Generation of activated OTI T cells in vitro

OTI T-cell receptor transgenic mice were used in this study, and these were of the C57BL/6 strain, male in gender, and between 12 and 15 wk of age. Spleen and lymph nodes (LN) were dissected from OTI TCR transgenic mice, and single-cell suspensions were prepared by both mashing the organs through a 70-μm filter and washing with complete medium. Red blood cell lysis was performed on splenocytes and LN-derived cells, using a premade RBC lysis buffer (Cat #420302; BioLegend). Splenocytes were pulsed with ovalbumin peptide (SIINFEKL), for 45 min at 37°C, and washed with complete RPMI three times. LN-derived cells were then added to the splenocytes, and cells were cultured for 1 wk in RPMI supplemented with 10% heat-inactivated FCS, 1% penicillin–streptomycin, 2.5 mM L-glutamine, and 50 μm beta-mercaptoethanol. 10 U/ml of recombinant murine IL-2 was added to the expanding culture every 2 d.