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 c....

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 PB....

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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