Recombinant mouse gm csf and il 4

The following reagents, antibodies and kits were used in the present study: recombinant mouse GM-CSF and IL-4 (R&D Systems, Minneapolis, MN); recombinant human interleukin 2 (IL-2) (Chiron Corp., Emeryville, CA); high purity TLR4 ligand (with less than 3% impurities), Escherichia coli serotype 055:B5 lipopolysaccharide (LPS) (part No 7193, lot No GL1457; Lonza, Walkersville, MD); synthetic TLR1/2 agonist Pam₃CSK4 (Pam₃, InvivoGen, San Diego, CA); phycoerythrin (PE)-conjugated rat anti-mouse TNF, CD14 and TLR4 monoclonal antibodies (BD-Pharmingen, CA, USA); unconjugated rat anti-mouse TNF (XT22, Pierce-Endogen, Rockford, IL), human TNFR2-Fc fusion protein (ENBREL, etanercept; Amgen, Thousand Oaks, CA), hamster anti-mouse TNFR1 and TNFR2 (BD-Pharmingen) and isotype control monoclonal antibodies (BD-Pharmingen); dominant negative TNF constructs (DNTNF1, XPro1595; and DNTNF2, XENP550; Xencor, Monrovia, CA); Limulus Amebocyte Lysate (LAL) Chromogenic Assay kit (Thermo Fisher Scientific Inc, Pittsburgh, PA); mouse Quantikine IFN-γ enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems); and mouse TNF DuoSet ELISA kits (R&D Systems).

Mouse immature DCs were produced in vitro as previously described (28 (link)). Briefly, bone marrow cells were obtained from wild-type, TNFko or TNFR2ko mouse femur and tibia, depleted of mature hematopoietic cells and cultured for 6 days in RPMI-1640 medium supplemented with 0.1 mM nonessential amino acids, 2 mM sodium pyruvate, 1 mM L-glutamine, 100 μg/mL streptomycin, 100 U/mL penicillin, 20 mM HEPES buffer, 10% heat-inactivated fetal bovine serum (FBS) (all from Life Technologies, Grand Island, NY), 50 μM 2-mercaptoethanol (Bio-Rad, Hercules, CA) and 15 ng/mL recombinant mouse GM-CSF and IL-4 (R&D Systems, Minneapolis, MN). DCs were 95% CD11c⁺CD205^+/-lineage marker^-CD80⁺CD86⁺CD40⁺MHC class I⁺MHC class II^lo+. In some experiments, wtDCs and/or TNFko DCs were stimulated with recombinant mouse sTNF (Alecxis Biochem, San Diego, CA) as indicated in Results section.