Rapid flow cytometry method for quantitation of LFA-1-adhesive T cells.
Adhesion molecules are important for leukocyte endothelial attachment and migration to sites of inflammation. The LFA-1 (CD11a and CD18) integrin molecule is constitutively expressed on the T-cell surface. Following T-cell activation, a rapid conformational change of LFA-1 to an "adhesive" state occurs, allowing LFA-1 binding to intracellular cell adhesion molecule type 1 (ICAM-1)-expressing targets, such as antigen-presenting cells. For this study, a rapid flow cytometry method for the quantitation of LFA-1-adhesive T cells following activation was developed. Purified ICAM-1 was bound to 4.5-microm-diameter beads. Following peripheral blood mononuclear cell activation culture (phorbol myristate acetate and ionomycin), the cells were incubated with the ICAM-1 beads, which allowed attachment to occur. The T cell-bead complexes were then resolved from unbound T cells by flow cytometry. Multicolor analysis allowed a complete phenotypic analysis of the adhesive T-cell subsets. Experimental controls indicated that the T cell-bead attachment was LFA-1 and ICAM-1 specific. Very little binding between unactivated T cells and ICAM beads or between activated T cells and plain beads was observed. The kinetics of the response was extremely rapid, with nearly maximal numbers of adhesive T cells observed following 5 min of activation. Scanning electron microscopy analysis was used to characterize legitimate bead-cell binding. By using multicolor cytometry, the responding adhesive T-cell population was usually identified as a distinct subset of T cells with the following phenotype: CD3+ CD4+ or CD8+ CD19- CD16- CD45RO+ CD62L+ CD27+ CD57-. A rapid and simple method for the scoring of LFA-1-adhesive T cells was developed and may have significant utility for immune function studies.
Crucian
Brian
B
Wyle Laboratories, Space Phystiology and Countermeasures Department, NASA-Johnson Space Center, Human Adaptation and Countermeasures Office (SK3), Houston, TX 77058, USA.
Nelman-Gonzalez
Mayra
M
Sams
Clarence
C
D002448:Cell Adhesion / Q000276:immunology; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping; D007700:Kinetics; D018655:Lymphocyte Count / Q000379:methods; D016169:Lymphocyte Function-Associated Antigen-1 / Q000096:biosynthesis / Q000276:immunology*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008863:Microspheres; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism / Q000648:ultrastructure
D016169:Lymphocyte Function-Associated Antigen-1