This work was supported by Project Grants #1010395 (BQ and CP) and #525431 (CR), and a Program Grant #455395 (CP) from the National Health and Medical Research Council of Australia, an Australian Centre for Hepatitis and HIV Virology EOI 2012 grant (CR and RJJ) and a grant from the Gordon and Gretel Bootes Foundation (BQ and CR). We wish to thank Harpreet Vohra and Michael Devoy for their excellent maintenance of the JCSMR FACS laboratory, the Australian Cancer Research Foundation Biomolecular Resource Facility, JCSMR, ANU, for peptide synthesis, and Dr. David Boyle, CSIRO Animal Health Laboratories, Geelong, Australia for providing the parent HIV vaccine stocks.

Note: Mice used under this protocol were handled according to the guidelines of the Australian National University Animal Experimentation Ethics Committee and mice were euthanized by cervical dislocation.
1. Dye and Peptide Preparation
<ol>
	<li>Dye preparation 
	Note: Dyes are prediluted at different concentrations to allow labeling of cells at discrete fluorescence intensities. CFSE is used at seven concentrations made through 3.5-fold serial dilutions, and CTV and CPD are used at six different concentrations made through 3.7-fold serial dilutions (see Tables 2-4). The stock concentrations of dyes listed in Tables 2-4 will be used to label target cells at the final dye concentrations listed in Tables 2-4. Dyes react upon exposure to aqueous solution. It is therefore important that vials be equilibrated to RT prior to opening to minimize exposure of dyes to condensation.
	<ol>
		<li>CFSE 
		Note: CFSE is purchased as carboxyfluorescein diacetate succinimidyl ester (CFDA, SE; MW 557.47), typically at 25 mg/vial.
		<ol>
			<li>Resuspend 25 mg of CFSE in 4.48 ml&#160;DMSO to obtain a 10 mM stock solution.</li>
			<li>Serially dilute CFSE: Add 35 &#956;l of 10 mM CFSE stock into 87.5 &#956;l of DMSO and repeat this with diluted stock solution to give each dye concentration in Table 3.</li>
		</ol>
		</li>
		<li>CTV 
		Note: CTV is typically purchased in packs of 9 vials, each of which can be reconstituted with 10 &#956;l of DMSO to generate a 10 mM solution of the dye.
		<ol>
			<li>Reconstitute and pool all 9 vials of CTV with 90 &#956;l of DMSO to give a 10 mM solution.</li>
			<li>Serially dilute CTV: Add 11 &#956;l of 10 mM CTV stock into 29.7 &#956;l of DMSO and repeat this with diluted stock solution to give each dye concentration in Table 2.</li>
		</ol>
		</li>
		<li>CPD 
		Note: CPD (MW 792.6) is typically purchased as 0.5 mg/vial.
		<ol>
			<li>Resuspend 0.5 mg of CPD in 63 ml of DMSO to get a 10 mM solution.</li>
			<li>Serially dilute CPD: Add 11 &#956;l of 10 mM CPD stock into 29.7 &#956;l of DMSO and repeat this with diluted stock solution to give each dye concentration in Table 4. 
			Note: Dye stocks can be stored at -20 &#176;C for several months and can be thawed and refrozen several times without significant loss in function.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Peptide preparation 
	Note: MHC class-I-binding peptides are generally used to pulse target cells at 6 different concentrations made using 10 fold dilutions from a starting concentration of 1 &#956;M (Table 5). MHC class II-binding peptides are generally used to pulse target cells at 6 different concentrations made using 3 fold dilutions from a starting concentration of 400 &#956;M (Table 5). Each peptide is made at 2x final concentration in PBS such that every peptide concentration has a minimum volume of 250 &#956;l. Peptide stocks can be prepared prior to FTA construction and stored at -20 &#176;C without significant loss in function.
	<ol>
		<li>Preparing stock MHC class-I-binding peptides</li>
		<li>Prepare the highest concentration of each peptide epitope to 2 &#956;M stock solutions (2x 1 &#956;M)</li>
		<li>Serially dilute MHC class-I-binding peptides: add 44.4 &#956;l of 2 &#956;M peptide stock solution into 400 &#956;l of PBS. Repeat this with diluted stock solution to give each peptide concentration in Table 5.</li>
		<li>Preparing stock MHC class-II-binding peptides</li>
		<li>Prepare the highest concentration of each peptide epitope to 800 &#956;M stock solutions (2x 400 &#956;M).</li>
		<li>Serially dilute MHC class-II-binding peptides: add 150 &#956;l of 800 &#956;M peptide solution into 300 &#956;l of PBS. Repeat this with diluted stock solution to give each peptide concentration in Table 5.</li>
	</ol>
	</li>
</ol>
2. FTA Preparation
