Rescue of T-cell Function During Persistent Pulmonary Adenoviral Infection by Toll-like Receptor 9 Activation.

Increasingly, it is being recognized that the lung harbors persistent subclinical viral infections with unknown effects on immunity. Pulmonary adenoviral infections can become lethal, and yet adenoviruses are detected in the lungs of otherwise healthy subjects at a relatively high frequency, similar to cytomegalovirus (CMV),1Vlaminck I.D. Martin L. Kertesz M. Patel K. Kowarsky M. Strehl C. et al.Noninvasive monitoring of infection and rejection after lung transplantation.Proc Natl Acad Sci U S A. 2015; 112: 13336-13341Crossref PubMed Scopus (206) Google Scholar, 2Humar A. Doucette K. Kumar D. Pang X.-L. Lien D. Jackson K. et al.Assessment of adenovirus infection in adult lung transplant recipients using molecular surveillance.J Heart Lung Transplant. 2006; 25: 1441-1446Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar suggesting viral persistence. However, it is currently unknown whether and, if so, how persistent adenoviral infections localized in the lung affect immune responses because suitable experimental models are lacking. 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. Intratracheal inoculation of AdLGO, as described in the Methods section in this article's Online Repository at www.jacionline.org, resulted in a dose-dependent infection of the respiratory tract (Fig 1, A) that steadily decreased in a CD8 T cell–dependent manner until about 16 days postinfection (dpi; Fig 1, B). Beyond that time point, the infectious load after instillation with 5 × 108 infection units remained stable for at least 100 dpi (Fig 1, C and D). Most of the persistent infection localized to bronchial and alveolar epithelial cells, as demonstrated by using confocal microscopy and flow cytometry with a similar adenovirus expressing tdTomato (tdT; Fig 1, E, and see Fig E1 in this article's Online Repository at www.jacionline.org). Mice displayed normal appearance, behavior, and weight (see Fig E2 in this article's Online Repository at www.jacionline.org), indicating a subclinical course of chronic infection. Although virus-specific CD8 T cells were detected locally at high frequencies (see Fig E3 in this article's Online Repository at www.jacionline.org). They were clearly protective during the acute phase of AdLGO infection and failed to fully eradicate infected cells (Fig 1, B), suggesting an impairment in their effector function. To test whether a persistent adenoviral infection induced functional impairment of CD8 T cells, carboxyfluorescein succinimidyl ester (CFSE)–labeled, AdLGO-specific CD45.1+ OT-I CD8 T cells were adoptively transferred into chronically infected B6 mice that were also challenged intratracheally with their cognate antigen OVA. Although persistent adenoviral infection did not impair the proliferative response of OT-I CD8 T cells toward the additional OVA challenge, it inhibited expression of granzyme B (GzmB; Fig 1, F, left versus middle panel), a hallmark of CD8 T-cell cytotoxic function.3Voskoboinik I. Whisstock J.C. Trapani J.A. Perforin and granzymes: function, dysfunction and human pathology.Nat Rev Immunol. 2015; 15: 388-400Crossref PubMed Scopus (599) Google Scholar In addition, OT-I CD8 T cells were also impaired in terms of CD25 expression and their capacity to produce IFN-γ and TNF-α (see Fig E4 in this article's Online Repository at www.jacionline.org), indicating a general dysfunction. We next investigated whether the cytotoxic lymphocyte (CTL) response was impaired also against pulmonary antigens unrelated to the AdLGO infection. For this, we challenged AdLGO-infected mice by means of intratracheal instillation of a lymphocytic choriomeningitis virus (LCMV) antigen and then monitored the immune response of adoptively transferred P14 CD8 T cells (specific for H2-Db/LCMV-GP33-41). Although