We would like to thank David Watkins for supporting the experiments that enabled the optimization of the present methodology. Research reported in this publication was supported in part by a pilot grant provided by the Miami Center for AIDS Research of the National Institutes of Health under award number P30AI073961. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

The peripheral blood mononuclear cell (PBMC) samples utilized in this manuscript were obtained from Indian rhesus macaques housed at the Wisconsin National Primate Research Center. These animals were cared for in accordance with the Weatherall Report under a protocol approved by the University of Wisconsin Graduate School Animal Care and Use Committee33. All animal procedures were performed under anesthesia and all efforts were made to minimize potential suffering.
1. PKI Treatment
NOTE: This is optional, but recommended for Mamu-B*017:01 tetramers. See Results and Discussion Sections.
<ol>
	<li>Resuspend PBMC in R10 medium at a concentration of 1.6 x 107 cells/ml.</li>
	<li>Add 50 &#181;l of this cell suspension to the corresponding flow cytometry tubes. Add 50 &#181;l of a 100 nM solution of PKI to each tube.</li>
	<li>Vortex each tube. Incubate at 37 &#176;C for 30 min. By the end of this step, each tube should have 100 &#181;l of a cell suspension containing 8.0 x 105 PBMC and 50 nM of PKI.</li>
	<li>Proceed to the pMHC-I tetramer staining step.</li>
</ol>
2. Staining with Fluorochrome-labeled pMHC-I Tetramers
<ol>
	<li>Before preparing the pMHC-I tetramer master mix, centrifuge the pMHC-I tetramer tubes at 20,000 x g for at least 15 min at 4 &#176;C. The goal of this step is to pellet protein aggregates in the pMHC-I tetramer solution that may increase background staining. Once the centrifugation step is done, avoid pipetting from the bottom of the tube where the protein aggregates will have accumulated.</li>
	<li>Prepare enough pMHC-I tetramer master mix to stain all experimental tubes. Prepare an excess of 15% of the total volume of this master mix to account for pipetting error.</li>
	<li>Dilute pMHC-I tetramers in stain buffer (e.g., Brilliant Stain Buffer) so that 25 &#181;l of the pMHC-I tetramer master mix are added to each test.
	<ol>
		<li>Label PBMC with two pMHC-I tetramers per test; one conjugated to allophycocyanin (APC) and the other to Brilliant Violet (BV) 421.</li>
	</ol>
	</li>
	<li>Add 8.0 x 105 PBMC to the corresponding flow cytometry tubes in a final volume of 100 &#181;l. If the cells were subjected to the PKI treatment described above, they should already be resuspended in 100 &#181;l at this step.</li>
	<li>Add 25 &#181;l of pMHC-I tetramer master mix to the corresponding flow cytometry tubes. By the end of this step, the final volume in each tube should be 125 &#181;l.</li>
	<li>Vortex each tube in order to homogenize the cell suspension.</li>
	<li>Incubate in the dark at room temp for 45 min. Prepare the surface staining mAb cocktail during this 45 min incubation.</li>
</ol>
3. Surface Staining
<ol>
	<li>Prepare enough mAb master mix to stain all experimental tubes. Prepare an excess of 15% of the total volume of this master mix to account for pipetting error.</li>
	<li>Adjust the volume of the master mix with stain buffer so that 50 &#181;l are added per test.</li>
	<li>For proper exclusion of non-CD8+ T-cells and delineation of memory subsets, use titrated amounts of mAbs directed against the following molecules in the surface staining master mix. 
	NOTE: The fluorochromes conjugated to each mAbs are provided as a reference: CD14 BV 510, CD16 BV 510, CD20 BV 510, CD8&#945; BV 785, CD28 PE Cy7, and CCR7 FITC. Note that the mAbs against CD14, CD16, and CD20 are conjugated to the same fluorochrome (i.e., BV 510) since they will be included in the &#34;dump&#34; gate.</li>
