Abstract
Major Histocompatibility Complex (MHC) tetramers have been used for two decades to detect, isolate and characterize T cells specific for various pathogens and tumor antigens. In the context of Human Immunodeficiency Virus (HIV) infection, antigen-specific CD8+ T cells have been extensively studied ex vivo, as they can be readily detected by HIV peptide-loaded MHC class I tetramers. In contrast, the detection of HIV-specific CD4+ T cells has proven more challenging, due to the intrinsically lower clonal expansion rates of CD4+ T cells, and to the preferential depletion of HIV-specific CD4+ T cells in the course of HIV infection.In the following protocol, we describe a simple method that facilitates the identification of CD4+ T cells specific for an HIV-1 capsid epitope using peptide-loaded MHC class II tetramers. Tetramer labeled CD4+ T cells can be analyzed for their cell surface phenotype and/or FACS-sorted for further downstream applications. A key point for successful detection of specific CD4+ T cells ex vivo is the choice of a peptide/MHC II combination that results in high-affinity T Cell Receptor (TCR) binding (Benati et al., 2016). A second key point for reliable detection of MHC II tetramer-positive cells is the systematic use of a control tetramer loaded with an irrelevant peptide, with the sample and control tubes being processed in identical conditions.
Keywords: Major histocompatibility complex class II, Tetramer, T cell receptor, CD4+ T cell, HIV
Background
Rare HIV-specific MHC II tetramer-positive cells have been detected in purified CD4+ T cells, after magnetic enrichment of tetramer-PE labeled cells with anti-PE microbeads (Seth et al., 2005). We found that with validated peptide/MHC II tetramer combinations, a simpler protocol based on direct tetramer labeling of 5 x 106 patient Peripheral Blood Mononuclear Cells (PBMC), followed by acquisition of all events on a flow cytometer, resulted in reliable detection of HIV-specific CD4+ T cells. Exclusion of irrelevant cells (CD14+, CD20+, CD8+) and dead cells (Fixable Viability dye+) through an appropriate gating strategy improved labeling specificity.
Materials and Reagents
Equipment
Software
Procedure
Ex vivo tetramer labeling of CD4+ T cells from HIV infected patients
Data analysis
Flow cytometry data was analyzed with the Flowjo v10.2 software (FlowJo, LLC.).
Recipes
Acknowledgments
This protocol was adapted from Benati et al. (2016). We acknowledge funding from Agence Nationale de la Recherche sur le SIDA et les Hépatites Virales (ANRS EP36-8) and Agence Nationale de la Recherche (ANR PD1VAX).
References
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