persistent AdLGO infection did not affect proliferation of P14 cells, it again inhibited GzmB expression (Fig 1, G). Collectively, these results indicate that persistent adenoviral infection localized to the lung induces a suppressive environment that impairs acquisition of effector function by pulmonary CTLs. On the basis of these results, we hypothesized that reverting the suppressive inflammatory environment in the lung to a proinflammatory setting might promote the accumulation of protective virus-specific effector CTLs. We used CpG-rich immunostimulatory oligodeoxynucleotide (CpG) for this purpose because it was shown to promote protective T-cell responses in a different setting of unresolved inflammation elicited by tumor growth,4Garbi N. Arnold B. Gordon S. Hämmerling G.J. Ganss R. CpG motifs as proinflammatory factors render autochthonous tumors permissive for infiltration and destruction.J Immunol. 2004; 172: 5861-5869Crossref PubMed Scopus (114) Google Scholar, 5Kawarada Y. Ganss R. Garbi N. Sacher T. Arnold B. Hämmerling G.J. NK− and CD8(+) T cell-mediated eradication of established tumors by peritumoral injection of CpG-containing oligodeoxynucleotides.J Immunol. 2001; 167: 5247-5253Crossref PubMed Scopus (189) Google Scholar and in addition, it is proposed for clinical use as an adjuvant because of its ability to promote protective immune responses.6Scheiermann J. Klinman D.M. Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer.Vaccine. 2014; 32: 6377-6389Crossref PubMed Scopus (231) Google Scholar CpG intratracheal instillation was able to modulate the pulmonary inflammatory context during chronic AdLGO infection, as demonstrated by enhanced expression of the type 1 cytokines IFN-γ and IL-12 and other proinflammatory cytokines, such as TNF-α, IL-1α, IL-6, IL-5, IL-13, and IL-17, in the bronchoalveolar lavage fluid (see Fig E5 in this article's Online Repository at www.jacionline.org). Strikingly, there was a rapid decrease in pulmonary adenoviral load, decreasing to less than the detection limit within 10 days after onset of CpG immunotherapy (Fig 2, A). In contrast, systemic CpG administration did not result in viral clearance (Fig 2, A), indicating that modulation of the lung inflammatory context was required for the therapeutic effect. Depletion of CTLs from chronically infected mice completely abrogated CpG-induced AdLGO clearance (Fig 2, B), demonstrating a crucial role of CTLs in this therapeutic setting. Intratracheal CpG administration remarkably increased virus-specific CTL numbers in the lung by approximately 60-fold (Fig 2, C). This increase in pool size was mediated both by enhanced antigen-specific proliferation in the mediastinal lymph nodes (Fig 2, D, and see Fig E6 in this article's Online Repository at www.jacionline.org) and by a reduction in their apoptotic rate in the lung (Fig 2, E). The coinhibitory receptors T-cell immunoglobulin and mucin 3 (Tim-3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)7Wherry E.J. T cell exhaustion.Nat Immunol. 2011; 12: 492-499Crossref PubMed Scopus (2434) Google Scholar were expressed by virus-specific CTLs during the persistent phase of adenoviral infection independently of CpG application (see Fig E7, A, in this article's Online Repository at www.jacionline.org), indicating that they did not play a dominant role during CpG immunotherapy. Although CpG instillation resulted in a partial decrease in programmed cell death protein 1 (PD-1) expression (see Fig E7, B), our results indicate that this decrease was not sufficient to mediate full viral clearance because blockade of programmed cell death ligand 1 (PD-L1) resulted in only a minor and nonstatistically significant decrease in adenoviral signal (see Fig E7, C). We next investigated whether local CpG therapy can also improve CTL functionality. Most virus-specific effector CTLs in the lung expressed