Note: The procedure below outlines the construction of a FTA comprised of cells pulsed with 7 different peptide epitopes at 6 different concentrations (i.e.&#160;42 cell clusters) repeated 6 times to generate 252 discernable cell clusters. Within each repeat, a cell cluster not pulsed with any epitope (Nil), is included as a control. It is helpful for FTAs to have a CD45 allotype difference from host animals to allow their discrimination from recipient cells by antibody labeling at the time of analysis. Otherwise FTAs can be labeled with other dyes such as PKH-26 for this purpose (described in step 2.6). Typically, the FTA preparation procedure is carried out from start to finish during one sitting.
<ol>
	<li>Label 42, 10 ml conical bottomed plastic tubes 1-42 (as in Table 1 for example).</li>
	<li>Preparation of cells
	<ol>
		<li>Isolate spleen and/or lymphnodes from mice. Prepare single cell suspension from tissues by mashing through a 70 &#956;m pored sieve with a 5 ml syringe plunger and count cells using a hemocytometer.</li>
		<li>Resuspend lymphocytes at up to 200 x 106 cells/ml in 11.5 ml of Rochester Park Memorial Institute 1640 (RPMI, or equivalent) containing 5% fetal calf serum (FCS). Note: It is important to use a buffer with high amine content to minimize dye toxicity to cells3.</li>
	</ol>
	</li>
	<li>CTV labeling 
	Note: Cells are initially labeled with 6 concentrations of CTV.
	<ol>
		<li>Thoroughly resuspend the cells by inverting the tube several times. Add 1.9 ml of cell suspension to 6 of the 10 ml tubes labeled 37-42, taking care not to wet the top half of the tubes.</li>
		<li>To label cells with CTV, remove the tube cap and lay the tube horizontally.</li>
		<li>Add 83 &#956;l of PBS to the non wetted portion at the top of the tube, and to this add 17 &#956;l of stock CTV (see Table 2 for which stock solution is assigned to which tube). Note: A non wetted tube is important to prevent cell suspension movement and premature mixing of the cell solution with the dye solution.</li>
		<li>Cap the tube and mix the cell suspension with the dye quickly and thoroughly by vortexing.</li>
		<li>Repeat steps 2.3.2-2.3.4 for the stock solutions of CTV in the designated tubes described in Table 2.</li>
		<li>Incubate cells for a minimum of 5 min at RT (20 &#176;C).</li>
	</ol>
	</li>
	<li>CFSE labeling
	<ol>
		<li>After CTV labeling, add 5 ml of RPMI containing 5% FCS to each tube and thoroughly resuspend the cells by vortexing.
		<ol>
			<li>From tube 37 transfer 1 ml of cell suspension to tubes 31, 25, 19, 13, 7, and 1.</li>
			<li>From tube 38 transfer 1 ml of cell suspension to tubes 32, 26, 20, 14, 8, and 2.</li>
			<li>From tube 39 transfer 1 ml of cell suspension to tubes 33, 27, 21, 15, 9, and 3.</li>
			<li>From tube 40 transfer 1 ml of cell suspension to tubes 34, 28, 22, 16, 10, and 4.</li>
			<li>From tube 41 transfer 1 ml of cell suspension to tubes 35, 29, 23, 17, 11, and 5.</li>
			<li>From tube 42 transfer 1 ml of cell suspension to tubes 36, 30, 24, 18, 12, and 6 
			Note: Take care not to wet the top half of the tubes during steps 2.4.1.1-2.4.1.6.</li>
		</ol>
		</li>
		<li>To label cells with CFSE, remove the tube cap and lay the tube horizontally.</li>
		<li>Add 103 ml of PBS to the non wetted portion at the top of the tube, and to this add 7 ml of stock CFSE (see Table 3 for which stock solution is assigned to which tube).</li>
		<li>Cap the tube and mix the cell suspension with the dye quickly and thoroughly by vortexing.</li>
		<li>Do steps 2.4.2-2.4.4 for the stock solutions of CFSE in the designated tubes described in Table 3.</li>
		<li>Incubate cells for a minimum of 5 min at RT&#160;(20 &#176;C).</li>
		<li>Wash cells: Dilute cell suspension with 9 ml of 20 &#176;C RPMI containing 5% FCS, sediment by centrifugation at 300 x g for 10 min at 20 &#176;C and remove supernatants by aspiration with a transfer pipette.</li>
	</ol>
	</li>
	<li>Peptide pulsing and cell washing 
	Note: After cells have been labeled with CTV and CFSE, they are pulsed with MHC class-I and/or MHC class-II binding peptides (prepared in 1.2). One of the cell populations must also not be pulsed with peptide (e.g.&#160;Nil in Table 1) and used as a negative control for calculation of T cell responses.
	<ol>
		<li>Peptide pulsing
		<ol>
			<li>Resuspend cells in a total volume of 250 &#956;l of RPMI containing 5% FCS. Note: Typically 50 &#956;l of cell suspension remains after aspiration of the supernatant at the end of step 2.4.7, therefore add 200 &#956;l of medium to cell pellet.</li>
			<li>Add 250 &#956;l of pre prepared peptide stocks (as in Table 5) to appropriately designated tubes (as in Table 1) and be sure to include a control tube of PBS added alone without peptide as a Nil control.</li>
