Female BALB/c (BALB/cAnNCr) mice were purchased from the National Cancer Institute (Frederick, MD). Male and female IFN-γ [C.129S7(B6)-Ifngtm1Ts/J]-deficient mice on BALB/c background were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice aged 6 to 8 weeks were used for all experiments. All experimental procedures were approved by the University of Iowa Animal Care and Use Committee.
All peptides were purchased from BioSynthesis (Lewisville, TX). Previously reported peptides used in our studies included RSV F 51-70 (GWYTSVITIELSNIKENKCN) (10
), RSV F 146-160 (VSVLTSKVLDLKNYI) (45
), and RSV F 184-199 (SAIASGIAVSKVLH) (45
). The peptide sequences for all other peptides used in the present study are listed in Fig. and .
FIG. 1. RSV F51-66 contains one or more I-Ed-restricted CD4 T-cell epitope(s). (A) Lung mononuclear cells isolated from vvβ-gal- and vvF-immunized wild-type mice 5 days after challenge RSV infection were stimulated ex vivo with the indicated peptides. (more ...)
FIG. 2. RSV F56-64 represents a single immunodominant epitope contained within RSV F51-66. (A) Alanine-substituted peptides within RSV F51-66. (B) Peptides from panel A were tested within an ICS as described in Fig. . The results are shown as mean (more ...) Viruses, infection of mice, and lung and BAL cell collection.
Recombinant vv stocks were gifts from Gail W. Wertz and Thomas J. Braciale (University of Virginia, Charlottesville), and Judy A. Beeler (U.S. Food and Drug Administration, Bethesda, MD). All vv's were grown in BSC-40 cells (American Type Culture Collection [ATCC], Manassas, VA). Mice were immunized with 3 × 106 PFU of a recombinant vv expressing either β-galactosidase (vvβ-gal) or the fusion protein of RSV (vvF) by scarification with a 25-gauge needle at the base of the tail. RSV (A2 strain) was a gift from Barney S. Graham (National Institutes of Health, Bethesda, MD) and was grown in HEp-2 cells (ATCC). After 3 weeks, immunized mice were anesthetized with 30% halothane (Halocarbon Laboratories, River Edge, NJ) in mineral oil (Fisher Scientific, Fair Lawn, NJ) and were given 1 × 106 to 3 × 106 PFU RSV intranasally. The weight of each mouse was determined and recorded daily. Each mouse was given an illness score daily based on the following scale: 0, no apparent illness; 1, slightly ruffled fur; 2, ruffled fur, active; 3, ruffled fur, inactive; 4, ruffled fur, inactive, hunched posture; and 5, moribund or dead. Cells in the BAL were collected by performing three washes of the airspace of the lung, each with 1 ml of phosphate-buffered saline (PBS). BAL cells were then counted, cytospun, and stained with Diff-Quik (Dade Behring, Inc., Newark, DE) to determine the percentage and total number of eosinophils. The lung vascular bed was perfused by injecting 5 ml of RPMI medium (Gibco, Grand Island, NY) supplemented with 10% bovine growth serum (HyClone, Logan, UT), 10 U of penicillin G/ml, 10 μg of streptomycin sulfate/ml, 2 mM l-glutamine (Gibco), 5 × 10−5 M 2-mercaptoethanol, 1 mM sodium pyruvate (Gibco), 0.1 mM nonessential amino acids (Gibco), and 10 mM HEPES (Gibco) into the right ventricle of the heart. The lungs were then dissected, and single-cell suspensions were prepared by pressing the lungs though a wire mesh screen (Bellco Glass, Inc., Vineland, NJ).
Intracellular cytokine staining.
Lung cells (2 × 106 cells/ml) were stimulated with 1 μM peptide in the presence of 10 μg of brefeldin A (Sigma, St. Louis, MO)/ml for 5 h at 37°C in RPMI medium supplemented as described above. In some experiments, 10 μg of an anti-I-Ad antibody (clone 34-5-3; BD Pharmingen, San Diego, CA) or an anti-I-Ad/I-Ed antibody (clone M5/114.15.2; eBioscience, San Diego, CA)/ml was included during incubation. After incubation, the cells were blocked with purified anti-FcγRII/III monoclonal antibody (clone 93; eBioscience) and stained with anti-CD4 MAb (allophycocyanin or fluorescein isothiocyanate-conjugated, both from eBioscience [clone RM4-5]). For determination of T-cell receptor (TCR) chain expression, anti-T-cell receptor α- or β-chain antibodies were used. FACS lysing solution (Becton Dickinson, San Jose, CA) was used to fix the lung mononuclear cells and lyse the red blood cells. Cells were then washed with permeabilization buffer (staining buffer containing 0.5% saponin; Sigma) and stained with a phycoerythrin-conjugated, anti-cytokine MAb, or an isotype control. Cells were collected on either a FACSCalibur or FACSCanto (Becton Dickinson). Single color controls were used for compensation. Lymphocytes were gated based on forward-scatter and side-scatter properties and were then analyzed by using FlowJo software (Tree Star, Inc., Ashland, OR). Background staining was determined by using either isotype controls or no peptide controls.
