This research was supported by the Singapore Ministry of Health&#8217;s National Medical Research Council under its CBRG/0064/2014 to N.R.J.G., by CREATE under its R571-002-012-592 to P.A.M., National Research Foundation Investigatorship R571-000-272-281 to P.A.M., and by a Singapore Translational Research (STaR) Investigator Award (NMRC/STaR/013/2012) to A.B. We are grateful to Dr. Paul Hutchinson and Mr. Teo Guo Hui from NUS Immunology Programme Flow Core facility for the expert cell sorting. We are grateful to Elijah Chen for critical reading of the manuscript.

NOTE: All steps involving use of live, non-fixed cells should be performed in a biosafety hood, and any biohazardous waste should be discarded according to the local health and safety regulations.
1. CHO Cell Transfection and Generation of Stable CHO Cell Lines
<ol>
	<li>CHO cell transfection 
	NOTE: This section describes CHO cell transfection using polyethylenimine (PEI). However, alternative methods can be used, as CHO cells are relatively easy to transfect using commercially available lipid transfection reagents.
	<ol>
		<li>Culture CHO cells in complete F12 (cF12, F12 supplemented with 5% FBS, 100 U/mL penicillin, 100 &#956;g/mL streptomycin and 10 &#181;g/mL blasticidin to maintain expression of the tetracycline repressor). Passage the cells every 3-4 days at a 1:10 ratio.</li>
		<li>One day before transfection, prepare the CHO cell suspension. Wash CHO cells in the flask using PBS (10 mL for PBS for a T75 flask, 5 mL PBS for a T25 flask). Add 0.05% Trypsin/0.02% EDTA in PBS (2 mL for a T75 flask, 1 mL for a T25 flask) to the flask and incubate for 5-10 min at room temperature (RT). Using a light microscope, check that the cells have detached, and stop trypsinization by adding cF12 to the flask (8 mL for a T75 flask, 4 mL for a T25 flask).</li>
		<li>Transfer the CHO cell suspension to a 15 mL tube, and centrifuge at 300-400 x g for 5 min at 4 &#176;C or RT. Remove supernatant, re-suspend in cF12 (10 mL for a T75 flask, 5 mL for a T25 flask) and count using a hemocytometer.</li>
		<li>Adjust cell concentration to 60,000-100,000 CHO cells/mL. Add 5 mL of CHO cell suspension (300,000-500,000 CHO cells) per well in a 6 well plate. Incubate overnight at 37 &#176;C, 5% CO2.</li>
		<li>On the day of the transfection, add 400 &#956;L of serum-free F12 media or low serum media to a 1.5 mL tube. Add 3 &#956;g of plasmid to 400 &#956;L of media. Mix by pipetting. Add 6 &#956;g of PEI (6 &#956;L of 1 mg/mL PEI solution) to the media/DNA mix, and vortex immediately for 10 s.</li>
		<li>Incubate the media/DNA/PEI mix at RT for 10-15 min. During this incubation, replace CHO cell media in the 6 well plate with 5 mL of fresh cF12 media.</li>
		<li>Add the media/DNA/PEI solution to CHO cells. Add the solution dropwise and evenly over the well. Incubate overnight at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
	<li>Generation of stable CHO cell lines
	<ol>
		<li>One day after transfection, remove media from the wells and add 5 mL of cF12 containing the appropriate selection antibiotics to each well. Use 0.3 mg/mL hygromycin for pcDNA5TO-based plasmids, and 1 mg/mL geneticin for pcDNA3-based plasmids.</li>
		<li>If the cells reach more than 90% confluency at 24 h post transfection, trypsinize the cells.</li>
		<li>Wash wells with 1 mL of PBS per well, add 1 mL of 0.05% trypsin/0.02% EDTA in PBS and incubate for 5-10 min at RT. Using a light microscope, check that the cells have detached, and stop trypsinization by adding 3-4 mL of cF12 per well.</li>
		<li>Transfer the CHO cell suspension to a 15 mL tube, and centrifuge at 300-400 x g for 5 min at 4 &#176;C or RT. Remove the supernatant and re-suspend in 10 mL of cF12. Add 5 mL of cell suspension per well in 6 well plate. Use two wells to maximize yield of the transfected cells.</li>
		<li>Incubate the 6 well plates at 37 &#176;C, 5% CO2. Monitor cell death and the growth of resistant colonies using a light microscope. Remove media and replace it with fresh media containing the appropriate antibiotics after 4-5 days, or when significant cell death is observed.</li>
		<li>Once the resistant cells reach more than 70% confluence, trypsinize the cells as described in step 1.2.2 and transfer to a T25 flask for expansion. The antibiotic selection takes 1-2 weeks.</li>
		<li>Once the antibiotic-resistant CHO cells in the T25 flask reach 70-80% confluence, perform antibody staining to verify cell surface expression of the constructs of interest. One day before antibody staining, trypsinize the cells as described in step 1.2.2 and adjust the cell concentration to 200,000 cells/mL, and add 1 mL of cell suspension to 12 well plate. For testing of tetracycline-inducible constructs, add 50-60 ng/mL of tetracycline. Incubate overnight at 37 &#176;C, 5% CO2.</li>
		<li>Use cell scraper to detach CHO cells from bottom of the well. This method is preferable to trypsinization, as it reduces the risk of epitope damage due to proteolytic cleavage.</li>
		<li>Transfer the 1 mL of media containing the CHO cells to a FACS tube. Spin down at 300-400 x g, 4 &#176;C for 5 min. Re-suspend in 100 &#956;L of anti-HLA antibody diluted in FWB. Incubate 30 min at 4 &#176;C on ice. Perform this staining with anti-A2 antibody to detect total A2 cell surface expression levels, and TCR-like antibody specific for A2 in complex with E183 peptide to detect agonist pMHC-I cell surface expression levels.</li>
