Name: spatial and temporal control of t cell activation using a photoactivatable agonist
Uploaded: 2018-04-25T13:00:00
Description: Watch this Scientific Journal Video about Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist at JoVE.com

We thank members of the Huse lab for advice and assistance. Supported by the US National Institutes of Health (R01-AI087644 to M.H. and P30-CA008748 to Memorial Sloan-Kettering Cancer Center).

1. Preparation of Stimulatory Glass Surfaces
<ol>
	<li>Coat eight-well chambered coverglass with biotinylated poly-L-lysine (Bio-PLL) diluted 1:500 in distilled, deionized water (ddH2O). Incubate for 30 min at room temperature (RT).</li>
	<li>Wash with H2O.</li>
	<li>Dry for 2 h at RT.</li>
	<li>Block Bio-PLL coated surfaces with blocking buffer (HEPES-buffered saline [10 mM HEPES pH 7.4, 150 mM NaCl], with 2% BSA) for 30 min at RT.
	<ol>
		<li>Dissolve 5 mg of poly-L-lysine hydrobromide in 1 mL o...

<ol>
	<li>Dustin, M. L., Chakraborty, A. K., Shaw, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+structure+and+function+of+the+immunological+synapse.">Understanding the structure and function of the immunological synapse.</a> Cold Spring Harb.Perspect.Biol. 2, a002311 (2010).</li><li>Liu, X., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+Synapse+Formation:+Cell+Polarity+During+T+Cell-APC+Interaction.">Immunological Synapse Formation: Cell Polarity During T Cell-APC Interaction.</a> Cell Polarity 1. , 247-275 (2015).</li><li>Bunnell, S. C., Kapoor, V., Trible, R. P., Zhang, W., Samelson, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+actin+polymerization+drives+T+cell+receptor-induced+spreading:+A+role+for+the+signal+transduction+adaptor+LAT.">Dynamic actin polymerization drives T cell receptor-induced spreading: A role for the signal transduction adaptor LAT.</a> Immunity. 14, 315-329 (2001).</li><li>Dustin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insights+into+Function+of+the+Immunological+Synapse+from+Studies+with+Supported+Planar+Bilayers.">Insights into Function of the Immunological Synapse from Studies with Supported Planar Bilayers.</a> Current topics in microbiology and immunology. 340, (2010).</li><li>Chauveau, A., Le Floc'h, A., Bantilan, N. S., Koretzky, G. a., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diacylglycerol+kinase+&#945;+establishes+T+cell+polarity+by+shaping+diacylglycerol+accumulation+at+the+immunological+synapse.">Diacylglycerol kinase &#945; establishes T cell polarity by shaping diacylglycerol accumulation at the immunological synapse.</a> Sci. Signal. 7, ra82 (2014).</li><li>Huse, 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=Spatial+and+Temporal+Dynamics+of+T+Cell+Receptor+Signaling+with+a+Photoactivatable+Agonist.">Spatial and Temporal Dynamics of T Cell Receptor Signaling with a Photoactivatable Agonist.</a> Immunity. 27, 76-88 (2007).</li><li>Quann, E. J., Merino, E., Furuta, T., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+diacylglycerol+drives+the+polarization+of+the+microtubule-organizing+center+in+T+cells.">Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells.</a> Nat Immunol. 10, 627-635 (2009).</li><li>Basu, R., Chen, Y., Quann, E. J., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+variable+hinge+region+of+novel+PKCs+determines+localization+to+distinct+regions+of+the+immunological+synapse.">The variable hinge region of novel PKCs determines localization to distinct regions of the immunological synapse.</a> PLoS One. 9, 1-8 (2014).</li><li>Liu, X., Kapoor, T. M., Chen, J. K., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diacylglycerol+promotes+centrosome+polarization+in+T+cells+via+reciprocal+localization+of+dynein+and+myosin+II.">Diacylglycerol promotes centrosome polarization in T cells via reciprocal localization of dynein and myosin II.</a> Proc. Natl. Acad. Sci. U. S. A. 110, 11976-11981 (2013).</li><li>Quann, E. J., Liu, X., Altan-Bonnet, G., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cascade+of+protein+kinase+C+isozymes+promotes+cytoskeletal+polarization+in+T+cells.">A cascade of protein kinase C isozymes promotes cytoskeletal polarization in T cells.