Anti cd3ε clone 145 2c11

7-17 cells were stimulated by T cell receptor (TCR) crosslinking. Anti-CD3ε (clone 145-2C11, # 16-0031, eBioscience, San Diego, CA, USA) was coated onto 24- or 96-wells. 1x10⁵ 7-17 cells per 24-well or 5x10⁴-1x10⁵ cells per 96-well (metabolic analysis) were plated on the anti-CD3ε coated wells or control-wells pre-treated with phosphate buffered saline (PBS). Then 2 ng/ml anti-CD28 (clone 37.51, # 16-0281, eBioscience) was added and cells were incubated for indicated stimulation times.

Cells obtained from spleens and livers were resuspended in RPMI-1640 medium supplemented with 10% FCS (Sigma-Aldrich) and 1% penicillin/streptomycin (Life Technologies). A total of 5 × 10⁵ cells were plated in 96-well plates precoated with anti-CD3ε (clone 145-2C11; eBioscience), and anti-CD28 (clone 37.51; eBioscience) was added at a final concentration of 1 μg/ml. For cell stimulations using heat-killed STm, STm SL3261 was grown in LB broth and washed twice in PBS before heat inactivation (75°C for 1 h, with killing confirmed by plating onto LB agar plates) and addition to plates at 10⁷ killed bacteria per well. Cells with medium only were included as nonstimulated controls. Cells were incubated at 37°C with 5% CO₂ for 48 h. Supernatants were harvested and aliquots were stored at −80°C until analysis. Cytokines were detected by ELISA following the manufacturer’s instructions (mouse IFN-γ and IL-17 Ready-Set-Go Kits; eBioscience).