This project was funded by the FWF Austrian Science Fund (project number P 25499-B13) and the European Union&#39;s Horizon 2020 research and innovation program under grant agreement No. 681032. The following reagent was obtained through the NIH Tetramer Core Facility: Class I MHC Tetramer for vesicular stomatitis virus nucleoprotein (RGYVYQGL).

All methods described were performed in compliance with the Austrian National Animal Experimentation Law (&#8220;Tierversuchsgesetz&#8221;), and animal trial permission was granted by Austrian National Authorities.
1. Buffer Preparation and Sample Collection
NOTE: The mouse strain used depends on the epitope analyzed. Choose an appropriate tetramer that binds to an MHC type expressed in the mice, e.g., H-2Kb for C57BL/6 mice. The gender and age of the animals will depend on the scientific question. For most of the experiments described here, use female mice at 6&#8211;8 weeks of age at the start of the experiment, i.e., first immunization.
<ol>
	<li>Prepare FACS Buffer (Phosphate-buffered saline (PBS) + 1% fetal calf serum (FCS) + 0.1% sodium azide + 2 mM Ethylenediaminetetraacetic acid (EDTA) and FACS fixing buffer (1.5% (v/v) formaldehyde in PBS). 
	NOTE: It is recommended that both buffers are prepared in advance. Stored at 4 &#176;C until use.</li>
	<li>Blood: Collect 20 &#181;L of blood per mouse from the tail vein of the mouse in EDTA-coated tubes, as described previously24. 
	NOTE: Blood may also be collected by other routes, e.g., vena facialis or retro-orbital sinus. However, the method of blood collection has to be in compliance with the national animal experimentation law and animal trial applications. Collection of blood from the tail vein is ideal for studies where repeatedly small amounts of blood are needed. Additional material is required for compensation controls and the non-stained control.</li>
	<li>Spleen: Isolate the organ and, with the help of the plunger of a syringe, press through a 70 nm and 40 &#181;m cell strainer. Perform lysis of erythrocytes, as described in step 6 and count. Adjust the concentration to 1 x 107 cells/mL in PBS. Per sample, 1 x 106 cells are required. 
	NOTE: Always include some mock immunized or control vector immunized animals as negative control. For ovalbumin (OVA)-tetramer, a sample from OT-1 mice might be used as positive control. Do not forget unstained and compensation controls in the calculation. If necessary, samples from different animals in the experiment might be pooled for this.</li>
	<li>After sample collection, directly proceed with the staining.</li>
</ol>
2. Staining Set-up
<ol>
	<li>Prepare one FACS tube for each sample. Label tubes properly and transfer 100 &#956;L of organ suspension (1 x 106 cells) or 20 &#956;L of blood into each tube.</li>
	<li>Spleen: Centrifuge for 5 min at 600 x g at 4&#8211;8 &#176;C and discard the supernatant. Vortex to resuspend the cell pellet. 
	NOTE: This will result in a remaining volume of around 20 &#181;L, similar as the volume for the blood samples.</li>
	<li>For each channel to be used also prepare one FACS tube for a compensation sample. Prepare one additional sample as an unstained control.</li>
</ol>
3. Tetramer Staining
<ol>
	<li>For each sample, use 50 &#956;L of tetramer dilution. For suggested tetramers and optimized dilutions, refer to Table 1. 
	NOTE: When working with tetramers or antibodies, turn off the light of the safety cabinet and protect samples from light.
	<ol>
		<li>Prepare a tube with FACS buffer (volume = 50 &#956;L &#215; number of samples plus additional 10% of the total volume to compensate for pipetting errors).</li>
		<li>Add tetramer(s) at optimal dilution, as listed in Table 1. Vortex the solution. 
		NOTE: When using the whole antibody panel (CD3, CD8, CD43, CD44, CD62L) with listed fluorophores, two tetramers (in PE and APC) can be included. Both tetramers can be combined in a single staining, i.e., staining of cells with both tetramers can be performed simultaneously.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tetramer</td>
			<td>Peptide sequence</td>
			<td>Allele&#160;</td>
			<td>Fluorophore</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>MHC I/OVA</td>
			<td>SIINFEKL</td>
			<td>H-2Kb</td>
			<td>APC</td>
			<td>1:25</td>
		</tr>
		<tr>
			<td>MHC I/VSV-NP</td>
			<td>RGYVYQGL</td>
			<td>H-2Kb</td>
			<td>PE</td>
			<td>1:25</td>
		</tr>
		<tr>
			<td>MHC I/EGFP</td>
			<td>HYLSTQSAL</td>
			<td>H-2Kd</td>
			<td>PE</td>
			<td>1:25</td>
		</tr>
		<tr>
			<td>MHC I/LCMV-GP</td>
			<td>KAVYNFATC</td>
			<td>H-2Db</td>
			<td>APC</td>
			<td>1:25</td>
		</tr>
		<tr>
			<td>MHC I/HPV 16 E7</td>
			<td>RAHYNIVTF</td>
			<td>H-2Db</td>
			<td>APC</td>
			<td>1:10</td>
		</tr>
	</tbody>
</table>
Table 1: Suggested tetramers and optimal dilutions. Recommended tetramers for some immunodominant peptides of model antigens (Ovalbumin (OVA) and enhanced green fluorescent protein (eGFP)) or pathogen components (Vesicular stomatitis virus (VSV) nucleoprotein (NP), Lymphocytic Choriomeningitis Virus (LCMV) Glycoprotein (GP) and human papillomavirus (HPV) E7 oncoprotein (E7)). For each, the peptide sequence and corresponding allele, as well as recommended fluorophore and optimized dilution is listed.
<ol start="2">
	<li>Add 50 &#956;L of tetramer dilution to each sample and vortex gently. Add FACS buffer only (without tetramer) to compensation controls and to the unstained sample.</li>
	<li>Incubate samples for 20 min at 37 &#176;C, protected from light. To ensure a seamless transition from tetramer to antibody staining, prepare the antibody mix as described in step 4 during the incubation time. 
	NOTE: For each individual tetramer, optimal conditions (dilution, incubation time and temperature) need to be adjusted.</li>
</ol>
4. Preparation of Antibodies
<ol>
	<li>For each sample, prepare 50 &#181;L of antibody mix.
	<ol>
		<li>Prepare a tube with FACS buffer (volume = 50 &#956;L &#215; number of samples plus additional 10% of the total volume to compensate pipetting errors).</li>
		<li>Add antibodies in the dilutions as listed in Table 2. 
		NOTE: Depending on the scientific question, other marker combinations apart from the one described here might be used. Make sure to always include antibodies against CD3 and CD8 in the panel.</li>
		<li>Vortex the solution.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td>Fluorophore</td>
			<td>Dilution</td>
			<td>&#181;g/sample</td>
			<td>Marker</td>
		</tr>
		<tr>
			<td>CD3</td>
			<td>PE-Cy7</td>
			<td>1:200</td>
			<td>0.05</td>
			<td rowspan="3">CTLs (CD3+CD8+)</td>
		</tr>
		<tr>
			<td rowspan="2">CD8</td>
			<td>Pacific Blue</td>
			<td>1:750</td>
			<td>0.013</td>
		</tr>
		<tr>
			<td>V450</td>
			<td>1:100</td>
			<td>0.1</td>
		</tr>
		<tr>
			<td>CD43</td>
			<td>FITC</td>
			<td>1:100</td>
			<td>0.25</td>
			<td>Activation (CD43+)</td>
		</tr>
		<tr>
			<td>CD44</td>
			<td>PE-Cy5</td>
			<td>1:250</td>
			<td>0.04</td>
			<td rowspan="2">Naive (CD44-CD62L+) &#38; effector (CD44+CD62L-)</td>
		</tr>
		<tr>
			<td>CD62L</td>
			<td>APC-Cy7</td>
			<td>1:500</td>
			<td>0.02</td>
		</tr>
	</tbody>
</table>
Table 2: Antibodies used in this protocol and optimal dilutions. Recommended surface markers (CD3, CD8, CD43, CD44 and CD62L) are listed in the first column. For each, the recommended fluorophore, optimized dilution and amount of antibody/sample are listed. In the last column, the cell type identified with each marker is specified.
<ol start="2">
	<li>Prepare antibodies for compensation controls. For each compensation control, use an antibody against CD8 in the respective color.
	<ol>
		<li>For each channel, prepare a tube with 200 &#956;L of FACS buffer and add 1 &#956;L of a 1:200 dilution antibody against CD8 in the respective color.</li>
		<li>Vortex the tubes.</li>
	</ol>
	</li>
	<li>Immediately proceed with staining.</li>
</ol>
5. Staining of Samples
<ol>
	<li>Wash samples once by adding ~1 mL of FACS buffer and centrifuge for 5 min at 600 x g at 4&#8211;8 &#176;C. After centrifugation, discard the supernatant and drain off remaining liquid on a stack of paper towels. 
	NOTE: When working with blood, be cautious when draining off remaining liquid. Prior to the lysis of erythrocytes, blood will not stick to the bottom of the FACS tube. Alternatively, aspirate the supernatant.</li>
	<li>Add 50 &#956;L of the antibody mix to each cell pellet and vortex gently.</li>
	<li>Add 50 &#956;L of each compensation mix to the cell pellet of the corresponding compensation control and vortex gently.</li>
	<li>Add 50 &#956;L of FACS buffer to the cell pellet of the unstained control and vortex gently.</li>
	<li>Incubate all samples for 30 min at 4 &#176;C, protected from light.</li>
	<li>When working with organs: skip step 6. Wash once by adding ~1&#8211;2 mL of FACS buffer and centrifuge for 5 min at 600 x g at 4&#8211;8 &#176;C. After centrifugation, discard the supernatant and drain off remaining liquid on a stack of paper towels.</li>
	<li>When working with blood: proceed to step 6 (lysis of erythrocytes).</li>
</ol>
6. Lysis of Erythrocytes
<ol>
	<li>Add 500 &#956;L of ACK (Ammonium-Chloride-Potassium) buffer25 to each sample and gently vortex. 
	NOTE: ACK buffer will lead to osmotic swelling and lysis, specifically of erythrocytes.</li>
	<li>Incubate for 5 min at room temperature in the dark.</li>
	<li>Add 1 mL of FACS buffer and centrifuge for 5 min at 600 x g at 4&#8211;8 &#176;C. After centrifugation, discard the supernatant and drain off the remaining liquid on a stack of paper towels. 
	Note: When the pellet is rather red, repeat the lysis of erythrocytes.</li>
	<li>Wash once by adding ~1&#8211;2 mL FACS buffer and centrifuge for 5 min at 600 x g at 4&#8211;8 &#176;C. After centrifugation, discard the supernatant and drain off remaining liquid on a stack of paper towels.</li>
</ol>
7. Flow Cytometric Measurement and Analysis
<ol>
	<li>Add 150&#8211;300 &#956;L of FACS fixing buffer to each tube and mix by vortexing. For 20 &#181;L of blood, 150 &#181;L of buffer is sufficient. 
	NOTE: Before fixation, make sure that cells are well re-suspended in order to prevent formation of clumps. Proceed with flow cytometric measurement as quickly as possible.</li>
	<li>Measure the compensation controls and correct any spectral overlaps.</li>
	<li>Set up sequential gates, as depicted in Figure 1 to select for CD3+/CD8+ cells.
	<ol>
		<li>Gate on lymphocytes using forward and sideward scatter (Area) (non-logarithmic scale).</li>
		<li>Within the lymphocyte population, gate on single cells using forward scatter width vs area (non-logarithmic scale).</li>
		<li>Plot single cell lymphocytes using CD3 and CD8 channels (logarithmic scale). Identify CD8+ T cells by gating on CD3+/CD8+ cells.</li>
		<li>Plot CD8+ vs Tetramer+ cells and gate on CD8+ Tetramer+ cells, as depicted in Figure 2.</li>
	</ol>
	</li>
	<li>If possible, record 20,000 cells (at least 5,000 cells from blood) in the CD3+/CD8+ gate for each sample and save as a FCS file. 
	NOTE: The amount of cells to record might need to be adjusted according to the frequency of the antigen-specific cells of interest.</li>
	<li>Analyze FCS files with appropriate analysis software. Use the gating strategy, as described above (section 7.3) and quantify CD8+ Tetramer+ cells.</li>
</ol>

