Siinfekl

Purified CD8⁺ T cells were labelled with 1 µM CFSE (Invitrogen) at 37°C for 20 min in serum-free RPMI. OT1 CFSE-labelled splenocytes were stimulated with OVA (SIINFEKL) peptide for 5 days in the presence or absence of recombinant human βig-h3 (rβig-h3) at a final concentration of 5 µg/mL. The antigen-specific suppression of CD8⁺ T cells was evaluated in coculture assays in which splenocytes obtained from OT-1 transgenic mice (antigen-specific assays) were seeded in triplicate in 96-well round bottom plates (5×10⁵ cells/well). The splenocytes were cultured in the presence of CAF SN that was treated with or without anti-βig-h3 Ab and then stimulated with a cognate antigen, the OVA-derived peptide SIINFEKL (1 mg/mL; New England Peptide) for 3 days. Alternatively, mitomycin-treated KC cells were cocultured with CFSE-labelled pancreatic lymph node cells in the presence of a depleting anti-βigh3 Ab or control Ab (BioXCell, USA) at a final concentration of 6 µg/mL for 5 days. Proliferation was evaluated at the end of the culture period using flow cytometry for CFSE dilution.