Flow cytometry‐based TCR‐ligand K_off‐rate assay for fast avidity screening of even very small antigen‐specific T cell populations ex vivo

Abstract

High epitope-specific sensitivity of CD8 1 T cells is required for optimal immune protection against intracellular pathogens as well as certain malignancies. The quality of antigen recognition of CD8 1 T cells is usually described as "avidity" to its cognate peptide MHCI complex. T cell avidity is mainly dependent on the structural qualities of the T cell receptor (TCR), as convincingly demonstrated by recombinant TCR reexpression experiments. Based on reversible MHCI multimer staining and k off -rate measurements of monomeric peptide MHCI complexes, we recently established a microscopic assay for determining the structural avidity of individual CD8 1 T cells. Here we demonstrate that this assay can be adapted for rapid flow-cytometric avidity screening of epitope-specific T cell populations. Furthermore, we show that-in combination with conventional nonreversible MHCI multimer staining-even very small epitope-specific CD8 1 T cell populations can be analyzed directly ex vivo without the need for previous TCR cloning or T cell sorting. This simplified approach provides highly accurate mean TCR-ligand k off -rate values for poly-or oligoclonal T cell populations and is ideally suited for high-throughput applications in basic research as well as clinical settings. V C 2016 International Society for Advancement of Cytometry Key terms TCR-ligand-k off -rate; structural TCR avidity; dissociation kinetics; direct avidity screening; T cell functionality; T cell therapy; online injection; uninterrupted acquisition; temperature controlled analysis; MHC multimer double staining THE importance of cellular adaptive immunity in the control of viral and some bacterial infections has been convincingly demonstrated by depletion or adoptive transfer experiments, as well as in genetic mouse models. Thereby, antigen-specific CD8 1 T cells seem to play a dominant role for protection against intracellular pathogens (1). Also in certain cancers, the presence of functional CD8 1 T cells could be associated with the induction of tumor regression (2,3). Different immunotherapeutic approaches therefore aim at the reconstitution of a functional CD8 1 T cell response in patients. If antigen-specific T cells are still present but silenced by the micromilieu, application of "checkpoint inhibitors" can serve to re-activate the efficacy of existing T cells (4). However, if endogenous antigen-specific T cells are lacking or existing cells carry T cell receptors (TCRs) with weak antigen recognition, adoptive T cell transfer of more potent T cells might represent a promising approach to restore a protective antigen-specific CD8 1 T cell response. Research over the last decades has indicated that not only the quantity but also the quality of prevalent or adoptively transferred T cells correlates with the success of T cell-based immunotherapies (5-7).

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition)

“…With directly fluorochrome‐labeled MHC molecules, the dissociation can be precisely measured and serves as an important parameter for TCR avidity (Fig. C, “dye‐conjugated reversible pMHC”) . Reversible staining has been further transferred to low affinity Ab‐derived Fab fragments (Fab Streptamer), extending the applicability of this labeling technology to virtually any surface antigen .…”

TCR repertoire evolution during maintenance of CMV‐specific T‐cell populations

“…Much work has therefore been put into the establishment of standardized assays to characterize TCR structural avidity. Using reversible MHC multimers, such as MHC streptamers, it is possible to precisely determine the k off ‐rate of surface‐bound pMHC monomers and thereby calculate the half‐life of TCR‐pMHC/CD8 interactions . k off ‐rate assays do not take into account the k on ‐rate and thereby do not measure TCR avidity per se, but the k off ‐rate has been shown to be predictive of both structural avidity and in vivo functionality, although this correlation may not necessarily be completely linear.…”

“…More work is needed to decipher the exact relationship of dissociation time and in vivo functionality, but at least within a physiological range of TCR dissociation times, the 2 parameters often do correlate well. Beyond these considerations, modern TCR structural avidity assays are in particular valuable with regard to their practical applications, as they enable high‐throughput analysis of T‐cell populations directly ex vivo . TCR structural avidity can be expected to represent, for example, a highly relevant parameter for the field of adoptive T‐cell therapy, as most promising TCRs have to be somehow selected out of often highly diverse sources for subsequent genetic engineering of therapeutic T cells …”

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

“…Yet, robust techniques allowing for the rapid identification and isolation of CD8 + T cells of highest avidity and functions directly ex vivo from tissues or blood samples and at the single-cell level are still required. In that regard, Nauerth and colleagues (67) proposed that small polyclonal virus-specific CD8 + T cell populations could be analyzed directly ex vivo without the need of previous TCR cloning or T cell sorting. The recent implementation of an ex vivo platform allowing for the single-cell serial determination of 2D TCR-pMHC affinity (based on micropipette adhesion frequency) and TCR clonotyping is also highly promising (68).…”