	<li>Include a fixable dye for discriminating dead cells in the surface staining master mix [i.e., amine reactive dye (ARD)]. Make sure that the ARD reagent is conjugated to a fluorochrome with a similar emission spectrum as the ones used in the &#34;dump&#34; gate. In this case, use ARD Aqua.</li>
	<li>Add 50 &#181;l of the staining master mix described in 3.1-3.4 to the corresponding flow cytometry tubes. By the end of this step, the final volume in each tube should be 175 &#181;l.</li>
	<li>Vortex each tube. Incubate in the dark at room temp for 25 min.</li>
	<li>Wash cells with wash buffer (PBS solution containing 0.1% of bovine serum albumin and 0.45 g/L NaN3). 
	CAUTION: Sodium azide is a toxic substance. Exposure to even minute amounts can cause symptoms. Handle this substance according to the guidelines specified by the Environmental Health and Safety (EHS) Office at the institution where these experiments are being performed.</li>
	<li>Centrifuge tubes at 510 x g for 5 min. Carefully decant supernatant into a separate waste container. Be sure not to disturb the pellet. 
	CAUTION: Do not decant wash buffer supernatant into reservoirs containing bleach as it can react with the sodium azide present in the wash buffer and result in the formation of a toxic gas34. Contact the EHS Office at the institution where these experiments are being performed for guidelines on how to dispose of sodium azide.</li>
	<li>After decanting, vortex the cells in the leftover liquid retained in each tube.</li>
</ol>
4. Cell Fixation
<ol>
	<li>Add 250 &#181;l of a 2% paraformaldehyde (PFA) solution to all tubes to fix the cells. 
	CAUTION: PFA is a toxic substance. Exposure to even minute amounts can cause symptoms. Handle this substance according to the guidelines specified by the EHS Office at the institution where these experiments are being performed.
	<ol>
		<li>Since the 2% PFA solution must be isotonic to the cells, use PBS to prepare this solution. One hundred milliliters of a 2% PFA solution is enough for multiple experiments. Thoroughly vortex all tubes immediately after the addition of 2% PFA in order to prevent the formation of cell aggregates.</li>
	</ol>
	</li>
	<li>Incubate in the dark at 4 &#176;C for 20 min.</li>
	<li>Wash cells by repeating steps 3.7-3.9. Once this step is completed, the cells can be stored in the dark at 4 &#176;C for 24-48 hr. Before proceeding to the Permeabilization phase, vortex the tubes thoroughly.</li>
</ol>
5. Permeabilization of Cells
<ol>
	<li>Add 500 &#181;l of permeabilization buffer. Vortex all tubes.</li>
	<li>Incubate in the dark at room temp for 10 min.</li>
	<li>Wash cells by repeating steps 3.7-3.9.</li>
</ol>
6. Intracellular Staining
<ol>
	<li>Prepare enough mAb master mix to stain all experimental tubes.</li>
	<li>Adjust the volume with PBS or stain buffer so that 50 &#181;l of the intracellular mAb master mix are added per test.</li>
	<li>Prepare the mAb master mix described in 6.1 and 6.2 using titrated amounts of mAbs directed against CD3 and Gzm B. 
	NOTE: TCR engagement by pMHC-I tetramers can result in CD3 internalization, which can interfere with the detection of tetramer+ CD3+ CD8+ T-cells if the anti-CD3 mAb is added to the surface staining master mix. To avoid this, add the anti-CD3 mAb at this stage, that is, after the cells are permeabilized. The fluorochromes conjugated to each mAb are listed as a reference: CD3 PerCP Cy5.5 and Gzm B PE.</li>
	<li>Add 50 &#181;l of mAb cocktail to the corresponding tubes. Incubate in the dark at room temp for 30 min.</li>
	<li>Wash cells by repeating steps 3.7-3.9. The tubes are ready to be acquired in a flow cytometer.</li>
</ol>