		<li>After verifying MHC expression, FACS-sort the positive cells, and maintain the sorted cells in media with antibiotics to reduce transgene loss. For CHO cells expressing agonist pMHC, single cell sorting is recommended, to ensure comparable agonist pMHC expression with and without supertransfection with co-agonist pMHC. 2-3 weeks are required for CHO cell growth following the single cell sorting.</li>
	</ol>
	</li>
</ol>
2. HBV-specific CTL Culture
<ol>
	<li>Preparation of PBMCs as feeders for HBV-specific human CTL re-stimulation 
	NOTE: For the A2-E183-specific CTL clone, re-stimulation with freshly isolated PBMCs, rather than thawed PBMCs, gives the best results. 
	CAUTION: Obtain all the necessary ethical approvals before recruiting volunteers for blood donations. For the purpose of this work, PBMC were collected from healthy volunteers under the protocol approved by NUS IRB. Informed consent was obtained from all donors. Follow all the necessary precautions when working with human blood to reduce risk of transmission of blood borne diseases.
	<ol>
		<li>Before starting, set the centrifuge temperature at 19-20 &#176;C and pre-warm the density gradient (e.g., Ficoll) to RT.</li>
		<li>Add 15 mL of room temperature density gradient to 50 mL tube. Centrifuge at 400 x g for 5 min at 19-20 &#176;C. This is to ensure there is no density gradient on the side of the tube.</li>
		<li>Collect 20 mL of blood from a healthy volunteer donor through venipuncture. Add 15 mL of PBS to 20 mL of blood in a 50 mL tube. Mix by pipetting up and down.</li>
		<li>Slowly, add the 35 mL of blood diluted in PBS on top of the 15 mL of density gradient from step 2.1.2. Make sure that the blood and the density gradient do not mix. Centrifuge the tubes at 19 &#176;C, 1,200 x g for 20 min with the brakes off.</li>
		<li>Remove approximately 70% of the plasma layer. Carefully aspirate the layer containing the PBMC (the cloudy layer on top of the clear density gradient layer) using a Pasteur pipette and place it in a new 50 mL tube.</li>
		<li>Add PBS to PBMCs to obtain a 50 mL total volume. Centrifuge tube at 4 &#176;C, 300-400 x g for 10 min. Carefully remove the supernatant using the vacuum aspirator and sterile glass Pasteur pipette, or by decanting.</li>
		<li>Re-suspend the cells in 50 mL of PBS. Centrifuge tube at 4 &#176;C, 300 x g for 10 min. Carefully remove the supernatant using the vacuum aspirator and sterile glass Pasteur pipette, or by decanting.</li>
		<li>Re-suspend cells in 2 mL of complete human T cell media (serum free media supplemented with 2% human serum). Count the cells using hemocytometer. This protocol gives on average 1-2 x 106 PBMCs per 1 mL of blood used.</li>
	</ol>
	</li>
	<li>CTL re-stimulation using PBMCs as feeder cells
	<ol>
		<li>Irradiate PBMCs at 30 Gy to inhibit proliferation in response to subsequent stimulation. 
		CAUTION: Ensure adequate training and compliance with the laboratory safety requirements when using the irradiator.</li>
		<li>Re-suspend the irradiated PBMCs in complete human T cell media at 2 x 106 cells/mL. Add cytokines and lectin from Phaseolus vulgaris (PHA) for the CTL re-stimulation at 2x concentration into the PBMC suspension. The final cytokine and PHA concentrations are: 20 U/mL recombinant human IL-2, 10 ng/mL recombinant IL-7, 10 ng/mL recombinant human IL-15 and 1.5 mg/mL PHA.</li>
		<li>Thaw the frozen CTL vial in a 37 &#176;C water bath. Transfer the thawed cells into a 15 mL tube with 10 mL of complete human T cell media. Spin down at 300-400 x g at 4 &#176;C for 5 min. Carefully remove the supernatant using the vacuum aspirator and sterile glass Pasteur pipette, or by decanting.</li>
		<li>Re-suspend CTLs in 2 mL of complete human T cell media. Count the cells using hemocytometer. Add complete human T cell media to obtain cell concentration of 106 cells/mL.</li>
		<li>In a 24 well plate, add 0.5 mL of irradiated PBMCs (106 cells) with 2x cytokines and PHA (step 2.2.2), and 0.5 mL of CTLs (0.5 x 106 cells) per well. The number of wells used depends on the total number of CTLs or PBMCs available. Incubate at 37 &#176;C, 5% CO2.</li>
		<li>After 4-5 days, when CTL blasts are visible and the media becomes yellow, transfer the cultures into 6 well plates, and supplement the initial 1 mL volume with 4 mL of complete human T cell media with 20 U/mL IL-2, 10 ng/mL IL-7 and 10 ng/mL IL-15 (no PHA).</li>
		<li>After 2-3 days, transfer the cultures into a T75 flask, add 40 mL of complete human T cell media with cytokines (step 2.2.2, no PHA) and culture in an upright position.</li>
		<li>Maintain CTLs at a concentration of 1-2 x 106 cells/mL by adding fresh media supplemented with cytokines (step 2.2.2, no PHA) at a volume equal to that of the existing culture volume at every other day. CTLs can be used for experiments 7-14 days after re-stimulation.</li>
	</ol>
	</li>
</ol>
3. T Cell Activation Experiments
NOTE: A typical experiment requires several different T-RExCHO lines: untransfected T-REx CHO cells (negative control), T-REx CHO cells expressing non-stimulatory pMHC only (scA2-GAG; negative control), T-REx CHO cells expressing low amounts of agonist pMHC-I (scA2-ENV without tetracycline, experimental sample), T-REx CHO cells expressing low amounts of agonist pMHC and high amounts of non-stimulatory co-agonist pMHC (scA2-ENV and scA2-GAG, without tetracycline, experimental sample), and T-REx CHO cells expressing high amounts of agonist pMHC-I scA2-ENV with tetracycline, positive control), as shown on Figure 1C,D.
<ol>
