Analysis, Isolation, and Activation of Antigen-Specific CD4+ and CD8+ T Cells by Soluble MHC-Peptide Complexes

Abstract

T cells constitute the core of adaptive cellular immunity and protect higher organisms against pathogen infections and cancer. Monitoring of disease progression as well as prophylactic or therapeutic vaccines and immunotherapies call for conclusive detection, analysis, and sorting of antigen-specific T cells. This is possible by means of soluble recombinant ligands for T cells, i.e., MHC class I-peptide (pMHC I) complexes for CD8+ T cells and MHC class II-peptide (pMHC II) complexes for CD4+ T cells and flow cytometry. Here we review major developments in the development of pMHC staining reagents and their diverse applications and discuss perspectives of their use for basic and clinical investigations.

Identification of Rare High-Avidity, Tumor-Reactive CD8+ T Cells by Monomeric TCR–Ligand Off-Rates Measurements on Living Cells

“…We previously developed a method for the isolation and analysis of antigen-specific cytotoxic T cells by reversible NTAmers (10,15). Upon addition of imidazole at low, nontoxic concentration, the SA-PE-NTA 4 moieties rapidly decay (average dissociation half-life of 2.5 seconds), thereby releasing the pMHC monomers bound at the cell surface.…”

“…Consequently, NTAmers allow FACS-sorting of CD8 þ T cells without inducing adverse effects on the cell integrity (e.g., activation-induced cell death; refs. 10,15). Taking advantage of the fact that NTAmers can be switched from stable binding to rapid dissociation, a two-color NTAmer was engineered to assess monomeric TCR-pMHC dissociation kinetics directly on living CD8 þ T cells (Fig.…”

“…Binding and dissociation measurements should ideally be performed using monomeric pMHC complexes. Because TCRpMHC interactions typically exhibit weak affinities and fast dissociation rates, this has long precluded conclusive measurements with monomeric pMHCs (10). Nauerth and colleagues (11) recently measured monomeric TCR-pMHC dissociation kinetics using reversible Streptamers and reported that virus-specific CD8 þ T cells with longer half-lives (low k off ) conferred increased functional avidity and better in vivo protection than T cells exhibiting shorter t 1/2 (high k off ).…”

Quantitative TCR:pMHC Dissociation Rate Assessment by NTAmers Reveals Antimelanoma T Cell Repertoires Enriched for High Functional Competence

“…Whereas pMHC multimers were often used in binding and dissociation assays, results are biased by the multivalent nature of multimers and the ensuing use of rebinding antagonists (15,16). Conversely, three-dimensional soluble TCR:pMHC affinity measurements by surface plasmon resonance (SPR) omit the impact of CD8 coreceptor binding and membrane fluidity (15).…”

“…Whereas pMHC multimers were often used in binding and dissociation assays, results are biased by the multivalent nature of multimers and the ensuing use of rebinding antagonists (15,16). Conversely, three-dimensional soluble TCR:pMHC affinity measurements by surface plasmon resonance (SPR) omit the impact of CD8 coreceptor binding and membrane fluidity (15). The twodimensional techniques have offered important membraneassociated kinetics insights (17) and led to the elaboration of mathematical models (14) predicting TCR:pMHC interactions and their association with T cell functions.…”