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<li>Roederer, M., et al. FACS Analysis of Leukocyes. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aRoederer%2C+M+%22Weir%27s+Handbook+of+Experimental+Immunology%2C+5th+ed%22">Weir's Handbook of Experimental Immunology, 5th ed</a>. Herzenberg, L. A., Weir, D. M., Blackwell, C. , Blackwell Science. Boston. 1-49 (1996).</li>
<li>Colantonio, A. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((KIR+polymorphisms+modulate+peptide-dependent+binding+to+an+MHC+class+I+ligand+with+a+Bw6+motif%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">KIR polymorphisms modulate peptide-dependent binding to an MHC class I ligand with a Bw6 motif.</a> PLoS Pathog. 7 (3), e1001316(2011).</li>
<li>Schafer, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Suppression+of+a+Natural+Killer+Cell+Response+by+Simian+Immunodeficiency+Virus+Peptides%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides.</a> PLoS Pathog. 11 (9), 1005145(2015).</li>
</ol>

The authors declare that they have no competing financial interests.

A few steps in this procedure merit discussion as they are crucial for yielding optimal results. First, since the quality of the biological specimens is a strong predictor of the success of any flow cytometric assay38, all care must be taken to ensure that the cells are viable and in suspension during the staining procedure. This is particularly relevant when working with cryopreserved samples since they are more prone to clumping and typically contain higher numbers of dead cells. In these cases, passing the ...

