The killing of NIT-1 islet cells was dependent on the specific recognition of the IGRP peptide/MHC I Kdcomplex by 8.3 TCR. (A) To verify that the cytotoxic assay is dependent on the 8.3 TCR’s specific recognition of its ligand (i.e. the IGRP peptide/Kd complex), a control peptide, TUM (aa sequence KYQAVTTTL) was included in parallel to IGRP; both IGRP and TUM at 20 ug/ml were tested. And NIT-1 cells at 2x103/well and an E/T ratio of 5/1 were used in the assay. (B) BALB/3T12-3, a mouse fibroblast cell line derived from BALB/c mice and thus has the correct allele (Kd), was also tested with the addition of exogenous IGRP peptide or the control TUM peptide. (C) In addition, the NIH3T3 cells, a mouse fibroblast that lacks expression of Kd/IGRP, was tested in the same conditions as NIT1 cells. After overnight incubation, 8.3 CD8+ T cells were removed by washing three times with cell culture medium while the adherent NIT-1 or fibroblast cells remained in the testing wells. Subsequently, 200 μl of 50% Cell Titer Glo was added to each well followed by measurement of luminescent signals on a luminometer, and the % killing was calculated based on the RLU measured. The data shown are the representative of three experiments.