			<li>Mix cell suspensions with a vortex. Note: It is critical that each peptide epitope and each peptide concentration is assigned to a single tube and this recorded clearly based on the expected fluorescence of the cells in this tube, since the fluorescence signature of this cluster will define this peptide (as in Table 1 for example).</li>
			<li>Incubate the cells at 37 &#176;C for 1 hr.</li>
		</ol>
		</li>
		<li>Cell washing
		<ol>
			<li>Add 5 ml of ice cold (4 &#176;C) RPMI containing 5% FCS to the cell suspension and resuspend cells by inverting tube. Carefully underlay the cell suspension with 3 ml of ice cold (4 &#176;C) FCS.</li>
			<li>Sediment the cells by centrifugation at 300 x g for 10 min at 4 &#176;C. Use slow acceleration and braking to ensure the interface of FCS and the cell suspension solution is maintained.</li>
			<li>Carefully aspirate off the RPMI and then the FCS with a transfer pipette, leaving the washed cell pellets undisturbed. Note: The use of a FCS underlay to wash cells helps to ensure as much peptide solution is removed from the cells as possible and thereby limiting exposure of cell populations to multiple free peptides when cells are pooled.</li>
			<li>Wash cells again: Resuspend the cell pellets in 10 ml of 4 &#176;C RPMI containing 5% FCS. Sediment the cells by centrifugation at 300 x g for 10 min at 4 &#176;C. Pour off supernatants.</li>
			<li>Pool all cell populations together into a single tube with a pipette using 6 ml of 4 &#176;C RPMI containing 5% FCS. Sediment pooled cells by centrifugation at 300 x g for 10 min at 4 &#176;C. Aspirate off supernatant&#160;with a transfer pipette.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>CPD Cell labeling 
	Note: At this point six intra assay replicates can be generated by labeling peptide pulsed cells with 6 different concentrations of CPD.
	<ol>
		<li>Add 11.4 ml of 20 &#176;C RPMI containing 5% FCS to the pooled cell pellet and resuspend thoroughly using a pipette.</li>
		<li>Add 1.9 ml of cell suspension to 6, 10 ml tubes labeled A-F, taking care not to wet the top half of the tubes.</li>
		<li>To label cells with CPD, remove the tube cap and lay the tube horizontally.</li>
		<li>Add 92 &#956;l of PBS to the non wetted portion at the top of the tube, and to this add stock CPD (see Table 4 for the quantity of stock solution assigned to each tube).</li>
		<li>Cap the tube and mix the cell suspension with the dye quickly and thoroughly by vortexing.</li>
		<li>Do steps 2.6.3-2.6.5 for the stock solutions of CPD in the designated tubes described in Table 4. Note: Unlike CFSE and CTV, the CPD labelling concentration is not precisely linearly related to the resulting fluorescence intensity of labelled cells, and so the labelling concentration used to obtain equidistant fluorescent peaks of several labelled populations has been determined empirically (see Table 4).</li>
		<li>Incubate cells for a minimum of 5 min at RT&#160;(20 &#176;C).</li>
		<li>Wash cells twice: Resuspend cells to 10 ml with 20 &#176;C RPMI containing 5% FCS. Sediment the cells by centrifugation at 300 x g for 10 min at 20 &#176;C. Aspirate off supernatant&#160;with a transfer pipette. Repeat.</li>
		<li>Pool all cells together into a single tube using 8 ml 4 &#176;C RPMI containing 5% FCS with a pipette. Sediment pooled cells by centrifugation at 300 x g for 10 min at 4 &#176;C and aspirate off the supernatant with a transfer pipette.</li>
	</ol>
	</li>
	<li>(Optional) PKH-26 Cell labeling 
	Note: If FTAs cannot be constructed with cells expressing a CD45 allotypic difference to host mice, they can be labeled with PKH-26 to allow their discrimination from recipient cells.
	<ol>
		<li>Add 2.9 ml of 20 &#176;C PBS to the pooled cell pellet and resuspend thoroughly with a pipette.</li>
		<li>Add cell suspension to a non wetted 10 ml tube, taking care not to wet the top half of the tube.</li>
		<li>Remove the tube cap and lay the tube horizontally.</li>
		<li>Add 58 &#956;l of diluent C (in the PKH-26 dye kit) to the non wetted portion at the top of the tube, and to this add 42 &#956;l of 1 mM stock PKH-26.</li>
		<li>Cap the tube and mix cell solution with the dye thoroughly by vortexing.</li>
		<li>Incubate cells for a minimum of 10 min at RT (20 &#176;C).</li>
		<li>Wash cells twice: Resuspend cells to 10 ml with 20 &#176;C RPMI containing 5% FCS. Sediment the cells by centrifugation at 300 x g for 10 min at 20 &#176;C. Aspirate off supernatant&#160;with a transfer pipette. Repeat.</li>
