Multimeric Major Histocompatibility Complex Reagents for the Detection and Quantitation of Specific T-Cell Populations

Abstract

T cells play essential effector and regulatory roles in adaptive immune responses. For accurate measurements of T-cell immunity as it exists in vivo, the act of in vitro expansion creates at least two significant problems. First, the efficiency of the expansion is highly variable and therefore the results are semiquantitative at best. Second, the stimuli required for in vitro expansion of specific T cells unavoidably alter their phenotype, inducing changes in the patterns of cell surface molecules involved in crucial functions such as cell adhesion and trafficking, as well as potentially altering effector functions such as cytokine secretion profiles. The major histocompatibility complex (MHC) tetramer version was the first to be described and has the largest publication record behind it. Identification of antigen-specific T cells by antigen-binding methods requires no assumptions with respect to the potential functions of the target cells. By far the most popular fluorophore labels for MHC tetramers are the algae-derived phycobiliproteins R-phycoerythrin and allophycocyanin. MHC tetramer stains are nearly always combined with an anti-CD3 antibody as well as either a CD8 antibody (for class I tetramers) or a CD4 antibody (for class II tetramers). Human leukocyte antigens (HLA) tetramer staining for flow cytometry can be performed using relevant single-cell suspensions from any source. A flow cytometer with a minimum of three fluorescent channels is required for collection of data from HLA tetramerstained cells. In addition to the flow cytometer, a variety of standard laboratory equipment-tabletop centrifuges, pipettes, vortex mixers, microscopes and hemacytometers is required.