Periodic Reporting for period 4 - SELFOR (How our adaptive immune system separates “self” from “foreign” – a physicochemical study of binding in cell contacts)
Período documentado: 2022-07-01 hasta 2023-12-31
The main goal of this project was to illuminate the problem of antigen discrimination by developing a physicochemical understanding of the interaction between T-cell receptors (TCRs) and peptide-loaded major histocompatibility complexes (pMHCs), and how this differs for pMHC with self and foreign peptides. The interaction between TCRs and pMHCs on contacting immune cells is the first, and arguably most important, step in initiating an adaptive immune response. It is also the interaction between TCR and pMHC that is the initial filter for our adaptive immune system to distinguish between our own cells and foreign pathogens, so called antigen discrimination. However, how this interaction differs between self and foreign pMHC, and how the binding kinetics is affected by TCR and pMHC being anchored to membranes, has been missing. I have in SELFOR been able to quantify this in physicochemical terms, with self being 2-3 orders of magnitude weaker than cognate antigen binding, but still detectable. This is important because it gives a first estimate of the binding window in which our immune system function, a binding window that is frequently missed when using directed antibodies for cell stimulation. I also discovered that small differences in binding affinity can still give rise to large differences in cell activation, stressing that it is not only the binding per see that gives rise to T-cell signalling but also the binding environment. In doing this I have had to develop a new means of measuring binding affinity in cell contacts as well as better understanding how auxiliary binding molecules can modulate the binding affinity of other ligand-receptor pairs by at least an order of magnitude, thus being able to significantly reduce the stimulating potency of an antigen. This again stresses the important of the binding environment. Finally, I have managed to study the CD4-pMHC-TCR interaction, showing that neither does CD4 influence TCR-pMHC binding nor does TCR influences CD4-pMHC binding. This is important in understanding the role of the co-receptor CD4, indicating that its role is to bring in activating kinases to already formed TCR-pMHC complexes instead of stabilizing them.
The main scientific objective of the first half of SELFOR has been twofold: (i) to work on a method to measure binding kinetics (WP1) and (ii) to study TCR/pMHC binding and the role of auxiliary molecules (WP2). Below is a summary of the key research and technological achievements for these two objectives:
A method to measure binding kinetics (WP1)
* We have developed a single-cell method to measure the lifetime and affinity of ligand-receptor interaction in the contact between a supported lipid bilayer (SLB) and a living cell (Junghans et al. JCS, 2020; Chouliara et al. unpublished).
TCR/pMHC binding and the role of auxiliary molecules (WP2)
* We have investigated how auxiliary binding molecules such as CD2 can modulate binding affinity of other ligands/receptors such as TCR to pMHC (Junghans et al, JCS 2020). We found that CD2 has a concentration range where its interaction with its corresponding ligand on the meeting cell is optimal to promote TCR/pMHC interaction.
* We have also investigated how the composition of the SLB influences the behaviour and signalling of binding cells, and how this can be avoided when measuring binding kinetics in SLB-cell contacts (Tommy Dam unpublished).
* Measurements have also been done between the co-receptor CD4 and pMHC using the methodology developed in WP1 (Junghans et al. JCS 2020; Chouliara unpublished) where it was confirmed that the affinity of the CD4-pMHC interaction is several orders of magnitude weaker than that of agonistic TCR binding pMHC, but detectable.
* Finally, a system to investigate the two-dimensional binding kinetics between TCR and self pMHC has been setup (Junghans et al. unpublished).
A method to measure binding kinetics (WP1)
* We have developed a single-cell method to measure the lifetime and affinity of ligand-receptor interaction in the contact between a supported lipid bilayer (SLB) and a living cell (Junghans et al. JCS, 2020; Chouliara et al. unpublished).
TCR/pMHC binding and the role of auxiliary molecules (WP2)
* We have investigated how auxiliary binding molecules such as CD2 can modulate binding affinity of other ligands/receptors such as TCR to pMHC (Junghans et al, JCS 2020). We found that CD2 has a concentration range where its interaction with its corresponding ligand on the meeting cell is optimal to promote TCR/pMHC interaction.
* We have also investigated how the composition of the SLB influences the behaviour and signalling of binding cells, and how this can be avoided when measuring binding kinetics in SLB-cell contacts (Tommy Dam unpublished).
* Measurements have also been done between the co-receptor CD4 and pMHC using the methodology developed in WP1 (Junghans et al. JCS 2020; Chouliara unpublished) where it was confirmed that the affinity of the CD4-pMHC interaction is several orders of magnitude weaker than that of agonistic TCR binding pMHC, but detectable.
* Finally, a system to investigate the two-dimensional binding kinetics between TCR and self pMHC has been setup (Junghans et al. unpublished).
WP1: To develop a general method to measure binding kinetics.
WP2: To characterize the interaction between TCR and pMHC.
WP3: To clarify how our adaptive immune system separates "self" from "foreign".
WP2: To characterize the interaction between TCR and pMHC.
WP3: To clarify how our adaptive immune system separates "self" from "foreign".