	</ol>
	</li>
	<li>Injecting FTAs into host animals 
	Note: To measure T cell responses in vivo, FTAs are injected into animals that have an active immune response and left in situ for up to 24 hr . It is critical that the FTAs are also injected into a na&#239;ve animal as a negative control.
	<ol>
		<li>Count and resuspend cells at 2.5 x 108 cells per ml in PBS. Inject 200 &#956;l of cells intravenously into host mice, including a na&#239;ve animal as a control.</li>
	</ol>
	</li>
</ol>
3.&#160;Flow Cytometry
<ol>
	<li>18-24 hr after FTA injection harvest blood or spleen (or other tissues of interest) from host mice.</li>
	<li>Prepare single cell suspension from tissues by mashing through a 70 &#956;m pored sieve with a 5 ml syringe plunger.</li>
	<li>Resuspend cells at up to 65 x 106 cells/ml in PBS containing 0.1% BSA.</li>
	<li>Dispense 100 &#956;l aliquots of cell suspension into wells of a microtitre plate for antibody labeling.</li>
	<li>Label cells with fluorochrome labeled antibodies and fluorescent viability probes with spectrally compatible fluorescence to CFSE, CTV and CPD. Note: Antibodies to B cell markers such as B220 and activation markers such as CD69 are essential to measure B cell activation if using the FTA to measure TH cell responses2. Include an antibody to CD45.1 and/or CD45.2 if the FTAs and host mice have a CD45 allotypic difference.</li>
	<li>Add 100 &#956;l of 2x stock solution of antibodies/viability dyes (in PBS containing 0.1% BSA) to 100 &#956;l aliquots of cells, mix well and incubate on ice (4 &#176;C) for 30 min.</li>
	<li>Wash cells: Sediment cells by centrifugation at 300 x g for 5 min at 4 &#176;C and remove supernatant.&#160;Resuspend cells in 200 &#956;l of PBS containing 0.1% BSA, sediment cells by centrifugation at 300 x g for 5 min at 4 &#176;C and remove supernatant.</li>
	<li>Resuspend cells in a total volume of 400 &#956;l&#160; of PBS containing 0.1% BSA, filter cells through a 70 &#956;m&#160;mesh and analyze by flow cytometry in a flow cytometer capable of detecting the relevant fluorescent dyes and conjugates. Collect up to 3 x 106 lymphocyte events in order to resolve each FTA cell cluster and gain enough cells for statistical analysis. Note: Ensure all the typical controls (such as single stained controls) for flow cytometry are employed. Typically, CFSE requires excitation from a blue laser source (typically at 488 nm) and detection with band pass filters centered over 520 nm; CTV requires excitation from a violet laser source (typically at 405 nm) and detection with band pass filters centered over 450 nm; and CPD requires excitation from a red laser source (typically at 633 nm or 640 nm) and detection with band pass filters centered over 670 nm.</li>
</ol>
4.&#160;Data Analysis
<ol>
	<li>Analyze flow cytometry data using standard flow cytometry software (see representative result for an example of the type of gating strategy employed).</li>
	<li>For % specific killing, calculate the number of cells in each FTA cell cluster pulsed with MHC class-I-binding peptides and the FTA clusters that were not peptide pulsed (&#8220;Nil&#8221;) and using the following formula to calculate % Specific Killing. 
	% Specific Killing<img alt="Formula" fo:content-width="2in" src="/files/ftp_upload/51627/formula.jpg" width="200" /> 
	Note: In the above formula &#8220;primed&#8221; refers to targets from animals that are thought to have an immune response against the target peptides, &#8220;na&#239;ve&#8221; refers to targets from na&#239;ve animals, &#8220;peptide&#8221; refers to targets pulsed with peptides and &#8220;nil&#8221; refers to targets not pulsed with any peptides.</li>
	<li>For B cell activation as a measure of TH activity, calculate the geometric mean fluorescence intensity (GMFI) of CD69 antibody fluorescence on FTA B cells pulsed with MHC class-II-binding peptides in &#8220;primed&#8221; animals and from this subtract the GMFI of CD69 expression on the corresponding FTA B cells in &#8220;na&#239;ve&#8221; animals to give a measure of TH activity.</li>
	<li>From the statistics generated in steps 4.2 and 4.3, use mathematical spreadsheet software such as GraphPad Prism to calculate other quantitative and qualitative parameters such as area under curve and EC50 (an in depth description of how these calculations are performed for AUC can be found at 
	http://graphpad.com/guides/prism/6/statistics/index.htm?stat_area_under_the_curve.htm, and for EC50: http://www.graphpad.com/guides/prism/6/curve-fitting/index.htm?reg_the_ec50.htm).</li>
</ol>

