Ifn γ

Isolated PBMCs were transferred into a flask containing RPMI medium (Bioidea, BI-1006-05). After incubation at 37 ^oC in a 5% CO2 atmosphere for 2–4 h, the non-adherent cells were then cultured in 24-well plate (at the density of 2 × 10⁶ cell/well containing RPMI medium supplemented with 100 IU/ml penicillin/streptomycin (Bioidea, BI-1203), 1000 IU/ml IFN-γ (Miltenyi Biotech, 130-096-482), and 10%, 5%, and 2.5% of one of the following sera: FBS, hPL and HS on day 0. Hence, this study compares the cell expansion kinetics of 9 groups based on the mentioned serum concentrations and serum source (Fig. 1).
On the following day (day 1), 300 IU/ml of rh-IL-2 (Thermo Fisher Scientific, 130-097-748) and 50 ng/ml of functional-grade anti-CD3 antibody (Miltenyi Biotech, 130-093-387) were added to the medium of all wells. Thenceforward, every 2–3 days, the wells were replenished with a fresh medium containing 300 U/ml IL-2. After reaching the confluence, the cells were subcultured into new wells with the same seeding density. The cell culture continued up to 30 days. Every day, cell density and morphological characteristics were inspected under an inverted microscope.

PBMCs were seeded in 12 well plates at a concentration of 1 x 10⁶ cells/mL in Aim V serum free media (Life Technologies). PBMCs were treated with increasing doses (0, 50, 100, 500, 1000 IU/mL) of IFN-γ (Miltenyi Biotec) for 24, 48 or 72 hours. Cell free supernatants were removed for KP metabolite quantification by HPLC and GC-MS and stored at ^-80°C. PBMCs were harvested from wells, with a 5 minute incubation with 0.05% Trypsin/EDTA (Life Technologies) used to detach all adherent monocytic cells. PBMCs were then washed in PBS and fixed in 3.7% paraformaldehyde for 10 minutes at 4°C.

Bone marrow-derived macrophages were obtained by flushing from murine (OPA1^f/f or OPA1^M/M) femur and tibia and differentiated into macrophages in RPMI 1640, 10% FBS (Superior, Millipore), Glutamine (300 mg/L), sodium pyruvate (1 mM), 2-mercaptoethanol (0.01 mM), HEPES (25 Mm) in the presence of M-CSF (Miltenyi Biotec), 40 ng/ml for 5 days and then refilled with 2 mL more with M-CSF (20 ng/mL) for 2 more days. At day 7, cells were differentiated to: M1 with Lipopolysaccharide from E. coli O111:B4 (Sigma-Aldrich) (LPS) (500 ng/mL) and IFNγ, 25 ng/mL (Miltenyi Biotec) or M2 with IL-4 (Miltenyi Biotec) 25 ng/mL; for 24 h at 37 °C, 5% CO₂. Different treatments were performed blinded to the mouse genotypes.

To expand antigen-specific T cells, PBMCs were adjusted to 3 × 10⁶ cells/2 ml and cultured in complete RPMI-1640 media supplemented with 1,000 U of IFN-γ (PeproTech, Cranbury, NJ, USA; cat. no. AF-300-02) and 1 μg of CMV pp65 peptide pool (JPT, Berlin, Germany; cat. no. PM-PP65-2). On day 1, 600 U of IL-2 (Proleukin) was added to each well. PBMC density was adjusted to 1 × 10⁶ cells/2 ml on days 4, 7, and 11, and the cells were re-stimulated with 300 U/ml of IL-2 and 5 ng/ml of IL-15 (PeproTech; cat. no. AF-200-15). On day 13, stimulated PBMCs were harvested and stimulated with 10 μM of CMV pp65 peptide pool pulsed on each matched aAPCs expressing a single HLA class I allotype for 24 h (APC : PBMC ratio; 1:10). Stimulated PBMCs captured IFN-γ-secreting cells by using IFN-γ Secretion Assay Detection Kits (Miltenyi; cat. no. 130-054-202) according to the manufacturer’s instructions and stained with anti-CD3-BV421, anti-CD8α-APC-Cy7, and anti-4-1BB-APC for 30 min at 4°C. In another test tube, stimulated PBMCs were stained with CMV pp65_495-503 Tetramer-PE, anti-CD3-BV421, and anti-CD8α-APC-Cy7 for 30 min at 4°C. In CD3-positive and CD8-positive cells, tetramer, IFN-γ, or 4-1BB-positive cells were sorted using SH800S Cell Sorter (Sony, San Jose, CA, USA). Sorted cells were used for the analysis of TCR repertoires by TCR sequencing.

Regulation of OPN and CD44 was assessed in the Ishikawa endometrial epithelial cell line (ECACC 99040201, STR authentication, Public Health England, UK), a well-differentiated human endometrial adenocarcinoma cell line, expressing both ERα and PR A & B receptors, regulated in a manner similar to that of normal endometrium [23 (link)]. Ishikawa cell lines at low passage number (passage number ≤ 22) were cultured at 37 °C with 5% CO₂; at least 24 h prior to experiments, the medium was changed to phenol red-free media with 10% charcoal stripped FCS. Cells were stimulated with pro-inflammatory cytokines TNF-α (25 ng/ml, 4 h) (Miltenyi Biotec UK, Cat no. 130-094-014) and IFN-γ (2 IU, 24 h) (Miltenyi Biotec UK, Cat no. 130-096-872) separately, or steroid hormones 17-β-oestradiol (E₂; 10 nM, 48 h) (Sigma-Aldrich, UK Cat-No E8875) and progesterone (P₄; 1 μM, 48 h) (Sigma-Aldrich UK, Cat-No M1629) alone or in combination as previously described [4 (link), 24 ]. Following stimulation, cell pellets were collected for mRNA and chromatin immunoprecipitation analysis. The presence of ERα, PRA and PRB receptors in Ishikawa cells was confirmed by immunoblots (data not shown).