CD8+ T-cells comprise a crucial component of the adaptive immune system as they participate in tumor immune surveillance and contribute to the eradication of intracellular pathogens1. In simple terms, CD8+ T-cells express T-cell receptors (TCRs) that specifically recognize peptide-major histocompatibility complex class I (pMHC-I) molecules present on the plasma membrane of host cells. Since these peptides are derived from the proteolysis of endogenously synthesized proteins, cell surface pMHC-I complexes provide a window into the intracellular environment. Upon virus infection, for example, infected cells will display MHC-I molecules containing virus-derived peptides that can serve as ligands for TCRs expressed by patrolling CD8+ T-cells. In the event a virus-specific CD8+ T-cell encounters an infected cell presenting its pMHC-I ligand, TCR engagement will result in CD8+ T-cell activation and ultimately lead to target cell lysis. Given the critical nature of these TCR/pMHC-I interactions, determining the magnitude, specificity, and phenotype of responding CD8+ T-cells can often reveal important clues about human diseases.
Until the early 1990s, quantification of Ag-specific CD8+ T-cells relied on the technically demanding limiting dilution assay (LDA)2,3. Not only did the LDA require several days to be completed, it also failed to detect cells that lacked proliferative potential. As a result, the LDA vastly underestimated the actual frequency of antigen (Ag)-specific CD8+ T-cells participating in an immune response. Although the development of ELISPOT and intracellular cytokine staining assays greatly improved the ability to measure cellular immunity, these methods still required in vitro stimulation for quantifying Ag-specific T-cells4. It was not until 1996 that Altman, Davis, and colleagues published their landmark article reporting the development of the pMHC-I tetramer technology5. Critical to the success of this technique was the multimerization of pMHC-I molecules, which extended the half-life of TCR/pMHC-I interactions, thereby reducing the probability of pMHC-I tetramers falling off during the washing steps of flow cytometric assays. The main advantage of pMHC-I tetramers over the aforementioned assays is the ability to accurately detect Ag-specific CD8+ T-cells directly ex vivo without the need for in vitro re-stimulation. Moreover, the combination of pMHC-I tetramer staining with multi-color flow cytometry has allowed detailed analyses of the differentiation stage, memory phenotype, and activation status of Ag-specific CD8+ T-cells2-4. In light of recent technical advances for characterizing CD8+ T-cell repertoires by pMHC-I multimer staining6, the breadth of applications for this methodology is likely to continue expanding.
Few areas in biomedical research have benefited more from pMHC-I tetramer staining than the field of HIV immunology7. Although CD8+ T-cells had been temporally associated with the initial control of HIV viremia by the time of the publication by Altman, Davis, and colleagues8,9, the use of pMHC-I tetramers in the ensuing years significantly expanded our understanding of the HIV-specific CD8+ T-cell response. For example, pMHC-I tetramer staining helped confirm the robust size of virus-specific CD8+ T-cell responses in most HIV-infected individuals10-12. This methodology also facilitated the characterization of HIV- and SIV-specific CD8+ T-cell responses restricted by MHC-I molecules associated with spontaneous control of viral replication in the absence of antiretroviral therapy&#8212;a phenomenon known as &#34;elite control&#34;13-15. Furthermore, pMHC-I tetramers were instrumental in establishing the programmed death 1 (PD-1)/PD ligand 1 (PD-L1) axis as a reversible pathway for the dysfunctional phenotype of HIV-specific CD8+ T-cells in uncontrolled chronic infection16,17. Collectively, these studies underscore the utility of pMHC-I tetramers for monitoring CD8+ T-cell responses against the AIDS virus.
Experimental SIV infection of rhesus macaques (Macaca mulatta) remains the best animal model for evaluating immune interventions against HIV/AIDS18,19. In the past 25 years, substantial progress has been made in the identification and characterization of SIV-specific CD8+ T-cells in this monkey species, including the discovery of MHC-I alleles and the definition of peptide binding motifs13,20-27. As a result, pMHC-I tetramers have been developed for the analysis of SIV-specific CD8+ T-cell responses in this animal model28. Most of these reagents are made of the gene products of four rhesus macaque MHC-I alleles: Mamu-A1*001, Mamu-A1*002:01, Mamu-B*008:01, and Mamu-B*017:01. Of note, rhesus macaques do not express an MHC-C locus29. The vast majority of the pMHC-I tetramers used in the present experiments were produced at the NIH Tetramer Core Facility at Emory University. Nevertheless, some of these reagents, including Mamu-A1*001 tetramers bound to the immunodominant Gag CM9 epitope, can only be obtained from commercial sources due to licensing agreements. Using pMHC-I tetramers of the four rhesus macaque alleles listed above, we have successfully enumerated CD8+ T-cells against a total of 21 SIV epitopes (Table 1), which were induced by vaccination or primary SIV infection30,31 (Martins et al., unpublished observations).
The present manuscript provides an optimized pMHC-I tetramer staining protocol for determining the frequency and memory phenotype of SIV-specific CD8+ T-cells in rhesus macaques. The assay begins with an elective 30 min incubation with a protein kinase inhibitor (PKI; here, Dasatinib is used) in order to decrease TCR internalization and thereby improve pMHC-I tetramer staining32. As described below, this treatment is especially useful when using Mamu-B*017:01 tetramers. Instructions on how to label the cells with fluorochrome-conjugated pMHC-I tetramers and monoclonal antibodies (mAbs) are also provided. This protocol also includes a cell permeabilization step for the intracellular detection of the cytolysis-associated molecule granzyme B (Gzm B). The mAb against CD3 is added at this step as well to improve detection of this TCR signaling molecule. As a reference, all fluorochromes employed in this staining panel are listed.