<ol><li>Tober, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((VSV-GP%3A+a+potent+viral+vaccine+vector+that+boosts+the+immune+response+upon+repeated+applications%5BTitle%5D)+AND+%22Journal+of+virology%22%5BJournal%5D)">VSV-GP: a potent viral vaccine vector that boosts the immune response upon repeated applications.</a> Journal of virology. 88 (9), 4897-4907 (2014).</li>
<li>Muik, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Re-engineering+vesicular+stomatitis+virus+to+abrogate+neurotoxicity%2C+circumvent+humoral+immunity%2C+and+enhance+oncolytic+potency%5BTitle%5D)+AND+%22Cancer+Reseaerch%22%5BJournal%5D)">Re-engineering vesicular stomatitis virus to abrogate neurotoxicity, circumvent humoral immunity, and enhance oncolytic potency.</a> Cancer Reseaerch. 74 (13), 3567-3578 (2014).</li>
<li>Eales, L. J., Farrant, J., Helbert, M., Pinching, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peripheral+blood+dendritic+cells+in+persons+with+AIDS+and+AIDS+related+complex%3A+loss+of+high+intensity+class+II+antigen+expression+and+function%5BTitle%5D)+AND+%22Clinical+and+Experimental+Immunology%22%5BJournal%5D)">Peripheral blood dendritic cells in persons with AIDS and AIDS related complex: loss of high intensity class II antigen expression and function.</a> Clinical and Experimental Immunology. 71, 423-427 (1988).</li>
<li>Altman, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phenotypic+analysis+of+antigen-specific+T+lymphocytes%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Phenotypic analysis of antigen-specific T lymphocytes.</a> Science. 274 (5284), 94-96 (1996).</li>
<li>Tetramers and Monomers. , Available from:
 <a target="_blank" href="https://www.mblintl.com/products/research/monomer-tetramers/filter/product_type/monomer,peptide,tetramer ">https://www.mblintl.com/products/research/monomer-tetramers/filter/product_type/monomer,peptide,tetramer </a> (2016).</li>
<li>NIH Tetramer Core Facility. , Available from:
 <a target="_blank" href="http://tetramer.yerkes.emory.edu/">http://tetramer.yerkes.emory.edu/</a> (2010).</li>
<li>Class I MHC Tetramer Preparation: Overview. , Available from:
 <a target="_blank" href="http://tetramer.yerkes.emory.edu/support/protocols">http://tetramer.yerkes.emory.edu/support/protocols</a> (2010).</li>
<li>Wolfl, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitation+of+MHC+tetramer-positive+cells+from+whole+blood%3A+evaluation+of+a+single-platform%2C+six-parameter+flow+cytometric+method%5BTitle%5D)+AND+%22Cytometry+A%22%5BJournal%5D)">Quantitation of MHC tetramer-positive cells from whole blood: evaluation of a single-platform, six-parameter flow cytometric method.</a> Cytometry A. 57 (2), 120-130 (2004).</li>
<li>Burrows, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peptide-MHC+class+I+tetrameric+complexes+display+exquisite+ligand+specificity%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">Peptide-MHC class I tetrameric complexes display exquisite ligand specificity.</a> The Journal of Immunology. 165 (11), 6229-6234 (2000).</li>
<li>Sims, S., Willberg, C., Klenerman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MHC-peptide+tetramers+for+the+analysis+of+antigen-specific+T+cells%5BTitle%5D)+AND+%22Expert+Review+of+Vaccines%22%5BJournal%5D)">MHC-peptide tetramers for the analysis of antigen-specific T cells.</a> Expert Review of Vaccines. 9 (7), 765-774 (2010).</li>
<li>Legoux, F. P., Moon, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peptide%3AMHC+tetramer-based+enrichment+of+epitope-specific+T+cells%5BTitle%5D)+AND+%22The+Journal+of+Visualized+Experiments%22%5BJournal%5D)">Peptide:MHC tetramer-based enrichment of epitope-specific T cells.</a> The Journal of Visualized Experiments. (68), (2012).</li>
<li>Xie, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+T+cell-based+vaccine+capable+of+stimulating+long-term+functional+CTL+memory+against+B16+melanoma+via+CD40L+signaling%5BTitle%5D)+AND+%22Cellular+%26+Molecular+Immunology%22%5BJournal%5D)">A novel T cell-based vaccine capable of stimulating long-term functional CTL memory against B16 melanoma via CD40L signaling.</a> Cellular & Molecular Immunology. 10 (1), 72-77 (2013).</li>
<li>Nanjundappa, R. H., Wang, R., Xie, Y., Umeshappa, C. S., Xiang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+CD8%2B+T+cell-based+vaccine+stimulates+Gp120-specific+CTL+responses+leading+to+therapeutic+and+long-term+immunity+in+transgenic+HLA-A2+mice%5BTitle%5D)+AND+%22Vaccine%22%5BJournal%5D)">Novel CD8+ T cell-based vaccine stimulates Gp120-specific CTL responses leading to therapeutic and long-term immunity in transgenic HLA-A2 mice.</a> Vaccine. 30 (24), 3519-3525 (2012).</li>
<li>Bowers, E. V., Horvath, J. J., Bond, J. E., Cianciolo, G. J., Pizzo, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antigen+delivery+by+alpha%282%29-macroglobulin+enhances+the+cytotoxic+T+lymphocyte+response%5BTitle%5D)+AND+%22Journal+of+Leukocyte+Biology%22%5BJournal%5D)">Antigen delivery by alpha(2)-macroglobulin enhances the cytotoxic T lymphocyte response.</a> Journal of Leukocyte Biology. 86 (2), 1259-1268 (2009).</li>
<li>Guo, H., Baker, S. F., Martinez-Sobrido, L., Topham, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+CD8+T+cell+heterologous+protection+by+a+single+dose+of+single-cycle+infectious+influenza+virus%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">Induction of CD8 T cell heterologous protection by a single dose of single-cycle infectious influenza virus.</a> The Journal of Immunology. 88 (20), 12006-12016 (2014).</li>
<li>Sakai, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dendritic+cell-based+immunotherapy+targeting+Wilms%27+tumor+1+in+patients+with+recurrent+malignant+glioma%5BTitle%5D)+AND+%22Journal+of+Neurosurgery%22%5BJournal%5D)">Dendritic cell-based immunotherapy targeting Wilms' tumor 1 in patients with recurrent malignant glioma.</a> Journal of Neurosurgery. 123 (4), 989-997 (2015).</li>
<li>Rosaely, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+responses+to+WT1+in+patients+with+AML+or+MDS+after+chemotherapy+and+allogeneic+stem+cell+transplantation%5BTitle%5D)+AND+%22International+Journal+of+Cancer%22%5BJournal%5D)">Immune responses to WT1 in patients with AML or MDS after chemotherapy and allogeneic stem cell transplantation.</a> International Journal of Cancer. 138 (7), 1792-1801 (2016).</li>
<li>Shane, H. L., Reagin, K. L., Klonowski, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Respiratory+Environment+Diverts+the+Development+of+Antiviral+Memory+CD8+T+Cells%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">The Respiratory Environment Diverts the Development of Antiviral Memory CD8 T Cells.</a> The Journal of Immunology. , (2018).</li>
<li>Li, S., Mwakalundwa, G., Skinner, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+Situ+MHC-tetramer+Staining+and+Quantitative+Analysis+to+Determine+the+Location%2C+Abundance%2C+and+Phenotype+of+Antigen-specific+CD8+T+Cells+in+Tissues%5BTitle%5D)+AND+%22The+Journal+of+Visualized+Experiments%22%5BJournal%5D)">In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues.</a> The Journal of Visualized Experiments. (127), e56130(2017).</li>
<li>De Vries, I. J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+situ+detection+of+antigen-specific+T+cells+in+cryo-sections+using+MHC+class+I+tetramers+after+dendritic+cell+vaccination+of+melanoma+patients%5BTitle%5D)+AND+%22Cancer+Immunology+Immunotherapy%22%5BJournal%5D)">In situ detection of antigen-specific T cells in cryo-sections using MHC class I tetramers after dendritic cell vaccination of melanoma patients.</a> Cancer Immunology Immunotherapy. 56 (10), 1667-1676 (2007).</li>
<li>Tan, H. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+vaginal-resident+HIV-specific+CD8+T+cells+with+mucosal+prime-boost+immunization%5BTitle%5D)+AND+%22Mucosal+Immunology%22%5BJournal%5D)">Induction of vaginal-resident HIV-specific CD8 T cells with mucosal prime-boost immunization.</a> Mucosal Immunology. , (2017).</li>