high levels of GzmB during the AdLGO acute infection (7 dpi; Fig 2, F), which is consistent with their ability to eliminate most infected cells (Fig 1, B). However, virus-specific CTLs expressed only residual amounts of GzmB during the chronic phase of adenoviral infection (28 dpi; Fig 2, F), which is consistent with their failure to clear infection. CpG instillation rescued GzmB production, resulting in a large majority of the specific CTLs having regained GzmB expression (Fig 2, F). Adenovirus-specific CTLs from CpG-treated mice that had cleared the infection showed even stronger antigen-specific killing than those from acutely infected mice (see Fig E8 in this article's Online Repository at www.jacionline.org), demonstrating competent cytotoxic function. Effector CTL function is characterized by a metabolic bias toward aerobic glycolysis.8Chang C.-H. Curtis J.D. Maggi Jr., L.B. Faubert B. Villarino A.V. O’Sullivan D. et al.Posttranscriptional control of T cell effector function by aerobic glycolysis.Cell. 2013; 153: 1239-1251Abstract Full Text Full Text PDF PubMed Scopus (1323) Google Scholar 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. Thus 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. In summary, we present a novel murine model of subclinical persistent adenoviral infection in the lung that has a profound and detrimental effect on CTL responses. Modulation of the local inflammatory context resulted in recovery of CTL function and elimination of infected cells. Although adenoviral pulmonary infections in patients are common,1Vlaminck I.D. Martin L. Kertesz M. Patel K. Kowarsky M. Strehl C. et al.Noninvasive monitoring of infection and rejection after lung transplantation.Proc Natl Acad Sci U S A. 2015; 112: 13336-13341Crossref PubMed Scopus (206) Google Scholar, 2Humar A. Doucette K. Kumar D. Pang X.-L. Lien D. Jackson K. et al.Assessment of adenovirus infection in adult lung transplant recipients using molecular surveillance.J Heart Lung Transplant. 2006; 25: 1441-1446Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar the extent of T-cell immunosuppression and the responsible mechanisms are not well defined yet because of the lack of clinical trials and animal models. The model presented here will be instrumental for delineating the underlying mechanisms and facilitating the development of potential therapeutic strategies for viral clearance from the lung. We thank Melanie Eichler and the Flow Cytometry Core Facility of the Bonn university hospitals for technical assistance. All mice were on a C57BL/6 background. C57BL/6J (B6), B6 albino (B6[Cg]-Tyrc-2J/J),E1Townsend D. Witkop C.J. Mattson J. Tyrosinase subcellular distribution and kinetic parameters in wild type and C-locus mutant C57BL/6J mice.J Exp Zool. 1981; 216: 113-119Crossref PubMed Scopus (39) Google Scholar CD45.1 OT-I,E2Hogquist K.A. Jameson S.C. Heath W.R. Howard J.L. Bevan M.J. Carbone F.R. T cell receptor antagonist peptides induce positive selection.Cell. 1994; 76: 17-27Abstract Full Text PDF PubMed Scopus (2267) Google Scholar and P14E3Pircher H. Bürki K. Lang R. Hengartner H. Zinkernagel R.M. Tolerance induction in double specific T-cell receptor transgenic mice varies with antigen.Nature. 1989; 342: 559-561Crossref PubMed Scopus (856) Google Scholar mice were bred and maintained under specific pathogen-free conditions at the animal facility of the University of Bonn Hospitals. OT-I mice harbor CD8 T cells carrying a transgenic T-cell receptor that recognizes the OVA-derived epitope SIINFEKL in the context of H-2Kb. P14 mice harbor CD8 T cells carrying a transgenic T-cell receptor that recognizes the LCMV glycoprotein–derived epitope KAVYNFATC in the context of H-2Db. Experiments were performed with sex- and age-matched mice according to institutional and governmental guidelines of animal welfare. Recombinant adenoviral vectors deleted of the early transcribed regions E1 and E3 were as follows: AdLGO (expressing click-beetle luciferase CBG99, eGFP, and OVA fragment containing the CD4 and CD8 T cell–immunodominant epitopes under the CMV promoter),E4Stabenow D. Frings M. Trück C. Gärtner K. Förster I. Kurts C. et al.Bioluminescence imaging allows measuring CD8 T cell function in the liver.Hepatology. 