T cell Activation Assay

The T cell activation experiment is a multi-cell in vitro test of the biological potential resultant from modification of the native peptide sequence. The assay utilized L929 cells as an antigen presenting cell (APC). These cells have been transfected with I-A^u MHC II protein [15] specific to MBP Ac1–11 and present antigen to the 172.10 T cell hybridoma [16]. The 172.10 cells express a TCR that specifically recognizes MBP Ac1–11 peptide in the context of I-A^u MHC II molecules. The 172.10 T cell hybridomas produce the cytokine IL-2 if they are activated through sufficient recognition of their antigenic peptide. IL-2 production by the 172.10 cells was used as an indication of the degree of stimulation through the TCR in response to the APL being tested. Aza-peptides from the binding experiment as well as unlabeled APLs with and without aza-amino acid substitutions were tested. Serial dilution of test peptides were added to 3 × 10⁴ L929 cells and 6.5 × 10⁴ 172.10 cells in 96 well plates (Corning, Corning, NY) and incubated at 37°C for 48 hrs. Supernatant samples were then taken and frozen at −80°C until the cytokine analysis was performed. The IL-2 concentration was determined by sandwich ELISA (BD Bioscience, San Diego, CA) in a 96 well plate format following standard protocols. The absorbance of the ELISA plates was read with a Vmax absorbance plate reader (Molecular Devices, Sunnyvale, CA) at 450 nm. Samples were quantified by comparing absorbance readings to a standard curve prepared with known concentrations of IL-2 following the kit directions.