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Immunological synapse derived ectosomes in T cell effector function

Periodic Reporting for period 3 - SYNECT (Immunological synapse derived ectosomes in T cell effector function)

Reporting period: 2018-11-01 to 2020-04-30

"The SYNECT team investigates the role of synaptic ectosomes in T cell biology. Synaptic ectosomes are defined as vesicle that bud from the plasma membrane of the T cell toward antigen presenting cells or model substrates with appropriate antigenic structures, for example, in the context of T lymphocytes recognizing virally infected cells. We discovered production of these small extracellular vesicles in the immunological synapse and published this in 2014. We hypothesize that these small ~ 80 nm vesicles that bud into the immunological synapse in response to antigen recognition play an important role in modulation of the immune response. 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 and potentially extend these signals. We further anticipate that this mode of communication has unique advantages for therapeutic targeting that have heretofore been overlooked. We have succeeded in developing 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 where this mode of communication contribute to errors.

The first significant research accomplishment funded by SYNECT is a collaboration between groups in German, Italy and Australia. Specifically, Carola Vinuesa from Australia and Claudio Doglioni from Italy discovered that human T follicular helper cells produce dopamine in response to antigen receptor engagement. The Vinuesa lab established the dopamine acts to rapidly up-regulated ICOSL on germinal center B cells. We determined that the dopamine containing granules are concentrated at the immunological synapse and that the resulting up-regulation of ICOSL enhances the release of CD40L into punctate structures in the central region of the immunological synapse, which we have previously shown is populated by synaptic ectocomes. Modeling work from Michael Meyer-Herman in Germany makes a specific prediction that the ability of dopamine to rapidly upregulate ICOSL and in term trigger CD40L delivery will accelerate the production of plasma cells by the germinal center reaction and thus allows more rapid antibody responses in humans. This was key data for the overall story and thus the ERC supported post-doc in my lab, David Saliba is co-second author on the study, which was published in 2017. This work is currently being taken forward by Pablo Cespedes, Stefan Balint and Ewaldus Compeer.

SYNECT has also supported Viveka Mayya, PhD, who published a well received review on factors that link input signals like antigen amount and duration into T cell proliferation to determine the scale of immune responses. The scale of immune responses is very important as it needs to be large enough to eliminate the pathogen, but should not be much larger than this as the response also causes tissue damage that can itself become fatal. The paper concludes that the duration of antigen recognition sets in motion the accumulation of mediators and cytokine production that drive transcriptional networks leading to a graded output that can be set to an appropriate level in most situations. This paper was published in Trends in Immunology.

Dr. Mayya also led work published in Cell Reports that established a new in vitro system that recapitulates key characteristics of in vivo T cell dynamics learned from mouse models with human T cells. While the approach has caveats it make provides a model for analysis of dynamics of human T cells that could not otherwise be studied at all. It provides insights into cell autonomous aspects of T cell behaviour that also helps explain puzzling in vivo observations. The paper are also identified a pronounced different in motility behaviour 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. Cells that form kinapses and continue to move can remain with antigen presenting cells because while they continue to move around, the movement can be highly confined to exploration of the antigen presenting cell surface.

Dr. Compeer has a paper that was supported by SYECT 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. Addition of cholesterol corrects the defect, confirming other recent work the synaptic ectosome formation requires cholesterol and may be modulated by targeted reduction of plasma membrane cholesterol in T cells."
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.
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.