MHC Tetramer and Anti-CD3 Stimulation of J.OT1 Cells

The stimulation method of J.OT1 cells is based on a previously established study^{21 (link)}. For each replicate per condition, 15 million J.OT1 cells were washed and resuspended in serum-free RPMI at a concentration of 50 million cells per mL. Cells were then incubated with either 10 nM of H-2K^b MHC tetramer (Class I, human beta-2-microglobulin) or ~1 ug/mL of anti-CD3 IgM antibody C305 on ice for 1 h. C305 was diluted in Dulbecco’s phosphate-buffered saline (DPBS) prior to stimulation. MHC tetramers^{30 (link)} were synthesized by the NIH Tetramer Core Facility (Atlanta, GA) using custom peptides from GenScript. The peptide sequences are SIINFEKL (OVA), SIITFEKL (T4), SIIGFEKL (G4), and RGYVYQGL (VSV). Stimulation was initiated by transferring the cells from ice to 37°C. After 3 mins of stimulation at 37°C, cells were lysed with equal volume of lysis buffer (pH 7.6) containing 1% (w/v) sodium dodecyl sulfate (SDS), 100 mM Tris-HCl and 1X MS-SAFE Protease and Phosphatase Inhibitor cocktail (Sigma-Aldrich MSSAFE). PV treatment was performed by incubating J.OT1 cells with 500 μM PV (prepared by mixing equal volume of 1 mM sodium orthovanadate and 1 mM hydrogen peroxide) for 10 minutes at 37°C and lysed with the lysis buffer as mentioned.