Four- to 6-week-old male and female C57BL/6 and BALB/c mice were purchased from Charles River Laboratories and used at 6–10 weeks of age. BALB/c TCR-transgenic DO11.10 (OVA323–339
), C57BL/6 OT-I (OVA357–364
) which were bred onto the Thy1.1 background, and OT-I OX40-deficient mice on the C57BL/6 background (kindly provided by Dr. Michael Croft, La Jolla Institute for Allergy and Immunology, La Jolla, CA) [49
] were bred and maintained at the Earle A. Chiles Research Institute. An institutional animal care and use committee approved all animal studies. All mice were bred and maintained under specific pathogen-free conditions in the Providence Portland Medical Center animal facility. Experimental procedures were performed according to the National Institute of Health Guide for the Care and Use of Laboratory Animals.
Preparation of antibodies
Control Rat Ig antibody was purchased from Sigma (St. Louis, MO), whereas rat anti-OX40 antibody (OX86) and anti-CTLA-4 (9H10) were produced in the laboratory from hybridomas and affinity purified over protein G columns.
Adoptive transfer and immunization
Spleens and DLNs were harvested from DO11.10, OT-I or OT-I OX40 deficient mice and processed by crushing between two frosted glass microscope slides and red blood cell (RBC) lysed with Ammonium chloride-based RBC lysis buffer (ACK, Cambrex, East Rutherford, NJ). The percentage of DO11.10 were identified by FACS using KJ1-26, which recognizes the specific DO11.10 T cell receptor [64
] or Thy1.1 antibodies for identifying the OT1 specific T cells (BD Biosciences, San Jose, CA). A total of 2–3×106
transgenic TCR T cells were adoptively transferred intravenously (i.v.) into BALB/c or C57BL/6 WT recipients. One day later, mice were immunized subcutaneously (s.c.) with 500μg ofOVA (Sigma-Aldrich, St. Louis, MO) and 50μg of anti-OX40 (OX86), 100μg of anti-CTLA-4 (9H10), or 50μg Rat Ig control (Sigma-Aldrich, St. Louis, MO). The following day, mice were given a second injection of anti-OX40, anti-CTLA-4 or Rat Ig.
Purification of Ag-specific T cells
DLNs were collected at indicated times after immunization and processed into a single-cell suspension. CD4 T cells were stained for 0.5h on ice with biotinylated KJ1-26 antibody (0.5mg/106 cells) and then washed. Cells were harvested using on an AutoMACS using anti-biotin microbeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. CD8 T cells were initially purified on an AutoMACS by negative selection, using a CD8 purification kit (Miltenyi Biotec, Auburn, CA), followed by negative selection with anti-Thy1.2 microbeads. The purity (>90%) of KJ1-26+ (CD4) or Thy1.1+ cells (CD8) was verified by flow cytometry FACsCalibur, Becton Dickinson, Franklin Lakes, NJ). The average numbers of CD4/KJ+ T cells recovered per mouse from the DLN were as follows: d3 Rat, 0.5 × 106; d3 anti-OX40, 3 × 106; d4 OX40, 6 × 106; d6 OX40, 1.5 × 106; d3 anti-CTLA-4, 1.75 × 106, d4 anti-CTLA-4, 3 × 106. The average numbers of CD8/Thy1.1+ T cells recovered per mouse from the DLN were as follows: d4 Rat, 1.5 × 106, d4 anti-OX40, 4 × 106.
DLNs were isolated 3, 4 or 6 days after immunization and Ag-specific T cells were purified as described above. Approximately 5-10 × 106 cells were collected by centrifugation and resuspended in Laemmli buffer and boiled at 98°C for 10 min. Lysates were quantitated using DC Protein Assay (BioRad, Hercules, CA), run on 12% polyacrylamide gels (equivalent microgram quantities of protein) (Ready Gel Tris-HCl Precast Gels, BioRad, Hercules, CA), and transferred onto nitrocellulose or PVDF membranes (Millipore, Billerica, MA). The following antibodies were using to detect these proteins: goat anti-Mxd4 (clone N-19, Santa Cruz Biotechnology, Santa Cruz, CA,), rabbit anti-Mnt (clone M-132, Santa Cruz Biotechnology), rabbit anti-c-Myc (clone N-262, Santa Cruz Biotechnology), rabbit-Lamin A/C (clone H-110, Santa Cruz Biotechnology), rabbit anti-PARP 1/2 (clone H-250, Santa Cruz Biotechnology), mouse anti-GFP (clone B-2, Santa Cruz Biotechnology), mouse anti-GAPDH (clone 6C5, Millipore, Billerica, MA) or rabbit anti-Actin (A5060, Sigma, St. Louis, MO). For secondary detection, anti-mouse-HRP, anti-rabbit-HRP (Cell signaling, Danvers, MA) or anti-goat-HRP (sc-2060, Santa Cruz Biotechnology) were used. For chemilluminescent detection, blots were incubated in Lumiglo (Cell Signaling, Danvers, MA), exposed to film and developed. Blots were stripped using stripping buffer containing 62. 5 mM Tris, pH 6.8, 2% SDS and 100 mM 2-mercaptoethanol and reprobed for loading controls. Images were prepared using Adobe Photoshop and the ECL signals were quantitated using ImageJ software by normalizing protein expression to the respective loading control (National Institute of Health, Bethesda, MD).
