Tracking Antigen-Specific CD8+ T Cells Using MHC Class I Multimers

Abstract

The tracking of epitope-specific T cells is a useful approach for the study of adaptive immune responses. This protocol describes how Major Histocompatibility Complex Class I (MHC-I) multimers can be used to stain, enrich, and enumerate (rare) populations of CD8(+) T cells specific for a given antigen. It provides the detailed steps for multimer labeling, magnetic enrichment, and cytometric analysis. Additionally, it provides informations for multiplexing experiments in order to achieve simultaneous detection of multiple antigenic specificities, and strategies for coupling the protocol with functional assays (e.g., intracellular cytokine staining). Future developments in cytometric systems (e.g., mass spectroscopy-based cytometry) and gene expression studies (e.g., single cell PCR) will extend these approaches and provide an unprecedented assessment of the immune repertoire.

Bystander hyperactivation of preimmune CD8+ T cells in chronic HCV patients

“…To evaluate more precisely the impact of these perturbations on antigen-specific populations, we applied recently developed strategies to detect, quantify and phenotype rare inexperienced antigen-specific CD8 + T cells ( Klenerman and Thimme, 2012 ; Alanio et al, 2013 ). Specifically, we utilized TAME to enumerate and subdivide Mart1-specific T cell populations.…”

“…TAME was performed as previously described ( Alanio et al, 2010 ; Alanio et al, 2013 ; Kyewski and Klein, 2006 ). Briefly, purified PBMCs (2x10 7 to 4x10 8 ) were incubated with FcR blocking reagent (Miltenyi, France), then stained with PE and/or APC pMHC-multimers at 20nM final concentration for 30 min.…”

“…cDNA were generated using the Supercript II enzyme (Invitrogen, France). RT-PCR reactions, thermal cycling conditions, calculations for relative usage of each Vβ family, and immunoscope profiles were performed as previously described ( Alanio et al, 2013 ; Bouvier et al, 2011 ) ( Supplementary file 2 ).…”