title: Nanobody-induced perturbation of LFA-1/L-plastin phosphorylation impairs MTOC docking, immune synapse formation and T cell activation.
abstract: The T cell integrin receptor LFA-1 orchestrates adhesion between T cells and antigen-presenting cells (APCs), resulting in formation of a contact zone known as the immune synapse (IS) which is supported by the cytoskeleton. L-plastin is a leukocyte-specific actin bundling protein that rapidly redistributes to the immune synapse following T cell-APC engagement. We used single domain antibodies (nanobodies, derived from camelid heavy-chain only antibodies) directed against functional and structural modules of L-plastin to investigate its contribution to formation of an immune synapse between Raji cells and human peripheral blood mononuclear cells or Jurkat T cells. Nanobodies that interact either with the EF hands or the actin binding domains of L-plastin both trapped L-plastin in an inactive conformation, causing perturbation of IS formation, MTOC docking towards the plasma membrane, T cell proliferation and IL-2 secretion. Both nanobodies delayed Ser(5) phosphorylation of L-plastin which is required for enhanced bundling activity. Moreover, one nanobody delayed LFA-1 phosphorylation, reduced the association between LFA-1 and L-plastin and prevented LFA-1 enrichment at the IS. Our findings reveal subtle mechanistic details that are difficult to attain by conventional means and show that L-plastin contributes to immune synapse formation at distinct echelons.
authors.affiliation: Department of Medical Protein Research, VIB, 9000, Ghent, Belgium.
authors.forename: Sarah
authors.initials: S
authors.lastname: De Clercq
authors.forename: Olivier
authors.initials: O
authors.lastname: Zwaenepoel
authors.forename: Evelien
authors.initials: E
authors.lastname: Martens
authors.forename: Joël
authors.initials: J
authors.lastname: Vandekerckhove
authors.forename: Aude
authors.initials: A
authors.lastname: Guillabert
authors.forename: Jan
authors.initials: J
authors.lastname: Gettemans
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D000938:Antigen-Presenting Cells / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D002147:Calmodulin / Q000276:immunology / Q000378:metabolism; D002450:Cell Communication; D002460:Cell Line; D002478:Cells, Cultured; D020832:EF Hand Motifs; D006801:Humans; D007376:Interleukin-2 / Q000276:immunology; D019169:Jurkat Cells; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D008213:Lymphocyte Activation; D016169:Lymphocyte Function-Associated Antigen-1 / Q000276:immunology* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D008840:Microfilament Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D022101:Microtubule-Organizing Center / Q000276:immunology* / Q000378:metabolism / Q000648:ultrastructure; D008958:Models, Molecular; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D061905:Single-Domain Antibodies / Q000276:immunology*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism
chemical_list: D000199:Actins; D002147:Calmodulin; D007376:Interleukin-2; D016169:Lymphocyte Function-Associated Antigen-1; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D061905:Single-Domain Antibodies; C026742:plastin