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A., Nygren, H., Ouchterlony, O., Tarkowski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+solid-phase+enzyme-linked+immunospot+(ELISPOT)+assay+for+enumeration+of+specific+antibody-secreting+cells.">A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells.</a> J Immunol Methods. 65, 109-121 (1983).</li><li>Klinman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ELISPOT+assay+to+detect+cytokine-secreting+murine+and+human+cells.">ELISPOT assay to detect cytokine-secreting murine and human cells.</a> Curr Protoc Immunol. , (2008).</li><li>Yerly, D., 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=Increased+cytotoxic+T-lymphocyte+epitope+variant+cross-recognition+and+functional+avidity+are+associated+with+hepatitis+C+virus+clearance.">Increased cytotoxic T-lymphocyte epitope variant cross-recognition and functional avidity are associated with hepatitis C virus clearance.</a> J Virol. 82, 3147-3153 (2008).</li></ol>

The authors declare that they have no competing financial interests.

The advantage of FTA-based assays is that they allow the discrimination of &#62;250 viable and fully functional target cell populations from a single host animal by flow cytometry. This provides a level of complexity to in vivo flow cytometry based assays that has not been possible before. This is highlighted in the 2 animal experiments shown above, where responses to 7 distinct viral epitopes at 6 concentrations could be monitored in replicates of 6 simultaneously in a single animal allowing parameters such as ...

