Recombinant mouse tgf β1

Cells were cultured in E medium with 15% fetal bovine serum (FBS) and 50 µM CaCl₂ (10 MKs) or 1.5 mM CaCl₂ (human SCC lines) at 37°C, 7.5% CO₂. For stimulation experiments, media were supplemented with either recombinant mouse TGF-β1 (100 pM = 2.5 ng/ml, R&D Systems), 4-hydroxytamoxifen (1 µM, Sigma), cisplatin (20 µM, Sigma), H₂O₂ (1–1,000 µM, Fisher), or ethacrynic acid (50 µM, Abcam). For colony formation assay, FACS-isolated 1.03 10⁴ α6^hiCD44⁺mCherry^neg and α6^hiCD44⁺mCherry⁺ primary tumor basal cells were plated onto mitomycin-treated mouse 3T3 fibroblasts in 6-well dishes in E medium with 15% FBS and 300 µM CaCl₂. Colony number was counted after 14 day culture.

FACS isolated tumor YFP+EpCam+ cells were plated on
g-irradiated 3T3 feeder cells in 6-well plates. For stimulation experiments,
media were supplemented with recombinant mouse TGF-β1 and
TGF-β2 (10ng/ml) (catalog number #76 66-MB and
#7346-B2 respectively, R&D Systems; resuspended with 4mM
HCl, 0,1% BSA).

The following antibodies were purchased from BioLegend (San Diego, CA): anti-CD8a (53-6.7), anti-CD44 (IM7) and anti-CD62L (MEL-14). H-2D^b restricted gp100 tetramer was provided from The National Institutes of Health (NIH) Tetramer Core Facility. Goat anti-mouse TGFBR2, goat IgG isotype control and recombinant mouse TGF-β1 were purchased from R&D Systems (Minneapolis, MN). Rabbit anti-goat IgG was purchased from Abcam (Cambridge, MA). Recombinant human IL-2 was purchased from PeproTech (Rocky Hill, NJ). Human gp100_25-33 (KVPRNQDWL) peptide was synthesized by the University of Pittsburgh Peptide Synthesis Facility.

Bone marrow derived DCs (BMDCs) were generated from bone marrow cells of 5-7 week old B6 or B6.MHCII KO mice. Bone marrow cells were cultured under maturation conditions for 10 days in full medium supplemented with GM-CSF (hybridoma supernatant, LUTZ-GMCSF, kindly provided by V.Horejsi). Autologous mixed lymphocyte reactions (auto-MLRs) were performed by co-culturing 1x10⁵ syngeneic (B6 or MHCII KO) BMDCs with 3x10⁵ CFSE labeled (Life Technologies, Invitrogen) magnetic bead enriched CD4 cells (Dynabeads, Invitrogen) in 96-well-U-shaped plates for 5 days. For in vitro, T_reg cell development experiments, 1x10⁵ thymocytes from 3BK508TCR-tg mice were co-cultured with 1x10⁵ B6 BMDCs in the presence of IL-2 (25U/ml, hybridoma X63 supernatant) and recombinant mouse TGF-β1 (10ng/ml, R&D Systems) for 48h with or without 1μM 3K (FEAQKAKANKAV), P2A (FEAAKAKANKAVD) or P-1A (FAAQKAKANKAVD) peptides (all obtained from Eurogentec). Re-aggregated thymic organ cultures were performed as previously described.58 In brief, RTOC were established from B3K508TCR-tg, MHCII KO thymocytes and thymic epithelial cells from B6 mice and were cultured in presence of P-1A (20 μM), P2A (2 μM) or 3K (0.2 μM) peptides for 7 days before analysis. All in vitro assays were performed at 37°C in 5% CO₂ using complete RPMI medium (GIBCO, Life Technologies).