Optogenetic Tuning of Ligand Binding to The Human T cell Receptor Using The opto-ligand-TCR System

T cells are one major cell type of the immune system that use their T cell antigen receptor (TCR) to bind and respond to foreign molecules derived from pathogens. The ligand-TCR interaction half-lives determine stimulation outcome. Until recently, scientists relied on mutating either the TCR or its ligands to investigate how varying TCR-ligand interaction durations impacted on T cell activation. Our newly created opto-ligand-TCR system allowed us to precisely and reversibly control ligand binding to the TCR by light illumination. This system uses phytochrome B (PhyB) tetramers as a light-regulated TCR ligand. PhyB can be photoconverted between a binding (ON) and non-binding (OFF) conformation by 660 nm and 740 nm light illumination, respectively. PhyB ON is able to bind to a synthetic TCR, generated by fusing the PhyB interacting factor (PIF) to the TCRβ chain. Switching PhyB to the OFF conformation disrupts this interaction. Sufficiently long binding of PhyB tetramers to the PIF-TCR led to T cell activation as measured by calcium influx. Here, we describe protocols for how to generate the tetrameric ligand for our opto-ligand-TCR system, how to measure ligand-TCR binding by flow cytometry and how to quantify T cell activation via calcium influx.


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[Background] Life depends to a large extent on the precise spatial and temporal coordination of molecular events. This is particularly important in cellular decision processes, for which cells constantly interpret signals from their environment in order to decide how to respond. Due to the lack of appropriate approaches, the impact of kinetics and localization of signaling processes on cellular decisions is still not well understood. Now the emerging field of optogenetics enables to perform the experiments required to fill this knowledge gap (Kolar and Weber, 2017; Goglia and Toettcher, 2019). As an example, we use T cells stimulated via their T cell antigen receptor (TCR) in this protocol. recognized by the TCR in the form of pathogen-derived peptides presented on major histocompatibility complex (MHC) proteins. These peptide-MHC conjugates serve as high affinity ligands for the TCR (Davis et al., 1998). Importantly, self peptides derived from endogenous proteins are presented on MHC as well. Self peptide-MHCs also bind to the TCR, but with low affinity and thus do not result in activatory signaling. It is therefore clear that TCRs are able to distinguish between ligands of different affinity (Holler and Kranz, 2003) and it has been proposed that T cells are able to make this differentiation based on the ligand binding time to the TCR (McKeithan, 1995).
So far, the majority of T cell researchers used peptides with point mutations, presented on MHC to investigate the effect of varying ligand affinity (and indirectly binding time) on T cell activation (Matsui et al., 1991 and1994;Weber et al., 1992;Sykulev et al., 1994;Corr et al., 1994;Lyons et al., 1996;Daniels et al., 2006). Alternative methods for changing ligand-TCR interaction time are the use of mutated superantigens (Andersen et al., 2001) or the mutation of the TCR itself (Tan et al., 2017) All those approaches have in common that they fail to exclusively manipulate the TCR-ligand binding time without affecting other properties of the binding event, such as on-rate, enthalpy, entropy, geometry of binding, Gibbs energy or ability to withstand forces.
To overcome these experimental restrictions we have developed the opto-ligand-TCR system , by making use of the light-dependent interaction between phytochrome B (PhyB) and PhyB interacting factor (PIF) (Levskaya et al., 2009;Toettcher et al., 2013;Kolar and Weber, 2017).
We chose the PhyB-PIF system as the optogenetic switch for our system, since it allows for active, light- Biotinylated PhyB molecules were tetramerized via streptavidin and these PhyB tetramers (PhyBt) were used as multimeric TCR ligands. 660 nm light illumination of PhyB led to a switch to the PIF-binding ON state (usually referred to as Pfr state) and 740 nm illumination reverses PhyB to the non-binding OFF state (usually referred to as Pr state) (Mancinelli, 1994). Hence, our opto-ligand-TCR system enabled us to specifically control ligand binding times via light illumination using the same ligand-receptor pair and without introducing mutations to the TCR or its ligands.
Our novel system allows high spatiotemporal control over reversible ligand binding to the TCR. This unique feature of the opto-ligand-TCR system could enable researchers to locally or timely restrict ligand-receptor interaction. Fusing PIF to other receptors would allow to control ligand binding to those receptors as well, as we previously demonstrated for integrins (Baaske et al., 2019). Further, our system could be used to investigate the signaling events that happen after ligand dissociation, which have been mostly neglected due to the lack of appropriate methods. 3 www.bio-protocol.org/e3540

Data analysis
The Unicorn software suite was used to control the Äkta chromatography system and analyze the resulting data. FlowJo was used to analyze all flow cytometry data. Only the living cell population was used for the depicted flow cytometry results. Intracellular calcium was quantified by the ratio of