EXploring T cell Activation Signal Integration in 4D – EXTASI 4D

1- Description of the dynamic orchestration of TCR signaling during the whole T cell activation process through transient and sequential interactions with APCs:
To this aim, in collaboration with a microfluidician, we generated microwells that will allow the confinement of T cell activation process under the microscope field, in a 3D environment provided by collagen lattice. Using a panel of fluorescent probes, we are currently analyzing TCR signaling in primary CD4 T cells activated in this ex vivo system.
2- Characterization of the influence of the stimulation parameters of T cell on TCR signaling:
To address this point, an optigenetically modified stimulation system for T cells will be developed. This will enables to determine i) the parameters of stimulation required to trigger a sustained signaling imprint (strength, duration), ii) the half-life of this imprint after interruption of the stimulation, and iii) how pulsed/short-lived activation signals are translated into signaling molecule activity. These experiments will also unveil molecular actors involved in the signal integration process.
3- Characterization of the impact of the dynamics of T cell/APC interactions on T cell activation outcome:
This study will be performed by coupling the 4D ex vivo stimulation system with T cell phenotyping procedures, as well as by controlling T cell stimulation temporal pattern using a light-controlled TCR stimulation system based on optogenetics. Together, these approaches will permit to demonstrate the putative causal link between T cell interaction dynamics and activation outcomes.

We have been able to follow the first time naïve T cell activation through transient and sequential interactions with APC at the individual living cell level, recapitulating the 3 phase of activation described in vivo.
We have also shown that during this transient interactions, different signaling pathways down-stream TCR exhibit specific kinetics of activity, rising new interesting information on signal integration process and temporal coordination of the TCR signaling pathways.

At short term, we will pursue the analysis of TCR signaling regulation during T cell activation through sequential short-lived interaction with APCs.
We started the development of light controllable stimulation system for T cells. We will provide strong effort to finalize rapidly this tool to determine how the stimulation parameters influence TCR signaling and T cells activation outcome.
Considering the EXTASI4D project evolution, we are still following the initial research plan developed in the original proposal.

Poster presentation at the «Stochastic Single-Cell Dynamics in Immunology - Experimental and Theoretical Approaches« colloquium in Amsterdam

T cells are central in adaptive immune response. They permanently patrol throughout the body in search of their cognate antigen at the surface of antigen presenting cells (APCs). Their activation process is initiated following engagement of their antigenic receptor, called T Cell Receptor (TCR) by its ligand, an antigenic peptide presented on Major Histocompatibility Complex (MHC) molecules on APCs.
Until recently, T cell activation was considered to require the formation of stable and highly structured cell junction called immune synapse. Pioneering studies however revealed that T cell activation in vivo may be initiated by series of transient interactions with APCs. To date, little is known about the mechanisms which allow T cells to integrate (or sum up) sequential and transient signals. Another important finding of these in vivo studies is that the dynamics of T cell interactions with APCs (duration, frequency) seems to impact on the T cell activation outcome (Immunity vs tolerance and possibly Th1 vs Th2 response).

Main objectives and work hypotheses of EXTASI 4D:
Through this project, I aim:
-at characterizing the molecular basis of the sequential signal-integration process during naïve CD4 T cell activation. I hypothesize that the spatiotemporal regulation of signaling molecules activities allows T cells to maintain an imprint of the successive stimulations they perceive.
-at determining the influence of T cell/APCs interaction dynamics on TCR signaling and T cell activation outcome. My hypothesis is that the frequency and the intensity of T cell stimulations received through interactions with APCs, provide an additional layer of information allowing the T cell to better evaluate the danger and to develop the appropriate response.

Specific aims and methodology:
1- Description of the dynamic orchestration of TCR signaling during the whole T cell activation process through transient and sequential interactions with APCs: To this aim, in collaboration with a microfluidician, we are generating microwells that will allow the confinement of T cell activation under the microscope field, in a 3D environment provided by collagen lattice. Using a panel of fluorescent probes, we will analyze TCR signaling in primary CD4 T cells activated in this ex vivo system as well as in vivo.
2- Characterization of the influence of the dynamic parameters of T cell/APC interactions on TCR signaling: To address this point, holographic optical tweezers developed in our team will be used to control the duration and the frequency of T cell/APC interactions. This enables to determine i) the condition of interaction required to trigger a sustained signaling imprint, ii) the half-life of this imprint after the cell separation, and iii) how pulsed/short-lived activation signals are translated into signaling molecule activity. These experiments will also unveil molecular actors involved in the signal integration process.
3- Characterization of the impact of the dynamics of T cell/APC interactions on T cell activation outcome: This study will be performed by coupling the 4D ex vivo stimulation system with T cell phenotyping procedures, as well as by controlling T cell stimulation temporal pattern using a light-controlled TCR stimulation system based on optogenetics. Together, these approaches will permit to demonstrate the putative causal link between T cell interaction dynamics and activation outcomes.

This challenging and interdisciplinary program will for the first time provide a description of the whole history of individual T cell interactions with APCs and the associated signaling events during the activation process. It will unravel new molecular mechanisms underlying activation signal integration in T cells, as well as their influence on activation outcomes. This concept of signal dynamics and integration is conceivably not restricted to T cells. Thus the data obtained from EXTASI 4D are likely to impact other fields of biomedical research.