	<li>Plating CHO cells as antigen presenting cells
	<ol>
		<li>Culture CHO cells in T75 flask, as described in step 1.1.1. Use cells at 70-90% confluence for best results. One day before the T cell activation experiment, trypsinize the cells as described in section 1.1.2. After trypsinization, transfer the cell suspension into a 15 mL tube, centrifuge at 400 x g for 5 min at 4 &#176;C or RT, remove supernatant by pipetting or decanting.</li>
		<li>Re-suspend the cell pellet in 5 mL of cF12. Count CHO cells using a hemocytometer.</li>
		<li>Adjust the cell concentration to 2 x 105/mL using cF12. Add 50-60 ng/mL of tetracycline to the agonist only samples to induce high amounts of agonist pMHC-I expression as positive control (Figure 1C). CHO cells will settle down in 15 mL tubes, so make sure to mix them by pipetting or vortexing before plating.</li>
		<li>For T cell activation assay, add 100 &#956;L of the CHO cell suspension per well to U-bottom 96 well plate, and use triplicates per CHO cell line. For CHO cell anti-MHC staining, add 1 mL of cell suspension to 12 well plate, and use triplicates per cell line. Incubate overnight at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
	<li>T cell activation assay 
	NOTE: This T cell activation assay allows simultaneous quantification of T cell degranulation (CD107a staining, a measure of T cell cytotoxicity) and cytokine production.
	<ol>
		<li>The day before the experiment, centrifuge CTLs cells at 400 x g at 4 &#176;C for 5 min, count using hemocytometer and re-suspend the cells at 106 cells/mL in complete human T cell media without cytokines or PHA. Incubate at 37 &#176;C, 5% CO2. The purpose of this rest step is to reduce CTL sensitivity, as we have observed maximum activation in response to low amounts of antigen without this step.</li>
		<li>On the day of the experiment, centrifuge T cells at 400 x g at 4 &#176;C for 5 min, remove the supernatant by decanting or pipetting, and re-suspend the cells in 2 mL complete serum-free media (without cytokines or PHA). Count live T cells using a hemocytometer, and adjust T cell concentration to 106 live cells/mL using complete human T cell media (without cytokines or PHA).</li>
		<li>Add anti-human CD107a antibody at a 1:100 dilution (final concentration of 2.5 &#956;g/mL), add brefeldin A at a 1:1000 dilution (final concentration of 1 &#956;g/mL).</li>
		<li>Remove media from the CHO cells in the 96 well plate by aspirating with glass Pasteur pipette or by flicking the plate. Per well, add 200 &#956;L of the T cell suspension (0.2 x 106 cells) containing anti-CD107a and brefeldin A. Co-culture T cells with CHO cells for 3-4 h.</li>
		<li>At the end of the co-culture, perform T cell surface antigen staining. Spin down the 96 well plate at 300-400 x g, 5 min at 4 &#176;C, and remove the supernatant by aspirating or flicking. Add 2.5 &#956;L of CD3 and 2.5 &#956;L of CD8 antibodies in 50 &#956;L of 0.5% BSA in PBS (flow wash buffer, FWB) per well for cell surface antigen staining. Incubate samples on ice for 30 min in the dark.</li>
		<li>Wash samples by adding 150 &#956;L of FWB per well. Spin down the 96 well plate at 300-400 x g, 5 min at 4 &#176;C, and remove the supernatant by aspirating or flicking.</li>
		<li>Perform fixation/permeabilization steps using a fixation/permeabilization kit. Add 100 &#181;L of fixation/permeabilization solution (from the fixation/permeabilization kit) per well, mix by pipetting. Incubate on ice for 20 min.</li>
		<li>Centrifuge at 300-400 x g and 4 &#176;C for 5 min, and remove the supernatant by aspirating or flicking. Add 200 &#956;L of 1x perm/wash buffer (dilute the 10x perm/wash stock buffer from the kit prior to use) per well. Repeat this step.</li>
		<li>Re-suspend the cell pellet in 50 &#956;L of 1x perm/wash buffer containing anti-IFN-&#947; antibody at 0.3 &#956;g/mL. Incubate on ice for 30 min in dark.</li>
		<li>Add 150 &#956;L of 1x perm/wash buffer. Centrifuge at 300-400 x g for at 4 &#176;C for 5 min, and remove the supernatant by aspirating or flicking. Add 200 &#956;L of 1x perm/wash buffer, centrifuge 300-400 x g for at 4 &#176;C for 5 min, and remove the supernatant by aspirating or flicking.</li>
		<li>Re-suspend each sample in 200 &#956;L of FWB. Proceed with flow cytometric analysis.</li>
	</ol>
	</li>
	<li>CHO cell MHC class I staining 
	NOTE: It is critical to verify scMHC-I cell surface levels on cells used on each experiment, as cell lines may lose transgene expression. When preparing 96 well plate for T cell stimulation, set up 12 well plate for CHO cell staining, as outlined in Section 3.1.4.
	<ol>
		<li>Use a cell scraper to detach CHO cells from the bottom of the well. This method is preferable to trypsinization, as it reduces the risk of epitope damage due to proteolytic cleavage.</li>
		<li>Transfer the 1 mL of media containing the CHO cells to the FACS tube. Spin down at 300-400 x g at 4 &#176;C for 5 min. Re-suspend in 100 &#956;L of anti-HLA antibody diluted in FWB. Incubate for 30 min at 4 &#176;C on ice.
		<ol>
			<li>Perform this staining with anti-A2 antibody to detect total A2 cell surface expression levels, and TCR-like antibody specific for A2 in complex with E183 peptide32 to detect agonist pMHC-I cell surface expression levels.</li>
		</ol>
		</li>
		<li>Add 1 mL of FWB to each CHO sample, spin down at 300-400 x g, 4 &#176;C, 5 min, and then re-suspend the sample in 0.3 mL of FWB. Proceed with flow cytometry analysis.</li>
	</ol>
	</li>
</ol>

