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

Summary

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.

Abstract

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.

Introduction

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-cells^2-4. 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 colleagues^8,9, 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 individuals^10-12. 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—a phenomenon known as "elite control"^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 infection^16,17. 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/AIDS^18,19. 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 motifs^13,20-27. 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 infection^30,31 (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.

Protocol

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.

1. Resuspend PBMC in R10 medium at a concentration of 1.6 x 10⁷ cells/ml.
2. Add 50 µl of this cell suspension to the corresponding flow cytometry tubes. Add 50 µl of a 100 nM solution of PKI to each tube.
3. Vortex each tube. Incubate at 37 °C for 30 min. By the end of this step, each tube should have 100 µl of a cell suspension containing 8.0 x 10⁵ PBMC and 50 nM of PKI.
4. Proceed to the pMHC-I tetramer staining step.

2. Staining with Fluorochrome-labeled pMHC-I Tetramers

1. 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 °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.
2. 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.
3. Dilute pMHC-I tetramers in stain buffer (e.g., Brilliant Stain Buffer) so that 25 µl of the pMHC-I tetramer master mix are added to each test.
   1. Label PBMC with two pMHC-I tetramers per test; one conjugated to allophycocyanin (APC) and the other to Brilliant Violet (BV) 421.
4. Add 8.0 x 10⁵ PBMC to the corresponding flow cytometry tubes in a final volume of 100 µl. If the cells were subjected to the PKI treatment described above, they should already be resuspended in 100 µl at this step.
5. Add 25 µl 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 µl.
6. Vortex each tube in order to homogenize the cell suspension.
7. Incubate in the dark at room temp for 45 min. Prepare the surface staining mAb cocktail during this 45 min incubation.

3. Surface Staining

1. 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.
2. Adjust the volume of the master mix with stain buffer so that 50 µl are added per test.
3. 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, CD8α 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 "dump" gate.
4. 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 "dump" gate. In this case, use ARD Aqua.
5. Add 50 µl 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 µl.
6. Vortex each tube. Incubate in the dark at room temp for 25 min.
7. 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.
8. 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.
9. After decanting, vortex the cells in the leftover liquid retained in each tube.

4. Cell Fixation

1. Add 250 µl 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.
   1. 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.
2. Incubate in the dark at 4 °C for 20 min.
3. Wash cells by repeating steps 3.7-3.9. Once this step is completed, the cells can be stored in the dark at 4 °C for 24-48 hr. Before proceeding to the Permeabilization phase, vortex the tubes thoroughly.

5. Permeabilization of Cells

1. Add 500 µl of permeabilization buffer. Vortex all tubes.
2. Incubate in the dark at room temp for 10 min.
3. Wash cells by repeating steps 3.7-3.9.

6. Intracellular Staining

1. Prepare enough mAb master mix to stain all experimental tubes.
2. Adjust the volume with PBS or stain buffer so that 50 µl of the intracellular mAb master mix are added per test.
3. 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.
4. Add 50 µl of mAb cocktail to the corresponding tubes. Incubate in the dark at room temp for 30 min.
5. Wash cells by repeating steps 3.7-3.9. The tubes are ready to be acquired in a flow cytometer.

Results

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...

Discussion

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 ...

Materials

List of materials used in this article

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