Hirschil Trust, Michael Saperstein Medical Scholars Research Funds, and the NYU Whitehead fellowship supported this work.

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.
<ol>
	<li>Prepare the following solutions:
	<ol>
		<li>Prepare the coating solution by resuspending recombinant ICAM-1 with phosphate buffered saline (PBS) solution (137 mM NaCl,&#160;2.7 mM KCl,&#160;10 mM Na2HPO4,&#160;2 mM KH2PO4,&#160;pH 7.4) enriched with calcium (1 mM CaCl2) and magnesium (2 ...

<ol>
	<li>Dustin, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membranes+as+messengers+in+T+cell+adhesion+signaling.">Membranes as messengers in T cell adhesion signaling.</a> Nat Immunol. 5 (4), 363-372 (2004).</li><li>Mor, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GTPases+and+LFA-1+reciprocally+modulate+adhesion+and+signaling.">GTPases and LFA-1 reciprocally modulate adhesion and signaling.</a> Immunol Rev. 218, 114-125 (2007).</li><li>Rose, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+modulation+and+signaling+in+leukocyte+adhesion+and+migration.">Integrin modulation and signaling in leukocyte adhesion and migration.</a> Immunol Rev. 218, 126-134 (2007).</li><li>Alon, R., Feigelson, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokine-triggered+leukocyte+arrest:+force-regulated+bi-directional+integrin+activation+in+quantal+adhesive+contacts.">Chemokine-triggered leukocyte arrest: force-regulated bi-directional integrin activation in quantal adhesive contacts.</a> Curr Opin Cell Biol. 24 (5), 670-676 (2012).</li><li>Griffith, J. W., Luster, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+cells+in+motion:+migrating+toward+improved+therapies.">Targeting cells in motion: migrating toward improved therapies.</a> Eur J Immunol. 43 (6), 1430-1435 (2013).</li><li>Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+molecules+and+actin+cytoskeleton+at+immune+synapses+and+kinapses.">Cell adhesion molecules and actin cytoskeleton at immune synapses and kinapses.</a> Curr Opin Cell Biol. 19 (5), 529-533 (2007).</li><li>Springer, T. A., Dustin, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+inside-out+signaling+and+the+immunological+synapse.">Integrin inside-out signaling and the immunological synapse.</a> Curr Opin Cell Biol. 24 (1), 107-115 (2012).</li><li>Mor, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D1+regulates+lymphocyte+adhesion+via+upregulation+of+Rap1+at+the+plasma+membrane.">D1 regulates lymphocyte adhesion via upregulation of Rap1 at the plasma membrane.</a> Mol Cell Biol. 29 (12), 3297-3306 (2009).</li><li>Bivona, T. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rap1+up-regulation+and+activation+on+plasma+membrane+regulates+T+cell+adhesion.">Rap1 up-regulation and activation on plasma membrane regulates T cell adhesion.</a> J Cell Biol. 164 (3), 461-470 (2004).</li><li>Zeltzer, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diminished+adhesion+of+CD4++T+cells+from+dialysis+patients+to+extracellular+matrix+and+its+components+fibronectin+and+laminin.">Diminished adhesion of CD4+ T cells from dialysis patients to extracellular matrix and its components fibronectin and laminin.</a> Nephrol Dial Transplant. 12 (12), 2618-2622 (1997).</li><li>Bhattacharyya, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Both+adhesion+to+immobilized+vitronectin+and+Fc+epsilon+RI+cross-linking+cause+enhanced+focal+adhesion+kinase+phosphorylation+in+murine+mast+cells.">Both adhesion to immobilized vitronectin and Fc epsilon RI cross-linking cause enhanced focal adhesion kinase phosphorylation in murine mast cells.</a> Immunology. 98 (3), 357-362 (1999).</li><li>Dustin, M. L., Groves, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+signaling+clusters+in+the+immune+synapse.">Receptor signaling clusters in the immune synapse.</a> Annu Rev Biophys. 41, 543-556 (2012).</li></ol>

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&#8217; 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....

