BALB/c By mice were purchased from the Jackson Laboratory, Bar Harbor, ME. IFN-γ−/−
mice on the BALB/c background (45
) and BALB/c Thy1.1 mice, originally obtained from Jonathan Sprent (The Scripps Research Institute, La Jolla, CA) were maintained at the Trudeau Institute Animal Breeding Facility. T cell receptor (TCR) transgenic (Tg) mice in which CD4 cells recognize the hemagglutinin peptide from residues 126 to 138 (HA126-138
) from H1N1 influenza virus A/Puerto Rico 8/34 (PR8) were a gift from D. Lo (The Scripps Research Institute, La Jolla, CA) and have been described in detail (32
). HA-specific TCR Tg mice, which we refer to as HNT mice (because the amino acid sequence of the peptide recognized by these cells begins with HNT), were bred to IFN-γ−/−
and BALB/c Thy1.1 mice at the Trudeau Institute Animal Breeding Facility to generate HNT IFN-γ−/−
mice and HNT Thy1.1 mice, respectively. Perforin-deficient (Pfn−/−
) mice on the BALB/c background were a gift from J. Harty (University of Iowa, Iowa City, IA) and were bred to HNT mice at the Trudeau Institute Animal Breeding Facility (7
). B cell-deficient mice homozygous for a disruption at the JH locus (JHD) (30
) backcrossed to BALB/c mice were also maintained at the Trudeau Institute Animal Breeding Facility. All experimental animal procedures were conducted in accordance with the Trudeau Institute Animal Care and Use Committee guidelines.
Influenza virus and infections.
PR8 was grown in the allantoic cavity of embryonated hen's eggs as previously described (28
). BALB/c or BALB/c IFN-γ−/−
mice were infected intranasally with 500 egg infectious units (EIU) PR8 in 50 μl of phosphate-buffered saline (PBS) as a sublethal dose for analysis of CD4 effector functions or 5,000 to 10,000 EIU as a lethal dose for survival experiments.
Isolation of cell populations, adoptive transfer, and lethal infection.
Influenza virus-infected BALB/c mice were sacrificed at various times postinfection (p.i.) with a lethal dose of tribromoethanol (Avertin) anesthesia intraperitoneally. Lungs were perfused via the right ventricle of the heart with PBS, minced, and incubated with collagenase D at a 2.5-mg/ml final concentration to dissociate lung tissue. Single-cell suspensions were filtered through 70-μm-mesh-size mesh and used in various assays or further incubated with anti-CD4-conjugated magnetic beads (clone GK1.5) according to the manufacturer's protocol (Miltenyi Biotec). We typically observed a >80% enrichment in CD4+ T cell effectors from the lung, with each mouse yielding approximately 2 × 106 to 4 × 106 CD4 cells. DLN cells were dissociated, and single-cell suspensions were used in various assays or further enriched with anti-CD4 magnetic beads as described above. Total CD4 cells (1 × 107) were injected into naïve host mice and 18 h later infected with 5,000 to 10,000 EIU PR8 in 50 μl PBS intranasally (i.n.). Mice were then monitored for weight loss and survival over the course of the experiment. Mice were euthanized when they became moribund, according to the Trudeau Institute Animal Care and Use Committee guidelines. Kaplan-Meier survival curves were generated, and statistical significance was analyzed by the log-rank test.
Isolation of TCR Tg CD4 cells and adoptive transfer.
Naïve CD4 cells were isolated from TCR Tg mice using a positive-selection protocol with anti-CD4 beads (clone GK1.5) according to the manufacturer's protocol (Miltenyi Biotec, Cambridge, MA). Naïve HNT or HNT γ−/− CD4 cells (1 × 106 to 2 × 106) were injected via the tail vein into BALB/c and BALB/c IFN-γ−/− mice, respectively. In some experiments, wild-type (WT) HNT or HNT Pfn−/− CD4 cells were injected into naïve host mice via the tail vein. At 18 to 24 h after adoptive transfer, mice were infected i.n. with 500 EIU PR8. At 7 to 10 days postinfection, mice were sacrificed and CD4 cells were isolated from DLN and lung as described above. This population included TCR Tg CD4 cells as well as endogenous CD4 T cell effectors and was used for in vitro assays or survival studies where noted in the figure legends. Typically, 2 to 5% of the CD4 cells isolated from the DLN were HNT specific, while a range of from 6 to 12% of the CD4 cells in the lung were HNT specific.