The authors declare no competing interests.

The presented protocol provides a robust tool for investigation of molecular interactions during human CD8+ T cell activation. A critical step for investigation of co-agonism during human T cell activation is control over agonist pMHC cell surface expression to ensure equal agonist pMHC presentation on the APCs with or without co-agonist pMHC expression. In our experimental system, this is achieved by using a single cell clone expressing low amounts of agonist pMHC, followed by supertransfection with a co-agonist pMHC construct and agonist pMHC quantification using TCR-like antibody10,32. As agonist sc pMHC expression can change over time, it is critical to quantify agonist pMHC surface expression on APCs used in each experiment using TCR-like antibodies. If no specific TCR-like antibody is available, agonist scMHC-I can be expressed as fluorescent protein fusion, and its cell surface expression can then be quantified using a sensitive microscopy method, such as total internal reflection fluorescence microscopy35,36. Moreover, cell surface expression of co-agonist pMHC decreases over time, due to methylation-dependent and -independent silencing of the CMV promoter37, and should be monitored using antibody staining and flow cytometry analysis, with repeated FACS-sorting when needed. We have cultured CHO cells for up to 5 weeks with relatively limited loss of sc pMHC expression. We highly recommend freezing the CHO cells soon after selection/sorting to ensure a reliable stock of cells with known sc MHC expression.
The several steps of the presented protocols can be modified, depending on the availability of equipment, or depending on the specific research aims. For example, PBMC proliferation potential can be inhibited (Step 3.2.1) using alternative methods that do not require a gamma (or X-) irradiator, such as treatment with mitomycin C38. A non-enzymatic cell dissociation buffers containing metal chelators39 can be used instead of cell scraping in step 3.3.1. Moreover, the antigen presenting system can be modified to make it more suitable for human CTL clones expressing different TCRs. CHO cells express hamster MHC class I molecules, and although these are irrelevant for the CTL line studied here (Figure 2A and Figure 3A, we observed no T cell responses to untransfected CHO cells), there is a possibility that other human TCRs may show some xenoreactivity. In that case, hamster MHC class I expression can be reduced by knocking out hamster &#946;2-microglubulin or TAP using CRISPR/Cas9; or a human APC line can be used after CRISPR/Cas9-mediated &#946;2-microglubulin or TAP knock out.
The experimental system presented here allows precise control of TCR and CD8 interactions with MHC molecules presenting pre-defined peptides. For example, we have previously shown that mutations abolishing CD8 binding40 to coagonist pMHC eliminated coagonist-mediated activation enhancement in murine and human CD8+ T cells9,10, whereas abrogating TCR interaction with co-agonist pMHC had no effect on coagonist-mediated activation enhancement10. However, the use of single chain MHC constructs has several limitations. For example, it is time and labor-consuming to test multiple co-agonist peptide sequences using this experimental system, as this involves generation of multiple plasmids encoding sc MHC with different peptides, and subsequent transfections and cell sorting. Previously, we used the murine TAP-deficient cell line RMA-S to test multiple co-agonist peptides in mouse T cell activation8,9, but this approach was not successful with the TAP-deficient human T2 cell line10, most likely due to presentation of TAP-independent peptides41. Another limitation of our experimental system is that is does not provide a fast method for altering the amount of co-agonist pMHC molecules for the purpose of determining the critical amount of co-agonist pMHC required to trigger T cell activation. Moreover, the tetracycline-inducible system used does not allow titration of agonist pMHC quantity at the single cell level by changing the tetracycline concentration, as varying tetracycline concentration alters the proportion of HLA-A2 positive cells, rather than HLA-A2 levels on per cell basis. An alternative approach that we are currently investigating is to use supported lipid bilayers42, previously used to investigate co-agonism during murine CD4+ T cell activation4 or beads presenting fixed amount of agonist pMHC in the presence of varying amounts of co-agonist pMHC. When used in conjunction with UV-cleavable peptide technology4,43, these alternative experimental systems should allow fast testing of multiple co-agonist peptide sequences as well as different amounts of co-agonist pMHC.
The sc MHC technology can be also applicable to investigation of co-agonism in human or murine CD4 T cells, as single chain MHC class II molecules presenting pre-determined peptides have been successfully expressed on mammalian cell surfaces44. Moreover, the use of sc MHC technology can be extended to investigation of non-classical MHC molecules such as HLA-E. Sc HLA-E has been described before, and its expression has been shown to inhibit human T cell activation45. Another non-classical MHC molecule of interest is CD1, which presents lipids instead of peptides46. Sc constructs consisting of CD1 heavy chain and &#946;2-microglobulin have been shown to be expressed on the cell surface and to bind relevant lipid ligands47. Single chain MHC technology has great potential as a research tool for investigating molecular interactions during T and NK cell activation.