2010; 51: 1430-1437Crossref PubMed Scopus (32) Google Scholar AdLTO (eGFP from AdLGO replaced by tdT), or AdlacZ (expressing lacZ β-galactosidase under the CMV promoter). The indicated cDNAs were separated by P2A linker sites from the Porcine Teschovirus 1 followed by bGH poly(A) signal. Recombinant adenoviruses were generated with Gateway technology from Thermo Fisher Scientific (Waltham, Mass). Expression cassettes were cloned into Gateway pENTR 11 Dual Selection Vector (Thermo Fisher Scientific) and recombined into pAD/PL-DEST Gateway Vector (Thermo Fisher Scientific) in vitro through the LR Clonase Enzyme Mix (Thermo Fisher Scientific). 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.E5Sprinzl M.F. Oberwinkler H. Schaller H. Protzer U. Transfer of hepatitis B virus genome by adenovirus vectors into cultured cells and mice: crossing the species barrier.J Virol. 2001; 75: 5108-5118Crossref PubMed Scopus (134) Google Scholar For intratracheal administration of adenoviruses (5 × 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 × 1-inch cannula (B. Braun Medical, Bethlehem, Pa) with the help of a light-assisted rodent-laryngoscope (Penn-Century, Wyndmoor, Pa). Subsequently, 50 μL of solution was inoculated, and mice were actively ventilated for a further 60 seconds with a MiniVent Mechanical Ventilator (Harvard Apparatus, Holliston, Mass) by using 250 μL of tidal volume and 250 strokes/min. Measurement of AdLGO bioluminescence was performed with an IVIS Lumina System (Caliper Life Sciences, Hopkinton, Mass), as previously described,E6Tittel A.P. Heuser C. Ohliger C. Llanto C. Yona S. Hämmerling G.J. et al.Functionally relevant neutrophilia in CD11c diphtheria toxin receptor transgenic mice.Nat Methods. 2012; 9: 385-390Crossref PubMed Scopus (112) Google Scholar by injecting 4.5 mg of D-Luciferin intraperitoneally. Mice were kept at 37°C for 5 minutes, and then bioluminescence was measured for 120 seconds at 37°C. In some experiments, after in vivo bioluminescence, imaging organs were dissected out, incubated in 4.5 mg/mL D-Luciferin at 37°C for 5 minutes, and imaged ex vivo at 37°C. Image analysis was performed with Living Image software (version 4.3.1; Caliper Life Sciences) by defining a region of interest in the thorax region equal for all mice. Organs were dissected and disrupted in Dulbecco-PBS containing 3% FCS, 1 mg/mL collagenase IV (Sigma-Aldrich), and 50 U/mL DNase I (Roche, Mannheim, Germany) for 30 minutes at 37°C. Cell suspensions where then filtered and washed twice in PBS at 4°C. Bronchoalveolar lavage (BAL) fluid was obtained by means of 3 consecutive airway washes with 1 mL of PBS containing 0.1 mmol/L EDTA. For adoptive transfer of naive CFSE-labeled OT-I and P14 cells, splenic CD8 T cells were positively selected with CD8 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), labeled with 1 μmol/L CFSE, and transferred intravenously.E7Feuerer M. Beckhove P. Garbi N. Mahnke Y. Limmer A. Hommel M. et al.Bone marrow as a priming site for T-cell responses to blood-borne antigen.Nat Med. 2003; 9: 1151-1157Crossref PubMed Scopus (263) Google Scholar CD8 T cells (1 × 106) were transferred intravenously into mice that had been infected with 5 × 108 infection units of AdLGO 37 days earlier. Analysis was performed in mediastinal lymph nodes 3 days after transfer. Cells (105-106/well) were restimulated in vitro in 96-well U-bottom plates in 200 μL of RPMI containing 50 ng/mL phorbol 12-myristate 13-acetate, 500 ng/mL ionomycin, and