Medium and peptides.
All cells were grown in RPMI 1640 containing 2 mM l-glutamine, 100 IU penicillin, and 100 μg/ml streptomycin (all obtained from Invitrogen Life Technologies), 10 mM HEPES (Research Organics), 50 μM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and 8% fetal bovine serum (FBS) (HyClone). HA peptide from residues 126 to 138 (HA126-138; HNTNGVTAACSHE), HA peptide from residues 518 to 526 (HA518-526; IYSTVAASL), NP peptide from residues 146 to 160 (NP146-160; ATYQRTRALVRTGMD), and NP peptide from residues 216 to 230 (NP216-230; RIAYERMCNILKGKF) were synthesized by New England Peptide (Gardner, MA).
DLNs were harvested from influenza virus-infected mice at 7 to 10 days postinfection, dissociated to a single-cell suspension, and incubated with irradiated H-2d-expressing A20 B lymphoma cells as antigen-presenting cells (APCs) and specific influenza virus peptides at 37°C for 48 h. [3H]thymidine (Amersham Biosciences, Piscataway, NJ) at 0.2 μCi/well was added for 18 h, plates were harvested using a Filtermate harvester (Perkin Elmer, Shelton, CT), and incorporated 3H was measured using a Microbeta Trilux scintillation counter (Perkin Elmer). Results are expressed as a stimulation index, calculated by dividing the number of counts per minute obtained in wells stimulated with peptide or anti-CD3 by the number of counts per minute in medium control wells.
Enzyme-linked immunosorbent spot (ELISpot) assay for cytokine-secreting cells.
Multiscreen HA plates (Millipore) were coated overnight with anti-IFN-γ antibody (clone R4-6A2; BD Biosciences) or anti-IL-17 antibody (R&D Systems, Minneapolis, MN) at 5 μg/ml in PBS. Plates were washed with PBS–Tween 20 and blocked using complete RPMI 1640 medium with 10% FBS. DLN or lung cells were added at 106 cells/well and serially diluted 1:2 in RPMI medium. Irradiated naïve splenocytes were added as APCs with or without major histocompatibility complex class II-restricted PR8 HA126-138, HA110-120, NP147-160, and NP216-230. Plates were incubated overnight at 37°C, washed with PBS–Tween 20, and incubated with biotinylated anti-IFN-γ antibody (clone XMG1.2; BD Biosciences) or biotinylated anti-IL-17 antibody (R&D Systems) at 1 μg/ml overnight at 4°C. Plates were washed with PBS–Tween 20 and incubated with streptavidin-alkaline phosphatase (ExtraAvidin; Sigma-Aldrich) at 1:500 for 1 h at 25°C. Spots were developed using a 1-mg/ml solution of 5-bromo-4-chloro-3-indolyl phosphate–nitroblue tetrazolium (BCIP/NBT; Sigma-Aldrich). Spots were counted using an Immunospot counting system (Cellular Technologies, Ltd.). Data are plotted as the number of IFN-γ spots per input cell number.
Frozen lung sections (5 μm) from PR8-infected and uninfected mice were blocked with PBS containing Fc Block (10 μg/ml) and 5% donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at room temperature. To block endogenous biotin, lung sections were incubated with 100 μl of avidin (1 mg/ml) for 15 min at room temperature (RT), followed by 100 μl of biotin (1 mg/ml) for 15 min at RT (Sigma). Lung sections were then incubated with biotin-conjugated anti-mouse I-Ad and fluorescein isothiocyanate (FITC) anti-mouse Thy1.1 (BD biosciences) for 30 min at RT. As a control, lung sections were incubated with an irrelevant isotype control (biotin-conjugated IGg2a,k; BD Biosciences). Class II was detected with streptavidin conjugated to Alexa Fluor 594 and T cells with anti-FITC conjugated to Alexa Fluor 488 (Molecular Probes, Carlsbad, CA). Type II alveolar epithelial cells were detected using rabbit polyclonal anti-prosurfactant protein C (Abcam, Cambridge, MA) (data not shown). Bound antibodies were detected with donkey anti-rabbit antibody conjugated to Texas Red (Jackson ImmunoResearch). Sections were mounted with medium for fluorescence with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA).
