Tgf β

The GR9 cell line is derived from a mouse fibrosarcoma induced by methylcholanthrene in BALB/c mice and has been extensively characterized in our laboratory. It is composed of cell clones with different H-2 class I expression patterns. GR9-B9 and -B11 are two different clones obtained by limited dilution method from the GR9 tumor cell line and were recloned by capturing individual cells under phase contrast microscopy. CT26 is a BALB/c mouse-derived colon carcinoma cell line, and 4T1 is a BALB/c mouse-derived mammary carcinoma cell line; both were obtained from ATCC. All cell lines were characterized by PCR assay using short tandem repeats and were regularly tested for MHC-I surface expression. Cell lines were maintained in Dulbecco’s medium supplemented with 10% fetal bovine serum (Life Technologies, Thermo Fischer Scientific, Carsbald, CA, USA), 2 mM glutamine, and antibiotics. In some experiments, cell lines were treated with 50 U/mL IFN-γ or with different concentrations of TNF-α, TGF-β (Miltenyi-Biotech, Bergisch Gladbach, Germany), methotrexate, cisplatin, paclitaxel, and doxorubicin (Sigma-Aldrich, St. Luis, MO, USA) for 48 h.

For CD4⁺T cell isolation, spleens were mechanically disrupted through a 70 μm cell strainer (BD Biosciences; San Jose, CA). CD4⁺ cells were isolated by positive immunoselection using CD4 (L3T4) microbeads (Miltenyi Biotec; Bergisch Gladbach, DE). Purified CD4⁺T cells were stained with anti-CD62L and anti-CD44 and sorted on a FacsAria II resulting in a 98% pure population of naïve T cells. The purified T cells were stimulated with 1 μg/mL plate-bound anti-CD3ε (eBioscience; San Diego, CA), 0.1 μg/mL TLR ligands, 10 μg/mL cecal lysate, and 2 ng/mL recombinant TGF-β (R&D Systems; Minneapolis, MN), in the presence or absence of 10 nM RA. In some experiments 50 μg/ml anti-IL-10Rα antibody (clone 1B1.3a) or isotype rat IgG1 (BD Biosciences) control was added to the wells.
For dendritic cell isolation, spleens were digested with 400 units/mL collagenase type IV (Sigma-Aldrich). Cells were filtered, re-suspended in 22.5% Optiprep (Sigma-Aldrich), overlaid with Hank’s Buffered Saline (HBS; Sigma-Aldrich) and centrifuged at 670g for 30 minutes. Dendritic cells were sorted from the interphase using magnetic CD11c Microbeads (Miltenyi Biotec; Bergisch Gladbach, DE) and stimulated with 10 μg/mL cecal lysate and 2 ng/mL TGF-β in the presence or absence of 10 nM RA.

Rabbit CEnC (2.5 × 10⁵ cells) were seeded on an FNC-coated 6 well, 24 h prior to harvesting for the cytofluorimetric analysis. bFGF (Thermo Fisher Scientific, USA) and TGFβ (Miltenyi, Germany) were resuspended in MilliQ H₂0 and then used at a final concentration of 20 ng/mL and 10 ng/mL, respectively. Cells were treated at different concentrations of Quercetin (Q4951, Sigma-Aldrich, USA) and CHIR 99,021 (SML1046, Sigma-Aldrich, USA). The treatments were performed at 24 h as both drugs were previously shown to elicit their effect at this time point^{81 (link),82} . CHIR99021 was tested at 50, 500 nM, 1, 3, 6 and 10 µM, Quercetin at 10 and 25 µM. Both compounds were dissolved in DMSO (Sigma-Aldrich, USA) and used 0.1% v/v in culture medium. Cytofluorimetric analysis of treated and untreated cells (DMSO as a vehicle control) was performed as described in the flow cytometry section. All the treatments described were performed 3 h after plating and the cells harvested 24 h after the treatment.

Naive CD4⁺ T cells were cultured in 96-well round-bottomed plates (Corning, New York City, NY, USA) at a density of 5 × 10⁴ per well in X-VIVO 15 serum-free medium (Lonza, Walkersville, MD, USA) in the presence of Dynabeads CD3-CD28 T cell expander (one bead per cell; Life Technologies, Carlsbad, CA, USA) and indicated cytokines: IL-1β (10 ng/mL), IL-6 (20 ng/mL), TGF-β (1 ng/mL) and IL-23 (100 ng/mL) (Miltenyi) for Th17 differentiation, as previously described [11 (link),21 (link)]. After 5–6 days, cells were harvested and stained for flow cytometry analysis, or extensively washed, counted, and re-stimulated 1 × 10⁶ cells/mL with Dynabeads CD3-CD28 T cell expander (one bead per cell) for 24 h for cytokine quantification.

Growth media (GM) consisted of TexMACS medium supplemented with 5% heat-inactivated AB serum, 1,000 IU/ml IL-2, 100 ng/ml Sirolimus (SRL: Rapamycin; Sigma Aldrich) and 1 µg/ml TGF-β (Miltenyi). G-Rex bioreactor (Wilson Wolf,) cultures were initiated with 2 × 10⁷ to 3 × 10⁷ cells and Exp-Act® beads at a 4:1 bead: cell ratio. GM was added every two to three days to maintain a cell density of 1 × 10⁶/mL. On Day 7, the cells were restimulated with Exp-Act® beads, at a ratio of 1:1 beads. On Day 14, in-process testing was preformed: 14-day aerobic and anaerobic sterility cultures, 30-day fungal culture, mycoplasma, endotoxin detection, cell counts, viability, phenotyping, and the Treg suppression assay. SRL was not added after Day 9 and cells were harvested on Day 21.

Single-cell suspensions of splenocytes were harvested as described above. According to the manufacturer’s instructions, CD4⁺ T cells were purified with an EasySep Mouse CD4⁺ T cell enrichment kit (STEMCELL Technologies, Canada), and the purity was confirmed to be greater than 90% confirmed by FACS (Zhang et al., 2014 (link)). The purified naïve CD4⁺ T cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (HyClone) at a density of 1 × 10⁵ in a 5% CO₂ humidified incubator at 37°C. In addition, CD4⁺ T cells were induced by incubation with an anti-CD3 antibody (2.5 μg/ml, Invitrogen), an anti-CD28 antibody (5 μg/ml, Invitrogen), IL-2 (20 U/ml, Miltenyi Biotec) and TGF-β (2 ng/ml, Miltenyi Biotec) in the presence or absence of 10 nM RvD1 for 5 days (Chiurchiu et al., 2016 (link)). Cultures were supplemented with RvD1 every other day. After 5 days, cells were collected for FACS and real-time PCR analyses. In some cases, the purified naïve CD4⁺ T cells were preincubated with anti-GPR32 neutralizing antibodies (2 μg/ml, GeneTex) and/or anti-ALX/FPR2 neutralizing antibodies (2 μg/ml, Genovac) for 30 min before the incubation with RvD1 or vehicle and then stimulated with anti-CD3/CD28, IL-2, and TGF-β.