The following phycoerythrin-conjugated Vα-chain antibodies were used: Vα2 (clone B20.1; Caltag, Burlingame, CA), Vα8 (clone CTVA8; Caltag), and Vα8.3 (clone B21.14; Pharmingen). The following fluorescein isothiocyanate-conjugated Vβ-chain MAbs (all were from Pharmingen except as noted) were used: Vβ2 (clone B20.6), Vβ3 (clone KJ25), Vβ4 (clone KT4), Vβ5.1/5.2 (clone MR9-4), Vβ6 (clone R44-7), Vβ7 (clone TR310), Vβ8 (clone F23.1), Vβ8.1/8.2 (clone MR5-2), Vβ8.2/8.3 (clone CT8E; Caltag), Vβ8.3 (clone 1B3.3), Vβ9 (clone MR10-2), Vβ10b (clone B21.5), Vβ11 (clone RR3-15), Vβ12 (clone MR11-1), Vβ13 (clone MR12-3), Vβ14 (clone 14-2), and Vβ17a (clone KJ23). Cytokine antibodies and isotypes (all eBioscience unless otherwise noted) used were as follows: interleukin-4 (IL-4; clone 11B11), IL-5 (clone TRFK5; Pharmingen), IL-6 (clone MP5-20F3), IL-10 (clone JES5-16E3), IFN-γ (clone XMG1.2), rat immunoglobulin G1 (IgG1; clone R3-34), and rat IgG2b (clone KLH/G2b-1).
In vitro restimulation and cytokine enzyme-linked immunosorbent assay (ELISA).
Wells of a 48-well plate (Falcon, Franklin Lakes, NJ) were coated overnight with 10 μg of anti-CD3 antibody (clone 145-2C11; eBioscience)/ml in PBS. After several washes with PBS, 2 × 106 lung mononuclear cells were incubated with or without 1 μM peptide or placed on anti-CD3 coated or placed on uncoated wells in the presence or absence of 1 μM RSV F51-66 peptide for 48 h. Cell-free supernatant was collected and frozen at −80°C until further use. Nunc-Immuno Plates with MaxiSorp Surface (Nalge Nunc International, Rochester, NY) were coated overnight at 4°C with 2 μg of capture antibody/ml in 0.1 M Na2HPO4 (pH 9.0). After a wash with PBS-0.5% Tween 20 (Sigma), plates were blocked with RPMI media supplemented as described above for at least 2 h at room temperature. Plates were incubated with 100 μl of samples overnight at 4°C. Cytokine was detected by incubation with 0.1 μg of biotinylated anti-cytokine antibody (IL-13)/ml or 1 μg of biotinylated anti-cytokine antibody/ml (all others) for 2 h at room temperature. Avidin-peroxidase (1:400 dilution; Sigma) was added for 30 min before the plates were developed with 3,3′,5,5′-tetramethyl-benzidine dihydrochloride (Sigma). The reaction was stopped after 5 min with 2 N H2SO4 (Ricca Chemical Company, Arlington, TX). Plates were read at 450 nm using an ELx800 plate reader and analyzed by using KC Junior software (both from Bio-Tek Instruments, Winooski, VT).
The following anti-cytokine pairs were used (all from eBioscience unless otherwise noted): IL-4, purified clone 11B11 and biotinylated clone BVD6-24G2; IL-5, purified clone TRFK5 and biotinylated clone TRFK4; IL-13, purified clone 38213.11 and biotinylated goat anti-mouse IL-13 (both from R&D Systems); IFN-γ, purified clone R4-6A2 and biotinylated clone XMG1.2 (both from eBioscience). Recombinant murine IL-4 (eBioscience), IL-5 (Pharmingen), IL-13 (R&D Systems), and IFN-γ (eBioscience) were used to calculate standard curves.
Ex vivo chemokine ELISA.
