Chimeric γδ T Cell Receptors
γδ and αβ T cells are important parts of the vertebrate immune system, expressing a γδ T cell receptor (TCR) or αβ TCR, respectively. We plan to engineer chimeric γδ TCRs to unravel how γδ T cells respond to temporally varying ligand encounter and provide proof-of-principles of new approaches to improve cancer immunotherapy and to combat virus infection. To this end, we plan to use technologies that we have successfully developed for the αβ TCR, and apply them for the first time to the γδ TCR. The planned work is divided into two parts, whose common denominator is the use of novel chimeric γδ TCRs.
Firstly, we will ask how γδ T cells respond to temporally controlled ligand encounter. To this end, we will engineer γδ TCRs to bind to Phytochome B (PhyB) as their ligand by appending a small domain to the TCR that binds to PhyB. The protein PhyB is derived from plants and can be switched between two conformations using red and far-red light, thus either binding or not to the engineered γδ TCR. We will alter the half-life of the γδ TCR-ligand interaction by using different intensities of red light and study T cell activation. These experiments aim to answer an important unsolved question: whether γδ T cells can distinguish between ligands of different affinities using a mechanism known as kinetic proofreading. In fact, we have used this optogenetic system with the αβ TCR, to show that the half-life is the critical parameter in αβ T cell activation (Yousefi et al., eLife, 2019). Under physiological conditions, T cells are activated while exploring the putative target cells, thus having temporal contacts to the antigen-presenting cells separated by pauses of different duration. Our system is unique to find out whether pauses of ligand binding are tolerated by the γδ T cells, and if specific temporal patterns will lead to differential γδ T cell activation.
Secondly, we will equip γδ TCRs with new specificities by fusion of a single chain Fv fragment against tumour antigens to the γδ TCR. Although γδ T cells have the inherent property to recognize tumours, this is often not sufficient to successfully kill the malignant cells. We will evaluate whether this could be used as a novel T cell based therapy against cancer. We have already used this technology with the αβ TCR and shown that the re-programmed αβ T cells are superior in killing tumour cells in vivo than CAR-T cells (Baeuerle et al., Nature Comm, 2019). We will also explore whether chimeric γδ TCRs can be used to redirect γδ T cells to kill virus infected cells.
Understanding the mechanism of γδ TCR activation, in order to enhance tumour killing
The T cells of the adaptive immune system can be divided into the well-characterised αβ T and the less well understood γδ T cells, dependent of whether they express the αβ T cell receptor (TCR) or the γδ TCR. Recently, human Vγ9Vδ2 and other γδ T cell subsets have gained increasing attention, due to their capacity to recognize and lyse tumour cells in an HLA-independent manner. Since the γδ TCR is often involved in these processes, we aim here to obtain a detailed mechanistic understanding of the biochemical processes with which the γδ TCR transmits signals upon antigen-binding, i.e. the triggering mechanism of the γδ TCR.Thus, we will systematically analyse whether signalling events that are crucial for triggering the αβ TCR are also operative in γδ T cells in work package A. These are antigen-induced clustering of the γδ TCR, CD3 conformational changes in the γδ TCR, phosphorylation of CD3 in the γδ TCR and coupling to downstream signalling pathways. We will reconstitute the γδ TCR in TCRαβ-negative αβ T cells, to focus on differences between these receptors and not on differences in the cellular context, and apply sophisticated biochemical and synthetic biology approaches. The main results will be validated in short-term expanded γδ T cells from human blood.In work package B, we will use these novel insights for a translational purpose, namely to enhance the tumour killing activity of γδ T cells upon γδ TCR triggering by tumour antigens. Our recent finding that stabilisation of the active CD3 conformation in the γδ TCR enhances tumour lysis, will be a starting point. Here, we will engineer novel recombinant, monovalent anti-γδ TCR antibody variants that stabilize the active CD3 conformation. Using different tumour cells, such as melanoma, pancreatic and ovarian cancers as well as B cell lymphomas, we will test in vitro whether the antibody variants indeed increase tumour lysis by short-term expanded human γδ T cells. This project is part of the application for a Research Group FOR2799 “Receiving and Translating Signals via the γδ T Cell Receptor”. As detailed in the application, this project will benefit from the planned interactions with the other groups within the FOR2799.