GolgiPlug/GolgiStop (BD Biosciences, San Jose, Calif) for 5 hours at 37°C in a 5% CO2 atmosphere. For degranulation analysis, BV421-labeled mAb 1D4B directed against CD107a (BioLegend, San Diego, Calif) was added at a 1:100 dilution during the 5-hour restimulation in vitro. Cells were then washed twice and immediately processed for flow cytometric analysis. Flow cytometry of viable cells was performed by using standard techniques. Antibodies directed against the following antigens were used: CD8α (53-6.7), CD8β (YTS156.7.7), CD45.2 (104), CD45.1 (A20), CD4 (GK1.5), CD11b (M1/70), CD11c (N418), CD19 (6D5), CD44 (IM7), CD326 (G8.8), CD31 (MEC13.3), F4/80 (BM8), FoxP3 (FJK-16s), GzmB (GB11), I-A/I-E (M5/114.15.2), CD64 (X54-5/7.1), KLRG1 (2F1/KLRG1), F4/80 (BM8), Ly6-C (HK1.4), Ly6-G (RB6-8C5), NK1.1 (PK136), CD3 (145-2c11), CD115 (AFS98), PD-1 (RMP1-30), Tim-3 (B8.2C12), cytotoxic T lymphocyte–associated antigen 4 (UC10-4B9), CD160 (7HI), KLH (RTK2071, RTK4530; all from BioLegend), and Siglec-F (E50-2440; eBioscience, San Diego, Calif). For dextramer staining, cells were stained with Kb/SIINFEKL-Dextramer (Immudex, Copenhagen, Denmark) before antibody staining in PBS containing 5% FCS for 20 minutes at 4°C. For intracellular staining, the Foxp3/Transcription Factor Buffer Set (eBioscience) was used, according to the manufacturer's guidelines. Annexin V staining was done with the Annexin V apoptosis detection kit (BioLegend), according to the manufacturer's guidelines. After Annexin V staining, cells were stained with Kb/SIINFEKL-Dextramer in Annexin V binding buffer containing 5% FCS for 20 minutes at 4°C. For the glucose uptake assay, freshly isolated cells were cultured with 10 μg/mL 2-NBDG (Cayman Chemicals, Ann Arbor, Mich) in glucose-free RPMI for 15 minutes at 37°C, stained for surface markers, and analyzed for 2-NBDG uptake by means of flow cytometry. The mitochondrial membrane potential was determined by using the MitoProbe DilC1(5) Assay Kit (Thermo Fisher Scientific), and total mitochondrial mass was evaluated with the MitoTracker Deep Red Kit (Thermo Fischer Scientific), according to the manufacturer's guidelines. For staining of DNA content, after fixation in 4% methanol-free paraformaldehyde for 20 minutes at 4°C, cells were resuspended in FoxP3/Transcription factor permeabilization buffer (eBioscience) containing 3 μmol/L 4′-6-diamidino-2-phenylindole dihydrochloride (BioLegend), incubated for 15 minutes at room temperature, and immediately analyzed by using flow cytometry. Dead cells were excluded by using the fixable viability dye eFluor 780 (eBioscience), Hoechst 33342 (Sigma-Aldrich), or propidium iodide (Sigma-Aldrich). Samples were acquired on an LSR Fortessa (BD Biosciences) or FACSCanto II (BD Biosciences) and analyzed with FlowJo software (version 10.1; TreeStar, Ashland, Ore). Cell sorting was performed on a FACSAria, with cooling at 4°C. Vibratome sections were used to visualize AdLTO-infected cells. Briefly, lungs were kept in the inspiration phase by inflating them with 1 mL of 2% low-melting agarose solution (Promega, Madison, Wis) at 37°C. Lungs were dissected out, placed at 4°C for 30 minutes, and subsequently embedded in 4% low-melting agarose solution. Embedded lungs were cut with a Leica VT1000S vibrating-blade microtome (Leica, Wetzlar, Germany) at 100 μm in thickness. Sections were kept on ice and immediately processed for staining without fixation to preserve tdT fluorescence by blocking in PBS containing 3% FCS and 15 mg/mL human poly-immunoglobulin (Privigen, Hattersheim am Main, Germany) for 30 minutes at 4°C. Sections were then stained with antibodies in PBS containing 3% FCS for 16 hours at 4°C and washed 4 times in PBS. The following antibodies were used: anti-CD45–BV421 (30-F11), anti-CD326–allophycocyanin (G8.8), and anti-CD31–fluorescein isothiocyanate (MEC13.3) (BioLegend). Sections were