ELISA for chemokines was performed as described above for cytokines. Plates were coated with either 0.4 μg/ml (CCL11 and CCL22) or 2 μg/ml (CCL17) of capture antibody (R&D Systems). Whole lungs were harvested and homogenized by using glass homogenizers (Kontes Glass Co., Vineland, NJ) in 1 ml of RPMI media supplemented as described above and containing a 1:200 dilution of protease inhibitor cocktail (Sigma). BAL and lung cells were centrifuged, and 50 μl of supernatant was incubated on the precoated plates overnight at 4°C. Recombinant murine CCL11, CCL22, and CCL17 (R&D Systems) were diluted in PBS plus 10% fetal calf serum (FCS; Atlanta Biologicals, Norcross, GA) and used to calculate standard curves. Chemokine detection was performed with 0.1 μg biotinylated anti-chemokine antibody (R&D Systems)/ml.
Whole lungs with the heart attached were harvested from vvβ-gal- and vvF-immunized mice 7 days after RSV challenge. Lungs were fixed in 10% neutral buffered formalin (Fisher Scientific) prior to being processed and paraffin embedded at the University of Iowa Comparative Pathology Laboratory. Paraffin blocks were sectioned at a thickness of 5 μm. Sections were hematoxylin and eosin (H&E) stained at the University of Iowa Central Microscopy Core. Eosinophils were detected by immunoperoxidase reaction for the eosinophil-specific major basic protein (MBP) using the avidin-biotin complex method. Sections were deparaffinized in xylenes, rehydrated in graded alcohols, and rinsed with 1× Dako buffer (Dako, Carpinteria, CA). Proteolytic digestion was accomplished by using proteinase K (Dako) for 5 min at room temperature. Endogenous peroxidase activity was quenched by using a 3% hydrogen peroxide solution for 8 min at room temperature. Endogenous biotin staining was blocked by the application of 1.5% normal rabbit serum (Dako) for 30 min at room temperature. Sections were covered with rat anti-mouse monoclonal primary antibody (kindly provided by Nancy and James Lee, Mayo Clinic, Scottsdale, AZ) at a 1:500 dilution in 1.5% rabbit serum, whereas the negative control was rat IgG (Sigma) using a dilution of 1:2,760 in 1.5% rabbit serum, incubated for 60 min at room temperature, and rinsed. Sections were then covered with biotinylated anti-rat secondary antibody (Vector Laboratories, Burlingame, CA), using a dilution of 1:200 in 1.5% rabbit serum for 60 min at room temperature, rinsed, and then covered in avidin-biotin complex for 60 min at room temperature and rinsed. Sections were incubated with diaminobenzidine chromogen for 5 min to demonstrate the signal of the primary antibody. A counterstain of hematoxylin (Surgipath, Richmond, IL) was applied for 30 s at room temperature. The sections were then blued in tap water, dehydrated, cleared, and mounted. Slides were blinded and scored by a board-certified veterinary pathologist (David Meyerholz, University of Iowa). H&E-stained slides were scored by using the following scales according to the specified area of the lung: (i) airway—0, no detectable inflammation; 1, rare to uncommon inflammatory cells or debris; 2, small numbers of airway inflammatory cells or debris; 3, moderate numbers of inflammatory cells or debris; 4, moderate to severe inflammatory cells or debris with uncommon airway plugging; and 5, severe multifocal inflammatory cells or debris with airway plugging; (ii) alveolar—0, no detectable inflammation; 1, uncommon inflammatory cells; 2, small to moderate numbers of inflammatory cells mostly in septa; 3, moderate inflammation mostly in septa with some rare spillover into alveoli; 4, moderate to severe inflammation extending into alveoli; and 5, severe inflammation and consolidation with multifocal to lobar plugging by cells and fluid; (iii) peribronchiolar—0, no detectable inflammation; 1, rare individual inflammatory cells; 2, small localized numbers of inflammatory cells; 3, small aggregates of cellular inflammation; 4, moderate to severe aggregates of cellular inflammation; and 5, severely expanded aggregates of cellular inflammation; and (iv) perivascular—0, no detectable inflammation; 1, rare individual inflammatory cells; 2, small local numbers of inflammatory cells; 3, small to moderate aggregates of cellular inflammation with mild exocytosis; 4, moderate to severe aggregates of cellular inflammation and edema with active exocytosis; and 5, severely expanded aggregates, edema and exocytosis. Anti-MBP-stained slides were scored for aggregates according to the following scale: 0, no to rare perivascular staining; 1, detectable perivascular staining; 2, small clusters of staining; 3, moderately defined aggregates; and 4, well-defined aggregates. Eosinophils in the lung interstitium were quantified by averaging counts from five random high-power fields.