<li>Huang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD8%28%2B%29+T+Cell+Immune+Response+in+Immunocompetent+Mice+during+Zika+Virus+Infection%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">CD8(+) T Cell Immune Response in Immunocompetent Mice during Zika Virus Infection.</a> Journal of Virology. 91 (22), (2017).</li>
<li>Hensel, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Selective+Expression+of+CCR10+and+CXCR3+by+Circulating+Human+Herpes+Simplex+Virus-Specific+CD8+T+Cells%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">Selective Expression of CCR10 and CXCR3 by Circulating Human Herpes Simplex Virus-Specific CD8 T Cells.</a> Journal of Virology. 91 (19), (2017).</li>
<li>Diehl, K. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+good+practice+guide+to+the+administration+of+substances+and+removal+of+blood%2C+including+routes+and+volumes%5BTitle%5D)+AND+%22Journal+of+Applied+Toxicology%22%5BJournal%5D)">A good practice guide to the administration of substances and removal of blood, including routes and volumes.</a> Journal of Applied Toxicology. 21 (1), 15-23 (2001).</li>
<li>ACK Lysis Buffer. , Available from:
 <a target="_blank" href="http://www.cshprotocols.cshlp.org/content/2014/11/pdb.rec083295.short">http://www.cshprotocols.cshlp.org/content/2014/11/pdb.rec083295.short</a> (2014).</li>
<li>San Jose, E., Borroto, A., Niedergang, F., Alcover, A., Alarcon, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Triggering+the+TCR+complex+causes+the+downregulation+of+nonengaged+receptors+by+a+signal+transduction-dependent+mechanism%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Triggering the TCR complex causes the downregulation of nonengaged receptors by a signal transduction-dependent mechanism.</a> Immunity. 12 (2), 161-170 (2000).</li>
<li>Dietrich, J., Hou, X., Wegener, A. M., Geisler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD3+gamma+contains+a+phosphoserine-dependent+di-leucine+motif+involved+in+down-regulation+of+the+T+cell+receptor%5BTitle%5D)+AND+%22EMBO+Journal%22%5BJournal%5D)">CD3 gamma contains a phosphoserine-dependent di-leucine motif involved in down-regulation of the T cell receptor.</a> EMBO Journal. 13 (9), 2156-2166 (1994).</li>
<li>Schone, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunodominance+of+Adenovirus-Derived+CD8%28%2B%29+T+Cell+Epitopes+Interferes+with+the+Induction+of+Transgene-Specific+Immunity+in+Adenovirus-Based+Immunization%5BTitle%5D)+AND+%22Journal+of+Virology%22%5BJournal%5D)">Immunodominance of Adenovirus-Derived CD8(+) T Cell Epitopes Interferes with the Induction of Transgene-Specific Immunity in Adenovirus-Based Immunization.</a> Journal of Virology. 91 (20), (2017).</li>
<li>Dolton, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((More+tricks+with+tetramers%3A+a+practical+guide+to+staining+T+cells+with+peptide%E2%80%93MHC+multimers%5BTitle%5D)+AND+%22Immunology%22%5BJournal%5D)">More tricks with tetramers: a practical guide to staining T cells with peptide–MHC multimers.</a> Immunology. 146 (1), 11-22 (2015).</li>
<li>Whelan, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specificity+of+CTL+Interactions+with+Peptide-MHC+Class+I+Tetrameric+Complexes+Is+Temperature+Dependent%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">Specificity of CTL Interactions with Peptide-MHC Class I Tetrameric Complexes Is Temperature Dependent.</a> The Journal of Immunology. 163 (8), 4342-4348 (1999).</li>
<li>Wooldridge, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tricks+with+tetramers%3A+how+to+get+the+most+from+multimeric+peptide%E2%80%93MHC%5BTitle%5D)+AND+%22Immunology%22%5BJournal%5D)">Tricks with tetramers: how to get the most from multimeric peptide–MHC.</a> Immunology. 126 (2), 147-164 (2009).</li>
<li>Denkberg, G., Cohen, C. J., Reiter, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Critical+role+for+CD8+in+binding+of+MHC+tetramers+to+TCR%3A+CD8+antibodies+block+specific+binding+of+human+tumor-specific+MHC-peptide+tetramers+to+TCR%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">Critical role for CD8 in binding of MHC tetramers to TCR: CD8 antibodies block specific binding of human tumor-specific MHC-peptide tetramers to TCR.</a> The Journal of Immunology. 167 (1), 270-276 (2001).</li>
<li>Daniels, M. A., Jameson, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Critical+role+for+CD8+in+T+cell+receptor+binding+and+activation+by+peptide%2Fmajor+histocompatibility+complex+multimers%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers.</a> The Journal of Experimental Medicine. 191 (2), 335-346 (2000).</li>
<li>Tungatt, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Antibody+stabilization+of+peptide-MHC+multimers+reveals+functional+T+cells+bearing+extremely+low-affinity+TCRs%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs.</a> The Journal of Immunology. 194 (1), 463-474 (2015).</li>
<li>Lissina, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+kinase+inhibitors+substantially+improve+the+physical+detection+of+T-cells+with+peptide-MHC+tetramers%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">Protein kinase inhibitors substantially improve the physical detection of T-cells with peptide-MHC tetramers.</a> Journal of Immunological Methods. 340 (1), 11-24 (2009).</li>
<li>Rius, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peptide-MHC+Class+I+Tetramers+Can+Fail+To+Detect+Relevant+Functional+T+Cell+Clonotypes+and+Underestimate+Antigen-Reactive+T+Cell+Populations%5BTitle%5D)+AND+%22The+Journal+of+Immunology%22%5BJournal%5D)">Peptide-MHC Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations.</a> The Journal of Immunology. 200 (7), 2263-2279 (2018).</li>
<li>Xiao, Z., Mescher, M. F., Jameson, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detuning+CD8+T+cells%3A+down-regulation+of+CD8+expression%2C+tetramer+binding%2C+and+response+during+CTL+activation%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">Detuning CD8 T cells: down-regulation of CD8 expression, tetramer binding, and response during CTL activation.</a> The Journal of Experimental Medicine. 204 (11), 2667-2677 (2007).</li>
<li>McMichael, A. J., O'Callaghan, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+New+Look+at+T+Cells%5BTitle%5D)+AND+%22The+Journal+of+Experimental+Medicine%22%5BJournal%5D)">A New Look at T Cells.</a> The Journal of Experimental Medicine. 187 (9), 1367-1371 (1998).</li>
<li>Hunsucker, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Peptide%2FMHC+tetramer-based+sorting+of+CD8%28%2B%29+T+cells+to+a+leukemia+antigen+yields+clonotypes+drawn+nonspecifically+from+an+underlying+restricted+repertoire%5BTitle%5D)+AND+%22Cancer+Immunology+Research%22%5BJournal%5D)">Peptide/MHC tetramer-based sorting of CD8(+) T cells to a leukemia antigen yields clonotypes drawn nonspecifically from an underlying restricted repertoire.</a> Cancer Immunology Research. 3 (3), 228-235 (2015).</li>
<li>Pittet, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+analysis+of+tumor+antigen+specific+CD8%2B+T+cell+responses+using+MHC%2Fpeptide+tetramers+in+cancer+patients%5BTitle%5D)+AND+%22International+Immunopharmacology%22%5BJournal%5D)">Ex vivo analysis of tumor antigen specific CD8+ T cell responses using MHC/peptide tetramers in cancer patients.</a> International Immunopharmacology. 1 (7), 1235-1247 (2001).</li>
<li>Cohen, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+neoantigen-specific+T+cells+from+tumor+and+peripheral+lymphocytes%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Investigation%22%5BJournal%5D)">Isolation of neoantigen-specific T cells from tumor and peripheral lymphocytes.</a> The Journal of Clinical Investigation. 125 (10), 3981-3991 (2015).</li>
</ol>