MHC class I (MHC-I) molecules present short (8-10 amino acids) peptides derived from proteins synthesized by each cell. MHC-I molecules on healthy cells present self peptides derived from endogenous proteins. Viral infection leads to presentation of non-self virus-derived peptides on MHC-I, but even infected cells present non-self peptides in the presence of large amounts of self peptide-MHC (pMHC). MHC-I is recognized by the T cell receptor (TCR) and the CD8 co-receptor on T cells. TCR binding affinity to peptide pMHC is highly dependent on the sequence of presented peptides, whereas CD8 binding is peptide-independent. TCR binding to non-self agonist pMHC complex leads to T cell activation and effector functions. Non-stimulatory self 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-agonism1,2,3. Co-agonism has been observed in mouse CD4+ T cells4,5,6 as well as in mouse and human CD8+ T cells7,8,9,10,11,12,13. Under physiological conditions, T cells need to recognize limited quantities of agonist pMHC on antigen presenting cells (APCs) co-presenting high levels of non-stimulatory pMHC complexes, suggesting that co-agonism contributes to optimal T cell responses in vivo. Total cell surface MHC class I levels are frequently downregulated during viral infections14 and on tumors15. This immune evasion strategy decreases agonist pMHC presentation, but also reduces the amount of co-agonist pMHC I available for T cell activation enhancement. Moreover, high cell surface expression levels of the MHC class I molecule HLA-C have been shown to correlate with improved HIV control16, suggesting a critical role for total MHC class I expression in T cell activation. Despite the physiological significance of co-agonism, its molecular mechanism is still incompletely understood. This is largely due to technical challenges of experimentally controlling the amount of presented co-agonist pMHC while keeping agonist pMHC constant.
We developed an experimental system to investigate co-agonism during human CD8+ T cell activation by expressing human MHC class I molecules presenting pre-determined peptide as single polypeptide (single chain MHC-I, Figure 1A) in a xenogeneic cell line9,10. Single chain (sc) agonist pMHC was expressed under control of tetracycline-inducible promoter in the hamster-derived T-REx CHO cell line expressing tetracycline repressor; this allows very low &#8220;leaky&#8221; expression of sc agonist pMHC in the absence of tetracycline and high sc agonist pMHC expression after addition of tetracycline (Figure 1B,C). The sc agonist pMHC-expressing T-REx CHO cells were then supertransfected with non-stimulatory, co-agonist sc pMHC-I under control of a constitutively active promoter (Figure 1B,C). Use of this experimental system allowed us to compare CD8+ T cell responses to agonist pMHC in the presence or absence of high levels of non-stimulatory pMHC. Critically, since peptide and MHC-I heavy chain are covalently linked in the scMHC-I format, this allows introductions of mutations into MHC molecules presenting pre-determined peptides. Use of scMHC-I technology thus allowed us to specifically modulate TCR or CD8-binding properties of agonist or co-agonist pMHC.
Co-agonism in CD8+ T cell activation has been observed using purified MHC-I complexes presenting agonist and co-agonist peptides in the form of heterodimers11,17 or presented on quantum dots12,13. Early work on co-agonism in CD8+ T cell activation in the context of agonist pMHC recognition on APC used TAP2-deficient cell lines as APCs. TAP is required for peptide transport from the cytoplasm to the endoplasmic reticulum, and TAP-deficiency severely reduces the pool of available MHC class I-binding peptides. In the absence of peptides, the nascent complex of MHC class I heavy chain and &#946;2-microglobulin is unstable, resulting in very low MHC-I cell surface expression. Initially, comparison of T cells stimulated with antigen loaded on TAP-sufficient and -deficient mouse RMA and RMA-S cell lines, respectively, as APCs, did not reveal any evidence for co-agonism during mouse T cell activation18. However, use of this experimental system did not allow precise control over the amount of agonist pMHC presented independently of non-stimulatory self pMHC. Moreover, these two cell lines may also differ in expression of other molecules that modulate T cell activation, such as ligands to T cell co-stimulatory or inhibitory receptors, or adhesion molecules.
Subsequently, we developed a protocol for loading TAP2-deficient RMA-S cells with exogenous peptides to allow presentation of a fixed amount of agonist pMHC in the presence or absence of non-stimulatory pMHC. This was achieved through the following: incubation of TAP2-deficient RMA-S cells at lower temperatures (28 &#176;C, instead of the usual 37 &#176;C) to stabilize empty MHC class I heavy chain/&#946;2 microglobulin complexes19; incubation at 28 &#176;C with exogenous agonist peptide; incubation at 28 &#176;C in the presence or absence of exogenous non-stimulatory peptide; and incubation at 37 &#176;C to reduce cell surface expression of empty MHC class I complexes8,9. Use of this experimental set-up revealed that the presence of non-stimulatory pMHC enhanced activation of mouse thymocytes, na&#239;ve peripheral CD8+ T cells and CTLs8. Mechanistically, the presence of non-stimulatory pMHC can induce CD8 recruitment to the T cell:APC immune synapse even in the absence of agonist pMHC, and non-stimulatory pMHC can enhance TCR interaction with CD8 coreceptor7,8. However, this experimental system does not allow testing the relative contributions of TCR and CD8 coreceptor binding to agonist and co-agonist pMHC in mediating the activation enhancement, as any modifications altering TCR or CD8 binding to MHC class I would affect all MHC molecules, regardless of the presented peptide. We therefore used MHC class I single chain technology to overcome this problem.
The single chain (sc) MHC class I format has been used before to improve antigenic pMHC presentation for therapeutic strategies, and to answer fundamental questions on mechanisms of TCR and NK cell receptor activation and function20,21. scMHC-I consists of peptide, &#946;2-microglobulin and MHC-I heavy chain joined by two flexible serine/glycine-rich linkers (Figure 1A), several different linker sequences have been developed and tested22. scMHC-I can fold independently of the TAP complex and chaperone proteins required for conventional pMHC complex assembly20. scMHC-I have been shown to fold correctly, as determined using conformation-specific antibody staining22 and by solving sc structure with X-ray crystallography23. The basic scMHC-I design has been modified to improve binding of low affinity peptides24,25.
This protocol describes the use of human scMHC-I to investigate co-agonism during human CTL clone activation. The protocol has been optimized for human CTL clone specific for an epitope of Hepatitis B virus (HBV). HBV is a severe healthcare burden worldwide, as there are 350 million people infected with HBV and chronic HBV infection can lead to development of hepatocellular carcinoma (HCC). HCC cells resulting from HBV infection can usually present HBV-derived epitopes and can be recognized by specific human T cells. HBV and HCC clearance relies on the human T cell responses26, but HCC patients often suffer from T cell exhaustion and reduced T cell responses27. Introduction of HBV-specific TCR into T cells of HBV has been tried as a potential immunotherapy strategy to eliminate chronic HBV infection28. However, it is unknown if this method will be effective in a clinical setting, due to the low levels of surface MHC-I in human hepatocytes29. Therefore, understanding the mechanism of coagonism may provide alternative perspectives for immunotherapy of HBV, possibly by enhancing the presentation of endogenous pMHC to induce co-agonism-mediated T cell responses. The protocol covers all the necessary steps for research on human co-agonism: transfection of T-REx CHO cells with agonist and co-agonist sc pMHC, generation of stable T-REx CHO cell lines, culture of HBV-specific human CTL CD8+ T cell line and CTL activation experiments quantifying cytokine production and degranulation in response to T-REx CHO cells. Although we focus on using the sc MHC class I technology to investigate co-agonism during HBV T cell activation, it must be noted that the presented methods can easily be adapted for research on other aspects of T cell activation in human T cell systems with known pMHC-I specificity.
This protocol uses a human CD8+ CTL line specific for HBV-derived peptide E183-91 (FLLTRILTI: &#34;E183&#34;)30 presented on HLA-A2. sc HLA molecules consisting of a signal sequence from human &#946;2-microglobulin, a HLA-A2 binding peptide of interest, a glycine/serine linker, a human &#946;2-microglobulin, a glycine/serine linker and a A2 heavy chain can be commercially synthesized (Figure 1A). Plasmids encoding scA2 constructs with agonist E183 peptide, or coagonist HIV GAG (SLYNTVATL)31 peptides are available from the authors upon request. The peptide sequence can be changed using site directed-mutagenesis. A tetracycline-inducible vector pcDNA5/TO was used to express the agonist single chain HLA-A2 with E183 peptide (sc E183-HLA-A2). In the presence of tetracycline repressor, the pcDNA5/TO plasmid allows only very low, &#8220;leaky&#8221; expression of the protein of interest; high expression can be induced by the addition of tetracycline (Figure 1B,C). Use of a tetracycline-inducible expression system allows control over the amount of cell surface agonist pMHC expression. A constitutive expression plasmid pcDNA3.1 was used to express coagonist scA2 with GAG peptide (sc GAG-HLA-A2). The tetracycline-regulated expression (T-REx) CHO cell line (hamster cell line, no endogenous human MHC) was used to express agonist and co-agonist sc-HLA-A2 constructs. This experimental system allows precise control over pMHC presentation (Figure 1C): no pMHC presentation (untransfected T-REx), a high amount of co-agonist pMHC presentation (constitutive sc GAG-HLA-A2 expression), a low amount of agonist pMHC presentation (sc E183-HLA-A2 in the absence of tetracycline), a high amount of agonist pMHC presentation (sc E183-HLA-A2 in the presence of tetracycline), a low amount of agonist pMHC presentation and high expression of co-agonist pMHC (sc E183-HLA-A2 in the absence of tetracycline, and constitutive sc GAG-HLA-A2 expression). Use of this experimental system allows precise control of cell surface amounts of agonist and coagonist pMHC.