mounted in PBS and immediately analyzed on a 710 confocal microscope (Carl Zeiss Microimaging, Oberkochen, Germany). Data were analyzed with Imaris software (version 7.6.3; Bitplane, Zurich, Switzerland). Cytokines in BAL fluid were quantified by using the Mouse Th1/Th2 10-plex Flow Cytometric Bead Assay (eBioscience) or a custom-made mouse 7-plex bead assay (BioLegend), according to the manufacturer's guidelines. For this, mice were infected with 5 × 108 AdLGO infection units intratracheally and treated or not with 5 μg of CpG at the indicated times. The broncho-alveolar space was lavaged with 1 mL of PBS, and BAL fluid was frozen at −70°C until analysis. For antigen-specific kill activity of CTLs, total leukocytes were isolated by lavaging the broncho-alveolar space of mice infected with 5 × 108 AdLGO infection units intratracheally either 7 days earlier (acute infection) or 40 days earlier plus 5 μg of CpG administered intratracheally at 30, 33, and 38 dpi. Effector Kb/S8L-specific CTLs were adjusted to equal numbers in the different effector samples by staining an aliquot with the respective dextramer. Specific target cells were generated by pulsing B6 splenocytes with 10 μmol/L SIINFEKL (Xaia Custom Peptides) peptide for 30 minutes and further labeling with 3 μmol/L cell proliferation dye eFluor 670 (eBioscience) for 15 minutes (eFhi) at room temperature. Nonspecific target cells were unpulsed B6 splenocytes and labeled with 0.3 μmol/L of the cell proliferation dye eFluor 670 (eFlo). Specific and nonspecific target cells were mixed at a 1:1 ratio. BAL fluid CTLs were then mixed with 2 × 104 total target cells in a V-bottom 96-well plate (CELLSTAR, Frickenhausen, Germany) at different effector/specific target ratios (4:1, 1:1, 0.2:1; maximum number of Kb/S8L-specific CTLs was 4 × 104). As a negative control, target cells were incubated without CTLs. Specific cytotoxicity was calculated 5 hours later by using flow cytometry with the following equation:100−[100×(eFhi/eFlo)+CTLs/(eFhi/eFlo)−CTLs]. In vivo depletion of CD8 T cells was performed by using intraperitoneal administration of 300 μg of YTS169.4E8Ewart S.L. Gavett S.H. Margolick J. Wills-Karp M. Cyclosporin A attenuates genetic airway hyperresponsiveness in mice but not through inhibition of CD4+ or CD8+ T cells.Am J Respir Cell Mol Biol. 1996; 14: 627-634Crossref PubMed Scopus (11) Google Scholar antibody (BioXCell, Telangana, India) every 3 to 6 days. A respective concentration of anti-KLH mAb (LTF-2, BioXCell) was used as an isotype control. For blockade of the PD-1–PD-L1 axis, 200 μg of anti–PD-L1 mAb (10F.9G2, BioXCell)E9Zhang L. Gajewski T.F. Kline J. PD-1/PD-L1 interactions inhibit antitumor immune responses in a murine acute myeloid leukemia model.Blood. 2009; 114: 1545-1552Crossref PubMed Scopus (310) Google Scholar was administered intraperitoneally every 2 to 3 days. A respective concentration of anti-KLH mAb (LTF-2, BioXCell) was used as an isotype control. Statistical significance was set at a P value of less than .05 and determined by using a 2-tailed unpaired Student t-test or 1-way ANOVA, as appropriate, with GraphPad Prism 5.0a software (GraphPad Software; La Jolla, Calif). Data are expressed as means ± SEMs. Data shown are representatives of at least 3 experiments, with 3 to 6 mice per group.Fig E2Body weight progression in mice undergoing pulmonary adenoviral infection. if.u., Infection units; i.t., intratracheal.