Dorothee von Laer is an inventor of VSV-GP and holds minority shares in the biotech company ViraTherapeutics GmbH, which holds the intellectual property rights for VSV-GP. For the other authors, no competing financial interests exist.

Tetramer staining is a rather straightforward and uncomplicated protocol to analyze phenotype and peptide specificity of a T lymphocyte. The usage of blood for analysis, as described here, is minimally invasive and allows continuous monitoring, for example in vaccination studies. In the field of vaccination, the quantification of vector- and antigen-specific responses is of interest, as vector-specific responses might hinder an effective immune response against the vaccine antigen28. Of note is that with the protocol described here, both populations can be quantified simultaneously in a single tetramer staining, thereby reducing staining variability and sample amounts. However, a few steps need to be done carefully to ensure proper measurement and reliable data. If using blood from the tail vein for analysis, one should make sure to pre-warm the animals to induce vasodilation24. Thereby, sufficient blood can be collected in a short time, stress on the animals is reduced and the analysis is much better, as compared to if the blood is collected slowly. Also, after sample collection (either blood or organs), direct staining is recommended to avoid false negative results due to TCR downregulation. The same applies for all subsequent steps: the procedure should not be interrupted and all washing steps reduced to the minimal number (as stated in the protocol). To ensure proper staining, care should be taken to vortex all solutions and samples prior to and after incubation. This is especially important before fixing the samples to avoid clumping of cells.
In terms of modifying the protocol, other surface markers and tetramers can be used, depending on the aim of the analysis. However, all reagents then need to be titrated, optimally in combination with the whole staining panel. For some of the tetramers specified here, optimization revealed that we can increase the dilution recommended by the manufacturer (1:10 recommended, optimized 1:25) (Table 1). To compensate spectral overlap, compensation beads can be used instead of stained cells. Regarding the choice of the tetramer-coupled fluorochrome, one should envisage to use bright fluorochromes, as this facilitates detection - especially when the signal is low. As Dolton and colleagues29, we prefer to use PE- or APC-coupled tetramers, which can be perfectly combined in a single staining and CD8+ T cells with a single antigen specificity can be nicely detected (Figure 2). Regarding temperature and incubation times, a variety of tetramer staining conditions exist. In our optimization process, we addressed this issue and performed tetramer staining at different conditions (e.g. 4 &#176;C, room temperature or 37 &#176;C). From the results obtained, we recommend to stain for 20 min at 37 &#176;C, which is in concordance with literature30,31. Prolonged incubation should be avoided, as this can lead to internalization of the tetramer30 and false negative results.
The choice of the right antibody for detection of CD8+ cells is another important issue, which has to carefully considered (and potentially adapted). This arises from the fact that certain anti-CD8 antibody clones block binding of tetramers to the TCR, in human32 as well as mouse33 samples. For our tetramer staining protocol, we selected clone 53&#8211;6.7 to stain for murine CD8+ cells &#8212; a clone which does not block, but rather enhances tetramer staining.
Tetramer staining is rather uncomplicated when analyzing prominent immune responses at the peak of the T cell response, for example. However, there might be populations which are a bit more &#39;problematic&#39;. Such examples include cells specific for low affinity antigens (tumor, self), recently activated cells which subsequently down-regulated their receptors or rare cell subsets (e.g. na&#239;ve precursor or memory cell populations). In these cases, the classical tetramer staining protocol might need to be improved or combined with other methods. For example, the protein kinase inhibitor (PKI) dasatinib inhibits TCR internalization and might be included prior to tetramer staining. Tetramers can also be stabilized by including anti-fluorochrome unconjugated primary Abs after tetramer staining. Additionally, fluorescence intensity can be increased by addition of a second anti-Ab fluorochrome-conjugated Ab29,34,35,36. We optimized the conditions selectively for the tetramers specified in this protocol and did not include PKI or additional Abs. However, for any other tetramer, the optimal conditions have to be adjusted individually. With regard to rare populations, tetramer staining might need to be combined with magnetic enrichment11.
To facilitate and validate FACS analysis of tetramer staining, negative and positive controls should be included. As a negative control, we always stain cells of a na&#239;ve mouse of the same strain with our tetramer of interest. Alternatively, samples can be stained with tetramers with irrelevant peptides, but with the same fluorochrome as the tetramer of interest. Such controls are essential to exclude false positive signals, e.g., originating from dying cells. In addition to this, it is recommended to include a live/dead stain, such as propidium iodide (PI). This is of special importance if cells are not stained directly after isolation. Another strategy to remove autofluorescence background might be to include several non-T cell markers in one channel. By excluding cells positive in this channel, non-T cell populations can be excluded. As a positive control, a sample from an OT-1 mouse can be used for OVA tetramer, for example. For other tetramers, this has to be chosen individually (e.g., sample from a mouse, which was immunized several times). Alike others37, we also observe a down-regulation of the CD8 receptor during CTL activation at day 7 of the effector T cell response. Therefore, to avoid loss of activated tetramer+ effector T cells, we recommend to include the CD8low cells in the analysis (at least if measuring in the effector phase).
The quality and amount of information one can retrieve from this protocol is dependent on the knowledge about the antigen to be studied, the availability and specificity of tetramer and the quality of the FACS machine (number of lasers and available detectors). If working with animal samples, variation in the immune response is natural and inevitable. Therefore, to gain meaningful results from tetramer staining, at least 3&#8211;5 animals should be analyzed. If done so, the protocol described here will give reliable and reproducible results (exemplary result from one experiment can be found in Supplemental Table 1). As mentioned before, this method is perfectly suited to quantify the phenotype and antigen-specificity of CD8+ T cells (Figure 3, 4; Supplemental Figure 1), not only in the mouse but also in humans. However, to analyze CD8+ T cell effector functions, such as granzyme-induced cell death, ICS and/or ELISpot need to be performed. However, one should keep in mind that T cell functions, as measured by in vitro stimulation might not represent the actual situation in vivo. In vivo, a suppressive environment might prevent T cell functions which are measured in vitro.
On its own, tetramer staining does not provide all information, but it evolved to become an essential method to characterize T cell responses and quantify T cell subsets in vitro in a very sensitive manner38. Tetramers cannot only be used to quantify certain subsets, but also to isolate those39, localize them by in situ hybridization19,20 and study low-affinity antigens, such as tumor-associated40,41. Since the discovery of tetramer technology4, tetramer staining has become an essential tool in T-cell analysis and the range of applications.