</a> Nat. Immunol. 12, 647-654 (2011).</li><li>Boniface, J. J., Reich, Z., Lyons, D. S., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermodynamics+of+T+cell+receptor+binding+to+peptide-MHC:+evidence+for+a+general+mechanism+of+molecular+scanning.">Thermodynamics of T cell receptor binding to peptide-MHC: evidence for a general mechanism of molecular scanning.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 11446-11451 (1999).</li><li>Garboczi, D. N., Hung, D. T., Wiley, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HLA-A2-peptide+complexes:+refolding+and+crystallization+of+molecules+expressed+in+Escherichia+coli+and+complexed+with+single+antigenic+peptides.">HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides.</a> Proc. Natl. Acad. Sci. U. S. A. 89, 3429-3433 (1992).</li><li>Huse, M., Lillemeier, B. F., Kuhns, M. S., Chen, D. S., Davis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cells+use+two+directionally+distinct+pathways+for+cytokine+secretion.">T cells use two directionally distinct pathways for cytokine secretion.</a> Nat. Immunol. 7, 247-255 (2006).</li><li>Abeyweera, T. P., Merino, E., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+signaling+blocks+activating+receptor+clustering+and+induces+cytoskeletal+retraction+in+natural+killer+cells.">Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells.</a> J. Cell Biol. 192, 675-690 (2011).</li><li>Nguyen Duc, T., Huse, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Generalizable+Platform+for+the+Photoactivation+of+Cell+Surface+Receptors.">A Generalizable Platform for the Photoactivation of Cell Surface Receptors.</a> ACS Chem. Biol. 10, 2435-2440 (2015).</li><li>Airan, R. D., Thompson, K. R., Fenno, L. E., Bernstein, H., Deisseroth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally+precise+in+vivo+control+of+intracellular+signalling.">Temporally precise in vivo control of intracellular signalling.</a> Nature. 458, 1025-1029 (2009).</li><li>Xu, Y., 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=Optogenetic+control+of+chemokine+receptor+signal+and+T-cell+migration.">Optogenetic control of chemokine receptor signal and T-cell migration.</a> Proc. Natl. Acad. Sci. 111, 6371-6376 (2014).</li><li>Levskaya, A., Weiner, O. D., Lim, W. A., Voigt, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+cell+signalling+using+a+light-switchable+protein+interaction.">Spatiotemporal control of cell signalling using a light-switchable protein interaction.</a> Nature. 461, 997-1001 (2009).</li><li>Guntas, 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=Engineering+an+improved+light-induced+dimer+(iLID)+for+controlling+the+localization+and+activity+of+signaling+proteins.">Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.</a> Proc. Natl. Acad. Sci. 112, 112-117 (2015).</li><li>Wu, Y. I., 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=A+genetically+encoded+photoactivatable+Rac+controls+the+motility+of+living+cells.">A genetically encoded photoactivatable Rac controls the motility of living cells.</a> Nature. 461, 104-108 (2009).</li><li>Pathak, G. P., Vrana, J. D., Tucker, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+cell+function+using+engineered+photoreceptors.">Optogenetic control of cell function using engineered photoreceptors.</a> Biol. cell/under auspices Eur. Cell Biol. Organ. 105, 59-72 (2014).</li><li>Grusch, 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=Spatio-temporally+precise+activation+of+engineered+receptor+tyrosine+kinases+by+light.">Spatio-temporally precise activation of engineered receptor tyrosine kinases by light.</a> EMBO J. 33, 1713-1726 (2014).</li><li>Yi, J., 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=Centrosome+repositioning+in+T+cells+is+biphasic+and+driven+by+microtubule+end-on+capture-shrinkage.">Centrosome repositioning in T cells is biphasic and driven by microtubule end-on capture-shrinkage.</a> J. Cell Biol. 202, 779-792 (2013).</li></ol>

