T lymphocyte recognition

L. Limozin, PH. Puech, P. Robert

Collaboration: Y. Hamon (CIML), A. Van der Merwe (Oxford)

The detection of foreign material by T lymphocytes is both a key step of immune responses and a model of cell decision triggering. We addressed this model by several parallel approaches based on quantitative advanced biophysical methods. We measured the dynamics of T cell membrane interaction with planar surface with nearly nanometric and subsecond resolution, using reflection interference contrast microscopy (RICM) and evanescent wave illumination (TIRF). We found that T cells could detect the presence of foreign material within less than 10 seconds with highly motile microvilli and pulling motion (J. Immunol. 2013), starting ultimately active spreading, the earliest reporter of bona fide cell activation (J. Immunol. Methods 2011). T cell could quantitatively discriminate between specific antigens of varying potency within 45 seconds after initial contact (Eur. J. Immunol 2015). Quantitative analysis of TIRF images revealed that T cell plasma membranes displayed transverse undulations of several tens of nanometer amplitude at one hertz frequency (Cell Mol Bioengineering, 2015), an efficient way of mechanosensing the nearby surface through the TCR. The importance and significance of addressed problems was discussed in two review papers (Frontiers Immunol. 2012; Annual Rev. Immunol. 2015).

Pierre-Henri Puech et al. Force measurements of TCR / pMHC recognition at T cell surface. PloS ONE 2011;6(7):e22344 DOI: 10.1371/journal.pone.0022344

Coupling AFM force mode and fluorescence microscopy for mechano-transduction studies

PH. Puech

Collaboration: Y. Hamon, CIML.

We implemented single T cell simultaneous AFM force-based stimulation and activation recording, monitoring Ca2+ in real time. For that, we characterized and used an original internal timer signal, originating from a subtle physical effect of fluorescence excitation light onto the AFM lever. Aside, to control the shape and size of the contact, we developed micro-manipulations techniques to modify AFM levers with beads, and the chemistry to decorate them with biomolecules. Stimulation can then be performed with a continuous contact or periodic short ones to dissect the response to a given spatial or temporal distribution of the stimulation. To this goal, we developed optogenetics in T cells, using photoactivable small GTP-ases family members (eg. RacPA) in collaboration with M. Pelicot-Biarnes, and recorded the evolution of the mechanical properties upon photoactivation.

Synchronizing atomic force microscopy force mode and fluorescence microscopy in real time for immune cell stimulation and activation studies. Cazaux S, et al. Ultramicroscopy. 2016 Jan;160:168-81. DOI: 10.1016/j.ultramic.2015.10.014   Epub 2015 Oct 19.

T cells adapt mechanically to external cues, very rapidely, even before any strong internal signalling

PH. Puech

Collaboration: Y. Hamon, CIML., J. Husson LaDhyX

Together wirh the group of J. Husson, using the innovative micropipette rheometer that we validated with AFM, have shown that, when stimulated by microbeads mimicking target cells, leukocytes become up to 10 times stiffer and more viscous. These mechanical changes start within seconds after contact and evolve rapidly over minutes. Using our AFM/SCFS system, we demonstrated that the same type of behaviour was indeed happening at the interface between a T cell and an APC, thereby generalizing the observations to a more physiological context. These results indicate that simultaneously tracking both elastic and viscous properties during an active cell process provides a new way to investigate cell bio-mechanical processes and suggests that dynamic immunomechanical measurements can help discriminate between leukocyte subtypes during activation.

Single-cell immuno-mechanics: rapid viscoelastic changes are a hall-mark of early leukocyte activation Alexandra Zak et al., Biophys J. 2021 May 4;120(9):1692-1704. DOI: 10.1016/j.bpj.2021.02.042

Is there a mechanical link between the TCR/CD3 and the cytoskeleton ?

L. Limozin, PH. Puech

Collaboration: K. Sengupta (CINAM)

In order to validate some hypothesis made in modelling spreading of T cell on model soft susbtrates as reported in Wahl et al PNAS 2019, we developed techniques for pulling membrane tethers from living T cells using optical tweezers. We varied the « handle molecule » by using specific antibodies to quantitatively evaluate the anchoring of immune synapse membrane proteins to the cytoskeleton. in particular the TCR/CD3 complex, LFA1 in different conformations and CD45. We developed a refined mechanical model of full tether pulling sequence, with a theoretican physicist post doc, and quantitated our rich and important data set using model fitting. Coupling tube pulling with perturbation of actin using low doses of Latrunculin A, our data demonstrated that indeed IS proteins exhibit different degrees of interaction with actin, which we quantitated. This important result was very well received by the community, since it represents a link between molecular and cellular scales in t cell early activation. Aside,  we developped a user-friendly data, python based, processing software for force based optical tweezer data that we published & released as open-source on GitHub / pip / ReadTheDoc platforms

