Recombinant b7 1 fc chimeric protein

Naïve OT-I T cells were also purified as described above and stimulated for three days in vitro in flat-bottom, microtiter wells coated with antigen (DimerX H-2Kb:Ig fusion protein loaded with OVA_257–264 peptide) (BD Bioscience, San Jose, CA), and recombinant B7-1/Fc chimeric protein (R&D Systems, Minneapolis, MN) as previously described^{22 (link)}. 3 × 10⁵ cells in 1.5 mL Allos media were deposited in each well and 2.5 U/mL of IL-2 was added to wells in a 24-well plate. Where indicated, cultures were supplemented with 2 U/mL of murine rIL-12 (R&D Systems). Supernatant was harvested at the end of day three.

Flat-bottom microtiter wells in 24-well plates were coated with Dimer X-2Kb:Ig fusion protein loaded with OVA257–264 peptide (BD Pharmingen, San Jose, CA) and recombinant B7-1/Fc chimeric protein (R&D Systems, Minneapolis, MN) as previously described (42 (link)). For two signal (2SI) stimulation, purified naive OT-I CD8+ T cells were plated at a density of 3 × 10⁵ cells in 1.5 mL Allos medium in each well of a coated plate with 2.5 U/mL recombinant human IL-2 (R&D Systems, Minneapolis, MN). For three signal (3SI) stimulation, naive OT-I CD8 T cells were plated under 2SI conditions as described above and additionally supplemented with 2 U/mL of murine rIL-12 (R&D Systems, Minneapolis, MN). The cells were incubated for three days before further analysis.