Be0001 1

T cell hybridoma cells expressing GFP under a minimal NFAT promoter (Ise et al., 2010 (link)) were retrovirally transduced with TCRα chains of interest as previously described (Chai et al., 2017 ; Lathrop et al., 2011 (link)). Hybridoma cells (1.5×10⁴) were cultured with splenic CD11c⁺ DCs (5×10⁴) expanded in vivo with Flt3-ligand B16 melanoma cells in 200 μl D10 (200 μl complete DMEM (Thermo Fisher #SH3008101) with 10% FBS, Glutamax, 50 μM beta-mercaptoethanol, 1 mM sodium pyruvate, 10 mM HEPES, non-essential amino acids, and penicillin and streptomycin) in 96-well flat bottom plates with indicated concentrations of antigens for 40 hours at 37°C. TCRβ⁺ cells were analyzed for GFP expression by FACS. Where noted, 50 μg/ml soluble anti-CD3ε (BioXCell BE0001–1) or 20 μg/ml MHC Class II blocking antibody (BioXCell BE010). was added to cultures.

To assess the effects of 3-BrPa on T cell proliferation, T cells were isolated from spleens and lymph nodes of C57BL/6 mice using the R&D CD3⁺ T cell enrichment Column (MTCC-10), stained with 20 μM Cell Proliferation Dye eFluor450 (eBioscience #65-0842-85), and stimulated to divide in complete RPMI with platebound α-CD3 (BioXcell #BE0001-1) (1 μg/mL) and soluble α-CD28 (BioXcell #BE0015-5) (10 μg/mL) antibodies for 72hrs. After 24 hours of stimulation, a time when T cells have engaged glycolysis to fuel activation (19 (link)) but have not yet divided, 10 μM 3-BrPa or saline vehicle was added to the cells. Division was monitored for an additional 48 hours and determined every 24 hours by flow cytometric analysis of efluor450 dye dilution. The same T cell isolation procedure used for the proliferation assay was used to determine how 3-BrPa affects T cell cytokine production. Enriched T cells were stimulated with α-CD3 and α-CD28 in the presence of 3 μg/ml Brefeldin A (eBioscience #00-4506-51) as well as 10 μM 3-BrPa or saline vehicle for 6hrs at 37°C, 5% CO₂ and assessed for viability and cytokine production by flow cytometry.