<table><tbody><tr><td>Dasatinib</td><td>Axon medchem</td><td>Axon&amp;nbsp;1392</td><td>Must be resuspended in DMSO and immediately stored at -20 &amp;deg;C</td></tr><tr><td>RPMI w/ Glutamax</td><td>gibco/ Life Technologies</td><td>61870-036</td><td>Must be stored at 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Heat Inactivated FBS</td><td>gibco/ Life Technologies</td><td>10082-147</td><td>Must be stored at 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Penicillin-Streptomycinp-Amphotericin B</td><td>Lonza</td><td>17-745E</td><td>Must be stored at 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>DMSO, Anhydrous</td><td>Life Technologies</td><td>D12345</td><td>Store at room temperature.</td></tr><tr><td>5 ml Round-Bottom Polypropylene Tubes</td><td>VWR</td><td>60819-728</td><td></td></tr><tr><td>Fluorochrome-conjugated pMHC-I tetramers</td><td>NIH Tetramer Core or MBL, Inc.</td><td></td><td>Must be stored and maintained at&amp;nbsp; 4 &amp;deg;C. Centrifuge at 20,000 x g for 15 min before use. Do not freeze.</td></tr><tr><td>Fluorochrome-conjugated mAbs</td><td>Various companies</td><td></td><td>Must be stored and maintained at&amp;nbsp; 4 &amp;deg;C. Do not freeze.</td></tr><tr><td>LIVE/DEAD&amp;nbsp;Fixable Aqua&amp;nbsp;Dead&amp;nbsp;Cell Stain Kit</td><td>Life Technologies</td><td>l34957</td><td>Must be stored at -20 &amp;deg;C. Resuspend each aliquot in 50 &amp;mu;l of DMSO prior to use.</td></tr><tr><td>Brilliant Stain Buffer</td><td>BD Biosciences</td><td>563794</td><td>Must be stored at&amp;nbsp; 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Phosphate Buffered Saline</td><td>VWR</td><td>97064-158</td><td>Store at room temperature</td></tr><tr><td>Albumine Bovine</td><td>VWR</td><td>700011-230</td><td>Must be stored at&amp;nbsp; 4 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Sodium Azide</td><td>VWR</td><td>97064-646</td><td>Store at room temperature. Toxic substance. Do not mix with bleach.</td></tr><tr><td>Bleach</td><td>VWR</td><td>89501-620</td><td>Corrosive chemical, cannot be mixed with sodium azide. Handle with care</td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td>Flammable, corrosive, and toxic reagent. Handle with care</td></tr><tr><td>Polysorbate 20 (Tween-20)</td><td>Alfa Aesar</td><td>L15029</td><td>Store at room temperature</td></tr><tr><td>Permeabilization Solution 2&amp;nbsp;</td><td>BD Biosciences</td><td>340973</td><td>Toxic and corrosive reagent. Handle with care</td></tr><tr><td>Sarsdet Tubes 1.5 ml screw top</td><td>VWR</td><td>72.692.005</td><td></td></tr><tr><td>2.0 ml DNA/RNA Low bind Tubes</td><td>Eppendorf</td><td>22431048</td><td>The use of Sterile microtubes is preffered&amp;nbsp;</td></tr><tr><td>Vortex mixer</td><td>To discretion of scientist</td><td></td><td></td></tr><tr><td>Biosafety Cabinet&amp;nbsp;</td><td>To discretion of scientist</td><td></td><td></td></tr><tr><td>Milli Q Intergral Water Purification system</td><td>EMD Millipore</td><td>ZRXQ010WW</td><td>Molecular Biology grade water from any provider may be used</td></tr><tr><td>Microcentrifuge</td><td>To discretion of scientist</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>To discretion of scientist</td><td></td><td></td></tr><tr><td>4 &amp;deg;C&amp;nbsp; refrigerator</td><td>To discretion of scientist</td><td></td><td></td></tr><tr><td>BD LSR II</td><td>BD Biosciences</td><td></td><td>Flow cytometer must contain lasers and filters that are compatible with the staining panel used.</td></tr><tr><td>Deionized water</td><td></td><td></td><td></td></tr><tr><td>Aluminum Foil</td><td>VWR SCIENTIFIC INC.</td><td>89068-738</td><td></td></tr><tr><td>Incubator</td><td></td><td></td><td>Must be able to maintain 37 &amp;deg;C&amp;nbsp; internal temperature</td></tr><tr><td>FACS Diva software</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>Flowjo software version 9.6</td><td>Flowjo</td><td></td><td>Used to analyze FCS files generated by FACS Diva software</td></tr><tr><td>Micropippette tips</td><td></td><td></td><td></td></tr></tbody></table>

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.