In recent years, light has emerged as an excellent tool for spatiotemporally controlled activation of cellular processes. Various methodologies have been developed, each with associated advantages and disadvantages. The system described here, which is based on the decaging of immobilized, extracellular ligands, is ideally suited for the analysis of rapid, subcellular, polarized signaling responses. This approach has been applied to examine IS formation in T cells as described above. Additionally, caged ligands for other ...

T lymphocytes (T cells) play a central role in cellular immunity by recognizing antigenic peptides displayed in the context of cell surface MHC. Antigen recognition, which is mediated by the TCR, drives the differentiation of na&#239;ve T cells and promotes the delivery of cytolytic and communicative responses by effector populations. TCR engagement also induces dramatic changes in cellular architecture. Within minutes, the T cells gloms onto the side of the antigen-presenting cell (APC), forming a polarized interface known as the immunological synapse (IS)1,2. The IS potentiates T cell effector responses by enabling the directional release of cytokines or, in the case of cytotoxic T lymphocytes (CTLs), lytic proteins that destroy the APC.
TCR engagement by pMHC induces the rapid phosphorylation of multiple downstream adaptor molecules, including Linker for the Activation of T cells (LAT), which ultimately promotes robust remodeling of the synaptic cytoskeleton2. Cortical filamentous actin (F-actin) drives T cell spreading over the APC surface, and then resolves into an annular structure characterized by F-actin accumulation at the IS periphery and depletion from the center. F-actin ring formation is tightly coupled to the reorientation of the microtubule organizing center (MTOC, also called the centrosome in T cells) to a position just beneath the center of the interface. Both events occur within minutes of initial antigen recognition and establish the architectural context in which subsequent activation events and effector responses occur.
To study IS formation, various labs have developed approaches in which the APC is replaced by a glass surface that either contains immobilized TCR ligands or supports a lipid bilayer that itself contains the ligands3,4. T cells form IS-like contacts on these surfaces that can be imaged by total internal reflection fluorescence microscope (TIRF) or confocal microscopy, enabling high-resolution studies of early T cell activation and IS formation.
Although these approaches have allowed for excellent visualization of the fully assembled IS, much of the signaling following TCR:pMHC ligation occurs within seconds, complicating efforts to determine the sequence of events following TCR activation accurately. To circumvent this issue, a photoactivation approach has been developed, in which photoactivatable pMHC is used to achieve spatiotemporal control of TCR activation5,6,7. In this system, T cells are attached to glass surfaces containing photoactivatable pMHC that is non-stimulatory to the TCR until irradiated with ultraviolet (UV) light. UV irradiation of a micron sized region of the surface beneath the T cell removes the photocage creating a stimulatory zone that can be recognized by the T cell. Subsequent signaling events and cytoskeletal remodeling are then monitored using genetically encoded fluorescent reporters. Two photoactivatable versions of antigenic peptides, moth cytochrome c88-103 (MCC) and ovalbumin257-264 (OVA), which are presented in the context of the class II MHC I-Ek and the class I MHC H2-Kb, respectively, have been developed (Figure 1). This enables the analysis of both CD4+ T cells specific for MCC- I-Ek (expressing the 5C.C7, 2B4, or AND TCRs) and CD8+ T cells specific for OVA-H2-Kb (expressing the OT1 TCR).
Over the past decade, the TCR photoactivation and imaging approach has been utilized to establish the precise kinetics of early TCR signaling steps and also to identify the molecular pathways governing polarized cytoskeletal remodeling5,6,7,8,9,10. For example, the assay was instrumental in determining that centrosome reorientation toward the APC is mediated by a localized gradient of the lipid second messenger diacylglycerol centered at the IS. It is anticipated that this methodology will continue to be valuable for applications that demand high-resolution imaging analysis of T cell function.