The aim of this method is to assess the frequency of cytotoxic T lymphocyte (CTL) responses to (multiple) antigens in the mouse by flow cytometric analysis without the need for time-consuming peptide stimulation. This method analyses the phenotype and antigen specificity of CTL subsets, in a single staining. We optimized the Major Histocompatibility Complex I (MHC I) tetramer staining protocol to analyze efficacy of new vaccine approaches, such as VSV-GP, a new variant of the vesicular stomatitis virus (VSV), where the glycoprotein G of VSV has been replaced by the glycoprotein GP of the lymphocytic choriomeningitis virus (LCMV)1,2. In addition to the humoral response, an important read-out of the effectivity of a vaccine is the induction of a CTL response against one or several antigens. As the consistency and durability of the cellular response are important in this context, it is favorable to monitor kinetics of CTL responses from the same animal. This will also lead to a reduction of animal numbers, an important aspect regarding the principles of the &#34;3Rs&#34;3. Hence, analysis from as little as 20 &#181;L of blood is optimal for this purpose.
Tetramers were developed in the late 90s4 and became an important tool in the field of T cell immunology. Tetramers are fluorescently-labeled complexes of four MHC I/peptide molecules, which bind to TCRs, specific for a single peptide. Nowadays, they can be either bought ready-made5, custom-ordered free of charge at the NIH Tetramer Core Facility at Emory University6 or produced in the lab7. MHC I and II tetramers are available, i.e. for CD8+ and CD4+ T cells, respectively. The potency of tetramer staining lies in time-savings, rather simple and easy to standardize8 protocols, and sensitivity9. Also, if working with blood, animals do not need to be sacrificed and minimal amounts of sample are required. One measurement is not limited to a single antigen, but several antigens can be analyzed in one staining when combining tetramers conjugated with different fluorophores. Newly discovered antigens, e.g. from peptide screens, can easily be incorporated in tetramers and used for quantification of the T cell subset.
Tetramer staining will not give information about CTL functionality (i.e., cytokine production, effector functions), but only specificity. To gain information about T cell functionality, intracellular cytokine staining (ICS) or Enzyme Linked Immuno Spot (ELISpot) Assay needs to be performed8,10. Tetramer staining and ICS/ELISpot, however, are not redundant but rather complement each other. In vitro stimulation to induce cytokine production for ICS/ELISpot will alter the original T cell phenotype. Tetramer staining, in contrast, leaves the T cell untouched; the original phenotype is preserved and can be analyzed. Also, another big advantage of tetramers is that staining can be combined with magnetic sorting and enrichment of antigen-specific cells11. This allows for the analysis of rare populations, as well as culturing of sorted cells with defined antigen-specificities &#8212; a feature that is not be possible with other methods.
Using the protocol described here, tetramer staining, as well as ICS/ELISpot can be performed from one organ, because only very little material (blood: 20 &#181;L; spleen: 1 x 106 cells) is required for tetramer staining. However, depending on the frequency of the antigen-specific cells of interest, the strength of the respective TCR and the experimental context, the amount of cells required might need to be scaled up or magnetic enrichment might need to be applied.
Tetramers are widely used, for example to assess effectivity of (antitumor) vaccines12,13,14,15 or immunotherapy16,17, phenotypic analysis and spatial localization of antigen-specific T cell subsets18,19,20,21,22,23. The method described here is suited for studies, which aim to include the quantification and phenotypic analysis of murine antigen-specific CD8+ T cells in their analysis in a fast and convenient way.

