EL-4 lymphoma model
All animal experiments have been approved by a local ethics committee (“Malmö/Lunds Djurförsöksetiska nämnd”; DNR M 275–08 and M 60–10). C57BL/6 mice, (Taconic M&B, Ry, Denmark) mice were kept in an SPF animal facility at BMC, Lund. Twelve weeks old animals were injected subcutaneously with 50,000 EL4 lymphoma cells in 100 μl PBS. As control, 100 μl PBS alone was injected. After 14 days the animals were scored for tumor growth by palpation. Spleens and tumors were dissected, the cell suspension was thereafter passed through a 70 μm cell strainer and cells washed in Hank’s BSS (Invitrogen Life Technologies, Paisley, UK). When indicated 20,000 EL-4 cells were injected IP and after 10 days peritoneal cavity lavage were used as source for CD11b+ cells.
All TMPD animal experiments were conducted in the animal facility at Active Biotech AB. Eight to nine weeks old female BALB/c mice (Taconic, Denmark) were given a single intraperitoneal injection (0.5 ml) of 2,6,10,14-Tetramethyl-Pentadecane (TMPD, pristane; Sigma-Aldrich, US). Two weeks after TMPD administration the spleen, peritoneal cells and granulomas were harvested. The spleen was mashed through a 70 μm cell strainer. The peritoneal cavity was lavaged with 5 ml PBS and cells were collected by centrifugation. The granulomas were picked from the peritoneal lining and mashed through a 70 μm cell strainer. All cell samples were re-suspended in RPMI 1640 supplemented with 10% FCS.
Cell culture conditions
CD11b+ cells were enriched from C57Bl/6 or BALB/c spleens with magnetic cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany), performed according to the manufacturer’s protocol, using anti-CD11b magnetic beads (Miltenyi). The cells were separated on mini MACS columns (Miltenyi) and yielded approximately 90% pure cells. Splenic CD4 and CD11c positive cells were enriched in the same way using anti-CD4 or anti-CD11c antibody-conjugated beads.
Suppressor cell activity was assessed by co-culturing various numbers of CD11b
cells with 5 × 104 CD4+ cells and 3 × 103 CD11c+ cells in 200 μl cultures in round-bottom 96-well plates (Costar, Cambridge, MA). T cells were polyclonally stimulated by the addition of 1 μg/ml anti-CD3 antibodies (145.2C11) and 1 μg/ml anti-CD28 to the cultures. Cells were cultured in RPMI medium (Gibco) supplemented with 50 μM 2-ME, antibiotics, 10% FCS, 1 mM sodium pyruvate and 10 mM Hepes buffer (all supplements from Gibco) at 37°C, 5% CO2. Thymidine incorporation was measured on day 3 of culture after a 4-h pulse with 1 μCi [3H] thymidine (Amersham, Life Science).
Splenic CD11b+ cells were purified using anti-CD11b magnetic beads and LS-columns (Miltenyi Biotech, Bergisch Gladbach, Germany), as described above. Total RNA was extracted from CD11b+ cell preparations by use of the Purelink RNA mini Kit (Invitrogen). RNA was reverse transcribed to cDNA by use of the SuperScript III Platinum synthesis system (Invitrogen). Real-time PCR (RT-PCR) was performed for the detection of S100A9, Arginase and iNOS RNA and quantified using a SYBR GreenER kit (Invitrogen) in a MYIQ (Bio-Rad) PCR machine. The threshold cycle number was determined and relative expression level of each mRNA was determined using the formula 2(Rt– Et), where Rt and Et are the threshold cycles for the reference gene (β-actin) and the target gene, respectively.
Flow cytometric analysis was performed on spleen cell suspensions, as indicated. Primary antibodies used were: anti-mouse CD11b-APC (eBioscience), Ly6G-FITC (BD Pharmingen) and Ly6C-biotin (BD Pharmingen). Biotinylated antibodies were detected with streptavidin-QD605 (Invitrogen). Data were acquired using a FACS LSR II flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).
Tissues analyzed with immunohistology were embedded in OCT compound (Tissue-Tek®), and snap-frozen in liquid nitrogen. Cryosections (5–6 μm) were prepared on microscope slides, air dried and frozen at −20°C until staining procedures. Paraformaldehyde fixed sections were incubated with blocking 1% BSA 10% serum and FcRII/III blocker solution followed by Avidin/Biotin Blocking kit (Vector Laboratories, Inc. Burlingame, CA, USA). Thereafter the sections were incubated for 30 min at room temperature with primary antibodies: Rabbit-anti-murine S100A9, or the appropriate isotype controls (BD Pharmingen), followed by Donkey- anti- rabbit-Alexa488 (Molecular Probes) and anti-mouse CD11b-APC conjugate (eBioscience San Diego CA, USA), Ly6G-PE (BD Pharmingen), Ly6C-biotin (BD Pharmingen) followed by Streptavedin labeled with Alexa-647 (BD Pharmingen). The slides were mounted using ProLong Gold mounting media (Invitrogen, Oregon, USA) and inspected in a Zeiss microscope and analyzed with Volocity software.
Spleen cells were stained as described above and Ly6C+G+, Ly6C+G- and Ly6C++G- subpopulations were sorted using a FACSAria flow cytometer (BD Biosciences). For Western blot, 10 μg of proteins was loaded onto 12% polyacrylamide gels (C.B.S. Scientific, San Diego, CA, USA). Proteins were subsequently transferred to PVDF membrane (Roche), which was saturated with 1% dry milk in PBS. Thereafter, the membranes were incubated with Rat anti-Mouse S100A9 and Rat anti-Actin (RnD Systems) as primary antibody and Rabbit anti-Rat –HRP (SouthernBiotec Birmingham, Alabama, USA) as secondary antibodies and filters developed using ECL kit (GE Healthcare, UK).