A stepwise protocol to coat aAPC beads prevents out-competition of anti-CD3 mAb and consequent experimental artefacts

作者：Marraco Silvia A Fuertes; Baumgaertner Petra; Legat Amandine; Rufer Nathalie; Speiser Daniel E^*

来源：Journal of Immunological Methods, 2012, 385(1-2): 90-95.

DOI：10.1016/j.jim.2012.07.017

• Artificial antigen-presenting cells
• Protein coating protocol
• Quality control by flow cytometry
• T-cell activation

Artificial antigen-presenting cells (aAPC) are widely used for both clinical and basic research applications, as cell-based or bead-based scaffolds, combining immune synapse components of interest. Adequate and controlled preparation of aAPCs is crucial for subsequent immunoassays. We reveal that certain proteins such as activatory anti-CD3 antibody can be out-competed by other proteins (e.g. inhibitory receptor ligands such as PDL1:Fc) during the coating of aAPC beads, under the usually performed coating procedures. This may be misleading, as we found that decreased CD8 T cell activity was not due to inhibitory receptor triggering but rather because of unexpectedly low anti-CD3 antibody density on the beads upon co-incubation with inhibitory receptor ligands. We propose an optimized protocol, and emphasize the need to quality-control the coating of proteins on aAPC beads prior to their use in immunoassays.

• 出版日期2012-11-30