title: A stepwise protocol to coat aAPC beads prevents out-competition of anti-CD3 mAb and consequent experimental artefacts.
abstract: 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.
authors.affiliation: Clinical Tumor Biology and Immunotherapy Unit, Ludwig Center for Cancer Research of the University of Lausanne, CH-1011 Lausanne, Switzerland.
authors.forename: Silvia A
authors.initials: SA
authors.lastname: Fuertes Marraco
authors.forename: Petra
authors.initials: P
authors.lastname: Baumgaertner
authors.forename: Amandine
authors.initials: A
authors.lastname: Legat
authors.forename: Nathalie
authors.initials: N
authors.lastname: Rufer
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Speiser
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000938:Antigen-Presenting Cells / Q000276:immunology*; D016477:Artifacts; D017252:CD3 Complex / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000276:immunology; D051379:Mice; D008863:Microspheres; D016853:Muromonab-CD3 / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D016853:Muromonab-CD3