Recombinant mouse granulocyte macrophage colony stimulating factor gm csf

Mouse OCDC vaccine preparation was previously described^{5 (link)}. Briefly, bone marrow cells from mouse femurs and tibias were cultured at 1–2 × 10⁶ cells/ml in complete Iscove’s Modified Dulbecco’s Medium (IMDM) containing 10% FBS, 50 µM 2-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml streptomycin (all from Invitrogen), and 1000 IU/ml recombinant mouse granulocyte–macrophage colony-stimulating factor (GM-CSF; PeproTech, UK). Day 3, floating cells representing granulocytes were removed, and fresh complete IMDM and 1000 IU/ml GM-CSF were added. Day 5, recombinant mouse IL-4 (100 IU/ml; PeproTech, UK) was added. Day 6, HOCl-oxidized ID8 tumor cell lysate were cocultured with DCs at 1:1 ratio for 20 h. DCs were stimulated with lipopolysaccharide (LPS) [120 EU/ml, Escherichia Coli O:113; InvivoGen Europe, France] and IFN-γ (4000 IU/ml; PeproTech, UK) for 16 h and used. HOCl oxidation of ID8 tumor lysate was previously described (ref. ^{5 (link)}; main paper). Briefly, 60 µM HOCl solution was prepared by diluting the stock NaOCl reagent (Sigma-Aldrich Chemie GmbH) with PBS (Invitrogen) and adding immediately to ID8 cells to give 1 × 10⁶ cells/ml. ID8 cells were incubated for 1 h at 37 °C, 5% CO₂ to induce oxidation-dependent cell death, and then subjected to six freeze–thaw cycles to complete cell fragmentation before coculturing with DCs.

MDSCs were purified from the spleens of MC38-implanted mice when the average tumor volume reached approximately 800 mm³. Briefly, MDSCs from splenocytes were separated using the EasySep™ Mouse MDSC Isolation Kit (STEMCELL Technologies, Vancouver, Canada), and the sorted MDSCs were confirmed by flow cytometry analysis with anti-CD11b and anti-GR-1 antibodies. MDSCs were incubated in RPMI-1640 medium containing 10% FBS and 40 ng/ml recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF, PeproTech).

Fluorochrome‐conjugated mAbs were from eBioscience (Hatfield, UK). Anti‐mouse CD4 (YTS177) and anti‐mouse CD25 (PC61) mAbs were produced in‐house. Anti‐human CD4 mAb TRX1 was a gift from Prof. S. Cobbold, University of Oxford, Oxford, UK. Abatacept (CTLA4‐Ig; Orencia) was from Bristol Myers Squibb (Uxbridge, UK). Recombinant mouse granulocyte macrophage colony‐stimulating factor (GM‐CSF), human GM‐CSF, human TGF‐β1 and human IL‐4 were from Peprotech EC (London, UK).

BMDCs were harvested as previously described54 (link). Briefly, BM cells from the C57BL/6 mice, the TLR9 KO mice and the MyD88 KO mice were cultured at a density of 2 × 10⁵ cells/ml in petri dishes that contained 10 ml of complete RPMI-1640 medium with 200 U/ml (20 ng/ml) recombinant mouse granulocyte-macrophage colony-stimulating factor (GM-CSF; PeproTech Inc., New Jersey, USA). On day 3, another 10 ml of complete RPMI medium that contained 20 ng/ml GM-CSF was added. On day 6, cells from each dish were collected, washed and counted.

Primary bone marrow-derived mononuclear phagocytes were generated as previously described (100 (link), 101 (link)). Briefly, cells from the bone marrow of 6-10 week old C57BL/6 mice (Charles River) were cultured in RPMI 1640 (Gibco) complemented with 10% fetal bovine serum (FBS, Sigma), gentamicin (20 μg/ml; Gibco), glutamine (2 mM; Gibco) and HEPES (0.01 M; Gibco), referred to as complete media, and further supplemented with 10 ng/ml of recombinant mouse granulocyte-macrophage colony stimulating factor (GM-CSF) (Peprotech). Culture media was replenished on days two, four and six. Loosely adherent cells (102 (link)) were harvested on day seven or eight for experiments.
The parasite lines used include GFP-expressing RH-LDMluc (LDM, cloned from RH-GFPS65T) and GFP-expressing PTGluc (PTG, cloned from ME49/PTG-GFPS65T) (103 (link)). Tachyzoites were maintained by serial 2-day passaging in human foreskin fibroblast (HFF-1 SCRC-1041, American Type Culture Collection) monolayers cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) with 10% FBS, gentamicin (20μg/ml), glutamine (2mM), and HEPES (0.01 M).