The protocol described here has been used to determine the magnitude and memory phenotype of vaccine-induced, Gag CM9-specific CD8+ T-cell responses in a Mamu-A1*001+ rhesus macaque. For this analysis, an APC-conjugated Mamu-A1*001/Gag CM9 tetramer was used in an 8-color flow cytometric staining panel. Figure 1A-F shows the gating strategy used to analyze the data, which should be applied for both tetramers present in each test. Note t...

Watch this Scientific Journal Video about Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining at JoVE.com

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.

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 ...

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Research

JoVE Journal

Immunology and Infection

9.1K Views.  University of Miami.  Hier presenteren we een geoptimaliseerd protocol voor het opsommen en karakteriseren rhesus macaque CD8 + T cellen tegen het AIDS-virus. Dit artikel is niet alleen nuttig voor het gebied van immunologie HIV, maar ook op andere gebieden van biomedisch onderzoek waarbij CD8 + T cel responsen is bekend dat de ziekte uitkomst beïnvloeden. 

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

Multicolor Flow Cytometry Analyses of Cellular Immune Response in Rhesus Macaques

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development of vaccines and therapeutics1,2,3. Here, we describe a method for the detailed phenotypic and functional analyses of cellular immune responses, specifically intracellular cytokine production by CD4+ and CD8+ T cells as well as the individual memory subsets. We obtained precise quantitative and qualitative measures for the production of interferon gamma (INF-) and interleukin (IL) -2 in both CD4+ and CD8+ T cells from the rhesus macaque PBMC stimulated with PMA plus ionomycin (PMA+I). The cytokine profiles were different in the different subsets of memory cells. Furthermore, this protocol provided us the sensitivity to demonstrate even minor fractions of antigen specific CD4+ and CD8+ T cell subsets within the PBMC samples from rhesus macaques immunized with an HIV envelope peptide cocktail vaccine developed in our laboratory. The multicolor flow cytometry technique is a powerful tool to precisely identify different populations of T cells 4,5 with cytokine-producing capability6 following non-specific or antigen-specific stimulation 5,7.

We demonstrate the utility of multicolor flow cytometry for detailed phenotypic and functional characterization of total as well as memory subsets of CD4+ and CD8+ T cells in rhesus macaques, the ideal model for HIV/AIDS vaccine studies.

The rhesus macaque model is currently the best available model for HIV-AIDS with respect to understanding the pathogenesis as well as for the development ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Peptide:MHC Tetramer-based Enrichment of Epitope-specific T cells

A basic necessity for researchers studying adaptive immunity with in vivo experimental models is an ability to identify T cells based on their T cell antigen receptor (TCR) specificity. Many indirect methods are available in which a bulk population of T cells is stimulated in vitro with a specific antigen and epitope-specific T cells are identified through the measurement of a functional response such as proliferation, cytokine production, or expression of activation markers1. However, these methods only identify epitope-specific T cells exhibiting one of many possible functions, and they are not sensitive enough to detect epitope-specific T cells at naive precursor frequencies. A popular alternative is the TCR transgenic adoptive transfer model, in which monoclonal T cells from a TCR transgenic mouse are seeded into histocompatible hosts to create a large precursor population of epitope-specific T cells that can be easily tracked with the use of a congenic marker antibody2,3. While powerful, this method suffers from experimental artifacts associated with the unphysiological frequency of T cells with specificity for a single epitope4,5. Moreover, this system cannot be used to investigate the functional heterogeneity of epitope-specific T cell clones within a polyclonal population.
	The ideal way to study adaptive immunity should involve the direct detection of epitope-specific T cells from the endogenous T cell repertoire using a method that distinguishes TCR specificity solely by its binding to cognate peptide:MHC (pMHC) complexes. The use of pMHC tetramers and flow cytometry accomplishes this6, but is limited to the detection of high frequency populations of epitope-specific T cells only found following antigen-induced clonal expansion. In this protocol, we describe a method that coordinates the use of pMHC tetramers and magnetic cell enrichment technology to enable detection of extremely low frequency epitope-specific T cells from mouse lymphoid tissues3,7. With this technique, one can comprehensively track entire epitope-specific populations of endogenous T cells in mice at all stages of the immune response.

