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  • Project No: KIR-2023/12
  • Intake: 2023 KIR Non Clinical

project overview

Antigen recognition is a crucial process in health and disease. Over the last couple of years, a convincing body of evidence has emerged revealing that mechanical force is present at all stages of T-cell activation. An emerging body of evidence indicates that T cells sense and exploit mechanical force in the process of antigen recognition in T-cell activation.

However, the biological significance of mechanosensation for antigen recognition remains controversial. T cells discriminate between self- and non-self components of the human body by unique T-cell receptor (TCR) - peptide major histocompatibility complex (pMHC) interactions and a number of co-receptors and co-stimulatory receptors. Recent findings suggests that mechanical force contributes to the discriminating capabilities of this interaction, providing a possible explanation for differences in T-cell responses according to the physical properties of the antigen presenting cells (APC) presenting the pMHCs and the extracellular microenvironment1. The currently discussed hypothesis to understand the role of molecular force and hence the complete mechanism of pMHC-TCR interaction revolve around slip- and catch-bond dynamics. Studies to test these hypotheses remain inconclusive due to the inability to accurately measure molecular and cellular forces at the same time. Our findings demonstrate that the bond lifetime of the TCR-pMHC interaction does not scale with cellular generated mechanical force, hinting towards a force normalisation2. The co-stimulatory receptors CD2 and LFA-1 have been shown to enhance antigen discrimination by shielding TCR from forces that otherwise reduce the differences in synaptic off rates compared to solution off-rates5. Therefore, measuring the mechanical forces across length scales with consideration of these force-shielding co-stimulatory receptors is an absolute necessity to understand T-cell activation.

This project aims to quantify the mechanical forces on the molecular and cellular level in T-cell activation and further develop available methods to measure forces in the mechanosensitive T-cell response. With support of the Oxford-ZEISS Centre of Excellence at the Kennedy Institute and the custom-built super-resolution LLSM BioCOP project at the Rosalind-Franklin Institute further tool development could allow force measurement of tissue and cells inside tissue directly. To tackle these arising questions, we utilise our unique workflow including nouvelle astigmatic Traction Force Microscopy (aTFM)3 technique that allows to detect forces in the nN range in lateral and pN range in axial direction, capable to measure in the in the relevant force range. The custom-build optical setup additionally enables imaging with two-dimensional Total Internal Reflection Fluorescence Structured Illumination Microscopy (2D-TIRF-SIM)4 to correlate determined forces to super resolved cellular features, such as the TCR, co-receptor, and integrin location or the cellular cytoskeleton. Furthermore, super soft polyacrylamide hydrogels with stiffnesses below 1 kPa with various antigen densities will be required to simulate an APC with physiological relevant conditions. Our tools allow to quantify the influence of these integrins in molecular and cellular force generation and test for slip- and catch-bonds.

This study has the potential to resolve molecular and cellular forces with unprecedented sensitivity to illuminate the mechanosensation of T-cell activation exploiting advanced force probing and bioimaging technologies. These experiments promise to deepen our understanding of the TCR-pMHC recognition, allowing to accelerate engineering of T-cell activation platforms and drug design for auto immune disease and active health.

keywords

Live cell imaging, Traction Force Microscopy, T cells, Immunological Synapse, Mechanobiology.

training opportunities

  • Well-established DPhil programme (NDORMS) with defined milestones, ample training opportunities within the University and Department, and access to university/department-wide seminars by world leading scientists
  • The advanced imaging facility Oxford-ZEISS Center of Excellence (Oxford-Zeiss CoE) and Rosalind Franklin BioCOP project provide state of the art imaging techniques and training
  • Novel sensitive force probing technologies such as astigmatic Traction Force Microscopy (aTFM) and 2D Total-Internal-Reflection-Fluorescence Structured-Illumination-Microscopy (2D-TIRF-SIM)
  • Highly collaborative environment with expertise ranging from microscopy to molecular biology, as well as several other collaboration opportunities within the University of Oxford and worldwide

key publications

  1. Al-Aghbar, M. A.; Jainarayanan, A. K.; Dustin, M. L.; Roffler, S. R. The Interplay between Membrane Topology and Mechanical Forces in Regulating T Cell Receptor Activity. Commun Biol 2022, 5 (1), 1–16. https://doi.org/10.1038/s42003-021-02995-1.
  2. Colin-York, H.; Javanmardi, Y.; Skamrahl, M.; Kumari, S.; Chang, V. T.; Khuon, S.; Taylor, A.; Chew, T.-L.; Betzig, E.; Moeendarbary, E.; Cerundolo, V.; Eggeling, C.; Fritzsche, M. Cytoskeletal Control of Antigen-Dependent T Cell Activation. Cell Reports 2019, 26 (12), 3369-3379.e5. https://doi.org/10.1016/j.celrep.2019.02.074.
  3. Li, D.; Colin-York, H.; Barbieri, L.; Javanmardi, Y.; Guo, Y.; Korobchevskaya, K.; Moeendarbary, E.; Li, D.; Fritzsche, M. Astigmatic Traction Force Microscopy (ATFM). Nat Commun 2021, 12 (1), 2168. https://doi.org/10.1038/s41467-021-22376-w.
  4. Barbieri, L.; Colin-York, H.; Korobchevskaya, K.; Li, D.; Wolfson, D. L.; Karedla, N.; Schneider, F.; Ahluwalia, B. S.; Seternes, T.; Dalmo, R. A.; Dustin, M. L.; Li, D.; Fritzsche, M. Two-Dimensional TIRF-SIM–Traction Force Microscopy (2D TIRF-SIM-TFM). Nat Commun 2021, 12 (1), 2169. https://doi.org/10.1038/s41467-021-22377-9.
  5. Pettmann, J.; Awada, L.; Różycki, B.; Huhn, A.; Faour, S.; Kutuzov, M.; Limozin, L.; Weikl, T. R.; Merwe, P. A. van der; Robert, P.; Dushek, O. Mechanical Forces Impair Antigen Discrimination by Reducing Differences in T Cell Receptor Off-Rates. bioRxiv May 5, 2022, p 2022.05.05.490751. https://doi.org/10.1101/2022.05.05.490751.