<table border="0" cellpadding="0" cellspacing="0" width="1152">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Nunc Lab-Tek Chambered Coverglass</td>
			<td style="width:211px;">Thermofischer Scientific</td>
			<td align="right" style="width:119px;">155361</td>
			<td style="width:443px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine hydrobromide</td>
			<td>Sigma-Aldrich</td>
			<td>P2636</td>
			<td>Will need to make Biotinylated Poly-L-Lysine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZ-Link NHS-Biotin</td>
			<td>Thermofischer Scientific</td>
			<td align="right">20217</td>
			<td>Will need to make Biotinylated Poly-L-Lysine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin</td>
			<td style="width:211px;">Thermofischer Scientific</td>
			<td align="right">434301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BirA-500: BirA biotin-protein ligase standard reaction kit</td>
			<td>Avidity</td>
			<td>BirA500</td>
			<td>Will be used to biotinylate proteins</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated Hb I-EK&#160;</td>
			<td style="width:211px;"></td>
			<td style="width:119px;"></td>
			<td>For protein folding, see reference 6. For biotinylation, use BirA kit</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated NPE-MCC I-EK&#160;</td>
			<td>Anaspec</td>
			<td style="width:119px;"></td>
			<td>Custom NPE-MCC (H-ANERADLIAYL-K(Nvoc)-QATK-OH) can be purchased from Anaspec</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated &#945;H2-Kk antibody</td>
			<td style="width:211px;">BD Biosciences</td>
			<td align="right">553591</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated NPE-OVA H2-Kb</td>
			<td style="width:211px;">Anaspec</td>
			<td style="width:119px;"></td>
			<td>Custom NPE-OVA (H-SIINFE-K(Nvoc)-L-OH) can be purchased from Anaspec</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Biotinylated KAVY H2-Db&#160;</td>
			<td style="width:211px;">Anaspec</td>
			<td style="width:119px;"></td>
			<td>Custom synthesized protein (KAVYDFATL) can be purchased from Anaspec</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated ICAM1&#160;</td>
			<td style="width:211px;"></td>
			<td style="width:119px;"></td>
			<td>For protein folding, see reference in protocol. For biotinylation, use BirA kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Hand held UV lamp</td>
			<td style="width:211px;">UVP</td>
			<td style="width:119px;">UVGL-25</td>
			<td>Lamp is held &#60; 1 cm from the sample.&#160; 30 s of 365 light is sufficient for detectable decaging, 20 min for quantitative decaging.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Olympus IX-81 OMAC TIRF system.</td>
			<td style="width:211px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Additional information about the imaging system can be found in Figure 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Mosaic digital diaphragm</td>
			<td style="width:211px;">Andor</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:379px;">Slidebook software</td>
			<td style="width:211px;">Intelligent Imaging Innovations</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

spatial and temporal control of t cell activation using a photoactivatable agonist

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological synapse, a stereotyped cell-cell junction that regulates T cell activation and directionally targets effector responses. To study these processes effectively, an imaging approach that is tailored to capturing fast, polarized responses is necessary. This protocol describes such a system, which is based on a photoactivatable peptide-major histocompatibility complex (pMHC) that is non-stimulatory until it is exposed to ultraviolet light. Targeted decaging of this reagent during videomicroscopy experiments enables precise spatiotemporal control of TCR activation and high-resolution monitoring of subsequent cellular responses by total internal reflection (TIRF) imaging. This approach is also compatible with genetic and pharmacological perturbation strategies. This allows for the assembly of well-defined molecular pathways that link TCR signaling to the formation of the polarized cytoskeletal structures that underlie the immunological synapse.

