A method for the rapid isolation of human epidermal Langerhans cells using immunomagnetic microspheres.
Because Langerhans and indeterminate cells are the only epidermal cells that express the specific CD1a surface antigen T6, we have used immunomagnetic monodisperse polymer microspheres for positive selection of human epidermal Langerhans and indeterminate cells. Epidermal cells in suspension are successively incubated with a murine monoclonal anti-T6 antibody of the IgG1 subclass and then with magnetic beads coated with a sheep anti-mouse IgG1. Rosetted cells are obtained and then easily separated from the non-rosetted cells using a magnet. The two cell fractions are characterized by phase contrast microscopy, immunofluorescence, electron microscopy, and the skin cell-lymphocyte reaction. All the rosetted cells (1.5 to 5% of the total epidermal cells) express T6 antigen by indirect immunofluorescence and under the electron microscope possess all the ultrastructural characteristics of Langerhans cells. Moreover, the rosetted Langerhans cells remain functional: Under the electron microscope they internalize by receptor-mediated endocytosis gold labeled anti-T6 antibody, and in the skin cell-lymphocyte reaction they stimulate allogeneic lymphocytes. In contrast, the rosette depleted cell fraction is deprived of T6 positive cells and unable to stimulate allogeneic lymphocytes. The immunomagnetic depletion of epidermal cells is a simple and rapid method to isolate functional human Langerhans cells with good yield and high purity (97%). This technique should be of value in the study of the pharmacology of Langerhans cells and in the investigation of the interactions of Langerhans cells with keratinocytes or lymphocytes.
Hanau
D
D
INSERM, Université Louis Pasteur, Strasbourg, France.
Schmitt
D A
DA
Fabre
M
M
Cazenave
J P
JP
D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D000078404:Epidermal Cells; D006801:Humans; D007801:Langerhans Cells / Q000276:immunology / Q000648:ultrastructure; D008214:Lymphocytes / Q000276:immunology; D008280:Magnetics; D008863:Microspheres