Novel mechanisms of regulatory T cell mediated suppression: a fundamental role for VPS34

The immune system provides us with life-long protection against infectious agents. Specialised cells called T cells can both orchestrate the activities of other immune cells and kill infected cells directly. Remarkably, a small subset of T cells, called regulatory T cells (Tregs) are required to keep all the other T cells (conventional T cells, or Tcon) in check. Although Tregs comprise only about 1% of all the white blood cells, we cannot survive without them. Hence, children that lack a functional version of a gene called FOXP3 which is essential for Tregs do not live beyond two years, at which point their developing immune system turns against them, leading to a lethal syndrome known as IPEX. Mice that lack functional FOXP3 also die between 6 weeks because of an unrestrained immune attack against their organs and microorganisms in their skin and gut.

What remains a puzzle is how such a small immune cell population can have such enormous importance for maintaining health. How do Tregs control the activity of the much more abundant Tcon? We have discovered that an enzyme called VPS34, originally identified in yeast, is essential for the function of Tregs. This is an important distinction from Foxp3, which is required for the development of Tregs. Therefore, FOXP3-deficient mice lack completely Tregs, whereas if we deleted the gene encoding VPS34, and that only in Tregs, we find normal numbers of Tregs in immune-related organs, but the mice nonetheless do not survive beyond 4-6 weeks. Normally, mice can live for 2-3 years. We therefore hypothesise that by gaining a better understanding of what VPS34 does in Tregs, we can also learn more about how Tregs work and answer the question about which function is absolutely essential for their ability to restrain Tcon.

We will use several different experimental approaches to address this question. Most of the work will take advantage of a genetically modified mouse model in which the gene for VPS34 is deleted only in Tregs. Once mechanism through which Tregs are thought to work is by consuming certain stimulatory protein molecules so that they become unavailable for Tcon. We have already established that VPS34-deficient Tregs are still able to bind these proteins and internalise them. We will now follow what happens to these proteins once they have been transported inside the Tregs. Normally, they would be digested and degraded, but we suspect that this process is disrupted in Tregs lacking VPS34.

We will also use mice in which only about half the Tregs lack VPS34. Such mice live a normal life span. We will challenge such mice by either implanting tumour cells under the skin or by infecting them with bacteria. This will stimulate both Tcon and Tregs to multiply and change from a resting state to a highly activated state. We can then compare Treg with or without VPS34 in the same mouse and ask: does whether lack of VPS34 interfere with the ability of Tregs to be activated and perhaps reach their full immune suppressive potential.

Each T cell has about 6000-8000 different kinds of proteins. We have been able to measure these proteins in normal and VPS34-deficient Tregs. We can therefore determine whether without VPS34, you end up with more of some proteins, and less of others. Many of the proteins that are altered in absence of VPS34 are involved in cellular metabolism (i.e. processes that regulate how Tregs take up and use nutrients). We will determine if some of these metabolic activities affect Treg function and suppression. We believe that these complementary approaches will lead us to discover novel aspects of Treg function that might one day can be exploited for therapeutic purposes, for instance by designing drugs that either enhance or inhibit Treg function, and that could be used in the context of cancer or autoimmune diseases.

Regulatory T cells (Tregs) play an essential role in maintaining immune cell homeostasis and preventing autoimmunity. A mutant mouse strain called Scurfy, which lack the Treg-defining transcription factor Foxp3, die from a lymphoproliferative and autoimmune disease about 4 weeks after birth. How Tregs suppress the activity of CD4+ and CD8+ conventional T cells (Tcon) is not fully understood. We have used conditional gene-targeting to delete the phosphatidylinositol 3-kinase (PI3K) VPS34 in Tregs. VPS34 is an evolutionary conserved kinase, originally identified in yeast, which is known to control two distinct biological processes: autophagy and endocytosis.

