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 of 10 mM NaPO4, pH 8.5.</li>
		<li>Add 125 &#956;mol (0.55 mg) of NHS-biotin (from a 100 mg/mL stock in DMSO) and check the pH of the reaction. If the pH is below 8.5, add 2 &#956;L of 4 N NaOH to raise it to between pH 8.5 and 9.5.</li>
		<li>Vortex for 30 min.</li>
		<li>Quench the reaction with 50 &#956;L of 100 mM glycine dissolved in 20 mM Tris pH 8.0.</li>
		<li>Spin at full power for 10 min and transfer the supernatant to a new tube. 
		NOTE: Bio-PLL quality is typically compared to previous preparations by coating serial two-fold dilutions of the material onto a 96-well ELISA plate and quantifying biotin content after incubation with alkaline phosphatase coupled streptavidin.</li>
	</ol>
	</li>
	<li>Remove blocking buffer. Do not allow wells to dry. Add streptavidin (100 &#181;g/mL in blocking buffer). Incubate for 1 h at 4 &#176;C.</li>
	<li>Wash in HBS. Fill and invert chamber slide wells 4 - 5 times, removing HBS from the wells. Do not allow wells to dry.</li>
	<li>Add the biotinylated pMHC ligands and adhesion molecules to the surface. 
	NOTE: MCC and OVA can be photocaged by adding ortho-nitrobenzyl based protecting groups (e.g., nitrophenylethyl (NPE) or nitroveratryloxycarbonyl (NVOC)) to the &#949;-amino group of key lysines (K12 in MCC, K7 in OVA). Photocaged MCC and OVA can be obtained from commercial vendors. After reconstitution in aqueous buffer, they are refolded into bacterially expressed I-Ek and H2-Kb using established approaches11,12.</li>
	<li>Validation of photoactivatable MCC or OVA peptide
	<ol>
		<li>Decage NVOC-protected peptides by UV irradiating with a handheld UV lamp (see Table of Materials) for 20 min at RT.</li>
		<li>Pulse 1.0 x 105 APCs with 1 &#181;M of nonstimulatory peptide (negative control, e.g. Hb), unirradiated photoactivatable peptide, irradiated photoactivatable peptide, and agonist peptide (positive control, e.g., MCC) for 1 h to overnight at 37 &#176;C.</li>
		<li>To stimulate T cells expressing the 5C.C7/2B4/AND TCR, use CH12 or CH27 B cells. To stimulate OT1 T cells, use RMA-s or EL4 thymoma cells.</li>
		<li>Mix the pulsed APCs with an equal number of T cells in 96-well round bottom plates at a final volume of 200 &#181;L. Incubate at 37 &#176;C for 12 - 24 h.</li>
		<li>Recover the supernatants from each well and analyze IL-2 by ELISA with streptavidin-horseradish peroxidase colorimetric detection. 
		NOTE: Confirmation of the caging status of all peptides by electrospray mass spectrometry prior to refolding into MHC complexes, is strongly recommended.</li>
	</ol>
	</li>
	<li>Perform the folding and purification of MHC so as to minimize exposure to light. For example, wrap folding reactions in aluminum foil and carry out gel filtration chromatography with the UV lamp off. 
	NOTE: It has been found that photoactivatable MCC- I-Ek and OVA- H2-Kb induce UV independent T cell activation at high density, possibly due to incomplete functional caging. Hence, the photoactivatable pMHC is diluted into a 10 - 30x excess of nonstimulatory pMHC (e.g. I-Ek containing the hemoglobin64-76 peptide (Hb)) during the immobilization step. This enhances the signal to noise ratio of the subsequent imaging experiment.</li>
	<li>To photoactivate CD4+ T cells, collectively use a mixture of biotinylated Hb-I-Ek (3 &#181;g/mL), biotinylated photoactivatable MCC- I-Ek (0.1 &#181;g/mL), and biotinylated antibody against the class I MHC H2-Kk (0.5 &#181;g/mL). The anti-H2-Kk antibody encourages 5C.C7/2B4/AND T cells, which express H2-Kk, to spread onto the glass surface without undergoing activation.</li>
	<li>For CD8+ T cells, use a mixture of biotinylated H2-Db bearing the peptide KAVYDFATL (1 &#181;g/mL), biotinylated photoactivatable OVA-H2-Kb (0.1 &#181;g/mL), and the extracellular domain of the adhesion molecule ICAM-1 (2 &#181;g/mL produced by insect cell culture13). The ICAM-1 encourages close contact formation by engaging the integrin LFA1 on the T cell surface.</li>
	<li>Apply all protein mixtures in blocking buffer, followed by incubation for 1 h at RT or at least 2 h at 4 &#176;C. 
	NOTE: The molecular density on surfaces of this kind to be ~8000 per &#181;m2 has been previously determined6. Given that photoactivatable pMHC represents ~1/30th of the biotinylated protein on the surface, its density will be ~267 molecules per &#181;m2, prior to decaging.</li>
	<li>Wash as in step 1.6 and leave in HBS until ready to use.</li>
	<li>Add 200,000 CD4+ or CD8+ T cells expressing the appropriate TCR into each well and allow cells to adhere at 37 &#176;C for 15 min. Once cells have attached to and spread on the surface, they are ready for photoactivation and imaging. 
	NOTE: Retroviral transduction of effector T cells with fluorescent imaging probes is described in detail elswhere6,7. Calcium signaling can also be studied using untransduced T cells loaded with the calcium sensitive dye Fluo-46. The ratiometric dye Fura-2 is not recommended because it requires excitation in the UV range, which also induces decaging of the photoactivatable pMHC.</li>
</ol>
2. Image Acquisition
<ol>
	<li>Use an inverted TIRF microscope outfitted with a UV compatible 150X objective lens for image acquisition. UV irradiate user-defined regions using a digital diaphragm system attached to a 100 W mercury lamp (HBO). Direct UV light from this lamp onto the sample using a 400 nm long pass mirror.</li>
	<li>Use image analysis software for localized photoactivation and time-lapse acquisition. In most experiments, monitor probes in both the green and red channels using 488 nm and 561 nm excitation lasers, respectively. Laser light is directed onto the sample using a dual-bandpass dichroic mirror that also transmits in the UV range (to enable decaging). Please see the Table of Materials and Figure 6 for additional information on microscope configuration.</li>
	<li>After mounting the chamber slide containing the T cells, adjust settings to obtain TIRF or epifluorescence illumination, as necessary. In live mode, select a field of cells that are expressing the fluorescent probe(s) of interest. Establish micron-scale regions for photoactivation beneath individual cells using software control.</li>
	<li>Begin time-lapse acquisition. Typically, 80 timepoints are acquired, with an interval of 5 s between each time point. This leaves more than enough time for sequential 488 nm and 563 nm exposures, in the case of dual color experiments.</li>
	<li>After 10 timepoints, photoactivate the selected regions by opening the digital diaphragm shutter for 1 - 1.5 s.</li>
	<li>After the time lapse is complete, select a new field of cells and repeat the process.</li>
</ol>
3. Data Analysis
NOTE: Localized photoactivation of immobilized ligands creates a well-defined, stationary stimulatory region that can be used for quantitative analysis of signaling responses and cytoskeletal remodeling events. Analysis protocols typically involve either quantifying fluorescence intensity within the irradiated region or using the irradiated region as a positional endpoint for distance measurements (e.g. for assessing polarization of the centrosome to the irradiated region). Both analysis protocols are described below. Various interactive image analysis programs can be used to make intensity and distance measurements, which can then be output for additional processing and analysis.
<ol>
	<li>Fluorescence intensity
	<ol>
		<li>Determine the fluorescence intensity (FI) of a background region outside of the cell (FIb). This will be used for background correction.
		<ol>
			<li>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. 
			NOTE: Procedural details (e.g. 3.1.1.1) refer to Slidebook software (see Table of Materials). Implementation of this protocol using other software packages (e.g., FIJI) will be slightly different.</li>
		</ol>
		</li>
		<li>Determine the FI within the photoactivated region for each time point.
		<ol>
			<li>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.</li>
		</ol>
		</li>
		<li>Subtract the background FI from the measured FI values within the region. Then, normalize the corrected FI measurements by dividing by the average, background corrected FI of the first 9 frames before photoactivation. &#916;F/F = ((FI-FIb)/mean(FI1-9)-FIb)). 
		NOTE: Graph &#916;F/F as a function of time.</li>
	</ol>
	</li>
	<li>Distance
	<ol>
		<li>Obtain the x and y coordinates of the center of the photoactivated region.
		<ol>
			<li>To determine the x and y coordinates of the center of the photoactivated region, select 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.</li>
		</ol>
		</li>
		<li>Determine the x and y coordinates for the fluorescent probe of interest for each time point. This is typically achieved through either manual or automated particle tracking.
		<ol>
			<li>To 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 time points have been tracked, select Analyze | Mask Statistics | Center of Area and export the values.</li>
		</ol>
		</li>
		<li>Calculate the distance between the fluorescent protein of interest and the center of the photoactivated region for each time point using the equation: Distance = &#8730;((x2-x1)2+(y2-y1)2), in which x2 and y2 are the coordinates of the fluorescent protein of interest and x1 and y1 are the coordinates of the center of the photoactivated region.</li>
		<li>Graph the distance as a function of time.</li>
	</ol>
	</li>
</ol>