Lungs were harvested from vvβ-gal- and vvF-immunized wild-type and IFN-γ-deficient BALB/c mice 4 days after RSV challenge. Lungs were homogenized in 1 ml of TRIzol (Invitrogen, Carlsbad, CA), and supernatants were collected. RNA was purified by sequential chloroform (200 μl/lung; Fisher Scientific) and isopropyl alcohol (500 μl/lung; Fisher Scientific) extraction. Pellets were washed with 70% ethanol and air dried before resuspension in distilled water at 55°C for 10 min. RNA was cleaned by using the RNeasy Plus minikit (Qiagen, Valencia, CA). cDNA was prepared by using a SuperScript First-Strand synthesis kit for RT-PCR (Invitrogen) and used as a template for real-time PCR. Real-time PCRs to detect the nucleocapsid (N) gene of RSV were performed with TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA) on either an ABI 7000 or 7300 real-time PCR system (Applied Biosystems) using universal thermal cycling parameters. The N-gene primers and probe have been previously described (18
). The probe was synthesized to contain 5′ 6-carboxyfluorescein reporter dye and 3′ carboxytetramethylrhodamine quencher dye (Integrated DNA Technologies, Coralville, IA). Samples were compared to known standard dilutions of a plasmid containing the N gene of RSV. The results were analyzed by using sequence detection system analysis software (Applied Biosystems). The number of copies of the N gene per lung was calculated based on number of copies of N gene in the sample and the total RNA isolated from the lung.
Whole lungs were harvested from vvβ-gal- and vvF-immunized wild-type or IFN-γ-deficient mice, weighed, and homogenized by using an ULTRA-TURRAX T 25 basic homogenizer (IKA, Wilmington, NC). Supernatants were collected and stored at −80°C until further use. Vero cells (ATCC) were grown in minimal essential medium (Gibco) supplemented with 10% FCS (Atlanta Biologicals), 10 U of penicillin G/ml, 10 μg of streptomycin sulfate/ml, 2 mM l-glutamine (Gibco), 5 × 10−5 M 2-mercaptoethanol, 1 mM sodium pyruvate (Gibco), and 0.1 mM nonessential amino acids (Gibco) to 90 to 95% confluence in six-well plates (Falcon). Serial dilutions of supernatants were performed in serum-free minimal essential medium supplemented as described above, followed by incubation on Vero cells for 90 min at 37°C. Plates were rocked every 15 min and overlaid with Eagle minimum essential medium (Lonza, Walkersville, MD) supplemented with 10% FCS (Atlanta Biologicals), 10 U of penicillin G/ml, 10 μg of streptomycin sulfate/ml, and 2 mM l-glutamine (Gibco) and mixed 1:1 with 1% SeaKem ME agarose (Cambrex, North Brunswick, NJ). After 5 days, plates were stained with 1:1 mixture of supplemented Eagle minimum essential medium and 1% agarose with the addition of 1% neutral red (Sigma). Plaques were counted after 24 to 48 h with the assistance of a light box.
Spleen cell preparation and enzyme-linked immunospot (ELISPOT) analysis.
Spleens were harvested from wild-type and IFN-γ-deficient mice 3 weeks after immunization with vvF, and single-cell suspensions were prepared by pressing the tissue between the ends of two frosted glass slides. Red blood cells were lysed by the addition of 0.84% ammonium chloride to the cell suspension, followed by washing in supplemented RPMI media. Wells of a 96-well filtration plate (Millipore, Billerica, MA) were coated overnight with 2 μg of purified anti-tumor necrosis factor alpha (anti-TNF-α; clone 1F3F3D4; eBioscience) or anti-IL-5 (clone TRFK5; eBioscience) antibody/ml. Plates were washed with PBS and blocked with medium supplemented as described above. Cells were plated in duplicate twofold dilutions starting with 5 × 106 cells. Plates were incubated at 37°C for 48 h. After a wash with PBS-0.5% Tween 20 (Sigma), the plates were incubated with 2 μg of biotinylated anti-TNF-α (clone MP6-XT22/MP6-XT3; eBioscience) or anti-IL-5 (clone TRFK4; eBioscience) antibody/ml for 2 h. After a washing step, a 1:400 dilution of a 1-μg/ml avidin-peroxidase solution (Sigma) was added for 30 min. The plates were washed and developed with a 10-mg/ml solution of 3-amino-9-ethyl-carbazole (Sigma) in N,N-dimethylformamide (Sigma) diluted in 30 ml of 0.1 M phospho-citrate buffer and filtered by using a 0.45-μm-pore-size syringe filter (Whatman, Clifton, NJ). Plates were read on an ImmunoSpot reader (Cellular Technology, Ltd., Cleveland, OH) and counted by using ImmunoSpot Software (Cellular Technology).
All statistical analyses were performed by using GraphPad InStat software (San Diego, CA). A P value of <0.05 was considered significant.