T cells play a central role in the adaptive immune response and are often targeted for manipulation in immunotherapy. CD4+ effector T cells respond to foreign antigen by secreting cytokines that regulate many aspects of immunity and can also directly help B cells to manufacture antibodies. CD8+ cytotoxic T cells (CTLs) can also respond to foreign antigen by secreting cytokines as well as playing a central role in directly killing cells expressing a foreign antigen. The fundamental interaction that initiates these T cell effector functions involves the interaction of the T cell receptor (TCR) with foreign peptides displayed on MHC molecules on the surface of cells. CD4+ T cells recognize peptides displayed on MHC class-II molecules on antigen presenting cells and CD8+ T cells recognize peptides displayed on MHC class-I molecules that are typically displayed on microbe infected cells.
In order to assess the role T cells play in an immune response, it is essential that their effector functions are measured by reliable and sensitive techniques. Common methods for T cell response assessment include; MHC class-I/II/peptide tetramer reactivity; cytokine production by ELISPOT and intracellular cytokine staining; and killing capacity by 51Cr-release assays. These assays, however, are typically performed ex vivo with in vitro stimulation, or provide limited insight into T cell function. Ideally, when measuring T cell responses it would be beneficial to assess them in situ, in vivo as they occur, with no manipulation of T cells so as to avoid changes in functional parameters that may occur through in vitro stimulation. Some of the most commonly used in vivo T cell functional assays are based on measuring CTL mediated killing of target cells pulsed with MHC class I-binding peptides, that are enumerated in vivo via their detection through fluorescent labeling with vital dyes such as CFSE. While these types of assays can monitor CTL mediated killing of targets when they happen in vivo, they have previously had a relatively limited capacity to assess killing of multiple targets presenting different concentrations and different types of peptide epitopes, which is required to allow qualitative parameters such as functional avidity and epitope variant cross-reactivity to be assessed. These assays also do not provide any information on CD4+ T cell mediated responses.
To overcome many of the limitations with current methods used to assess T cell responses, we have recently developed a multiplex assay based on fluorescent target arrays (FTAs), which allows the monitoring of T cell responses against &#62;250 target cells simultaneously in one animal by flow cytometry 1,2. FTAs are comprised of lymphocytes labeled with several concentrations and combinations of vital dyes like CFSE, CTV and CPD allowing &#62;250 cell clusters of unique fluorescence to be generated. Since these cells remain viable and fully functional, they can be injected into animals to allow monitoring of their interaction with effector T cells in vivo3. For example, the FTA cell clusters can be pulsed with MHC class-I-binding peptides to allow assessment of antigen-specific CTL mediated killing of target cells1. In addition, the FTA cell clusters can also be pulsed with MHC class-II-binding peptides, allowing assessment of antigen specific T helper cell (TH) activity by assessing activation (by assessment of activation markers such as CD69, CD44 and/or CD62L) of B cells within the FTA bearing cognate peptide2. Since more than 250 targets can be detected simultaneously, it is possible to measure CTL and TH responses against many target cell clusters pulsed with numerous peptides at different concentrations and the inclusion of many replicates. The FTA assay therefore provides an unprecedented level of T cell effector response assessment in vivo.
Here we describe in detail the construction of a FTA and show how they can be applied to assessing T cell responses in vivo. The procedure describes the construction of a FTA comprised of 252 discernable cell clusters through the use of three vital dyes, comprised of 6 repeats of 42 cell clusters pulsed with MHC class-I and II-binding peptides. The labeling of 42 cell clusters occurs in 10 ml conical bottomed tubes and it is helpful to lay these out in a tube rack as depicted in Table 1. This method can be adjusted for smaller numbers of discernable clusters as required by reducing the amount of labeling of each dye performed1.
We highlight the utility of the assay by showing how it can measure responses generated by recombinant pox -virus vaccination against multiple epitopes in a small cohort of mice. This shows how the FTA assay can be used to measure cumulative responses and functional avidity through, respectively, the use of area under curve (AUC) assessments and measurement of effective peptide concentration required to generate half maximal responses (EC50).

