Rifn γ

To analysis chemokine production by CAF were plated at 4 x 10⁴/well in 6 well plates (Corning) and allowed establish prior replacing media with or without addition of 25 ng/ml rIFN-γ, 25 ng/ml rTNF-α (both Biolegend), or a combination of rIFN-γ and rTNF-α as indicated. After 48 hrs supernatants were harvested, centrifuged at 350 x g to remove cells and debris, supernatants were then stored at -20°C prior to analysis. For co-culture with T cells, CAF were plated at 2 x 10⁴/well in 48 well plates in complete media (DMEM plus 10% FBS with addition of Pen/Strep and L-Glutamine (All Life technologies)). T cells were purified from PBMC preps using an EasySep Human T cell isolation kit (STEMCELL technologies) according to the manufacturer's instructions. 4 hrs after plating, when CAF had adhered, purified T cells were plated alone or added to CAF containing wells at 5x10⁵/well in complete media with the additions indicated in individual experiments. T cells were stimulated with anti-CD3 (clone) anti-CD28 (clone) antibodies (both at 1 μg/ml) or with anti-CD3/anti-CD28 coated Beads (DYNAL) at a 1:1 ratio with T cells as indicated. The anti-IL-2 antibody (Clone 5334, R and D systems) was used at 2 μg/ml to neutralise IL-2. To inhibit TGF-β, the ALK inhibitor SB431542 was used at 10 µM (TOCRIS).

Femoral and tibial bone marrow cells were harvested from mice, and subsequently cultured in RPMI-1640 medium (R8758, Sigma-Aldrich) supplemented with 10% FBS, sodium pyruvate (Thermo Fisher Scientific), MEM NEAA (Thermo Fisher Scientific), and recombinant (r) M-CSF (40 ng/ml, BioLegend) for 6 days, with medium changes every 2 days. The differentiated BMDMs were treated with rIFN-γ (100 ng/ml; BioLegend) + LPS (50 ng/ml; L5273, Sigma-Aldrich) or rIL-4 (20 ng/ml; BioLegend) for 16 to 24 hours to achieve M1 or M2 polarisation, respectively. In order to evaluate M2 polarisation, we stained BMDMs treated with rIL-4 with PE-conjugated anti-CD11b (M1/70, BioLegend,), FITC-conjugated anti-F4/80 (BM8, eBioscience), and Alexa Fluor® 647-conjugated anti-CD206 (MR5D3, AbD Serotec), and analysed on a FACSCalibur™ cytometer (BD Biosciences).