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 that slows and arrests the rolling cell3. An immediate change in integrin avidity mediates this process. Migration requires dynamic interactions betweenTcells and endothelial cells with the formation of adhesions at the &#39;front end&#39; and breakage of adhesions at the &#39;rear end&#39; with an interval of about a minute between forming and breaking4. The IS forms inminutes, but needs to remain intact for hours6.
Interestingly, one adhesion molecule, the integrin family member lymphocyte function associated antigen (LFA)- 1, is essential for all these processes5. LFA-1 mediates adhesion through interactions with several members of the immunoglobulin superfamily. The most extensively studied ligand with the highest affinity to LFA-1 is the intercellular adhesion molecule (ICAM)-1. Non-activated circulating lymphocytes express low affinity LFA-1 on the cell surface, and therefore are unable to adhere to ICAM-1-coated surfaces. LFA-1 affinity is variable and regulated by several signaling events such as G protein coupled receptor activation, cytokine stimulation, and signals mediated by T cell receptors (TCR).The resulting high affinity form of LFA-1 conveys intracellular activation to the extracellular space through interactions with ICAM-1. This pathway is termed inside-out signaling7. Likewise, signaling through LFA-1 from the extracellular space is called outside-in signaling.
The intracellular signaling cascades involved in inside-out and outside-in signaling are a major focus of current research. The small GTPase Rap1 has recently emerged as the key component of inside-out signaling that is common to both TCR ligation and cytokine signaling8. The critical role of Rap1 in integrin activation is highlighted by the discovery that overexpression of Rap1 stimulates integrin-dependent adhesion of T cells, whereas T cell adhesion is blocked by expression of dominant negative Rap19. These advances in our understanding of integrin regulation by Rap1 have been accomplished using in&#160;vitro tools. Among them is the static adhesion assay described here.
The overall goal of this method is to study T cell adhesiveness to ICAM-1 coated surfaces. More specifically, it is used to objectively measure and quantify LFA-1 affinity toward its counter ligands in live cells at real time, under different conditions. This technique uses polystyrene wells coated with ICAM-1 to mimic the cellular surfaces that the T cells interact with. Many previously described static T cell adhesion assays were experimentally complex. These assays often required for T cells to be radioactively labeled, utilized cultured bovine corneal cells to create an extracellular matrix as the substrate for T cell adhesion, or called for non-physiological T cell stimulation over an extended duration to promote T cell adhesion10. The use of fluorometric measurement to quantify T cells following the adhesion incubation is a more sensitive and accurate method of quantification as compared to flow cytometry and microscopy, as are utilized in many other assay systems11. Additionally, single cell microscopic analysis of integrin localization does not allow for the broad, population based analysis in the same way as the fluorometric measurement. While activation state-specific LFA-1 antibodies are commercially available, these antibodies offer low sensitivity relative to the method outlined here. The main advantage over alternative techniques is its simplicity and the ability to examine multiple experimental conditions simultaneously. When considering this method for a specific application, one should take into account that T cells should be negatively selected, freshly isolated, and labeled with a fluorescent marker.

<table border="0" cellpadding="0" cellspacing="0" width="828">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Plate reader</td>
			<td style="width:160px;">BioTek&#160;</td>
			<td style="width:136px;">Synergy H1 Hybrid</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Multichannel pipette</td>
			<td style="width:160px;">Fisher</td>
			<td style="width:136px;">21-377-829</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">96-well microplate with optical bottom</td>
			<td style="width:160px;">Corning Costar</td>
			<td style="width:136px;">3603</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Recombinant ICAM-1</td>
			<td style="width:160px;">R&#38;D Systems</td>
			<td style="width:136px;">ADP4-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Anti-CD3 antibodies</td>
			<td style="width:160px;">Ancell</td>
			<td style="width:136px;">144-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">PMA</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:136px;">79346</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">BSA</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:136px;">A2058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">CFSE</td>
			<td style="width:160px;">Molecular Probes</td>
			<td style="width:136px;">C1157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">RPMI</td>
			<td style="width:160px;">Gibco</td>
			<td style="width:136px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">DPBS containing calcium and magnesium</td>
			<td style="width:160px;">Gibco</td>
			<td style="width:136px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Peripheral lymphocytes</td>
			<td style="width:160px;">e.g. mouse or human</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:444px;">Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>499609</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Open Gen 5 software</td>
			<td>BioTek</td>
			<td>Version 2.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ficoll-Paque PLUS</td>
			<td>GE Healthcare</td>
			<td>71-7167-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 and 50 mL centrifuge tubes</td>
			<td>Fisher</td>
			<td>352099, 352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable plastic Pasteur pipettes</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-75 culture flasks</td>
			<td>Fisher</td>
			<td>50-754-1366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RosetteSep T cell enricment kit</td>
			<td>StemCell Technologies</td>
			<td>15061</td>
			<td></td>
		</tr>
	</tbody>
</table>

static adhesion assay for the study of integrin activation in t lymphocytes

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 &#945;L and &#946;2, 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&#160;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.&#160;VLA-4 - fibronectin).

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...

Watch this Scientific Journal Video about Static Adhesion Assay for the Study of Integrin Activation in T Lymphocytes at JoVE.com

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

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.

T lymphocyte adhesion is required for multiple T cell functions, including migration to sites of inflammation and formation of immunological synapses with ...

static-adhesion-assay-for-study-integrin-activation-t

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

Cell-Matrix Interactions

SCIENTISTS IN ACTION

15.8K visualizações.  New York University School of Medicine.  Ensaio de adesão estático é uma ferramenta poderosa que pode ser utilizado para modelar as interacções entre linfócitos T e de outros tipos de células. Interacções são geradas por injecção de células T marcadas em poços revestidos com moléculas de adesão, enquanto que um leitor de placa é utilizada para quantificar o número de células aderentes a seguir lavagens em série. 