title: Phenotypic Approaches to Identify Inhibitors of B Cell Activation.
abstract: An EPIC label-free phenotypic platform was developed to explore B cell receptor (BCR) and CD40R-mediated B cell activation. The phenotypic assay measured the association of RL non-Hodgkin's lymphoma B cells expressing lymphocyte function-associated antigen 1 (LFA-1) to intercellular adhesion molecule 1 (ICAM-1)-coated EPIC plates. Anti-IgM (immunoglobulin M) mediated BCR activation elicited a response that was blocked by LFA-1/ICAM-1 specific inhibitors and a panel of Bruton's tyrosine kinase (BTK) inhibitors. LFA-1/ICAM-1 association was further increased on coapplication of anti-IgM and mega CD40L when compared to individual application of either. Anti-IgM, mega CD40L, or the combination of both displayed distinct kinetic profiles that were inhibited by treatment with a BTK inhibitor. We also established a FLIPR-based assay to measure B cell activation in Ramos Burkitt's lymphoma B cells and an RL cell line. Anti-IgM-mediated BCR activation elicited a robust calcium response that was inhibited by a panel of BTK inhibitors. Conversely, CD40R activation did not elicit a calcium response in the FLIPR assay. Compared to the FLIPR, the EPIC assay has the propensity to identify inhibitors of both BCR and CD40R-mediated B cell activation and may provide more pharmacological depth or novel mechanisms of action for inhibition of B cell activation.
authors.affiliation: Discovery Sciences, Janssen Research and Development LLC, La Jolla, CA, USA erex@its.jnj.com.
authors.forename: Elizabeth B
authors.initials: EB
authors.lastname: Rex
authors.affiliation: Discovery Sciences, Janssen Research and Development LLC, La Jolla, CA, USA.
authors.forename: Suzie
authors.initials: S
authors.lastname: Kim
authors.affiliation: Immunology, Janssen Research and Development LLC, La Jolla, CA, USA.
authors.forename: Jake
authors.initials: J
authors.lastname: Wiener
authors.affiliation: Immunology, Janssen Research and Development LLC, La Jolla, CA, USA.
authors.forename: Navin L
authors.initials: NL
authors.lastname: Rao
authors.affiliation: Discovery Sciences, Janssen Research and Development LLC, La Jolla, CA, USA.
authors.forename: Marcos E
authors.initials: ME
authors.lastname: Milla
authors.affiliation: Discovery Sciences, Janssen Research and Development LLC, La Jolla, CA, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: DiSepio
mesh_terms: D000888:Antibodies, Anti-Idiotypic / Q000494:pharmacology; D001402:B-Lymphocytes / Q000187:drug effects* / Q000276:immunology* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D055808:Drug Discovery / Q000379:methods*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008213:Lymphocyte Activation / Q000187:drug effects* / Q000276:immunology*; D010641:Phenotype; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism
chemical_list: D000888:Antibodies, Anti-Idiotypic; D015815:Cell Adhesion Molecules; D007166:Immunosuppressive Agents; D011947:Receptors, Antigen, B-Cell; C044203:anti-IgM; D018799:Intercellular Adhesion Molecule-1; D002118:Calcium
keywords: EPIC; FLIPR; LFA-1/ICAM-1 adhesion; calcium flux; label free