<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>

The authors have nothing to disclose.

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 ...

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

Research

JoVE Journal

Immunology and Infection

6.2K Views.  Memorial Sloan-Kettering Cancer Center. This protocol describes an imaging-based method to activate T lymphocytes using photoactivatable peptide-MHC, enabling precise spatiotemporal control of T cell activation.

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

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 ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...

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 ...

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and damaging the testes. During an infection with the ZIKV, immune cells have been shown to infiltrate into the tissues. However, the cellular mechanisms that define the protection and/or immunopathogenesis of these immune cells during a ZIKV infection are still largely unknown. Herein, we describe methods to evaluate the virus-specific T-cell functionality in these immunoprivileged organs of ZIKV-infected mice. These methods include a) a ZIKV infection and vaccine inoculation in Ifnar1-/- mice; b) histopathology, immunofluorescence, and immunohistochemistry assays to detect the virus infection and inflammation in the brain, testes, and spleen; c) the preparation of a tetramer of ZIKV-derived T-cell epitopes; d) the detection of ZIKV-specific T cells in the monocytes isolated from the brain, testes, and spleen. Using these approaches, it is possible to detect the antigen-specific T cells that have infiltrated into the immunoprivileged organs and to evaluate the functions of these T cells during the infection: potential immune protection via virus clearance and/or immunopathogenesis to exacerbate the inflammation. These findings may also help to clarify the contribution of T cells induced by the immunization against ZIKV.

Evaluation of Zika Virus-specific T-cell Responses in Immunoprivileged Organs of Infected Ifnar1-/- Mice

A protocol to evaluate antigen-specific T-cell responses in the immunoprivileged organs of the Ifnar1-/- murine model for the Zika virus (ZIKV) infection is described. This method is pivotal for investigating the cellular mechanisms of the protection and immunopathogenesis of ZIKV vaccines and is also valuable for their efficacy evaluation.

The Zika virus (ZIKV) can induce inflammation in immunoprivileged organs (e.g., the brain and testis), leading to the Guillain-Barr&#233; syndrome and ...