We deleted VPS34 specifically in Tregs using Foxp3-Cre and a floxed allele of VPS34. Mice lacking VPS34 in Tregs died between 4-6 weeks of age from a Scurfy-like disease. In contrast to Scurfy mice, however, the deletion of VPS34 did not interfere with the development of Tregs, which were found in normal numbers and looked phenotypically similar to normal Tregs with the exception that there was a lower proportion of VPS34-deficient Tregs expressing the activation marker CD44 suggesting a defect in Treg maturation.

We found that VPS34-deficient Tregs functioned normally during in vitro suppression assays. Furthermore, the consumption of CD80 (by CTLA-4) and IL-2 (by the IL-2 receptor) were not affected by VPS34-deficiency. However, since VPS34 has been associated with endo-lysosomal processes, we will determine whether the processing of endocytosed material is disrupted in VPS34-deficient Tregs.

We will also use experimental models of tumours and infection with Listeria monocytogenes to determine whether VPS34-deficient Tregs can mature and acquire suppressive functions in vivo. Finally, we will seek to discover novel Treg-mediated mechanisms by analysing the phospho-proteome and transcriptome of VPS34-deficient Tregs and testing candidate genes by CRISPR-Cas9.

Klaus Okkenhaug has an excellent track record the broad area of cell signalling, immunity and infection. Is evidenced by an extensive publication record, presentations at conferences, successful collaborations with industry, consultancies, public engagement activities, and where appropriate, coverage in the news. Through these activities, we have established a network which will help facilitate and deliver impact from our future research described in this proposal.

Knowledge exchange
We will disseminate our research findings through presentations at national and international meetings and discussing our work with academic, commercial and clinical colleagues. Klaus Okkenhaug is on the Steering Committee for the Cambridge Immunology Network which facilitates interaction and discussion among immunologist in the wider Cambridge are, including surrounding institutes. Klaus Okkenhaug will present at the Keystone meeting for PI3K in 2020: "PI3K and PTEN at the Interface of Cell Growth, Vesicular Trafficking and Disease". This will be an excellent forum to present our latest research on how VPS34 regulates vesicle trafficking and the impact this has on immune regulation.

Publication of our research
We will publish our data in reputable, open access journal with the aim to reach the widest possible audience. During the submission process, we will deposit our manuscripts to BioRxiv to achieve more immediate impact of our work. We will work with the University of Cambridge Press Office to generate press releases to reach a wider audience, including major stake holders from biopharmaceutical industries.

Membership of learned societies
Klaus Okkenhaug is a member of the British Society for Immunology and the Biochemistry Society. Both organisations play important roles by organising meetings, publishing journals and promoting bioscience to different stakeholders outside the immediate academic circles. We will use the opportunities offered by these societies to disseminate our research at national meetings and other fora.

Industrial collaborations
Klaus Okkenhaug has an active collaboration with GSK surrounding activated PI3Kdelta syndrome (APDS). He is also on the Scientific Advisory Board of Karus Therapeutics and had consulted for about a dozen different companies. Other avenues to reach industrial collaborative partners include through the Milner Therapeutics Institute at the University of Cambridge which enables early proof-of-concept studies in collaboration with major stakeholder from pharma. We will continue to work closely with Cambridge Enterprise who help draft confidentiality agreements, material transfer agreements and consultancy agreements. Moreover, Cambridge Enterprise can provide invaluable advice and mentorship once a commercial opportunity is recognised. Although the research described in this proposal is of a fundamental value without immediately obvious commercial development potential, the network is in place to act rapidly should opportunities present themselves.

Several pharmaceutical companies, including Novartis and Sanofi, are generating inhibitors against VPS34. Because VPS34 is ubiquitously expressed, we do not expect that such an inhibitor given systemically can affect Tregs preferentially. However, it is possible that modulating VPS34 activity in Tregs ex-vivo before the transfer into patients may be considered in the future. We may also uncover Treg-restricted targets that are affected by VPS34 that may be of interest for drug development.

Public engagement
Last year we had a record 800 members of the public visit the Department during our open day as part of the Cambridge Science Festival. We will develop an exhibit introducing regulatory T cells are and their role in health and disease. We believe such activities help us connect with the public who fund our science and garner support for scientific research more broadly.