Mice and Viruses
C57BL/6 mice were purchased from Taconic (Germantown, NY) and CD45.1 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). LMP2−/− were originally obtained from Luc Van Kaer (Vanderbilt Medical Center, Nashville, TN), who backcrossed them 10 times, and then bred at Taconic. LMP2−/− MECL1−/− and LMP7/MECL1−/− mice were bred at the Cincinnati Children’s Hospital Medical Center. Mice were maintained under specific pathogen-free conditions and all procedures involving mice were approved by the Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases. Influenza A virus (IAV; A/Puerto Rico/8/34) was propagated in 10-d-old embryonated chicken eggs. For all vaccinations, the virus was first inactivated by treating with paraformaldehyde (1:864 dilution) for 2 d at 4°C, and then ~100 hemagglutination units of inactivated IAV was injected i.p. At several time points after vaccination, mice were retro-orbitally bled, and RBCs were removed using serum-gel tubes (Sarstedt, Newton, NC). Serum was inactivated by incubating at 56°C for 30 min.
Hemagglutination inhibition titrations
Hemagglutination inhibition (HAI) titrations were performed in 96-well round polystyrene plates (Corning, Corning, NY). Serial 2-fold dilutions of sera in 100 ul PBS were mixed with four agglutinating doses of IAV in 100 μl of PBS. Next, 25 μl of a 2% (v/v) human RBC solution was added, and the reaction was allowed to proceed for 60 min at room temperature. The plates were held perpendicularly to the ground, and HAI was observed as the agglutinated RBCs formed streaks in each well. HAI titers are expressed as the reciprocal of the highest Ab dilution that was able to inhibit four agglutinating doses of virus in a total volume of 225 μl.
Flat bottom microtiter plates (Thermo Fisher Scientific, Waltham, MA) were coated with ~75 hemagglutination units of IAV per well in a 0.1 M sodium carbonate coating solution (BD Biosciences, San Jose, CA) overnight at 4°C and then blocked with PBS containing 10% FBS for 2 h at room temperature. Mouse sera was then added and incubated for 2 h at room temperature. Next, rabbit (IgG) anti-mouse secondary Abs of different isotypes (Calbiochem, San Diego, CA) were added and incubated for 1 h at room temperature, and then HRP conjugated goat anti-rabbit IgG (Calbiochem) was added and incubated for 1 h at room temperature. Finally, tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added, and then 1 M H3PO4 was quickly added to stop the reaction. OD was read at 450 nm. In between each step of the anti-IAV ELISAs, the plates were washed with PBS-Tween (0.05%).
Spleens and bone marrow were isolated, single cell suspensions were created, and ACK lysing buffer (BioWhittaker, Walkersville, MD) was added to lyse RBCs. The cells were then passed through a 70-μm nylon cell strainer (BD Falcon, San Jose, CA) and washed twice with balanced salt solution containing 0.1% BSA. Cells were incubated with different combinations of fluorescently labeled Abs (eBiosciences, San Diego, CA and BD Pharmingen, San Jose, CA) and samples were analyzed using an LSR II machine (BD Biosciences).
Lymphocyte Transfer Studies
Splenocytes were isolated as described above, and CD4+ T cells and B cells were purified by negative selection using commercially available kits (Miltenyi Biotec, Auburn, CA). For some experiments, APCs (CD11c+ and CD11b+) were removed from splenocytes preparations after staining the cells with anti-CD11c FITC and anti-CD11b FITC and then using a commercially available kit (Miltenyi Biotec) that selects FITC-negative cells. Mice were irradiated with ~800 rad; 6 h later, purified cell populations that were 83–98% pure were injected i.v. through the tail vein; 1.5 × 107 cells were injected for APC-depletion studies, and 8 × 106 B cells and 3 × 106 CD4+ T cells were injected for fractionated splenocytes studies. For all studies, equal numbers of C57BL/6 and LMP2−/− cells were injected. Two hours later, mice were injected i.p. with ~100 hemagglutination units of inactivated IAV, and anti-IAV Abs were determined at several time points as described above. For experiments involving CD45.1 mice, we did not inject IAV, but instead isolated the spleens from these irradiated mice 7 d after transferring cells. For these experiments, single cell suspensions were prepared as described above and cells were stained using Abs against CD45.1 and CD45.2 (eBiosciences) and analyzed on an LSR II flow cytometer (BD Biosciences).
Lymphocyte proliferation assays
Proliferation assays were performed as described previously (18
T cells and B cells were purified by negative selection as described above and plated in RPMI 1640 with 10% FCS in flat bottom polystyrene plates (Corning). For B cell assays, 1 × 105
cells per well were plated; for CD4+
T cell assays, 2 × 105
cells per well were plated. Various amounts of Con A or LPS were added to CD4+
T cells or B cells, respectively. The cells were pulsed with [3
H]thymidine 2 d later and [3
H] incorporation was determined 1 d after pulsing.
Bone marrow-derived DCs were cultured for 10 d in RPMI 1640 containing 20 ng/ml GM-CSF (PeproTech, Rocky Hill, NJ) and then reseeded at 2.5 × 105
cells per well in round-bottom polystyrene plates (Corning). IAV was added to the cells, and supernatant was collected 18 h later. Cytokine levels in supernatants were measured by Luminex technology (Bio-Rad, Hercules, CA) and by an IFN-α ELISA that was performed using a previously described protocol (22
B cells were purified by negative or positive selection using magnetic beads. For some experiments, cells were treated with LPS and then removed from culture and resuspended in lysis buffer (20 mM Tris pH 7.4, 150 mM mNaCl, 2mM sodium pyrophosphate, 25 mM β-glycerol phosphate, 5 mM NEM, 1% NP-40) and incubated on ice for 30 min. Nuclei and insoluble material were pelleted by centrifugation, and the resulting supernatants were mixed with SDS-PAGE buffer (Quality Biologicals, Gaithersburg, MD) and run on a 12% SDS-PAGE gel followed by blotting onto nitro-cellulose. For polyubiquitin and immunoproteasome subunit staining, total cell lysates were prepared. Blots were probed with rabbit polyclonal anti-IκBα (Santa Cruz Biotechnology, Santa Cruz, CA) and mouse monoclonal anti-actin (Abcam, Cambridge, MA) Abs or with mouse monoclonal anti-polyubiquitin (clone FK2; BIOMOL, Plymouth Meeting, PA) Ab followed by infrared dye coupled secondary Abs. For immunoproteasome subunit expression studies, blots were probed with rabbit anti-LMP2 (Abcam), rabbit anti-LMP7 (Abcam), or rabbit anti-MECL1 (BIOMOL), as well as a GAPDH loading control Ab (Abcam). Blots were analyzed using an Odyssey imaging system (LI-COR, Lincoln, NE) or a STORM phosphorimager (GE Healthcare Bio-Sciences, Uppsala, Sweden).
Cells were homogenized in ice cold homogenization buffer (50 mM Tris-HCl pH 7.5, 5 mM MgCl2, 25 mM KCl, 0.2 M sucrose, 0.5% NP-40, 100 μg/ml CHX, EDTA-free protease inhibitors [Roche, Basel, Switzerland], 10 U/Ml RNase Out [Invitrogen, Carlsbad, CA], diethyl pyrocarbonate water), and the lysate was centrifuged at 20,000 × g for 10 min at 4°C. The cleared lysate was layered on a 15–50% sucrose gradient in the same buffer. After centrifugation at 35,000 rpm (Beckman, SW41.Ti) for 2.5 h at 4°C, the gradient was fractionated. Each fraction was pelleted to concentrate proteins and analyzed by immunoblotting as described above.