This protocol describes the use of peptide:MHC tetramers and magnetic microbeads to isolate low frequency populations of epitope-specific T cells and analyze them by flow cytometry. This method enables the direct study of endogenous T cell populations of interest from in vivo experimental systems.

A  basic necessity for researchers studying adaptive immunity with in vivo experimental models is an  ability to identify T cells based on their T cell ...

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 ...

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 ...

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

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 ...

Isolation and Characterization of Antigen-Specific T Cells in Mouse Lungs

Identification of Rare Antigen-Specific T Cells from Mouse Lungs with Peptide:Major Histocompatibility Complex Tetramers

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune pathophysiology. The technical challenges of tracking antigen-specific T cell populations within the endogenous T cell repertoire have been greatly advanced by the development of peptide:MHC tetramer reagents. These fluorescently labeled soluble multimers of MHC class I or class II molecules complexed to antigenic peptide epitopes bind directly to T cells with corresponding T cell receptor (TCR) specificity and can, therefore, identify antigen-specific T cell populations in their native state without a requirement for a functional response induced by ex vivo stimulation. For exceedingly rare populations, tetramer-bound T cells can be magnetically enriched to increase the sensitivity and reliability of detection.
As the investigation of tissue-resident T cell immunity deepens, there is a pressing need to identify antigen-specific T cells that traffic to and reside in nonlymphoid tissues. In this protocol, we present a detailed set of instructions for the isolation and characterization of antigen-specific T cells present within mouse lungs. This involves the isolation of T cells from digested lung tissue followed by a general T cell magnetic enrichment step and tetramer staining for flow cytometry analysis and sorting. The steps highlighted in this protocol utilize common techniques and readily available reagents, making it accessible for nearly any researcher engaged in mouse T cell immunology, and are highly adaptable for a variety of downstream analyses of any low frequency antigen-specific T cell population residing within the lungs.

Author Spotlight: Optimized Protocol for Detecting Antigen-Specific T Cells in Mouse Lungs Using Tetramers

We provide a detailed protocol for isolating and identifying rare antigen-specific T cell populations in mouse lungs through magnetic bead-based T cell enrichment and peptide:major histocompatibility complex (MHC) tetramers.

The identification and characterization of antigen-specific T cells during health and disease remains a key to improving our understanding of immune ...

A Peptide-MHC-I Tetramer Staining Technique to Analyze SIV-Specific CD8+ Memory T Cells

Source: Gonzalez-Nieto, L., et al. <a href="https://app.jove.com/v/54881">Analysis of Simian Immunodeficiency Virus-specific CD8+ T-cells in Rhesus Macaques by Peptide-MHC-I Tetramer Staining.</a> J. Vis. Exp. (2016)
This video demonstrates a method for characterizing CD8+ memory T cells specific to simian immunodeficiency virus (SIV) using peptide-major histocompatibility complex class I (pMHC-I) tetramers. A pMHC-I tetramer is a complex of four MHC-I molecules bound to SIV-specific peptides and conjugated to a fluorophore. These tetramers selectively bind to T cell receptors (TCRs) on SIV-specific CD8+ T cells, facilitating the characterization of these cells.

1. PKI Treatment
NOTE: This is optional, but recommended for Mamu-B*017:01 tetramers.

	Resuspend PBMC in R10 medium at a concentration of 1.6 x 107 ...

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

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