title: Sensitive and viable quantification of inside-out signals for LFA-1 activation in human cytotoxic lymphocytes by flow cytometry.
abstract: An early step in immunosurveillance by cytotoxic lymphocytes is leukocyte functional antigen (LFA)-1-dependent adhesion to target cells, which is promoted by inside-out signals from receptors such as the T cell receptor and a variety of natural killer (NK) cell activating receptors. Inside-out signals induce a conformational change in LFA-1, resulting in an extension of the extracellular domain of the receptor. Here, we have evaluated several mAbs that specifically detect the extended conformation of LFA-1 and detail a protocol for flow cytometric quantification of β2-integrin activation in human peripheral blood cytotoxic T cells and NK cells in response to target cell recognition. By comparison to the markers of degranulation and chemokine synthesis, e.g. surface CD107a expression and intracellular MIP-1β expression, respectively, evaluation of LFA-1 conformational changes represent a sensitive and rapid parameter of primary cytotoxic lymphocyte activation that can facilitate isolation of viable cells. Potentially, combined with other read-outs of cytotoxic lymphocyte function, this technique is applicable to the assessment of various clinical conditions, including for the diagnosis of primary immunodeficiency syndromes and for evaluating the efficacy of tumor targeting by donor lymphocytes.
authors.affiliation: Center for Infectious Medicine, Department of Medicine, Karolinska Institute, Karolinska University Hospital Huddinge, Stockholm, Sweden.
authors.forename: Jakob
authors.initials: J
authors.lastname: Theorell
authors.forename: Heinrich
authors.initials: H
authors.lastname: Schlums
authors.forename: Samuel C C
authors.initials: SC
authors.lastname: Chiang
authors.forename: Terry Y
authors.initials: TY
authors.lastname: Huang
authors.forename: Anna
authors.initials: A
authors.lastname: Tattermusch
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Wood
authors.forename: Yenan T
authors.initials: YT
authors.lastname: Bryceson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D018821:CD18 Antigens / Q000378:metabolism; D015550:Cell Degranulation / Q000276:immunology; D054407:Chemokine CCL4 / Q000378:metabolism; D018925:Chemokines / Q000096:biosynthesis; D003587:Cytomegalovirus / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D007118:Immunoassay / Q000379:methods* / Q000706:statistics & numerical data; D066298:In Vitro Techniques; D007694:Killer Cells, Natural / Q000276:immunology; D008213:Lymphocyte Activation; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry / Q000378:metabolism*; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D051379:Mice; D011487:Protein Conformation; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000502:physiology
chemical_list: D000911:Antibodies, Monoclonal; D018821:CD18 Antigens; D054407:Chemokine CCL4; D018925:Chemokines; D016169:Lymphocyte Function-Associated Antigen-1; D052118:Lysosomal-Associated Membrane Protein 1