Synthetic peptides and reagents Ninety one HLA 2 binding peptides were derived cell assay from caspase cleaved fragments of ACTB, ROK, LAM1, MYH9, VIME, PSA1, GDIS, and RLA as previously described. Seventeen 21 mer overlapping peptides spanning the entire human MBP sequence were synthesized by Inhibitors,Modulators,Libraries high performance liquid chromatog raphy. The purity of peptides was determined by reverse phase HPLC. Cell preparations Peripheral blood mononuclear cells were isolated and T cell lines were generated as previously described. CD8 T cells were purified from PBMCs by positive Inhibitors,Modulators,Libraries selection coupled to magnetic beads. Flow cytometry analysis demon strated 99% CD8 cells in the positively purified population and 5% in the CD8 depleted population.
Spontaneous apoptosis of T cells from patients was determined by staining fresh PBMCs with fluorescein isothiocyanate labeled Annexin V, propidium iodide and allophycocyanin labeled anti CD3 monoclonal antibody. Immature DCs were derived from peripheral monocytes and generated as described. Inhibitors,Modulators,Libraries Monocyte derived iDCs were purified by positive selection with anti CD14 mAb coupled to magnetic beads. CD14 cells were in cubated for 5 days in Roswell Park Memorial Institute 1640 medium containing 10% FCS, 2 mM glutam ine, 1% nonessential amino acids, 1% sodium pyruvate, 50 ugml kanamycin, 50 ngmL recombinant GM CFS, and 1000 UmL rIL 4. Mature DCs were obtained by a 40 h stimulation of iDCs with soluble rCD40L molecules. The definition of monocyte derived DCs was based on their surface phenotype profile by staining with anti CD14, anti CD86, anti CD1a, anti CD1c, anti CD11c, anti CD32, anti CD80 mAbs.
Enzyme linked immunospot assay After stimulation with 12 independent pools of apoptotic peptides or 16 single MBP peptides PBMCs were tested by enzyme linked immunospot assay. Briefly, 96 well millimeter high affinity plates Inhibitors,Modulators,Libraries were coated with 10 ugmL of capture mAb against IFN at 4 C overnight. Plates were blocked for 2 h with blocking so lution. A total of 1 105 PBMCs were added to each well and stimulated for 18 h with peptides. Biotinylated anti IFN diluted to 5 ugmL in Blocking Solution was added and incubated for 2 h in 5% CO2 at 37 C. Plates were washed, incubated with alkaline phosphatase streptavidin and developed with Sigmafast BCIP NBT. The reaction was stopped by rinsing the plates with distilled water. Each well was then Inhibitors,Modulators,Libraries examined for positive dots.
The number of dots in each well was counted by an ELISPOT sellekchem reader system. IFN secreting cells were expressed as IFN spots per 1 106 cells. The IFN spot values were subtracted from the background, which was below 20 IFN spots in 1 106 cells for each test. Monoclonal antibody and dextramer staining PBMCs were incubated with APC labeled HLA A 0201 dextramer complexed to MYH9478 486, MYH9741 749, VIME78 87, VIME225 233 or ACTB266 274 peptides.