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Clinical and Vaccine Immunology, March 2006, p. 403-408, Vol. 13, No. 3
1071-412X/06/$08.00+0     doi:10.1128/CVI.13.3.403-408.2006

Rapid Flow Cytometry Method for Quantitation of LFA-1-Adhesive T Cells

Brian Crucian,1 Mayra Nelman-Gonzalez,1 and Clarence Sams2*

Wyle Laboratories, Space Physiology and Countermeasures Department, Houston, Texas,1 NASA-Johnson Space Center, Human Adaptation and Countermeasures Office, Houston, Texas2

Received 9 September 2005/ Returned for modification 31 October 2005/ Accepted 2 December 2005

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-µm-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.

* Corresponding author. Mailing address: NASA-Johnson Space Center, Human Adaptation and Countermeasures Office (SK3), Houston, TX 77058. Phone: (281) 483-7061. Fax: (281) 483-2888. E-mail: csams{at}ems.jsc.nasa.gov.

Clinical and Vaccine Immunology, March 2006, p. 403-408, Vol. 13, No. 3
1071-412X/06/$08.00+0     doi:10.1128/CVI.13.3.403-408.2006






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