<table><tbody><tr><td>Safety cabinet class 2</td><td>VWR</td><td>LBCP302411030</td><td></td></tr><tr><td>Flow cytometer (e.g. FACSCanto II)</td><td>BD</td><td>338962</td><td></td></tr><tr><td>Analysis platform for flow cytometry analysis (e.g. FlowJo)</td><td>Fisher Scientific Co. L.L.C.</td><td>NC0887833</td><td></td></tr><tr><td>Binocular microscopes, VisiScope 100</td><td>VWR</td><td>630-1553</td><td></td></tr><tr><td>Vortex mixer</td><td>Phoenix Instrument</td><td>RS-VA 10</td><td></td></tr><tr><td>Centrifuge suitable for FACS tubes (e.g. Rotanta 460R)</td><td>Hettich</td><td>5660</td><td></td></tr><tr><td>Sterile Scalpel Blades Nr. 10</td><td>Braun</td><td>BB510</td><td></td></tr><tr><td>Cell strainer 40 &amp;micro;m</td><td>Sigma</td><td>CLS431750</td><td></td></tr><tr><td>Cell strainer 70 &amp;micro;m</td><td>Sigma</td><td>CLS431751</td><td></td></tr><tr><td>Neubauer counting chamber</td><td>VWR</td><td>630-1506</td><td></td></tr><tr><td>Pipettes (20 &amp;mu;L, 200 &amp;micro;L and 1000 &amp;mu;L)</td><td>Eppendorf</td><td>4924000037, 4924000061, 4924000088</td><td></td></tr><tr><td>Pipette tips, sterile (20 &amp;micro;L, 200 &amp;micro;L, 1,000 &amp;micro;L)</td><td>Biozym</td><td>770050, 770200, 770400</td><td></td></tr><tr><td>Pipet Boy</td><td>Integra</td><td>155 000</td><td></td></tr><tr><td>Sterile pipettes (5 mL, 10 mL, 25 mL)</td><td>Sarstedt</td><td>86.1253.001, 86.1254.001, 86.1685.001</td><td></td></tr><tr><td>Multistep Pipette, HandyStep S</td><td>BRAND</td><td>705110</td><td></td></tr><tr><td>12.5 mL Combitips for Multistep Pipette</td><td>BrandTech Scientific</td><td>702378</td><td></td></tr><tr><td>Microvette CB 300 K2E</td><td>Sarstedt</td><td>16.444</td><td></td></tr><tr><td>Sterile reaction tubes (1.5 mL, 50 mL)</td><td>Sarstedt</td><td>72.692.005, 62.547.254</td><td></td></tr><tr><td>FACS tubes (non-sterile)</td><td>Szabo Scandic</td><td>BDL352008</td><td></td></tr><tr><td>PBS</td><td>Lonza</td><td>LONBE17-516F</td><td></td></tr><tr><td>Heat-inactivated FCS</td><td>ThermoFisher Scientific</td><td>10500064</td><td></td></tr><tr><td>Formaldehyde</td><td>Roth</td><td>4979.1</td><td></td></tr><tr><td>Sodium azide</td><td>Roth</td><td>K305.1</td><td></td></tr><tr><td>PE-Cy7 Rat Anti-Mouse CD3 Molecular Complex</td><td>BD</td><td>560591</td><td>Clone 17A2; Lot # 7235504</td></tr><tr><td>Pacific Blu Rat Anti-Mouse CD8a</td><td>BD</td><td>558106</td><td>Clone 53-6.7; Lot # 5058904</td></tr><tr><td>V450 Rat anti-Mouse CD8a</td><td>BD</td><td>560469</td><td>Clone 53-6.7; Lot # 5205945</td></tr><tr><td>FITC anti-mouse CD43</td><td>BioLegend</td><td>121206</td><td>Clone 1B11; Lot # B233778</td></tr><tr><td>PE-Cy5 Rat Anti-Mouse CD44</td><td>BD</td><td>553135</td><td>Clone IM7; Lot # 85660</td></tr><tr><td>APC-Cy7 Rat Anti-Mouse CD62L</td><td>BD</td><td>560514</td><td>Clone MEL-14; Lot # 7215801</td></tr><tr><td>OVA-tetramer/APC</td><td>MBL</td><td>TB-5001-2</td><td>SIINFEKL, H-2Kb; Lot # T1702008</td></tr><tr><td>VSV NP-tetramer/PE</td><td>MBL</td><td>TS-M529-1</td><td>RGYVYQGL, H-2Kb; Lot # 007</td></tr><tr><td>EGFP-tetramer/PE</td><td>MBL</td><td>TS-M525-1</td><td>HYLSTQSAL, H-2Kd; Lot # 004</td></tr><tr><td>LCMV-GP-tetramer/APC</td><td>MBL</td><td>TB-5002-2</td><td>KAVYNFATC, H-2Db; Lot # T1412006</td></tr><tr><td>HPV 16 E7-tetramer/APC</td><td>MBL</td><td>TB-5008-2</td><td>RAHYNIVTF, H-2Db; Lot # T1804003</td></tr></tbody></table>

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.