<table>
	<tbody>
		<tr>
			<td>Aim-V (serum free media)</td>
			<td>Gibco</td>
			<td>12055091</td>
			<td></td>
		</tr>
		<tr>
			<td>blasticidin</td>
			<td>Gibco</td>
			<td>R21001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytofix/Cytoperm (fixation/permealibisation kit)</td>
			<td>BD</td>
			<td>554714</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Gibco</td>
			<td>10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>HyClone</td>
			<td>SH3007103</td>
			<td></td>
		</tr>
		<tr>
			<td>geneticin</td>
			<td>Gibco</td>
			<td>10131035</td>
			<td></td>
		</tr>
		<tr>
			<td>GolgiPlug (Brefeldin A)</td>
			<td>BD</td>
			<td>555029</td>
			<td></td>
		</tr>
		<tr>
			<td>Human serum</td>
			<td>Sigma-Aldrich</td>
			<td>H4522</td>
			<td></td>
		</tr>
		<tr>
			<td>hygromycin</td>
			<td>Gibco</td>
			<td>10687010</td>
			<td></td>
		</tr>
		<tr>
			<td>Lectin from Phaseolus vulgaris (PHA)</td>
			<td>Sigma-Aldrich</td>
			<td>L4144</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM (low serum media)</td>
			<td>Bibco</td>
			<td>31985070</td>
			<td></td>
		</tr>
		<tr>
			<td>10 X PBS</td>
			<td>Vivantis</td>
			<td>PB0344 &#8211; 1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>polyethylenimine (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>408727</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human IL2</td>
			<td>R&#38;D Systems</td>
			<td>202-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human IL7</td>
			<td>R&#38;D Systems</td>
			<td>207-IL</td>
			<td></td>
		</tr>
		<tr>
			<td>recombinant human IL15</td>
			<td>R&#38;D Systems</td>
			<td>247-ILB</td>
			<td></td>
		</tr>
		<tr>
			<td>tetracycline</td>
			<td>Sigma-Aldrich</td>
			<td>T7660</td>
			<td></td>
		</tr>
		<tr>
			<td>T-REx CHO cell line</td>
			<td>Thermo Fisher Scientific</td>
			<td>R71807</td>
			<td></td>
		</tr>
		<tr>
			<td>10 x Trypsin/EDTA</td>
			<td>Gibco</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>BD</td>
			<td>347347</td>
			<td>Clone SK7, RRID:AB_400287</td>
		</tr>
		<tr>
			<td>CD8&#945;</td>
			<td>BD</td>
			<td>563795</td>
			<td>Clone RPA-T8, RRID:AB_2722501</td>
		</tr>
		<tr>
			<td>CD107a</td>
			<td>BD</td>
			<td>566261</td>
			<td>Clone H4A3, RRID:AB_2739639</td>
		</tr>
		<tr>
			<td>HLA-A2</td>
			<td>eBioscience</td>
			<td>17-9876-41</td>
			<td>Clone BB7.2, RRID:AB_11151522</td>
		</tr>
		<tr>
			<td>IFN-&#947;</td>
			<td>eBioscience</td>
			<td>12-7319-41</td>
			<td>Clone 4S.B3, RRID:AB_1311250</td>
		</tr>
	</tbody>
</table>

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.

