foal DCs and macrophages were comparable (p > 0.05) to adult horse cells.ConclusionCpG-ODN treatment did not induce specific maturation and cytokine expression in foal macrophages and DCs. Nevertheless, adult horse DCs, but not macrophages, increased their expression of IL-12 and IFNalpha cytokines upon CpG-ODN stimulation. Importantly, foals presented an age-dependent limitation in the expression of MHC class II in macrophages and DCs, independent of treatment.

Project description:BackgroundToll-like receptor 9 (TLR9) recognises unmethylated CpG DNA and activates a signalling cascade, leading to the production of inflammatory cytokines such as TNF-α, IL-1, IL-6 and IL-12 via the adaptor protein MyD88. However, the specific sequence and structural requirements of the CpG DNA for the recognition of and binding to TLR9 are unknown. Moreover, the 3D structures of TLR9 and the TLR9-ODN complex have not been determined. In this study, we propose a reliable model of the interaction of the TLR9 ECD with CpG ODN using bioinformatics tools.ResultsThe three-dimensional structures of two TLR9 ECD-CpG ODN complexes were constructed using a homology modelling and docking strategy. Based on the models of these complexes, the TLR9 ECD-CpG ODN interaction patterns were calculated. The results showed that the interface between the human TLR9 and the CpG ODN molecule is geometrically complementary. The computed molecular interactions indicated that LRR11 is the main region of TLR9 that binds to CpG ODN and that five positively charged residues within LRR11 are involved in the binding of the TLR9 ECD to the CpG ODN. Observations in the close-up view of these interactions indicated that these five positively charged residues contribute differently to the binding region within the TLR9 ECD-CpG ODN complex. 337Arg and 338Lys reside in the binding sites of ODN, forming hydrogen bonds and direct contacts with the CpG ODN, whereas 347Lys, 348Arg, and 353His do not directly contact the CpG ODN. These results are in agreement with previously reported experimental data.ConclusionIn this study, we present two structural models for the human and mouse TLR9 ECD in a complex with CpG ODN. Some features predicted by this model are consistent with previously reported experimental data. This complex model may lead to a better understanding of the function of TLR9 and its interaction with CpG ODN and will improve our understanding of TLR9-ligand interaction in general.

Dataset Information

CpG-ODN Facilitates Effective Intratracheal Immunization and Recall of Memory against Neoantigen-Expressing Alveolar Cells.

Publications

CpG-ODN Facilitates Effective Intratracheal Immunization and Recall of Memory against Neoantigen-Expressing Alveolar Cells.

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