Measuring 2D Affinity of Virus-Specific T Cells

The relative two-dimensional affinity (2D MP) of polyclonal IAb:GP_66–77 specific cells from C57BL/6-FOXP3 EGFP mice (JAX) was measured using the previously described 2D- MP assay (25 (link)–27 (link)). Seven days following LCMV infection and AIM assay culture, splenic CD4+ T cells were negatively enriched by magnetic separation (Miltenyi Biotec;130-104-454) and were stained and sorted to obtain FOXP3- (GFP-) CD25-OX40- AIM-negative CD4+ T cells and FOXP3- (GFP-) CD25+OX40+ AIM-positive CD4+ T cells. Human RBCs were coated with IAb:GP_66–77 monomer or with negative control H2Db:NP_366–374 monomer obtained from the NIH tetramer core. Quantification of pMHC density with anti-IA/IE Ab (M5/114/15/2; eBioscience), TCR surface density with anti-mouse TCRβ PE Ab (H57–597; BD Biosciences) were determined using QuantiBRITE PE quantification beads (BD Bio-sciences). Quantification of binding events and TCR:pMHC affinity calculations were calculated as previously described (25 (link)–27 (link)). Each cell was tested to the monomer of interest and then the control monomer to determine antigen specificity of the cell.