Name: analysis of simian immunodeficiency virus-specific cd8+ t-cells in rhesus macaques by peptide-mhc-i tetramer staining
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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.
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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.

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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dasatinib</td>
			<td style="width:236px;">Axon medchem</td>
			<td style="width:127px;">Axon&#160;1392</td>
			<td style="width:757px;">Must be resuspended in DMSO and immediately stored at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI w/ Glutamax</td>
			<td>gibco/ Life Technologies</td>
			<td>61870-036</td>
			<td>Must be stored at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat Inactivated FBS</td>
			<td>gibco/ Life Technologies</td>
			<td>10082-147</td>
			<td>Must be stored at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycinp-Amphotericin B</td>
			<td>Lonza</td>
			<td>17-745E</td>
			<td>Must be stored at 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO, Anhydrous</td>
			<td>Life Technologies</td>
			<td>D12345</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-mL Round-Bottom Polypropylene Tubes</td>
			<td>VWR</td>
			<td>60819-728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorochrome-conjugated pMHC-I tetramers</td>
			<td>NIH Tetramer Core or MBL, Inc.</td>
			<td></td>
			<td>Must be stored and maintained at&#160; 4 &#176;C. Centrifuge at 20,000 x g for 15 min before use. Do not freeze.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorochrome-conjugated mAbs</td>
			<td>Various companies</td>
			<td></td>
			<td>Must be stored and maintained at&#160; 4 &#176;C. Do not freeze.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LIVE/DEAD&#160;Fixable Aqua&#160;Dead&#160;Cell Stain Kit</td>
			<td>Life Technologies</td>
			<td>l34957</td>
			<td>Must be stored at -20 &#176;C. Resuspend each aliquot in 50 &#956;L of DMSO prior to use.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brilliant Stain Buffer</td>
			<td>BD Biosciences</td>
			<td>563794</td>
			<td>Must be stored at&#160; 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td>VWR</td>
			<td>97064-158</td>
			<td>Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumine Bovine</td>
			<td>VWR</td>
			<td>700011-230</td>
			<td>Must be stored at&#160; 4 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Bleach</td>
			<td>VWR</td>
			<td>89501-620</td>
			<td>Corrosive chemical, cannot be mixed with sodium azide. Handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td>Flammable, corrosive, and toxic reagent. Handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polysorbate 20 (Tween-20)</td>
			<td>Alfa Aesar</td>
			<td>L15029</td>
			<td>Store at room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Permeabilization Solution 2&#160;</td>
			<td>BD Biosciences</td>
			<td>340973</td>
			<td>Toxic and corrosive reagent. Handle with care</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sarsdet Tubes 1.5mL screw top</td>
			<td>VWR</td>
			<td>72.692.005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2.0ml DNA/RNA Low bind Tubes</td>
			<td>Eppendorf</td>
			<td>22431048</td>
			<td>The use of Sterile microtubes is preffered&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex mixer</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biosafety Cabinet&#160;</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Microcentrifuge</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 &#176;C&#160; refrigerator</td>
			<td>To discretion of scientist</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Deionized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum Foil</td>
			<td>VWR SCIENTIFIC INC.</td>
			<td>89068-738</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td></td>
			<td></td>
			<td>Must be able to maintain 37 &#176;C&#160; internal temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Diva software</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">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 ...

Research

JoVE Journal

Immunology and Infection

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 ...

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

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 ...

piggyBac Transposon System Modification of Primary Human T Cells

piggyBac Transposon System Modification of Primary Human T Cells

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

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 ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

Preparation of CD4+ T Cells for Analysis of GD3 and GD2 Ganglioside Membrane Expression by Microscopy

The methods described herein for activation of na&#239;ve CD4+ T cells in suspension and their adherence in coverslips for confocal microscopy analysis ...

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

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 ...

Zika Virus Specific Diagnostic Epitope Discovery

High-density peptide microarrays allow screening of more than six thousand peptides on a single standard microscopy slide. This method can be applied for ...

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