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Adenovirus-specific CD8 T cells are generated during the acute phase of infection. A, B6 mice were infected intratracheally with 5 × 108 AdLGO infection units (if.u.) intratracheally and the frequency and number of Kb/SIINFEKL-specific CD8 T cells identified by dextramer staining in the indicated samples. Flow cytometric density contours were pregated on live CD8 T cells. Dex, Dextramer; Kb/S8L+, Kb/SIINFEKL-Dex+; mLN, Lung-draining mediastinal lymph nodes; N.D., Not detected (CD8 T cells are not present in the bronchoalveolar space of naive mice). B, Frequency of total CD8 T cells in indicated samples of naive and AdLGO infected mice as in Fig E3, A. Shown is a representative of at least 3 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Adenovirus-specific CD8 T cells do not upregulate effector molecules and mechanisms during adenoviral persistence. AdLGO infection, OVA challenge intratracheally and adoptive transfer of CD45.1+ CFSE-labeled AdLGO-specific naive OT-I T cells were performed as in Fig 1, F. Analysis of CD25 (A), IFN-γ (B), and TNF-α (C) expression, as well as degranulation activity (D) by OT-I CD8 T cells in the mediastinal lymph nodes was performed 3 days after transfer. Shown is a representative of 3 independent experiments (n = 4 mice per group). *P < .05, **P < .01, and ***P < .001, ANOVA (n = 4 mice per group).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5CpG instillation intratracheally promotes expression of proinflammatory cytokines in the lungs of mice with AdLGO persistent infection. Quantification of cytokine content in the BAL fluid of mice infected or not with 5 × 108 AdLGO infection units intratracheally is shown. A, Mice were treated at 38 dpi with PBS or 5 μg of CpG intratracheally, and the BAL fluid was collected 48 hours later. B, Naive mice infected with 5 × 108 AdLGO infection units 7 days earlier or 40 days earlier and treated with PBS or 5 μg of CpG intratracheally 30, 33, and 38 dpi. BAL fluid was collected 48 hours later. Shown is a representative of 2 independent experiments (n = 4 mice per group). *P < .05, **P < .01, and ***P < .001, ANOVA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E6Enhanced virus-specific CTL proliferation after local CpG instillation in AdLGO persistently infected mice. Histograms (left panels) showing percentages of proliferating CD44hiKb/S8L-Dex+ CTLs in chronically infected mice recovered from the lungs or mediastinal lymph nodes (mLN). AdLGO, Infected with 5 × 108 infection units of AdLGO 40 days earlier and treated with PBS intratracheally at 30, 33, and 38 dpi; AdLGO + CpG, similarly infected and treated with 5 μg of CpG intratracheally. Right panel, Respective cumulative analysis for Dex+ and Dex−CD44hi CD8 T cells, as indicated. Dex, Kb/SIINFEKL dextramer; if.u., infection units; i.t., intratracheal. Shown is a representative of 3 independent experiments (n = 4 mice per group). **P < .01 and ***P < .001, Student t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E7Expression of inhibitory receptors on virus-specific CD8 T cells during adenoviral persistence. A and B, Expression of Tim-3, cytotoxic T lymphocyte–associated antigen 4 (CTLA4), and CD160 (Fig E7, A) and PD-1 (Fig E7, B) on Kb/SINNFEKL dextramer-positive (Dex+) CD8 T cells in the lungs of infected mice having received or not 5 μg of CpG administered intratracheally as in Fig 2, A. C, Mice were infected with 5 × 108 AdLGO infection units (if.u.) intratracheally and treated with anti−PD-L1 antibodies intraperitoneally, when indicated (arrows). Iso, Isotype control. Shown is a representative of 2 experiments using 5 mice per group. ***P < .001, Student t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E8CpG treatment leads to potent cytolytic activity of virus-specific CTLs in the airways. Ex vivo Kb/SIINFEKL-specific cytotoxic activity against splenocyte target cells by CTLs isolated from the BAL fluid of either mice infected with 5 × 108 infection units AdLGO 40 days earlier and treated with CpG intratracheally at 30, 33, and 38 dpi or mice infected with 5 × 108 infection units of AdLGO 7 days earlier. E, Kb/SIINFEKL-specific CTLs; T, Kb/SIINFEKL-loaded splenocyte targets. *P < .05; **P < .01, Student t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)