The sc MHC class I technology used here allows precise control of cell surface expression of MHC class I with known, covalently linked peptides, to induce T cell activation. We have generated an engineered xenogeneic APC system (Figure 1A,B,C) that allows presentation of low levels of agonist pMHC in the presence or absence of co-agonist pMHC (Figure 1D,E). Critically, we have used a TCR-like antibody specific for HLA-A2 presenting E183 peptide to show that presentation of the agonist sc E183-HLA-A2 is not altered by co-expression of co-agonist sc GAG-HLA-A2 (Figure 1E). However, it must be noted that the E183-HLA-A2 specific TCR-like antibody shows some binding to HLA-A2 presenting GAG peptide (Figure 1E). Similar engineered antigen presenting cell systems can be constructed using different peptides or MHC molecules to investigate molecular requirements for TCR and/or coreceptor interactions in human T cells with different specificities.
We used these engineered APCs to stimulate an E183-HLA-A2-specific human CD8+ T cell clone. Three negative controls were used: T cells only, untransfected T-REx CHO cells, and T-REx CHO cells expressing high amounts of co-agonist sc GAG-HLA-A2 to test for potential T cell activation by any molecules expressed on T-REx CHO cells (untransfected T-REx CHO cells as compared to T cell only), and for T cell activation by sc-GAG-HLA-A2 (T-REx CHO cells expressing high levels of sc-GAG-HLA-A2 as compared to untransfected T-REx CHO cells). Untransfected T-REx CHO cells or T-REx CHO cells expressing sc-GAG-HLA-A2 did not induce activation of E183-HLA-A2-specific CD8+ CTL clone, as determined by quantifying IFN-&#947; production and expression of degranulation marker CD107a as a measure of T cell cytotoxicity (Figure 2A,B). Expression of high levels of agonist sc E183-HLA-A2, used as positive control, induced very efficient IFN-&#947; production and degranulation (Figure 2A,B), demonstrating that the sc HLA-A2 construct can be recognized by a specific TCR to activate T cell effector functions. Expression of low levels of sc E183-HLA-A2 induced lower levels of IFN-&#947; production and degranulation, and this was enhanced by presence of co-agonist sc GAG-HLA-A2 (Figure 2A,B). It must be noted that very high percentages of CD107a+ T cells were observed even in response to low levels of antigenic pMHC (Figure 2B), consistent with previous reports of relatively low requirements for the amount of agonist pMHC for induction of T cell cytotoxicity33. The CD107a MFI, indicating the amount of degranulation on a single cell basis, provides a better dynamic range (Figure 2B). The T cell activation assay used allows simultaneous quantification of two major effector functions: cytokine production and cytotoxicity, and provides very sensitive readout with good dynamic range.
We used the single chain technology to test whether TCR binding to co-agonist pMHC is required for co-agonist pMHC-dependent activation enhancement. We mutated valine at position 152 at the edge of the HLA-A2 peptide binding groove (Figure 3B) to glutamic acid to create V152E mutant, as this mutation has been reported to abolish TCR binding to HLA-A234. We then tested if the V152E mutation can abolish TCR binding for the E183-HLA-A2 CTL clone used, by introducing this mutation into agonist sc E183-HLA-A2 and expressing the mutant agonist sc construct in T-REx CHO cells. Wild type, but not V152E, sc E183-HLA-A2 induced activation of the E183-HLA-A2-specific CTL clone used (Figure 3A). It must be noted that any TCR-binding mutation on MHC class I needs to be separately tested for each individual TCR/T cell clone used, as the effect of mutation will depend on the precise molecular interactions between TCR and pMHC complex, and these will differ between different TCRs. For example, we tested eight mutations, either previously reported TCR-binding mutations or mutations predicted to disrupt TCR binding without abrogating peptide binding into HLA-A2. Of these, V152E was the only mutation that abolished activation of the E183-specific T cell clone used10. We then introduced the V152E mutation into the co-agonist sc GAG-HLA-A2, and co-expressed WT or V152E sc GAG-HLA-A2 with low levels of agonist sc E183-HLA-A2. Both WT and V152E sc GAG-HLA-A2 enhanced IFN-&#947; production and degranulation (Figure 3C,D), suggesting that TCR binding to co-agonist pMHC is not required for co-agonist pMHC-dependent CD8+ T cell activation enhancement. Single chain MHC class I technology, as presented here, can be used to modify TCR and/or coreceptor binding to MHC class I with known peptide to investigate the molecular requirements for T cell antigen recognition and activation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59126/59126fig1.jpg" /> 
Figure 1: Single chain HLA-A2 constructs used to investigate co-agonist in human CD8+ T cell activation.&#160;(A) Schematic diagram of a scHLA-A2 construct, consisting of covalently signal sequence from fused human &#946;2 microglobulin, peptide of choice, flexible glycine-serine linker (GGGGSGGGGS GGGGS), human &#946;2 microglobulin, flexible glycine-serine linker and HLA-A2 heavy chain.&#160;(B) Schematic diagram of the plasmids used to generate engineered antigen presenting cells to investigate co-agonism in human CD8+ T cell activation: tetracycline repressor, agonist sc E183-HLA-A2 under control of tetracycline-inducible promoter, non-stimulatory co-agonist sc GAG-HLA-A2 under control of constitutively active promoter.&#160;(C) Schematic diagram of the engineered antigen presenting cells used to investigate co-agonism: T-REx CHO cells (no human MHC expression), T-REx CHO cells expressing constitutively high levels of sc GAG-HLA-A2 (non-stimulatory co-agonist pMHC), T-REx CHO cells expressing low &#8220;leaky&#8221; levels of sc E183-HLA-A2 in the absence of tetracycline (low level of agonist peptide), T-REx CHO cells expressing high levels of sc E183-HLA-A2 in the presence of tetracycline (high level of agonist peptide), T-REx CHO cells expressing low levels of sc E183-HLA-A2 and high levels of sc GAG-HLA-A2 (low level of agonist peptide and high level of co-agonist peptide).&#160;(D) Cell surface staining of the engineered antigen presenting cell lines using anti-HLA-A2 antibody. The numbers shown denote MFI values for MHC stain in the live cell gate. Data representative of 5 independent experiments (E) Cell surface staining of the engineered antigen presenting cell lines using TCR-like antibody specific against HLA-A2 presenting E183 peptide. The numbers shown denote MFI values for MHC stain in the live cell gate. Data representative of 5 independent experiments. <a href="https://www.jove.com/files/ftp_upload/59126/59126fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59126/59126fig2v3.jpg" /> 
Figure 2: Presence of co-agonist pMHC enhanced cytokine production and degranulation of E183-HLA-A2-specific human CD8+ CTL clone.&#160;(A) Intracellular IFN-&#947; staining of HLA-A2-specific human CD8+ CTL clone after 3h stimulation with the indicated engineered antigen presenting cells. The numbers shown denote the percentage positive for IFN- &#947; staining. Data representative of 3 independent experiments, each performed with technical triplicates.&#160;(B) Cell surface CD107a staining of HLA-A2-specific human CD8+ CTL clone after 3h stimulation with the indicated engineered antigen presenting cells. The numbers shown denote the percentage positive for CD107a staining or the CD107a MFI for the CD8+ T cell population. Data representative of 3 independent experiments, each performed with technical triplicates.&#160;<a href="https://www.jove.com/files/ftp_upload/59126/59126fig2v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59126/59126fig3.jpg" /> 
Figure 3: Co-agonist &#8211; mediated T cell activation enhancement does not require TCR binding to the co-agonist pMHC complex.&#160;(A) V152E mutation in sc E183-HLA-A2 abolishes activation of the E183-HLA-A2-specific human CD8+ CTL clone. T-REx CHO cells expressing high levels (tetracycline induction) of the WT or V152E mutant sc E183-HLA-A2 were used to stimulate E183-HLA-A2-specific human CD8+ CTL clone for 3h. T cell activation was quantified using intracellular IFN-&#947; staining. The numbers shown denote the percentage positive for IFN- &#947; staining. Data representative of 3 independent experiments, each performed with technical triplicates.&#160;(B) Location of V152E mutation within the peptide binding groove of HLA-A2.&#160;(C) V152E mutation on sc GAG-HLA-A2 co-agonist pMHC complex does not abolish co-agonist-dependent activation enhancement. Intracellular IFN-&#947; staining of HLA-A2-specific human CD8+ CTL clone after 4h stimulation with the indicated engineered antigen presenting cells. The numbers shown denote the percentage positive for IFN- &#947; staining. Data representative of 3 independent experiments, each performed with technical triplicates.&#160;(D) V152E mutation on sc GAG-HLA-A2 co-agonist pMHC complex does not abolish co-agonist-dependent activation enhancement. Cell surface CD107a staining of HLA-A2-specific human CD8+ CTL clone after 3h stimulation with the indicated engineered antigen presenting cells. The numbers shown denote the percentage positive for CD107a staining or the CD107a MFI for the CD8+ T cell population. Data representative of 3 independent experiments, each performed with technical triplicates. <a href="https://www.jove.com/files/ftp_upload/59126/59126fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation at JoVE.com

