How cholesterol and phospholipids fine tune T cell early mechanosensitivity?
It is crucial for fundamental, but also applied, reasons to identify physiological regulators of the adaptive immune response. In our work, we focus on the initiation and propagation of the initial antigenic signal in naïve T lymphocytes. How T cells translate rare, diverse, medium affine antigenic signals into a strong, specific and nearly instantaneous response is still imperfectly understood. We have shown that T Cell Receptor (TCR) triggering is intimately dependent on the lipid organization of the plasma membrane [Chouaki et al. 2018 see references] and others described that TCR acts as a mechanosensor molecule [Kim et al. 2009] making T cells sensitive to the magnitude as well as the kinetics of force application. Our hypothesis for the present project is that the lipid and cholesterol transporters, ABCA1 and ABCG1 are instrumental in regulating the initiation of the adaptive immune response at the T cell level [Wu et al. 2020]. To decipher this particular point, we propose to investigate in depth the phenotype of conditional KO mice defective for ABCA1/G1 in T cells (DKO T cells), focusing on the changes of membrane lateral organization, mechanics and its mechanotransduction properties of the TCR upon T cell contacts with B cells acting as antigen presenting cells. We will start from our recent observation of early mechanical changes occurring prior to intracellular signaling [Zak et al., 2021].
T cells, Cholesterol, membrane nano-organisation, cell biomechanics, cell signaling
We identified 3 main work packages for this project:
WP#1: How loss of ABCA1/G1 impact T cell biophysical homeostasis (membrane lipid composition and nano-organization, signal transduction, TCR configuration….)
WP#2: Do ABCA1/G1 loss alter the mechanical properties of DKO T cells vs WT T cells, at the resting state, in different T cell subsets?
WP#3: How changes evidenced in the first two WP impact on T cell responsiveness upon antigenic challenge? Are they, in turn, modulated by the early T cell activation?
Proposed approach (experimental / theoretical / computational)
The objectives will be tackled experimentally by combining diverse advanced biophotonic tools based on fluorescence correlation spectroscopy (FCS)/FLIM FRET/Super-resolution at the CIML. T cell forces and mechanics will be performed at in Lab. Adhesion & Inflammation (LAI) by atomic force microscopy (AFM) indentation or single cell force spectroscopy modes (SCFS) or optical tweezers , already available. We suspect that phase transitions may occur in the T cell membrane. A lot of modelling work is needed to shed new light on the experimental data. We wish to develop new experimental tools in both labs, based on image correlation or STED-FCS (@ CIML) and an innovative force sensing technique as the « lateral AFM » (LatAFM), based on SCFS, coupled to fluorescence microscopy that we are building (@ LAI).
How changes in T cell biophysics and biomechanics are converted into biological signals because of cell-to-cell contacts is of general importance in immunity, both in health and disease. Although lymphocytes are circulating cells, there task is to probe their antigenic environment through constant palpations of neighbouring cell in lymphoid organs, which may dysfunction in autoimmunity, cancer or atherosclerosis. The T cells integrate both biochemical and biophysical stimuli to read, understand and react to their microenvironment, in particular to the APCs. As such, this thematic at the border between biology and biophysics is multi- and inter-disciplinary thereby fitting exquisitely in the present call. We will highly benefit of the interaction with Centuri engineering team for data processing in order to extract quantitative data from the rich experiments we propose, coupling scalar measurement, resolved in time and space, with imaging techniques, to put in tight correlations and if possible, define clear causality links.
This topic provides for a cell biologist or an experimental physicist a unique approach at the interface of biology and physics in a young, highly cohesive and multidisciplinary environment (physicists, biologists and physicians) around a theme with strong applications in human health. The candidate should have bases in microscopy and computer programming since the richness of the data may imply to develop data analysis codes, or at least be willing to invest in learning it.
Is this project the continuation of an existing project or an entirely new one? In the case of an existing project, please explain the links between the two projects
This project is the direct continuation of a successful and productive thesis [cf Sadoun et al. 2021, Zak et al. 2021]. We now are seeking to identify physiological regulators (namely ABCA1/G1) of the T cell plasma membrane organization and biomechanical properties.
2 to 5 references related to the project
- Kim, S.T., et al., The αβ T cell receptor is an anisotropic mechanosensor. Journal of Biological Chemistry, 2009. 284(45): p. 31028-31037.
- Wu, A., et al., Do ABC transporters regulate plasma membrane organization? Cellular & Molecular Biology Letters, 2020. 25(1): p. 1-17.
- Courtney, A. H., et al. (2018). TCR signaling: mechanisms of initiation and propagation. Trends in biochemical sciences, 43(2), 108-123.
- Billaudeau, C., et al., Probing the plasma membrane organization in living cells by spot variation fluorescence correlation spectroscopy. Methods Enzymol, 2013. 519: p. 277-302.
3 main publications from each PI over the last 5 years
- Blouin, C.M., et al., Glycosylation-dependent IFN-γR partitioning in lipid and actin nanodomains is critical for JAK activation. Cell, 2016. 166(4): p. 920-934.
- Chouaki-Benmansour, N., et al., Phosphoinositides regulate the TCR/CD3 complex membrane dynamics and activation. Scientific reports, 2018. 8(1): p. 4966.
- Raducka-Jaszul, O., et al. (2021). Molecular Diffusion of ABCA1 at the Cell Surface of Living Cells Assessed by svFCS. Membranes, 11(7), 498.
- Sadoun, A., et al. (2021) Controlling T cells shape, mechanics and activation by micropatterning. Scientific reports, 11(1), 1-15.
- Zak, A., et al. (2021) Single-cell immuno-mechanics: rapid viscoelastic changes are a hallmark of early leukocyte activation. Biophysical Journal, 120(9), 1692-1704.
- Ndao, O., Puech, P. H., et al. (2020). Dynamics of Toxoplasma gondii oocyst phagocytosis by macrophages. Frontiers in cellular and infection microbiology, 10, 207.