The photoactivation and imaging approach allows for observation and facile quantification of rapid, polarized signaling responses. To illustrate its capabilities, reproduced here is an experiment examining the spatiotemporal correlation between TCR-induced DAG accumulation and centrosome reorientation. 5C.C7 T cell blasts were retrovirally transduced with two fluorescent reporters: a DAG biosensor containing the tandem C1 domains from protein kinase C-&#952; linked to GFP (C1-GFP) and RFP...

Watch this Scientific Journal Video about Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist at JoVE.com

Spatial and Temporal Control of T Cell Activation Using a Photoactivatable Agonist

This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

T lymphocytes engage in rapid, polarized signaling, occurring within minutes following TCR activation. This induces formation of the immunological ...

The overall goal of this protocol is to describe the use of photoactivatable peptide MHC to induce polarized signaling in T lymphocytes. This method can help answer key questions in immune cell signal transduction such as how lymphocytes transduce rapid localized signals and then mount polarized cellular responses to those signals. The main advantage of this technique is that it offers spatial temporal control of T cell signaling.It does this by fixing the precise time and location of T cell receptor activation. Demonstrating the procedure will be Elisa Sanchez, a grad student from my laboratory. Begin by adding biotinylated poly-L-lysine, or bio-PLL, diluted one to 500 in distilled deionized water to eight-well chambered cover glass.Incubate for 30 minutes at room temperature. Following the incubation, wash with distilled deionized water and then dry for two hours at room temperature. Block bio-PLL coated surfaces with blocking buffer consisting of HEPES buffered saline with 2%BSA for 30 minutes at room temperature.Following the incubation, remove the blocking buffer from the bio-PLL coated surfaces without allowing the wells to dry. Then add streptavidin and incubate at four degrees Celsius. After an hour, wash the coated surfaces in HBS.Fill and invert the chamber slide wells four to five times, removing HBS from the wells. Do not allow the wells to dry. Add the specific biotinylated photoactivatable peptide major histocompatibility complex ligands and adhesion molecules for either CD4 positive T cells or CD8 positive cells to the bio-PLL coated surfaces.Protect from light and incubate for one hour at room temperature. After the incubation, wash as before and leave in HBS until ready to seed. Finally, add 200, 000 CD4 positive or CD8 positive T cells expressing the appropriate TCR into the correct wells and allow the cells to adhere at 37 degrees Celsius for 15 minutes.Once the cells have attached to and spread on the surface, they are ready for photoactivation and imaging. Use an inverted total internal reflection fluorescence microscope outfitted with a UV-compatible 150X objective lens for image acquisition. Monitor probes in both the green and red channels using 488 nanometer and 561 nanometer excitation lasers, respectively.After mounting the chamber slide containing the T cells, adjust the microscope settings to obtain TIRF or epifluorescence illumination, as necessary. In Live mode, select a field of cells that are expressing the fluorescent probes of interest. Establish micron scale regions for photoactivation beneath individual cells using software control.Then, start the acquisition. Typically, 80 time points are required with an interval of five seconds between each. This leaves time for sequential 488 nanometer and 563 nanometer exposures in the case of dual color experiments.Then, after acquiring 10 time points, photoactivate the selected regions by opening the digital diaphragm shutter for one to 1.5 seconds. After the time lapse is complete, select a new field of cells and repeat the process. Begin by determining the fluorescence intensity of a background region outside of the cell to be used for background correction.Draw a micron-sized square region outside of the cell and make a mask. To determine the fluorescence intensity, click on Analyze, Mask Statistics. Select Mean fluorescence intensity and export the values.Determine the fluorescence intensity within the photoactivated region for each time point. Select Mask to highlight the region that was photoactivated. To determine the fluorescence intensity, click on Analyze, Mask Statistics.Select Mean fluorescence intensity and Export the values. Obtain the X and Y coordinates of the center of the photoactivated region by selecting Mask to highlight the region that was photoactivated. Once the region of photoactivation is highlighted, select Analyze, Mask Statistics, Center of Area, and Export the values.Determine the X and Y coordinates of the fluorescent probe of interest. Select Manual Particle Tracking and click on the fluorescent probe of interest over time. Once all of the time points have been tracked, select Analyze, Mask Statistics, Center of Area, and Export the values.T cells were attached to chambered cover glass containing photoactivatable MCC-IEFK. C1-GFP, a probe for the lipid second messenger DAG, was imaged using TIRF microscopy and RFP-Tubulin in epifluorescence mode. Localized photoactivation of the surface beneath the T cell induces the accumulation of DAG in the irradiated zone.This is followed within seconds by the reorientation of the centrosome to the same region. C1-GFP accumulation can be quantified by calculating the normalized fluorescence intensity after background correction at the center of the photoactivated region over time. Centrosome reorientation in response to photoactivation can be quantified by calculating the distance between the centrosome and the center of the photoactivated region as a function of time.Once mastered, this technique can be done in less than a day if it is performed properly. We typically generate 15 to 20 time lapse movies for analysis in this time. This approach paved the way for researchers to explore the precise kinetics and polarization of activating signaling in T cells.It was also adapted to study inhibitory signaling in natural killer cells. As a result, we now have a better understanding of how communicative immune cell cell interactions are assembled and dissolved.

