Cytotoxic T cells are successfully used in adoptive immunotherapy to specifically target and eliminate infected or malignant cells. This specific recognition relies on the binding of the T cell receptor (TCR) on the T cell surface (together with the coreceptor CD8) to major histocompatibility complex I molecules loaded with viral or abnormal peptides (pMHC) displayed on target cells. TCR-pMHC binding results in receptor clustering that subsequently initializes an intracellular signaling cascade in the T cell, leading to effector functions necessary for antigen-clearance. The efficiency, how the T cell reacts to antigen encounter, can be described as T cell avidity. Here, the binding strength of the TCR/CD8-pMHC interaction, often referred to as ‘structural avidity’ of the T cell, has a major impact on T cell functionality Citation[1,2]. It has been shown that T cells equipped with TCRs of higher avidity for their cognate ligand recognize their targets earlier and mediate superior effector functions Citation[3]. This turns T cells with high avidity into promising candidates for adoptive immunotherapy. As established methods did not enable fast and quantitative screening of TCR avidity on living T cells, we developed a novel assay system, which allows to determine a major component of TCR avidity, the TCR-ligand koff-rate. This assay is based on the cooperative binding of monomeric pMHC molecules multimerized on a StrepTactin backbone, so called MHC Streptamers Citation[4]. Addition of D-biotin to Streptamer-stained T cells displaces StrepTactin, leaving monomeric pMHC molecules bound to surface expressed TCRs. Fluorescence labeling of the pMHC molecules enables the monitoring of their subsequent dissociation from the T cell surface with real time microscopy. A slow dissociation (koff)-rate resulting in long half-life times (>100 s) indicates high structural avidity of the TCR/CD8 binding Citation[5]. When analyzing human virus-specific T cell populations or human T cell clones for cytokine secretion or cytotoxicity, we found that T cells with a long TCR-pMHC binding half-life time showed indeed better functionality in response to titrated amounts of their specific peptide Citation[5]. Moreover, we analyzed naïve virus-specific T cell clones that demonstrated no correlation between functionality and multimer staining intensity, although this is a parameter believed to characterize the T cell avidity of antigen-specific cells. In contrast, the koff-rate of these T cell clones correlated completely with their functionality Citation[6]. The high potential of TCR-ligand koff-rate measurements for immunotherapy became clear when analyzing the functionality of adoptively transferred murine T cells with known half-life times in two different infection models. These experiments revealed that only T cells with a long half-life time were able to efficiently respond to their target epitope in vitro (cytokine secretion and target cell lysis) and, more importantly, confer protection to subsequent (lethal) infection with Listeria monocytogenes or murine Cytomegalovirus Citation[5]. We recently demonstrated that even minimal numbers of functional T cells are able to establish a diverse and long-lasting immune response toward infection Citation[7], indicating that not only the number, but the quality of transferred cells determines the success of adoptive T cell immunotherapy. Therefore, the identification of T cells with optimal avidity could increase the effectiveness of immunotherapy while reducing the number of cells required for transfer.
How can we identify the optimal T cell avidity for adoptive immunotherapy? Can we assume that T cells with a short binding half-life time are not suitable? Experiments with OT-1 transgenic T cells, recognizing an ovalbumin-derived peptide presented on H2Kb, seem to contradict this interpretation. While these T cells confer good protection after infection with an ovalbumin-expressing Listeria monocytogenes strain even on the single cell level Citation[8], TCR-ligand koff-rate measurements revealed only a short half-life time (30 s) for the TCR-pMHC binding. To explain this correlation, we have not only to consider the half-life time, but also the on-rate of the TCR-pMHC binding, as both parameters define the affinity of the binding (KD = kon/koff). Discussions if one or the other parameter has a higher influence on T cell avidity led to the formation of the confinement time model Citation[9]. This model proposes that T cell activation is defined by the total time an individual TCR is bound to a pMHC-molecule, regardless of interruptions between rebinding. Here, a fast on-rate leads to serial rebinding instead of diffusion of the molecules and can thus compensate for a short half-life time. In the case of OT-1 T cells, we find a relatively fast on-rate Citation[10] and the high density of the transgenically expressed TCRs additionally favors rebinding events, potentially explaining the high functionality of these T cells. This, however, raises the question if we miss an important portion of highly functional T cells by assessing only the half-life time with the TCR-ligand koff-rate assay. So far, our analyses of many endogenous antigen-specific T cell populations and clones did not reveal a single example for a TCR with low ligand binding half-life time going along with high functionality. This indicates that the main proportion of highly functional