Detection, phenotyping, and quantification of antigen-specific T cells using a peptide-MHC dodecamer

Abstract

Here we report a peptide-MHC (pMHC) dodecamer as a "next generation" technology that is a significantly more sensitive and versatile alternative to pMHC tetramers for the detection, isolation, and phenotypic analysis of antigen-specific T cells. In particular, dodecamers are able to detect two-to fivefold more antigen-specific T cells in both human and murine CD4+ and CD8 + αβ T-cell compartments compared with the equivalent tetramers. The low-affinity, tetramernegative, dodecamer-positive T cells showed comparable effector cytokine responses as those of high-affinity, tetramer-positive T cells. Dodecamers are able to detect early stage CD4thymocytes on which T-cell receptors are 10-to 30-fold less dense than mature T cells. Dodecamers also show utility in the analysis of γδ T cells and in cytometry by time-of-flight applications. This construct has a simple structure with a central scaffold protein linked to four streptavidin molecules, each having three pMHC ligands or other molecules. The dodecamer is straightforward and inexpensive to produce and is compatible with current tetramer technology and commercially available streptavidin conjugates.T -cell receptors (TCRs) detect antigens in the form of peptides bound to peptide-major histocompatibility complex (pMHC) molecules on the surface of antigen-presenting cells. TCR-pMHC interactions determine the selection, development, differentiation, fate, and function of a T cell. However, TCRs bind monomeric pMHCs with very low binding affinities (K d , ∼1-200 μM, 1,000-to 200,000-fold weaker than a typical antibody-antigen interaction) and with fast dissociation rates (k off , ∼0.05 s −1 ) in solution (1). To increase the binding avidity and circumvent the problem of fast dissociation, we previously engineered a pMHC tetramer to detect antigen-specific T cells by conjugating four biotinylated pMHC monomers to a single fluorescent-labeled streptavidin (2). This fulfilled a critical need in both basic and clinical immunology to be able to identify and characterize often very rare specific T cells in a population. Subsequent improvements in sensitivity, manufacture, and combinatorial labeling have made this methodology even more useful (3-7). However, there is a sharp drop-off in tetramer binding in the lower affinity range (∼150 μM) (8) so there have been a number of higher valency alternatives including pentamers (ProImmune), lipid vesicles (9), octamers (10), dextramers (11), and quantum dot (QD)-based multimers (12). Some of these clearly have improved sensitivity, and this is important in detecting T cells with especially low affinities (13,14). For example, naive T cells and thymocytes, which express lowlevel and/or low-affinity TCRs, show little-to-no tetramer staining. Furthermore, αβ T cells and γδ T cells that do not bind antigen-specific tetramers can still produce significant antigenspecific cytokine responses (15, 16). MHC class II tetramers are also problematic in cytometry by time-of-flight mass spectrometry (CyTOF), which is an advanced version of flow...

TCR-ligand dissociation rate is a robust and stable biomarker of CD8+ T cell potency

“…Various reports have previously shown that certain functional antigen-specific CD8 + T cells fail to bind tetrameric MHC ligands, which could represent up to several percent of the CD8 + T cell subset (58)(59)(60). Moreover, this is of particular importance when staining tumor-specific CD8 + T cells, known to express lower TCR-pMHC affinity/avidity repertoires than virus-specific cells ( Figure 5 and refs.…”

Leveraging blood and tissue CD4+ T cell heterogeneity at the single cell level to identify mechanisms of disease in rheumatoid arthritis

“…Incorporating data on from multiple assays to define lymphocyte profiles will be essential for understanding their functional impact, as shown by multiple studies that utilize repertoire sequences or expression data in combination with single cell cytometry to identify disease-relevant populations [71••,105•,106,107]. The development of new peptide-MHC multimeric complexes supports the detection and isolation of antigen-specific lymphocytes at much lower frequencies [108] than was previously feasible. New methods have been recently developed to provide high-throughput single cell repertoire sequencing of B and T lymphocytes [109•,110].…”

A unique CD8+ T lymphocyte signature in pediatric type 1 diabetes

“…However, it is also conceivable that tetramer studies underestimate the number of islet-reactive CD8 þ T cells. Application of novel technologies such as Major Histo-Compatibility class I dodecamers [40] or micropipette adhesion [41] has revealed that the number of self-reactive T cells is much larger than previously assumed based on conventional tetramers. Whatever the antigenic specificity of the T cells carrying the expression signature, our results demonstrate that analysis of gene and protein expression by bulk memory/activated CD8 þ T cells uncovers a signature associated with onset of pediatric T1D.…”