Kevin J. Horgan

Roles of Adhesion Molecules in T‐Cell Recognition: Fundamental Similarities between Four Integrins on Resting Human T Cells (LFA‐1, VLA‐4, VLA‐5, VLA‐6) in Expression, Binding, and Costimulation

This review summarizes our recent work on expression and function of 4 integrins on resting human CD4+ T cells. Three themes are highlighted: multiplicity of molecular pathways of adhesion, regulation of adhesion, and costimulation by adhesion molecules. Four distinct receptor/ligand interactions have been elucidated: LFA-1/ICAM-1, VLA-5/fibronection, VLA-4/fibronectin, and VLA-6/laminin. Our studies indicate fundamental similarities in function and regulation of these four receptor/ligand interactions: 1) acute activation of the T cell (by CD3/TCR crosslinking or by PMA) induces rapid but transient integrin binding function; and 2) higher expression of each integrin on memory T cells compared to naive T cells results in greater binding of memory cells to each ligand. The identification of T-cell integrins (VLA-4, VLA-5, VLA-6) which interact with ECM components directs attention to the potential importance of T-cell interactions with ECM components which either may be immobilized in ECM or which may act as molecular bridges between cells. The existence of multiple adhesion pathways, of multiple ligands for a single receptor (such as LFA-1/ICAM-1 and LFA-1/ICAM-2), multiple receptors for a single ligand (such as VLA-4/FN and VLA-5/FN), and regulation of ligand expression (ICAM-1) provides opportunities for co-operativity, rebundancy and diversity which the T cell utilizes to exquisitely regulate its adhesive interactions. The thesis that adhesion molecules can be multifunctional receptors that also influence signalling is demonstrated by our findings that each of these integrin receptor/ligand interactions is capable of providing a potent costimulatory signal to CD3-mediated T-cell activation. The importance of interactions of T-cell integrins with their cell surface and ECM ligands is discussed with respect to T-cell migration, differentiation and recognition. Analysis of the precise mechanisms by which T cells regulate and exploit these multiple receptor/ligand interactions and the resulting functional consequences of those interactions will be exciting areas of future research.

Immunomagnetic Purification of T Cell Subpopulations

There are two types of magnetic cell isolation technologies, one column‐based and the other tube‐based. The column‐based technology utilizes nano‐sized particles that need to pass through a ferromagnetic spheres column to increase cell‐capture capacity. The tube‐based system utilizes micron‐sized beads that can be selected using a magnet applied to the tube. The beads are used for direct or indirect labeling of cells. Direct labeling is achieved with antibodies coupled to magnetic particles directly added to the cell suspension. For indirect labeling the cells are first labeled with the antibody of interest; the antibody can be simple, biotinylated, or fluorochrome‐conjugated. Subsequently, beads coated with streptavidin or anti‐immunoglobulin, anti‐biotin, anti‐fluorochrome antibodies are used to specifically mark the subpopulation of interest. Separation of target cells can be achieved using positive or negative selection or a combination of the two. Quality of the sample preparation is critical to obtain good purification and yield. Curr. Protoc. Immunol. 85:7.4.1‐7.4.9.

UNIT 7.4 Immunomagnetic Purification of T Cell Subpopulations

This unit describes a general method for physical separation of cell subpopulations from a heterogeneous mixture of cell types. The technique relies on having an appropriate monoclonal antibody or mix of monoclonal antibodies that distinguish between the cell types being separated. Its advantages over other antibody‐mediated selection techniques are purity of resulting cell preparation, reproducibility of separation, and ease of handling small to large numbers of cells (106 to 1010 cells). The basic protocol outlines preparation of purified human T lymphocytes. The method produces good yields of pure T cells prepared by negative selection (i.e., not labeled with antibody). The alternate protocol applies similar principles for purifying CD4+ T cells, and presents refinements of the basic protocol that minimize cost and improve convenience.