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

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 ...

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Immunology and Infection

9.6K Views.  National University of Singapore. 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.

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

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 ...

Use of Interferon-&gamma; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-&gamma; (IFN-&gamma;) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected1. A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- &gamma; secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-&gamma; secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- &gamma; ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)2 in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 105 cells per well typically used in ELISPOT assays1,3, 1,000 T-cell clone cells in the presence of 1 x 105 autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-&gamma;. This assures successful performance of IFN-&gamma; ELISPOT assay. Similarly, IFN- &gamma; ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- &gamma; ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes4.

Use of Interferon-&amp;#947; Enzyme-linked Immunospot Assay to Characterize Novel T-cell Epitopes of Human Papillomavirus

Characterizing T-cell epitopes of pathogens that cause localized infections such as human papillomavirus is a challenge because of limited number of T cells in circulation. A method is described in which rare T cells were isolated and were characterized starting with a very small number of cells.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human ...

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 ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

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.

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

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents directly bind to T-cell receptors on the surface of CD8+ T-lymphocytes, fluorochrome-labeled pMHC-I tetramers enable the accurate detection of antigen (Ag)-specific CD8+ T-cells without the need for in vitro re-stimulation. Moreover, when combined with multi-color flow cytometry, pMHC-I tetramer staining can reveal key aspects of Ag-specific CD8+ T-cells, including differentiation stage, memory phenotype, and activation status. These types of analyses have been especially useful in the field of HIV immunology where CD8+ T-cells can affect progression to AIDS. Experimental infection of rhesus macaques with simian immunodeficiency virus (SIV) provides an invaluable tool to study cellular immunity against the AIDS virus. As a result, considerable progress has been made in defining and characterizing T-cell responses in this animal model. Here we present an optimized protocol for enumerating SIV-specific CD8+ T-cells in rhesus macaques by pMHC-I tetramer staining. Our assay permits the simultaneous quantification and memory phenotyping of two pMHC-I tetramer+ CD8+ T-cell populations per test, which might be useful for tracking SIV-specific CD8+ T-cell responses generated by vaccination or SIV infection. Considering the relevance of nonhuman primates in biomedical research, this methodology is applicable for studying CD8+ T-cell responses in multiple disease settings.

Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining

Here, we present an optimized protocol for enumerating and characterizing rhesus macaque CD8+ T cells against the AIDS virus. This article is useful not only to the field of HIV immunology, but also to other areas of biomedical research where CD8+ T cell responses are known to affect disease outcome.

Peptide-major histocompatibility complex class I (pMHC-I) tetramers have been an invaluable tool to study CD8+ T-cell responses. Because these reagents ...

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled &#34;Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo.&#34; The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

Here, we describe a method that combines in situ MHC-tetramer staining with immunohistochemistry to determine localization, phenotype, and quantity of antigen-specific T cells in tissues. This protocol is used to determine the spatial and phenotypic characteristics of antigen-specific CD8 T cells relative to other cell type and structures in tissues.

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in ...

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs.
Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8+T cells and to discriminate between na&#239;ve and effector cells.

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

Here, we present a protocol for the ex vivo qualitative detection of antigen-specific CD8+ T cells. Analysis is possible with single cell suspensions from organs or from small amounts of blood. A broad range of studies require the analysis of cytotoxic T cell responses (vaccination and cancer immunotherapy studies).

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...