Figure 1&#160;demonstrates how to gate correctly on the target cells of this protocol, namely CD3+/CD8+ cells. It is to note that activated cells often downregulate the T cell receptor26,27 and, therefore, CD3low cells should also be included in the gating. After gating the CD3+/CD8+ cells, tetramer positive cells can be identified (Figure 2). Representative blots for a negative control (na&#239;ve) mouse, as well as animals either vaccinated with either OVA-secreting Adenovirus 5 (Adeno-OVA) or OVA-expressing VSV-GP (VSV-GP-OVA) are shown. As seen in the lower blots two different tetramers can be combined in the same tube for staining. This allows simultaneous quantification of two different CTL specificities: virus-specific (VSV N) and transgene-specific (OVA) CTLs. We confirmed that single and double tetramer stainings give similar percentages of positive cells for each tetramer. Using this protocol, other virus-specific (e.g., LCMV GP, HPV 16 E7) or transgene-specific (e.g., GFP) T cell populations can be analyzed (Supplemental Figure 1). In Supplemental Table 1, results for five animals after immunization with VSV-GP-OVA are shown &#8212; indicating robustness of tetramer staining.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58680/58680fig1.jpg" /> 
Figure 1: Representative gating strategy to analyze CD8+ T cells in blood. Schematic representation of the gating strategy used for flow cytometric analysis. After tetramer staining and flow cytometric measurement, data was analyzed. Lymphocytes were identified with forward and sideward scatter (Area) (non-logarithmic scale). From those, single cells were identified by applying forward scatter width vs area (non-logarithmic scale). CD8+ T cells were identified by gating on CD3+/CD8+ cells (logarithmic scale). <a href="https://www.jove.com/files/ftp_upload/58680/58680fig1large.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/58680/58680fig2.jpg" /> 
Figure 2: Representative gating strategy to quantify OVA- and N-specific CD8+ T cells in blood. Schematic representation of the gating strategy used for flow cytometric quantification of tetramer+ cells. CD3+/CD8+ cells were used for tetramer analysis. Upper and middle panel: the CD8 marker was plotted against respective tetramer (logarithmic scale). Lower panel: both tetramers were plotted against each other (logarithmic scale). Left: control (na&#239;ve) mouse; middle: mouse was immunized with OVA-secreting Adenovirus 5 (Adeno-OVA); right: mouse was immunized with ovalbumin (OVA)-expressing VSV-GP (VSV-GP-OVA). Blood was collected from tail vein at day 7 after immunization. <a href="https://www.jove.com/files/ftp_upload/58680/58680fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/58680/58680fig5.jpg" /> 
Supplemental Figure 1: Representative result of CD3+/CD8+ tetramer+ cells after vaccination. Schematic representation of flow cytometric quantification of tetramer+ cells. CD3+/CD8+ cells were used for tetramer analysis and the CD8 marker was plotted against respective tetramer (logarithmic scale). Left: mice were na&#239;ve, right: mice were immunized with VSV-GP (upper panel), enhanced Green Fluorescent Protein (eGFP)-expressing VSV-GP (middle panel) or VSV-GP expressing human papillomavirus (HPV) E7 oncoprotein (E7) (lower panel). Blood was collected from tail vein at day 7 after immunization and stained with tetramers (LCMV-GP, eGFP and HPV E7). <a href="https://www.jove.com/files/ftp_upload/58680/58680fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
One big advantage of the protocol described here is that T cell responses from the same mouse can be followed over time as only small amounts of blood are needed for each measurement. Figure 3 shows exemplary results for T cell responses over time. In addition to quantities of antigen-specific CTLs, their phenotype can also be analyzed using this protocol (Figure 4).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58680/58680fig3.jpg" /> 
Figure 3: Representative result of CD8+ T cell kinetic in blood after vaccination. Schematic representation of the gating strategy used for flow cytometric quantification of tetramer+ cells. CD3+/CD8+ cells were used for tetramer analysis and both tetramers were plotted against each other (logarithmic scale). Upper panels: mouse was immunized with OVA-secreting Adenovirus 5 (Adeno-OVA); lower panels: mouse was immunized with ovalbumin (OVA)-expressing VSV-GP (VSV-GP-OVA). Blood was collected from tail vein at day 3, 7, 10 and 14 after immunization. <a href="https://www.jove.com/files/ftp_upload/58680/58680fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58680/58680fig4.jpg" /> 
Figure 4: Representative result of CD8+ T cell activation and differentiation into na&#239;ve and effector cells after vaccination. Schematic representation of the gating strategy used for flow cytometric quantification of activated (CD43+), na&#239;ve (CD44-/CD62L+) and effector (CD44+/CD62L-) CD3+/CD8+ cells (logarithmic scale). Left: control (na&#239;ve) mouse; middle: mouse was immunized with OVA-secreting Adenovirus 5 (Adeno-OVA); right: mouse was immunized with ovalbumin (OVA)-expressing VSV-GP (VSV-GP-OVA). Blood was collected from tail vein at day 7 after immunization. <a href="https://www.jove.com/files/ftp_upload/58680/58680fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Mouse #</td>
			<td>% CD43+</td>
			<td>% VSV-N Tetramer+</td>
			<td>% OVA Tetramer+</td>
		</tr>
		<tr>
			<td rowspan="5">mock</td>
			<td>1</td>
			<td>1.7</td>
			<td>0.45</td>
			<td>0.41</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.77</td>
			<td>0.69</td>
			<td>0.15</td>
		</tr>
		<tr>
			<td>3</td>
			<td>1.34</td>
			<td>0.35</td>
			<td>0.31</td>
		</tr>
		<tr>
			<td>4</td>
			<td>1.68</td>
			<td>0.83</td>
			<td>0.26</td>
		</tr>
		<tr>
			<td>5</td>
			<td>0.84</td>
			<td>0.47</td>
			<td>0.23</td>
		</tr>
		<tr>
			<td rowspan="5">VSV-GP-OVA</td>
			<td>1</td>
			<td>23</td>
			<td>13.2</td>
			<td>3.69</td>
		</tr>
		<tr>
			<td>2</td>
			<td>32.6</td>
			<td>15.9</td>
			<td>3.54</td>
		</tr>
		<tr>
			<td>3</td>
			<td>22.1</td>
			<td>11.7</td>
			<td>2.43</td>
		</tr>
		<tr>
			<td>4</td>
			<td>15.1</td>
			<td>8.89</td>
			<td>1.79</td>
		</tr>
		<tr>
			<td>5</td>
			<td>29.1</td>
			<td>14.7</td>
			<td>5.64</td>
		</tr>
	</tbody>
</table>
Supplemental Table 1: Percentages of activated and antigen-specific CD3+/CD8+ cells after vaccination. Mice were either na&#239;ve or immunized with ovalbumin (OVA)-expressing VSV-GP (VSV-GP-OVA) (n = 5). Blood was collected from tail vein at day 7 after immunization and stained with tetramers (VSV-N and OVA). Activated (CD43+) and antigen-specific (tetramer+) CD3+/CD8+ cells were quantified by flow cytometry.

Watch this Scientific Journal Video about Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector at JoVE.

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).

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 ...

simultaneous-quantification-anti-vector-anti-transgene-specific-cd8-t

Research

JoVE Journal

Immunology and Infection

8.5K Views.  Medical University of Innsbruck. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van T-cel immunologie en de ontwikkeling van virale vectorvaccins. Het belangrijkste voordeel van deze methode is dat er slechts kleine hoeveelheden bloed nodig zijn, waardoor herhaalde metingen van dezelfde muis mogelijk zijn. Bovendien kunnen virus- en transgene-specifieke CTLs uit hetzelfde monster worden gedetecteerd.Het aantonen van de procedure zal Jasmine Rinnofner, een technicus, en Annika Rossler...

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

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

Biology

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in determining outcomes that range from clearance to chronic persistent infections. We describe here a method for assessing the spatial and quantitative relationships between immune effector (E) virus-specific CD8+ T cells and infected targets (T) that combines in situ tetramer (IST) staining to detect virus-specific CD8+ T cells and in situ hybridization (ISH) to detect viral-RNA+ cells in the tissues where the battle between immune defenses and infection takes place. The combination of IST staining and ISH, abbreviated ISTH, enables visualization and mapping of the locations of immune effector cells and targets, and facile determination of E:T ratios.  These parameters in turn can then be used to determine the relationships between spatial proximity, and the timing and magnitude of the immune response that predict outcomes in early infection.

