Supplemental Experimental Procedures Estimation of Available TCR (CD3ε)-Binding Sites

Antibody surface densities were calculated by coating our stimulatory substrates with Alexa-647-labeled OKT3. For coating densities from 3-30 µg/ml, protein retention was linear and corresponded to approximately 10 % of the input material. Antibody retention at lower coating densities was extrapolated assuming continued linear binding. The surface density of biologically active CD3ε-binding sites was estimated using a previously derived correction factor, which indicated that ~22 % of the randomly oriented antibodies retained on charged substrates were available for ligand recognition (Andersen et al., 2001). Using these calculations, the OKT3 concentration associated with maximal VLA-4-mediated costimulation (300 ng/ml) provides approximately 100 biologically active TCR binding sites per µm2. Derived Calculations (1) The pMHC ligands presented by lipid bilayers are laterally mobile; therefore, these ligands become enriched in domains apposed to TCR microclusters. Across a broad range of initial pMHC densities, the formation of pMHC clusters containing 60-350 pMHC per µm2 is associated with optimal T cell activation (Grakoui et al., 1999). Thus, VLA-4 facilitates the activation of Jurkat T cells in response to TCR ligand densities (100 sites per µm2) that are comparable to those that support the activation of antigen-specific primary T cells