Flanking residues are central to DO11.10 T cell hybridoma stimulation by ovalbumin 323-339.

T cell activation requires formation of a tri-molecular interaction between a major histocompatibility complex (MHC), peptide, and T cell receptor. In a common model system, the ovalbumin epitope 323-339 binds the murine class II MHC, I-A(d), in at least three distinct registers. The DO11.10 T cell recognizes the least stable of these, as determined by peptide-MHC dissociation rates. Using exogenous peptides and peptide insertions into a carrier protein in combination with IL-2 secretion assays, we show that the alternate registers do not competitively inhibit display of the active register four. In contrast, this weakly binding register is stabilized by the presence of n-terminal flanking residues active in MHC binding. The DO11.10 hybridoma is sensitive to the presence of specific wild-type residues extending to at least the P-3 peptide position. Transfer of the P-4 to P-2 flanking residues to a hen egg lysozyme epitope also presented by I-A(d) increases the activity of that epitope substantially. These results illustrate the inherent complexity in delineating the interaction of multiple registers based on traditional thermodynamic measurements and demonstrate the potential of flanking residue modification for increasing the activity of weakly bound epitopes. The latter technique represents an alternative to substitution of anchor residues within a weakly bound register, which we show can significantly decrease the activity of the epitope to a responding T cell.

Toxic concentrations of exogenously supplied methylglyoxal in hybridoma cell culture.

Concentrations at which methylglyoxal, a by-product of cellular metabolism, can be toxic to hybridoma cell cultures were determined using exogenously supplied doses. Trypan blue cell counts of 6-well cultures incubated for 24 h with various methylglyoxal concentrations revealed inhibition of cell growth at 300 muM and higher, with a median inhibitory concentration of 490+/-20 muM. The primary mode of death was apoptosis, as assessed by chromatin condensation, and the effects of methylglyoxal were observed to be complete by approximately eight hours. Yet, the impact of methylglyoxal was a function of the rate of dosing; stepwise addition of MG during the first 6 h of incubation inhibited growth but caused much less cell death than a comparable bolus dose. Inhibition of cellular metabolism by MG was found to coincide with inhibition of cell growth, with a comparable median inhibitory concentration of 360+/-20 muM. The effects on viable cell density and metabolism were both linear at doses approaching zero, with lowest observable effect levels of 54 and 77 muM, respectively. These results provide quantitative estimates for concentrations of methylglyoxal that may be inhibitory to biopharmaceutical-producing cell lines.