A Technique to Simultaneously Visualize Virus-Specific CD8+ T Cells and Virus-Infected Cells In situ

A technique combining in situ tetramer staining and in situ hybridization (ISTH) enables visualization, mapping and analysis of the spatial proximity of virus-specific CD8+ T cells to their virus-infected targets, and determination of the quantitative relationships between these immune effectors and targets to infection outcomes.

The numbers and locations of virus-specific CD8+ T cells relative to the numbers and locations of their infected cell targets is thought to be critical in ...

Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various nanoparticle systems have been engineered to mimic features of pathogens to improve antigen delivery to draining lymph nodes and increase antigen uptake by antigen-presenting cells, leading to new vaccine formulations optimized for induction of antigen-specific CD8+ T cell responses. In this article, we describe the synthesis of a &#8220;pathogen-mimicking&#8221; nanoparticle system, termed interbilayer-crosslinked multilamellar vesicles (ICMVs) that can serve as an effective vaccine carrier for co-delivery of subunit antigens and immunostimulatory agents and elicitation of potent cytotoxic CD8+ T lymphocyte (CTL) responses. We describe methods for characterizing hydrodynamic size and surface charge of vaccine nanoparticles with dynamic light scattering and zeta potential analyzer and present a confocal microscopy-based procedure to analyze nanoparticle-mediated antigen delivery to draining lymph nodes. Furthermore, we show a new bioluminescence whole-animal imaging technique utilizing adoptive transfer of luciferase-expressing, antigen-specific CD8+ T cells into recipient mice, followed by nanoparticle vaccination, which permits non-invasive interrogation of expansion and trafficking patterns of CTLs in real time. We also describe tetramer staining and flow cytometric analysis of peripheral blood mononuclear cells for longitudinal quantification of endogenous T cell responses in mice vaccinated with nanoparticles.

We describe whole-animal imaging and flow cytometry-based techniques for monitoring expansion of antigen-specific CD8+ T cells in response to immunization with nanoparticles in a murine model of vaccination.

Traditional vaccine adjuvants, such as alum, elicit suboptimal CD8+ T cell responses. To address this major challenge in vaccine development, various ...

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 ...

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 ...

A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine (BrdU) marks cells that are in or recently completed S-phase, and carboxyfluorescein succinimidyl ester (CFSE) detects daughter cells after division. However, these dyes do not allow identification of the cell cycle phase at the time of analysis. An alternative approach is to exploit Ki67, a marker that is highly expressed by cells in all phases of the cell cycle except the quiescent phase G0. Unfortunately, Ki67 does not allow further differentiation as it does not separate cells in S-phase that are committed to mitosis from those in G1 that can remain in this phase, proceed into cycling, or move into G0.
Here, we describe a flow cytometric method for capturing a &#34;snapshot&#34; of T cells in different cell cycle phases in mouse secondary lymphoid organs. The method combines Ki67 and DNA staining with major histocompatibility complex (MHC)-peptide-multimer staining and an innovative gating strategy, allowing us to successfully differentiate between antigen-specific CD8 T cells in G0, in G1 and in S-G2/M phases of the cell cycle in the spleen&#160;and draining lymph nodes of mice after vaccination with viral vectors carrying the model antigen gag of human immunodeficiency virus (HIV)-1.
Critical steps of the method were the choice of the DNA dye and the gating strategy to increase the assay sensitivity and to include highly activated/proliferating antigen-specific T cells that would have been missed by current criteria of analysis. The DNA dye, Hoechst 33342, enabled us to obtain a high-quality discrimination&#160;of the G0/G1 and G2/M DNA peaks, while preserving membrane and intracellular staining. The method has great potential to increase knowledge about T cell response in vivo and to improve immuno-monitoring analysis.

Clonal expansion is a key feature of antigen-specific T cell response. However, the cell cycle of antigen-responding T cells has been poorly investigated, partly because of technical limitations. We describe a flow cytometric method to analyze clonally expanding antigen-specific CD8 T cells in spleen and lymph nodes of vaccinated mice.

The cell cycle of antigen-specific T cells in vivo has been examined by using a few methods, all of which possess some limitations. Bromodeoxyuridine ...

Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique

T cell-mediated immunity plays an important role in controlling flavivirus infection, either after vaccination or after natural infection. The "quality" of a T cell needs to be assessed by function, and higher function is associated with more powerful immune protection. T cells that can simultaneously produce two or more cytokines or chemokines at the single-cell level are called polyfunctional T cells (TPFs), which mediate immune responses through a variety of molecular mechanisms to express degranulation markers (CD107a) and secrete interferon (IFN)-&#947;, tumor necrosis factor (TNF)-&#945;, interleukin (IL)-2, or macrophage inflammatory protein (MIP)-1&#945;. There is increasing evidence that TPFs are closely related to the maintenance of long-term immune memory and protection and that their increased proportion is an important marker of protective immunity and is important in the effective control of viral infection and reactivation. This evaluation applies not only to specific immune responses but also to the assessment of cross-reactive immune responses. Here, taking the Japanese encephalitis virus (JEV) as an example, the detection method and flow cytometry color scheme of JEV-specific TPFs produced by peripheral blood mononuclear cells of children vaccinated against Japanese encephalitis were tested to provide a reference for similar studies.

Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique

The present protocol combines ex vivo stimulation and flow cytometry to analyze polyfunctional T cell (TPF)&#160;profiles in peripheral blood mononuclear cells (PBMCs) within Japanese encephalitis virus (JEV)-vaccinated children. The detection method and flow cytometry color scheme of JEV-specific TPFs were tested to provide a reference for similar studies.

T cell-mediated immunity plays an important role in controlling flavivirus infection, either after vaccination or after natural infection. The "quality" ...

Deciphering Early Follicular Helper T-Cell Commitment in Acute LCMV Infection

Accessing Early Differentiation of Virus-Specific Follicular Helper CD4+ T Cell in Acute LCMV-Infected Mice

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, thus establishing long-term humoral immunity. During acute viral infection, the fate commitment of virus-specific TFH cells is determined in the early infection phase, and investigations of the early-differentiated TFH cells are crucial in understanding T cell-dependent humoral immunity and optimizing vaccine design. In the study, using a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection and the TCR-transgenic SMARTA (SM) mouse with CD4+ T cells specifically recognizing LCMV glycoprotein epitope I-AbGP66-77, we described procedures to access the early fate commitment of virus-specific TFH cells based on flow cytometry stainings. Furthermore, by exploiting retroviral transduction of SM CD4+ T cells, methods to manipulate gene expression in early-differentiated virus-specific TFH cells are also provided. Hence, these methods will help in studies exploring the mechanism(s) underlying the early commitment of virus-specific TFH cells.

Author Spotlight: Elucidating the Pathways of TFH Cell Differentiation in Acute LCMV Challenges

The current study showcases protocols for assessing the early fate commitment of virus-specific TFH cells and manipulating gene expression in these cells.

Follicular Helper T (TFH) cells are perceived as an independent CD4+ T cell lineage that assists cognate B cells in producing high-affinity antibodies, ...

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

Samenvatting

Meer video's verkennen