spatial-temporal-control-t-cell-activation-using-photoactivatable

Research

JoVE Journal

Immunology and Infection

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Artificial Antigen Presenting Cell (aAPC) Mediated Activation and Expansion of Natural Killer T Cells

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike classical T cells, NKT cells recognize lipid antigen in the context of CD1 molecules2. NKT cells express an invariant TCR&alpha; chain rearrangement: V&alpha;14J&alpha;18 in mice and V&alpha;24J&alpha;18 in humans, which is associated with V&beta; chains of limited diversity3-6, and are referred to as canonical or invariant NKT (iNKT) cells. Similar to conventional T cells, NKT cells develop from CD4-CD8- thymic precursor T cells following the appropriate signaling by CD1d 7. The potential to utilize NKT cells for therapeutic purposes has significantly increased with the ability to stimulate and expand human NKT cells with &alpha;-Galactosylceramide (&alpha;-GalCer) and a variety of cytokines8. Importantly, these cells retained their original phenotype, secreted cytokines, and displayed cytotoxic function against tumor cell lines. Thus, ex vivo expanded NKT cells remain functional and can be used for adoptive immunotherapy. However, NKT cell based-immunotherapy has been limited by the use of autologous antigen presenting cells and the quantity and quality of these stimulator cells can vary substantially. Monocyte-derived DC from cancer patients have been reported to express reduced levels of costimulatory molecules and produce less inflammatory cytokines9,10. In fact, murine DC rather than autologous APC have been used to test the function of NKT cells from CML patients11. However, this system can only be used for in vitro testing since NKT cells cannot be expanded by murine DC and then used for adoptive immunotherapy. Thus, a standardized system that relies on artificial Antigen Presenting Cells (aAPC) could produce the stimulating effects of DC without the pitfalls of allo- or xenogeneic cells12, 13. Herein, we describe a method for generating CD1d-based aAPC. Since the engagement of the T cell receptor (TCR) by CD1d-antigen complexes is a fundamental requirement of NKT cell activation, antigen: CD1d-Ig complexes provide a reliable method to isolate, activate, and expand effector NKT cell populations.

Artificial Antigen Presenting Cell aAPC Mediated Activation and Expansion of Natural Killer T Cells

Here we describe a method for activating and expanding human NKT cells from bulk T cell populations using artificial antigen presenting cells (aAPC). The use of CD1d-based aAPC provides a standardized method for generating high numbers of functional NKT cells.

Natural killer T (NKT) cells are a unique subset of T cells that display markers characteristic of both natural killer (NK) cells and T cells1. Unlike ...

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).