CD4 T cells were isolated from DLN, spleen, or lung and restimulated for 4 h with 10 ng/ml phorbol myristate acetate (PMA) and 500 ng/ml ionomycin (Sigma-Aldrich) or for 6 h with peptide-pulsed A20 cells as APCs. Brefeldin A (10 μg/ml; Sigma-Aldrich) was added for the final 2 h of culture and maintained throughout the intracellular cytokine staining (ICCS) procedure. In some experiments, donor T cells were surface stained with anti-Thy1.1 or anti-Thy1.2 and anti-CD4 as described previously (7
), fixed in 100 μl 4% paraformaldehyde, and stained in saponin buffer (PBS plus 1% FBS, 0.1% NaN3
, and 0.25% saponin; Sigma-Aldrich) containing anti-IFN-γ–APC, anti-IL-17–phycoerythrin (PE), anti-IL-2–FITC or anti-tumor necrosis factor alpha (anti-TNF-α)–PE (all antibodies were purchased from BD Biosciences). In some experiments, APC-conjugated mouse anti-human granzyme B (GrB; clone GB12; Caltag, Burlingame, CA) was used to measure intracellular levels of GrB protein in infected samples. APC-conjugated mouse IgG1 was used as an isotype control (Caltag).
Reverse transcription-PCR for transcription factors and cytolytic markers.
Mice were infected i.n. with 500 EIU PR8. Six to 8 days later, mice were sacrificed and CD4 cells were isolated from the lung and DLN by anti-CD4-conjugated magnetic beads. The flowthrough, enriched in CD8+ cells, served as a positive control. Cells were resuspended in TRIzol and RNA extracted with a RiboPure kit from Ambion (Applied Biosystems, Carlsbad, CA). cDNA was reverse transcribed with a high-capacity cDNA reverse transcription kit (Applied Biosystems) according to the manufacturer's instructions and amplified using the following validated TaqMan primer/probe sets: for Eomesodermin (Eomes), Mm01351985; for T cell-specific transcription factor (T-bet), Mm00450960; for GrA, Mm00439191; for GrB, Mm00442834; and for Pfn, Mm 00812512 (Applied Biosystems). Mouse GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an endogenous control for amplification. Amplification was carried out using a Step-One Plus real-time PCR machine from Applied Biosystems, and relative quantity was calculated by the delta delta threshold cycle method using Applied Biosystems software.
JAM assay for cytotoxicity.
Cytotoxicity was measured by the JAM (Just Another Method) assay (19
) as described previously (8
). CD4 cells were isolated from DLN or lung as described above and plated at decreasing concentrations in 96-well round-bottom plates. A20 cells, labeled for 4 h with 4 μCi/ml [3
H]thymidine (Amersham Biosciences), were added to effectors at a concentration of 1 × 104
was added at a final concentration of 2 μg/ml. In some experiments, a mixture of HA and NP peptides was added at a concentration of 2 μg/ml. Labeled A20 cells without peptide served as a negative control. After 4 h at 37°C, plates were harvested and incorporated [3
H]thymidine was measured using a Microbeta Trilux scintillation counter (Perkin Elmer). In some experiments, anti-FasL (clone MFL3) was added at 5 to 10 μg/ml to inhibit Fas-mediated killing (8
). Percent specific lysis was calculated as [(spontaneous counts per minute − experimental counts per minute)/spontaneous counts per minute] × 100 (19
Statistical analyses were performed using Prism software. The log-rank test was used for survival curves, and Student's two-tailed unpaired t test was used for analysis of statistical significance between experimental samples.