Wahl A, Dinet C, Dillard P, Nassereddine A, Puech PH, Limozin L, Sengupta K. Biphasic mechanosensitivity of T cell receptor-mediated spreading of lymphocytes. Proc Natl Acad Sci U S A. 2019 Mar 8. pii: 201811516. doi: 10.1073/pnas.1811516116

Fabio Manca, Gautier Eich, Omar N’Dao, Lucie Normand, Kheya Sengupta, Laurent Limozin, Pierre-Henri Puech. Probing mechanical interaction of immune receptors and cytoskeleton by membrane nanotube extraction. Scientific Reports 13, Article number: 15652 (2023). https://www.nature.com/articles/s41598-023-42599-9

Thierry Galliano, Guillaume Gay, Laurent Limozin, Pierre-Henri Puech*. OT_Analysis: a software for rich analysis of force curves when probing living cells with optical tweezers. Journal of Open Source Software, 8(90), 4877, https://doi.org/10.21105/joss.04877

Early cell mechanotransduction display spatial and time patterns

PH. Puech

Collaboration: K. Sengupta

Forces can be felt or applied : to quantify the stresses, forces and energies that T cells exert during the early stages of recognition and potential activation, using very soft PAA gels and traction force microscopy (TFM). During the thesis of F. Mustapha, we documented and published the detailled protocols for producing very soft PAA gels, well characterized mechanically, and reproducible , together with the open source  solutions and homemade scripts to quantify the obtained data. We quantified T cells spreading and mechanics (using AFM) of the cells when sitting on such soft, activating gels ; the observed behavior  appeared to be coherent with our previous reports. We observed, for the time to our knowledge, different stress and energy vs. time patterns among cells in a population, whose frequency and characteristics (maximum force, integrated energy) varied depending on the exact decoration of the substrate (activation signal alone, or in combination with coactivation signal or adhesion). Interestingly, we observed that for short contact times, during the spreading of the cells, forces were pointing outward from the cellular contact, while reversing for longer contact times typical of IS formation. We then obtained unique results on primary human T cells facing activating ultra-soft PAA gels. We observed the same main time-dependant patterns of stress and energy, and it clearly appeared that their frequence is depending on the cell subtype (naive, memory, activated – obtained using commercial kits from EFS blood bags), culminating in high forces & energy values for unactivated memory T cells. We concluded that the increased force exertion exhibited by memory T cells upon re-stimulation (in our case by aCD3 on soft-as-APC gels), in comparison to naive T cells, might contribute to their ability to undergo rapid and strong activation.

Cellular forces during early spreading of T lymphocytes on ultra-soft substrates. Farah Mustapha, Martine Biarnes-Pelicot, Remy Torro, Kheya Sengupta, Pierre-Henri Puech. bioRxiv. doi: link

Protocol for measuring weak cellular traction forces using well-controlled ultra-soft polyacrylamide gels. Mustapha F, Sengupta K, Puech PH. STAR Protoc. 2022 Jan 28;3(1):101133. doi: 10.1016/j.xpro.2022.101133

Role of cholesterol in T cell mechano-activation

PH. Puech

Collaboration: Y. Hamon, CIML.

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 and others described that TCR acts as a mechanosensor molecule 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. 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 (see Zak et al., Biophys. J. 2021).

PhD funded by Centuri Convergence Project.

Biosensors in T cell activation/migration

M. Biarnes-Pelicot

We determined that ZAP-70 is activated upon LFA-1 engagement during T cell migration. Furthermore, we demonstrated that the kinase is involved in regulating the recruitment of talin to high affinity LFA-1 and that this process requires binding of the ZAP-70 SH2 domains to a yet to be identified partner (Biophys J 2014, Biotech. J 2014, BBRC 2015). Aside of those reporters, we developped photoactivable proteins, such as PA-Rac, in order to use the power of optogenetics to perturb cell cytoskeleton and function.

Synchronizing atomic force microscopy force mode and fluorescence microscopy in real time for immune cell stimulation and activation studies. Cazaux S, et al. Ultramicroscopy. 2016 Jan;160:168-81. doi: 10.1016/j.ultramic.2015.10.014. Epub 2015 Oct 19.