MHC Class II Tetramer Labeling of Human Primary CD4⁺ T Cells from HIV Infected Patients

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.