TCR-Ligand k _off Rate Correlates with the Protective Capacity of Antigen-Specific CD8 ⁺ T Cells for Adoptive Transfer

Abstract

Adoptive immunotherapy is a promising therapeutic approach for the treatment of chronic infections and cancer. Thereby, T cells within a certain range of high avidity for their cognate ligand are believed to be most effective. T cell receptor (TCR) transfer experiments indicate that a major part of avidity is hard-wired within the structure of the TCR. Unfortunately, rapid measurement of structural avidity of TCRs is difficult on living T cells. We developed a technology, where dissociation (koff-rate) of truly monomeric peptide major histocompatibility complex (pMHC) molecules bound to surface expressed TCRs can be monitored by real-time microscopy in a highly reliable manner. A first evaluation of this method on distinct human Cytomegalovirus (CMV) -specific T cell populations revealed unexpected differences in the koff-rates. CMV-specific T cells are currently being evaluated in clinical trials for efficacy in adoptive immunotherapy; therefore, determination of koff-rates could guide selection of the most effective donor cells. Indeed, in two different murine infection models, we demonstrate that T cell populations with lower koff-rates confer significantly better protection than populations with fast koff-rates. These data indicate that koff-rate measurements can improve the predictability of adoptive immunotherapy and provide diagnostic information on the in vivo quality of T cells.

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

“…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 ). However, the Streptamer assay (11) needs a significant lag time until monomeric TCR-pMHC dissociation starts to become detectable, limiting thereby off-rate analyses to antigen-specific T cells of relative long half-lives, typically found in immune responses against pathogens.…”

“…However, the Streptamer assay (11) needs a significant lag time until monomeric TCR-pMHC dissociation starts to become detectable, limiting thereby off-rate analyses to antigen-specific T cells of relative long half-lives, typically found in immune responses against pathogens. Moreover, accurate measurements of TCR-pMHC binding parameters on living T cells (11)(12)(13)(14) require specialized equipment, which is currently not available for the high-throughput screen of antigen-specific T-cell populations.…”

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

“…pMHC molecules for generation of reversible multimers were refolded and multimerized as described previously (7). All pMHC molecules used for the k off -rate assays in this report were conjugated to Alexa488 maleimide fluorophore (Thermo fisher) and multimerized Streptactin APC (IBA).…”

“…Human CMV-specific T cell clones and murine T cell lines were generated as described previously (7). Peripheral blood was obtained from a healthy adult donor (male).…”

“…We recently developed a novel technology, the so-called "TCR-ligand k off -rate assay," to quantify an important component of structural TCR avidity (7). This assay is based on reversible multimers, so called Streptamers (11).…”