The 2D affinity of cultured HLA-DQ CD4+ T cell clones specific for DQ8cis (DQA1*0301/DQB1*0302) and DQ8trans (DQA1*0501/DQB1*0302) were measured using the previously characterized 2D micropipette adhesion frequency assay (33). Briefly, red blood cells (RBCs) were coated with Biotin-LC-NHS (BioVision), followed by streptavidin (Thermo Fisher Scientific) and with either covalently linked, biotinylated DQ8cis/GAD65250–266 or DQ8trans/GAD65250–266 monomers. In this 2D assay, the adhesion frequency between TCR on the T cell and ligand (pMHC on an RBC) aspirated on opposing pipettes was observed using an inverted microscope. An electronically controlled piezoelectric actuator repeated 50 T cell contact and separation cycles with the pMHC-coated RBC while keeping time (t) constant. Following retraction of the T cell, adhesion (binding of TCR:pMHC) was observed as distention of the RBC membrane, allowing for quantification of the adhesion frequency (Pa). Surface pMHC (ml) ligand and TCR (mr) densities were determined by flow cytometry and BD QuantiBRITE PE beads for standardization (BD Biosciences). The calculation of molecules per area was determined by dividing the number of TCRs and pMHCs per cell by the respective surface areas. The relative 2D affinities were calculated using the following equation: AcKa = −ln [1 − Pa (1)]/mrml. Normalized adhesion frequency was calculated using the equation [−ln(1 − Pa(s))/ml (pMHC)]. Geometric means of all measured single-cell affinities are reported ± SEM.

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