Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes

Summary

Static adhesion assay is a powerful tool that can be used to model the interactions between T lymphocytes and other cell types. Interactions are generated by injecting labeled T cells into wells coated with adhesion molecules, while a plate reader is used to quantify the number of adherent cells following serial washes.

Abstract

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with antigen presenting cells. T cells accomplish regulated adhesion by controlling the adhesive properties of integrins, a class of cell adhesion molecules consisting of heterodimeric pairs of transmembrane proteins that interact with target molecules on partner cells or extracellular matrix. The most prominent T cell integrin is lymphocyte function associated antigen (LFA)-1, composed of subunits αL and β2, whose target is the intracellular adhesion molecule (ICAM)-1. The ability of a T cell to control adhesion derives from the ability to regulate the affinity states of individual integrins. Inside-out signaling describes the process whereby signals inside a cell cause the external domains of integrins to assume an activated state. Much of our knowledge of these complex phenomena is based on mechanistic studies performed in simplified in vitro model systems. The T lymphocyte adhesion assay described here is an excellent tool that allows T cells to adhere to target molecules, under static conditions, and then utilizes a fluorescent plate reader to quantify adhesiveness. This assay has been useful in defining adhesion-stimulatory or inhibitory substances that act on lymphocytes, as well as characterizing the signaling events involved. Although described here for LFA-1 - ICAM-1 mediated adhesion; this assay can be readily adapted to allow for the study of other adhesive interactions (e.g. VLA-4 - fibronectin).

Introduction

T lymphocyte adhesion is a fundamental process in the immune response¹. It is required for T cell interaction with endothelial cells decorating the capillary walls, for scanning antigen presenting cells (APC) within lymph nodes, and for the formation of immunological synapses (IS) with target cells². These requirements are functionally and kinetically distinct. The process of lymphocytes extravasation consists of chemoattraction, rolling, firm adhesion, and transmigration. The transition from rolling to firm adhesion requires T cells to respond to a G protein coupled receptor signal rapidly. This response yields an integrin ligand interaction tha....

Protocol

1. Coating the Microplate Wells

Note: The goal of this step is to coat polystyrene surfaces with ICAM-1 to serve as ligands for T cell LFA-1.

1. Prepare the following solutions:
   1. Prepare the coating solution by resuspending recombinant ICAM-1 with phosphate buffered saline (PBS) solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO_4, 2 mM KH₂PO_4, pH 7.4) enriched with calcium (1 mM CaCl₂) and magnesium (2 nM MgCl₂). Make sure that the final concentration of ICAM-1 is 10 μg/ml, made from a 0.7 mg/ml stock solution and kept in a -80 °....

Results

Below is an example of adhesion assay using primary T cells stimulated with various concentrations of anti-CD3 antibodies. It is useful to know the fluorescence of unwashed cells (i.e. total loading). Unstimulated cells serve as a negative control and PMA treated cells are the positive control for anti-CD3 antibodies. Cells plated in uncoated wells serve as a control for ICAM-1. In a typical experiment the percentage of adherent PMA treated cells is between 40% to 50% while the percentage of unstimulated cells i.......

Discussion

There are several assays to study signals to LFA-1 activation and T cell adhesion¹². Flow cytometry is used to measure LFA-1 affinity states in living cells using monoclonal antibodies that bind selectively to either bended or extended LFA-1. One of the limitations of this method is that it does not take into account the integrins’ avidity. Migration assays are useful tools but they measure migration, and not measly adhesion at a specific time point. Mouse models to study T lymphocyte migration (e.g........

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Plate reader	BioTek	Synergy H1 Hybrid
Multichannel pipette	Fisher	21-377-829
96-well microplate with optical bottom	Corning Costar	3603
Recombinant ICAM-1	R&D Systems	ADP4-050
Anti-CD3 antibodies	Ancell	144-020
PMA	Sigma-Aldrich	79346
BSA	Sigma-Aldrich	A2058
CFSE	Molecular Probes	C1157
RPMI	Gibco	11875-093
DPBS containing calcium and magnesium	Gibco	14190250
Peripheral lymphocytes	e.g. mouse or human
Magnesium chloride	Sigma-Aldrich	M8266
Calcium chloride	Sigma-Aldrich	499609
Open Gen 5 software	BioTek	Version 2.01
Ficoll-Paque PLUS	GE Healthcare	71-7167-00
15 and 50 ml centrifuge tubes	Fisher	352099, 352070
Disposable plastic Pasteur pipettes	Fisher	13-711-7M
T-75 culture flasks	Fisher	50-754-1366
RosetteSep T cell enricment kit	StemCell Technologies	15061