Therefore it can be concluded that imaging or staining methods did not bias the results

tion, DO knockout mice have failed to demonstrate any clear Oleandrin immunological susceptibilities or advantages,,. Hence the data on DO and its functions have remained controversial. Much of the current knowledge on function of DO stems from studies with fibroblast cell lines transfected with MHC II related genes, including DO. A breakthrough was recently reported showing that when DO was overexpressed in dendritic cells of NOD mice; those mice gained an immunity to Type I diabetes, suggesting that DO expression eliminated presentation of self antigens that lead to diabetes. These findings were consistent with many studies that have linked the effects of HLA-DO to the dampening of peptide binding to MHC II molecules 8913470 through interaction with DM,. Nonetheless, there are also some reports to the contrary, suggesting an enhancing peptide Role for DO in Epitope Selection presentation for DO,,. All studies on DO have assumed that DO functions through editing the activity of DM. Here, using purified soluble MHC II HLA-DR1, DO, and DM we determined the effects of DO individually, and in conjunction with DM on binding and dissociation kinetics of multiple peptides from DR1 molecules. Experiments testing the simultaneous effect of both DM and DO molecules on DR1/ peptide complex formation suggests that DO interacts directly with DR molecules. We hypothesize that both DM and DO molecules work in concert to narrow down the peptide repertoire presented by MHC II molecules, with DO being the dominant editor having the final say over the selection process. The soluble DR1 heterodimers were kept in PBS pH 7.4 with 0.05% NaN3. Peptides Peptides used in our binding experiments at a minimum of 90% purity were derived from characterized immunodominant HLA-DR1 binding peptides. The peptides were Fluorescein labeled at N-Termini as previously described: HA: CPKYVKQNTLKLAT HA: PKAVKQNTLKLAT HA: CPKAVKANGAKAAT CII: CAGFKGEQGPKGEP H5N1-HA1: SNGNFIAPEYAYKIVK Materials and Methods Production of 7482723 Soluble MHC Class II Molecules Soluble HLA-DR1was expressed in Hi5 insect cells through a Baculovirus expression vector and purified via L243 coupled resin as originally described. For the purification of HLA-DM and HLA-DO, the protocol was modified to use the M2 FLAG and Ni-NTA affinity columns respectively using gravity flow. Upon elution from the columns DM and DO molecules were concentrated in Citrate Phosphate pH 6.0 buffer with 0.05% NaN3 and stored frozen at 280uC in small aliquots. Peptide Binding to DR1 Peptide binding experiments were performed as described previously,,, based on efforts to simulate physiological conditions of antigen processing compartments whenever practically possible. In brief, we performed all binding experiments at 37uC in Citrate-Phosphate buffer, pH 5.0. Wt DR1 or mutant DR1, DR1bG86Y was incubated with Role for DO in Epitope Selection fluorescent peptides in association experiments. For dissociation experiments we used DR1/fluorescent peptide complexes formed over 3 days incubation in 37uC, a competing nonfluoresceinated HA peptide was added to the dissociation reaction at a concentration of 50 mM. In kinetics experiments where DO and DM were included, concentrations of 1 mM for DM and 4 mM DO or DM/DO were used. For each of the experimental combinations of DR1 with DM and DO, the binding of peptide to DR1 was measured at eight different time points: 10 h, 5 h, 3 h, 2 h, 1 h, 0.5 h, 0.25 h, 0 h, unless otherwise indicated. Excess unbound peptid