CD96 a new immunoregulatory molecule - molecular mechanisms

Background
Special white blood cells, called regulatory T cells (Tregs) normally keep the immune system in check: too much control lets cancer arise, while too little regulation leads to autoimmunity. Special molecules on the surface of cells, called co-receptors, can mediate and alter Treg function. Co-receptors that share the same ligand can trigger either an increase or decrease in the immune response. These co-receptors appear to be good drug targets to stop cancer and/or autoimmunity with some already in use clinically (CTLA-4, PD1), and others being developed.
One such co-receptor that does regulate the immune system is CD96, and we want to find out how it works. CD96 shares its ligand CD155 with the co-receptors CD226, which turns the immune response up, and TIGIT, which lowers the immune response. Tregs do express all three co-receptors, but we do not yet know how they dictate Treg function and how exactly CD96 regulates the whole pathway.

Hypothesis and Aims
We believe CD96 regulates the immune responses by controlling the ligand CD155 for the other co-receptors in the same family and we think this affects how Tregs work.
We will test this overall hypothesis with three main questions in our aims:

Aim 1: How does CD96 actively take CD155 into the cell?
Aim 2: What other molecules inside the cell work with CD96 for its action?
Aim 3: How does CD96 change Treg functions?

Experimental Plan
We will use continuously growing cell line models as well as cells from the blood of healthy people that we can modify to only express specific co-receptors.
We have some data that suggests that CD96 works by taking CD155 from other cells. We will generate cell lines that express CD96 and CD155 with fluorescent tags that glow when specific light is shone on them. That way we can see and follow how CD96 and CD155 interact and move around inside the cells by using a powerful confocal microscope, flow cytometry to evaluate many parameters on each cell at once and biochemical assays where we can pull out CD96 and proteins bound to it and then measure them. By changing parts of CD96 before functional tests, we can find out which part of CD96 is important for its functions. We will also look at how CD96 competes for CD155 binding and/or interacts with the other co-receptor family members CD226/TIGIT.
We will look for the proteins that bind to CD96 inside the cell after it has bound ligand to enable CD96 actions. We have some candidates that likely bind to CD96 inside the cell, but we will also screen for new proteins interacting with CD96 inside the cell after it has seen its ligand CD155.
We will use cutting edge techniques like CRISPR-Cas9 to take away CD96 (CD96 knockout) from healthy human Tregs to find out for what cell functions CD96 is necessary. We will test how this knockout changes the activation, proliferation and signalling of Tregs. Tregs can supress other T cells from dividing, thus we will test also whether CD96 influences this crucial Treg function. Since effector T cells can also express CD96, we will test them in parallel to look for CD96 actions that are Treg-specific and ones that are universal for cells expressing CD96.

Importance
The immune system is tightly regulated in health, but too much control leads to cancer and too little to autoimmunity. By understanding how the co-receptor CD96 regulates the immune system and how it affects Treg functions, we will be able to harness that knowledge in the future to design new drugs targeting the co-receptor to treat cancer and/or autoimmunity.

The immune system is tightly regulated to maintain immune homeostasis. Activating and inhibitory co-receptors with shared ligands are important in regulating lymphocyte activation and shaping regulatory T cell (Treg) functions to effect immune regulation. Here we focus on CD96, a novel immunoregulatory receptor, part of the co-receptor family with CD226 and TIGIT and the shared ligand CD155. Currently, it is unclear whether CD96 is inhibitory or activating, we suggest CD96 might function by taking up ligand and depleting it for CD226/TIGIT interaction.
We hypothesise that CD96 on Tregs acts as an important immune regulator of the CD155 ligand to maintain immune homeostasis and will focus on 3 independent, distinct aims:
Aim 1: How does CD96 regulate CD155 uptake?
Aim 2: Defining CD96 cytoplasmic domain interactions upon ligation.
Aim 3: Defining the physiological role of CD96 in Treg activation phenotype, proliferation and function.
We will use cell line models (Jurkat, DG75, CHO) engineered to express fluorescently tagged CD96, CD96 mutants or CD155, and primary human Tregs knocked out for CD96, CD226/TIGIT by CRISPR-Cas9. Co-culture assays, flow cytometry, confocal microscopy and biochemical approaches will be utilised throughout. We will test whether CD96 uses endocytosis to take up CD155 in an organised manner via AP-2 (Aim1). We will evaluate the interactome of CD96's cytoplasmic tail after CD155 interaction by western blotting and mass spectrometry (Aim2). The effect of CD96-CD155 interaction on Treg signalling, activation, proliferation and functionality (i.e. suppression) will be examined in comparison to effector T cells. The importance of competition and interaction with CD226/TIGIT will be tested (Aim3).
This work will increase our understanding of CD96's immunoregulatory potential, its role in Treg function, and the cellular and molecular mechanisms involved. This might ultimately lead to specific targeting to fight cancer or dampen autoimmunity.