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Immunological synapse derived ectosomes in T cell effector function

Periodic Reporting for period 4 - SYNECT (Immunological synapse derived ectosomes in T cell effector function)

Reporting period: 2020-05-01 to 2021-04-30

The SYNECT team investigates synaptic ectosomes. Synaptic ectosomes are vesicles that bud from the plasma membrane of the T cell toward antigen presenting cells. We discovered these small extracellular vesicles in the immunological synapse in 2014. The key attributes of these structures that distinguish them from other signals generated by T cells is that their composition is determined by the specific molecular interactions with the antigen presenting cells and they are thus customized for this communication. They are uniquely suited to generate combinatorial signals that remain with the antigen-presenting cell after the T cell disengages to sustain these signals. We have developed a platform of methods for analysis, characterization and functional analysis of human synaptic ectosomes. The information that we gain from these studies will help design better vaccines and may also generate new targets for treatment of autoimmune diseases.

We collaborated with Carola Vinuesa from Australia and Claudio Doglioni from Italy on the ability of human T follicular helper cells produce dopamine in response to antigen receptor engagement. Dr. David Saliba determined that the dopamine containing granules are concentrated at the immunological synapse and that the resulting up-regulation of ICOSL triggered in B cells dopamine enhances the release of CD40L into punctate structures in the central region of the immunological synapse, the synaptic ectocomes.

Dr. Viveka Mayya published in Cell Reports that motility behaviour is different between effector CD8 T cells, which form stable synapse, and other major T cells subsets (CD4 Naive, CD4 memory and CD8 naive), that form motile "kinapses" while detecting antigen. Dr. Mayya used trails of synaptic ectosomes to follow movement of the kinapse forming T-cells. He has followed this up with a paper in Journal of Immunology that demonstrates superior competition by memory CD8 T cells for sources of antigen compared to naive CD8 T cells. This has implications for vaccines where memory T cell with inferior antigen receptors may sometimes beat out naive T cells with optimal antigen receptors.

Dr. Ewoud Compeer has a paper that was supported by SYNECT that was just accepted at Nature Immunology documenting the effects of enforced Foxo1 nuclear localisation on immunological synapse formation. Constitutively active Foxo1 keeps T cells in a metabolically quiescent state even when they are forced to enter cell cell cycle and attempt differentiation. This leads to T cells in which the plasma membrane composition is deficient in cholesterol. These quiescent effector cells have a defect in generation of a cSMAC- the structure containing the synaptic ectosomes.

Dr. Saliba, Cespedes, Balint and Compeer published in Elife on the composition, structure and function of synaptic ectosomes released from helper T cells. These structures combine T cell antigen receptors and the effect molecule CD40L in ~100 nm vesicles that are delivered to the antigen presenting cell.

Dr. Balint and colleagues published in Science on the discovery of supramolecular attack particles that contain cytotoxic proteins in a core/shell structure. This is a new mechanisms for T cell mediated killing and the entities may be useful in cancer immunotherapy.

Dr Compeer will complete and submit a paper focus on in vivo role of synaptic ectosomes and age dependent changes in TSG101 levels in T cells that may contribute to poor vaccine responses in the elderly.

Work on supramolecular attack particles will be continued with the ERC Synergy Grant ATTACK-
The objectives of the project were to:
1) isolate synaptic ectosomes from human T cells and determine their molecular composition.
Our initial proposal for a method to capture and isolate synaptic ectosomes was not successful, but we were able to work out an alternative approach based on use of plain glass beads and fluorescence based particle sorting, which can be carried out at a high speed. With this method, we have performed unbiased proteomics analysis of vesicles from human T cells and a candidate based analysis of antibody staining of the vesicles to determine their internal and surface composition. We have also used super-resolution microscopy to determine the composition of vesicles and the distributions of four proteins- the T cell antigen receptor, tetherin, CD40L and ICOS in relation to the vesicles. This has been done using 3-color storm, which is a state of the art methods that is superior to the correlative light and electron microscopy approach we applied in our initial description of synaptic ectosomes.
2) determine the functional impact of synaptic ectosomes on the antigen presenting cell.
We have also started to evaluate the function of the vesicles isolated from single T cells by imaging the response of human dendritic cells. We have been able to verify potent dendritic cell activation that is dependent upon CD40L carried in the synaptic ectosomes in the presence of CD40 in the eliciting bilayer.
3) use gene targeting to control the process in vivo to understand its role in T function of helper, cytotoxic and regulatory T cells.
We have obtained and backcrossed TSG101 conditional knockout mice onto the B6 background and will be ready to start in vivo experiments soon.
4) using the same methods are applied to synaptic ectosomes, we discovered supramolecular attack particles, which are glycoprotein based nanoparticles with a core-shell structure that can kill target cells independent of the T cell. We will explore applications of these particles for immunotherapy in the future.
The demonstration that CD40L is in synaptic ectosomes has immediate implications for vaccine design. There was already a notion in the field that CD40L, which exists in nature at a trimer, was insufficient for some functions and various artificial ways to increases its valence results in higher activity. Our results suggest that CD40L resides on vesicles with TCR that are transferred to B cells in the germinal center. Thus, the natural form of CD40L may have an even higher valence than various synthetic constructs with up to 12 CD40L monomers per unit.
The method that we are currently using allows us to capture vesicles that are otherwise normally directly consumed by antigen presenting cells. Thus, its an intercepted message that allows us to spy on the communication between T cells and B cells. This mode of communication was not previously appreciated and our ability to eavesdrop on this process will give us a new tool in developing new biomimetic therapies.
This work led to the discovery of supramolecular attack particles- a new mechanism for T cell mediated killing. This may also represent a new mode of communication and building block for organisation of extracellular matrix.
Immunological synapses with CD40L ± ICOSL and dSTORM example