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			<td height="22" style="height:22px;width:216px;">Fetal calf serum</td>
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			<td height="43" style="height:43px;width:216px;">CFDA, SE</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">C1157</td>
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			<td height="43" style="height:43px;width:216px;">CPD</td>
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			<td style="width:119px;">65-0840-90</td>
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			<td height="43" style="height:43px;width:216px;">anti-B220 PerCp-Cy5.5</td>
			<td style="width:71px;">eBioscience</td>
			<td style="width:119px;">110730</td>
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			<td height="43" style="height:43px;width:216px;">anti-CD69 brilliant violet 605</td>
			<td style="width:71px;">Biolegend</td>
			<td style="width:119px;">104529</td>
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			<td height="22" style="height:22px;width:216px;">PKH-26</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">PKH26GL-1KT</td>
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			<td height="22" style="height:22px;">EQUIPMENT:</td>
			<td></td>
			<td></td>
			<td></td>
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			<td height="43" style="height:43px;width:216px;">Material Name</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalogue Number</td>
			<td style="width:167px;">Comments (optional)</td>
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		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Vortex</td>
			<td style="width:71px;">Scientific Industries Inc&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Centrifuge</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Flow cytometer (Fortessa or equivalent with blue, red and violet laser source)</td>
			<td style="width:71px;">BD Bioscience</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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the use of fluorescent target arrays for assessment of t cell responses in vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

As an example of the use of the FTA assay, a BALB/c mouse was immunized with recombinant vaccinia virus (VV) expressing HIV-I epitopes (VV-HIV) and responses to the HIV-I CTL epitopes, Gag, Gag mut, Env and Pol, the VV CTL epitopes F2L and F2L mut, and the HIV-I TH cell epitope, Gag Th (as described in2) were assessed using a 252 parameter FTA assay (Figure 1B). Epitope variants of Gag (Gag mut) and F2L (F2L mut) are not expressed in the VV-HIV vector and therefore responses against...

Watch this Scientific Journal Video about The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo at JoVE.com

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

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Immunology and Infection

14.3K Views.  Australian National University. The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses >250 parameters simultaneously by flow cytometry.

Video: The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

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 ...

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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 ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

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 ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model offers accessibility to rare populations of antigen-specific T-cells representative of the cellular memory response as well as the cytokine microenvironment in situ.
This report describes the practical procedure of a cutaneous recall, an SB induction, and a harvest of antigen-specific T-cells. To exemplify the method, the tuberculin skin test is used for antigenic recall in individuals who, prior to this study, underwent a Bacillus Calmette-Gu&#233;rin vaccination against an infection with Mycobacterium tuberculosis. Finally, examples of multiplex and flow cytometric analyses of SB specimens are provided, illustrating high fractions of antigen-specific polyfunctional CD4+ T-cells available by this sampling method compared with cells isolated from the blood.
The method described here is safe and minimally invasive, provides a unique opportunity to study both innate and adaptive immune responses in vivo, and may be beneficial to a broad community of researchers working with cell-mediated immunity and human memory responses, in the context of vaccine development.

A Suction Blister Protocol to Study Human T-cell Recall Responses In Vivo

Here, we provide a demonstration of the suction blister cutaneous recall model. The model allows a simple access to study human in vivo adaptive immune responses, for instance in the context of vaccine development.

Cutaneous antigen-recall models allow for studies of human memory responses in vivo. When combined with skin suction blister (SB) induction, this model ...

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 ...

Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist pMHC, through a process termed co-agonism. This protocol describes an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptides as single polypeptides (single chain MHC) in a xenogeneic cell line. We expressed single chain MHCs under conditions where low levels of agonist single chain p-MHC complexes and high levels of non-stimulatory single chain p-MHC complexes were expressed. Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of non-stimulatory pMHC. The protocol describes cell line transfection with single chain MHC constructs, generation of stable cell lines, culture of hepatitis B virus-specific human CD8+ T cells and T cell activation experiments simultaneously quantifying cytokine production and degranulation. The presented methods can be used for research on different aspects of CD8+ T cell activation in human T cell systems with known peptide MHC specificity.

This protocol describes the use of single chain MHC class I complexes to investigate molecular interactions in human CD8+ T cell activation: generation of engineered antigen presenting cells expressing single chain constructs, culture of human CD8+ T cell clone and T cell activation experiments.

Non-stimulatory self peptide MHC (pMHC) complexes do not induce T cell activation and effector functions, but can enhance T cell responses to agonist ...