To establish a novel murine model of pulmonary adenoviral persistence, we performed intratracheal inoculation with AdLGO, a recombinant adenovirus coding for luciferase, enhanced green fluorescent ...protein (eGFP), and the model antigen ovalbumin (OVA), which allows convenient tracking of T-cell responses. Effector CTL function is characterized by a metabolic bias toward aerobic glycolysis.8 Consistent with a recovered cytotoxic function, CpG application promoted glucose uptake (Fig 2, G) and reduced mitochondrial membrane potential in virus-specific CTLs (Fig 2, H), suggesting a switch to aerobic glycolysis. ...we conclude that CpG instillation had a quantitative and qualitative effect on virus-specific effector CTLs, ultimately allowing clearance of persistent adenoviral infection in the lung. The resulting adenoviral DNA was propagated on human embryonic kidney (HEK 293) cells and purified according to standard protocols based on cesium chloride density-gradient centrifugation, as described previosuly.E5 Adenoviral infection and intratracheal instillation For intratracheal administration of adenoviruses (5 x 108 infection units, unless stated otherwise), unmethylated CpG-containing oligodeoxynucleotide #1668 (5 μg; TIB MOLBIOL, Berlin, Germany), OVA (100 μg, Sigma-Aldrich, St Louis, Mo), or extended LCMV GP26-48 peptide containing the minimal GP33-41 epitope LIVITGIKAVYNFATCGIFAFTL (25 μg; Xaia Custom Peptides, Göteborg, Sweden) or PBS, mice were anesthetized with 3% isoflurane/O2 (vol/vol) and intubated with a blunted indwelling 22-gauge x 1-inch cannula (B. Braun Medical, Bethlehem, Pa) with the help of a light-assisted rodent-laryngoscope (Penn-Century, Wyndmoor, Pa). Pang, D. Lien, K. Jackson, Assessment of adenovirus infection in adult lung transplant recipients using molecular surveillance, J Heart Lung Transplant, Vol. 25, 2006, 1441-1446 3 I. Voskoboinik, J.C. Whisstock, J.A. Trapani, Perforin and granzymes: function, dysfunction and human pathology, Nat Rev Immunol, Vol. 15, 2015, 388-400 4 N. Garbi, B. Arnold, S. Gordon, G.J. Hämmerling, R. Ganss, CpG motifs as proinflammatory factors render autochthonous tumors permissive for infiltration and destruction, J Immunol, Vol. 172, 2004, 5861-5869 5 Y. Kawarada, R. Ganss, N. Garbi, T. Sacher, B. Arnold, G.J. Hämmerling, J Immunol, Vol. 167, 2001, 5247-5253 6 J. Scheiermann, D.M. Klinman, Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer, Vaccine, Vol. 32, 2014, 6377-6389 7 E.J. Wherry, T cell exhaustion, Nat Immunol, Vol. 12, 2011, 492-499 8 C.-H.
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