Observing the T cell receptor (TCR) activation in real time: monitoring the intracellular domain orientation dynamics of the receptor complex in live cells with polarized time-resolved fluorescence imaging – ReceptOrient

We will first develop innovative real-time polarization imaging technologies including polarized single-molecule tracking (P-SMT) and polarized fluorescence correlation spectroscopy (P-FCS). In parallel, we will develop fluorescent probes via molecular genetic engineering that are specially designed for performing experiments of polarized imaging of TCR/CD3components. After these early experimental steps, then we will carry out measurements of molecular orientation and rotational diffusion for TCR/CD3 components, both before and after stimulation of the TCR by pMHC

Despite dramatic advances in genomics research, our understanding of the functioning of the immune system is still limited as shown by the slow progress in cancer immunotherapy or for the production of vaccines. A major reason for this stems from the fact that most of the proposed mechanistic scenarios, including those describing the process of T cell activation, remain unproven or even totally speculative. For this, it is essential to analyze the molecular events in living cells with spatiotemporal resolution sufficient thanks to the use of biophotonic methodologies at the state of the art. ReceptOrient aims to elucidate the most basic questions, but unresolved in adaptive immunity, namely how the activation of a T cell antigen receptor mediated by is initiated

The expected results will contribute not only to a better understanding of the mechanisms of the immune system, but also to develop new strategies against infectious diseases. This new understanding of the immune system should provide the means to be more effective against the emergence of new bacterial strains resistant to antibiotics as well as new types of pathogens. ReceptOrient is also relevant to autoimmune diseases, at both diagnostic and therapeutic levels. Finally, ReceptOrient should also contribute to make progresses in other important public health areas (manufacturing vaccines, immunotherapy cancer)

1. Kress A, Ferrand P, Rigneault H, Trombik T, He HT, Marguet D, Brasselet S
Probing Orientational Behavior of MHC Class I Protein and Lipid Probes in Cell Membranes by Fluorescence Polarization-Resolved Imaging
Biophysical Journal (2011) 101:468-76
2. He HT, Bongrand P
Membrane dynamics shape TCR-generated signaling
Frontiers in immunology (2012) 3:90

ReceptOrient is an interdisciplinary project with ambition to realize true breakthroughs at both conceptual and technological levels in the field of biology and health through strong collaborations between biologists and physicists. The project aims at monitoring in live T lymphocytes the dynamic changes in molecular orientation and rotational diffusion of T-cell receptor (TCR) complex components upon receptor engagement with peptide-MHC ligand.
We will implement innovative real-time polarized optics technologies. We anticipate that ReceptOrient would provide important and new insights on the earliest events in the TCR activation process, which would ultimately lead to novel perspectives of finely tuning the T cell responses in therapeutic strategies.
Despite spectacular progresses in genomic research, our understanding of immune system functioning is still very poor as exemplified by the slow progresses on cancer immunotherapy as well as on fabrication of vaccines. A major reason for this is that most of the proposed mechanistic scenarios, including those for the T cell recognition process, remain speculative and non-demonstrative. To solve this problem, it is crucial to be able to monitor molecular events at sufficient spatial-temporal resolutions in live cells, with the use of advanced and dedicated biophotonics methodologies.
T cell recognition is mediated by the TCR that detects the presence of antigens and transmits signals across the membrane and in turn initiates an activation program. Currently, the molecular basis of this transduction process, also called TCR triggering is still ill understood. It is proposed that the TCR/CD3 complex displays spatial orientation changes upon binding by its ligand: it is proposed that a key event is to free the CD3epsilon and CD3zeta cytoplasmic domain from their binding to the inner leaflet of the plasma membrane, such that the immunoreceptor tyrosine-based activation motifs become accessible for phosphorylation.
However, because of methodological limitations, no direct experimental observations have so far been reported to validate such model. Thus, it is of major interest to determine whether and how the reorientation of the cytoplasmic domain of CD3epsilon and CD3zeta happens upon receptor engagement by ligand in living cells. Molecular orientation information can generally be retrieved from an adequate fluorescence labeling and polarized fluorescence measurement. Whereas fluorescence anisotropy is a well-established technique in both time domain and imaging regimes, downscaling fluorescence anisotropy information to single molecules is still largely unexploited in addressing biological questions.
In addition to an innovative polarization imaging technique that we recently developed, two other complementary polarized photonics methodologies, namely polarized single-molecule tracking and polarized fluorescence correlation spectroscopy, will be implemented on a microscope equipped with holographic optical tweezers. In the mean time, dedicated probe molecules will be generated for the polarized fluorescence measurements of TCR/CD3 molecular orientation dynamics in the membrane of living T cells.
The ReceptOrient project addresses one of the most fundamental, but yet unresolved questions in adaptive immunity. Our approaches lie in the emergence of new biological concept based on the quantification of biological observables through the development of innovating technological tools. Ultimately, we expect to be able to predict and control the reactivity and the dynamics of the biological macromolecules and their complexes in specific cellular environment. By improving to our basic knowledge on the functioning of immune system, ReceptOrient project can further contribute significantly to setting up new therapeutic strategies.