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 ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Cortical Actin Flow in T Cells Quantified by Spatio-temporal Image Correlation Spectroscopy of Structured Illumination Microscopy Data

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell interaction known as the immunological synapse. F-actin is also speculated to play a role in regulating molecular distributions at the membrane of cells including sub-membranous vesicle dynamics and protein clustering. While standard light microscope techniques allow generalized and diffraction-limited observations to be made, many cellular and molecular events including clustering and molecular flow occur in populations at length-scales far below the resolving power of standard light microscopy. By combining total internal reflection fluorescence with the super resolution imaging method structured illumination microscopy, the two-dimensional molecular flow of F-actin at the immune synapse of T cells was recorded. Spatio-temporal image correlation spectroscopy (STICS) was then applied, which generates quantifiable results in the form of velocity histograms and vector maps representing flow directionality and magnitude. This protocol describes the combination of super-resolution imaging and STICS techniques to generate flow vectors at sub-diffraction levels of detail. This technique was used to confirm an actin flow that is symmetrically retrograde and centripetal throughout the periphery of T cells upon synapse formation.

To investigate flow velocities and directionality of filamentous-actin at the T cell immunological synapse, live-cell super-resolution imaging is combined with total internal reflection fluorescence and quantified with spatio-temporal image correlation spectroscopy.

Filamentous-actin plays a crucial role in a majority of cell processes including motility and, in immune cells, the formation of a key cell-cell ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Measurement of T Cell Alloreactivity Using Imaging Flow Cytometry

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal of immunosuppression. The mixed leukocyte reaction (MLR), limiting dilution assays, and trans-vivo delayed-type hypersensitivity (DTH) assay have all been applied to this question, but these methods have limited predictive ability and/or significant practical limitations that reduce their usefulness. Imaging flow cytometry is a technique that combines the multiparametric quantitative powers of flow cytometry with the imaging capabilities of fluorescent microscopy. We recently made use of an imaging flow cytometry approach to define the proportion of recipient T cells capable of forming mature immune synapses with donor antigen-presenting cells (APCs). Using a well-characterized mouse heart transplant model, we have shown that the frequency of in vitro immune synapses among T-APC membrane contact events strongly predicted allograft outcome in rejection, tolerance, and a situation where transplant survival depends on induced regulatory T cells. The frequency of T-APC contacts increased with T cells from mice during acute rejection and decreased with T cells from mice rendered unresponsive to alloantigen. The addition of regulatory T cells to the in vitro system reduced prolonged T-APC contacts. Critically, this effect was also seen with human polyclonally expanded, naturally occurring regulatory T cells, which are known to control the rejection of human tissues in humanized mouse models. Further development of this approach may allow for a deeper characterization of the alloreactive T-cell compartment in transplant recipients. In the future, further development and evaluation of this method using human cells may form the basis for assays used to select patients for immunosuppression minimization, and it can be used to measure the impact of tolerogenic therapies in the clinic.

This paper describes a method for measuring alloreactivity in a mixed population of T cells using imaging flow cytometry.

The measurement of immunological reactivity to donor antigens in transplant recipients is likely to be crucial for the successful reduction or withdrawal ...

Highly Multiplexed, Super-resolution Imaging of T Cells Using madSTORM

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and multiplexing ability. Using fluorescent nano-diamond fiducial markers, we describe the drift correction and alignment procedures required to obtain high precision in single molecule localization microscopy. In addition, a new multiplexing strategy, madSTORM, is described in which multiple molecules are targeted in the same cell using sequential binding and elution of fluorescent antibodies. madSTORM is demonstrated on an activated T cell to visualize the locations of different components within a membrane-bound, multi-protein structure called the T cell receptor microcluster. In addition, application of madSTORM as a general tool for visualization of multi-protein structures is discussed.

We demonstrate a method to image multiple molecules within heterogeneous nano-structures at single molecule accuracy using sequential binding and elution of fluorescently labeled antibodies.

Imaging heterogeneous cellular structures using single molecule localization microscopy has been hindered by inadequate localization precision and ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...