antigen-specific T cells within the endogenous repertoire is characterized by a long ligand binding half-life time, supporting the interpretation that measuring koff-rate values is most suitable for T cell/TCR screening approaches. In several studies, T cell avidity and effective functionality could be positively correlated. Whether this correlation is continuous or if a certain lower and upper avidity threshold for T cell effectivity exists is widely discussed. Analysis of engineered T cells expressing affinity enhanced TCRs (<1 μM) demonstrate the loss of specificity Citation[11], the development of alloreactivity Citation[12] as well as cross-reactivity Citation[13]. Interestingly, also the epitope-specific functionality of T cells decreases beyond this threshold Citation[14]. This functional decline might be explained by the productive hit rate model. This model for T cell activation describes that in addition to an optimal dwell time of TCR-pMHC binding Citation[15] serial triggering is necessary for full T cell activation Citation[14]. However, T cells expressing affinity enhanced TCRs are often characterized by high on-rates and very low off-rates, which may prevent serial triggering of the TCR. What does this mean for ‘optimal’ avidity T cells in adoptive immunotherapy? Can the structural avidity of individual T cells/TCRs be too high and then potentially problematic for adoptive immunotherapy due to negative regulatory effects? We currently believe that this concern is less relevant when working with T cells and unmodified TCRs derived from the normal T cell repertoire. Within physiological limits (100–1 μM) T cell function seems to improve with increasing avidity Citation[16]. This is in line with the current status of our results from the analysis of endogenous T cells, as we so far have not identified a single T cell with slow koff-rate and impaired functionality.
Our data strongly suggest that T cells most effective for adoptive immunotherapy can be identified by their TCR-ligand koff-rate. Thereby, half-life times beyond a certain lower threshold (∼100 s) might become a suitable selection criterion, as long as T cells expressing TCRs from the endogenous repertoire are analyzed (endogenous T cells should lie in the natural affinity range and are unlikely to break the upper avidity threshold that limits full functionality). Unfortunately, high avidity T cells are often rare or even absent in the context of malignancies or chronic infections, and therefore T cells with recombinantly expressed TCRs are probably the most promising strategy to supplement the repertoire with high avidity T cells in these clinical settings. Whereas first clinical trials indeed revealed enhanced on-target functionality of T cells expressing affinity engineered TCRs Citation[17], severe off-target effects occurred after the transfer of affinity-enhanced MAGE-A3-specific T cells, caused by cross-recognition of a different protein Citation[18]. This illustrates that beside reactivity, safety of the affinity enhanced TCRs has to be a major concern, and T cells expressing manipulated TCRs have to be analyzed intensively before a potential transfer. Also here the TCR-ligand koff-rate assay that allows for rapid screening of T cell clones expressing antigen-reactive TCRs, might offer an important parameter for the characterization of affinity enhanced TCRs.
For the future of adoptive immunotherapy, the TCR-ligand koff-rate might be an important parameter to enable the selection of optimal T cells with predictably high in vivo functionality. Furthermore, the described koff-rate assay is also applicable for the measurement of interactions other than TCR-pMHC binding. Theoretically, all receptor–ligand interactions that show dissociation of the monomeric bound partners could be analyzed. Preliminary data with chimeric antigen receptors (CARs) and their cognate ligands show the successful transfer of the method to other molecules. CARs, consisting of single chain variable fragments (scFv) of antibodies fused to intracellular signaling domains, enable a T cell to recognize antigens in an MHC-independent manner. Whereas the affinity of the originating antibodies is usually known, binding properties of the scFv and their effect on the CAR expressing T cell have to be determined. Also in this setting, the koff-rate assay could help to identify an optimal affinity of CAR-ligand binding to achieve high T cell functionality. As T cell expressing CARs gain impact on adoptive immunotherapy Citation[19], the clinical potential of the koff-rate assay seems manifold. Based on our findings, we can hypothesize that receptor–ligand koff-rate measurements using reversible staining reagents on living cells can become a helpful tool to optimize the quality of cells for immunotherapy.
Acknowledgements
The authors wish to thank R Knall for providing experimental data on OT-1 T cells, P Paszkiewicz and C Stemberger for carefully reading the manuscript.
Financial & competing interests disclosure
The authors were supported by SFB TR36 (TP-B10/13). Streptamer technology is covered by US Patent 4,776,562 (patent holder Dirk H. Busch and Hermann Wagner). Streptamer products are commercialized by IBA GmbH (Göttingen) and Stage Cell Therapeutics (Göttingen). The authors have no other relevant affiliations or other financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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