To isolate chromatin, approximately 5-10 × 106 cells were resuspended (100 × 106 cells/ml) in buffer A (10 mM HEPES, (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 100X protease inhibitor (Sigma), 0.1 mM phenylmethylsulfonyl fluoride). Triton X-100 (0.1%) was added, and the cells were incubated for 5 min on ice. Nuclei were collected by low-speed centrifugation (4 min, 1,300 × g, 4°C). The supernatant was further clarified by high-speed centrifugation (15 min, 20,000 × g, 4°C) to remove cell debris and insoluble aggregates and the supernatant was collected as the cytoplasmic fraction. To fractionate the chromatin, nuclei suspension was resuspended in buffer A plus 1mM CaCl2 and 0.2U micrococcal nuclease was added (Sigma, St. Louis, MO) at 37°C for 1 minute. The digestion was terminated by the addition of 1mM EGTA, the mixture was then centrifuged at 5,000 g for 3 min. Nuclei were lysed in buffer B (3 mM EDTA, 0.2 mM EGTA, 1 mM DTT, protease inhibitors as described above). Insoluble chromatin was collected by centrifugation (4 min, 1,700 × g, 4°C), washed once in buffer B, and centrifuged again under the same conditions. The final chromatin pellet (Chromatin fraction) was resuspended in Laemmli buffer and sonicated for 15 s. Proteins were analyzed by SDS-PAGE.
D011.10 T cells were purified from DLNs at day 4, as described above. 1-2×106 Ag-specific T cells were plated ex vivo in a 24 well plate and cells were treated with 50μg/ml cycloheximide for indicated times.
Mxd4 cDNA was cloned into a TOPO vector (Invitrogen). S145A mutant Mxd4 was generated by site directed mutagenesis (Stratagene, Agilent Technologies, Cedar Creek, TX) using the following primers: GTGTGCGCACAGACGCCACTGGCTCTGCTG and CAGCAGAGCCAGTGGCGTCTGTGCGCACAC and verified by sequencing (OHSU sequencing core, Portland, OR). The mutated Mxd4 gene was subsequently cloned into the modified pWPI-GFP vector (Trono Lab, Ecole Polytechnique Federale de Lausanne and modified by Dr. Hong Ming Hu, EACRI, Portland, OR) using the USER enzyme. HEK-293 cells were plated at 1.5 ×105 cells per well overnight in a 6 well plate and transfected with 2μg pWPI-GFP (empty vector), Wild-type pWPI-Mxd4-GFP (WT) or S145A mutant pWPI-Mxd4-GFP (S145A) using Metafectene (Biontex, Martinsried/Planegg, Germany) for 48h under 5% FBS conditions. Cells were treated with cycloheximide (CHX; 100μg/ml) and harvested at the indicated times as described above. The cell lysates were analyzed by Western blot with the indicated antibodies.
T cells were transfected by means of the nucleofection technique from Amaxa (Cologne, Germany) using the mouse T-cell nucleofection kit, according to Amaxa’s protocol. In brief, purified CD4+/KJ+ T cells isolated ex vivo at day 4 after mice after treatment with OVA and anti-OX40. The cells were resuspended in mouse T-cell nucleofection solution at a density of 2×106 cells per 100μL. Per transfection, 100μL of cell suspension were mixed with the respective amount of siRNA, transferred into a cuvette and pulsed in a Nucleofector I device using program X-01. Subsequently, the cells were diluted with 500μL of pre-warmed serum-free RPMI medium (37°C) and transferred into a microcentrifuge tube and incubated for 10 min at 37°C. Cells were then transferred to a 24-well-plate containing 1.5ml of pre-warmed Mouse T cell Nucleofector medium containing 5% FBS, 2mM glutamine and Medium Component A and B (Amaxa) (37°C) per well. Following transfection the cells were cultured at 37°C and 5% CO2. The following day, 0.5ml of media was added to each well. Cells were analyzed by flow cytometry at 48hrs post transfection. Transfection efficiency was assessed by transfection with pMax-GFP plasmid and gating on the live cells as determined by the FSC and SSC and determining the percentage of GFP positive CD4+/KJ+ cells by flow cytometry (FACs Calibur, Becton Dickinson, Franklin Lakes, NJ).
FACS analysis of cells from DLN or spleens
Cells were incubated for 30 min on ice with a combination of the following antibodies: CD4-FITC (BD Bioscience/BD Pharmingen), Biotin-KJ1-26, Strepavidin-APC (BD Bioscience/BD Pharmingen). After washing three times with PBS containing 0.1% w/v BSA (Sigma-Aldrich) and 0.02% w/v sodium azide, cells were resuspended in FACS buffer. 7AAD was added 5 minutes prior to analysis. Harvested samples were run on a FACScalibur and analyzed for cell survival (Becton Dickinson, Franklin Lakes, NJ).
Statistical significance was determined by the unpaired Student t test (for comparison between two groups), using Microsoft excel software; a p < 0.05 was considered significant. *, p<0.05; **, p<0.001; and ***, p <0.0001.