Molecular mechanisms enabling cDC2s to control Th2 cell priming

The intestine is a continuous tube that starts from the mouth and ends at the anus. It is home to the largest number of immune cells in the body. The reason for this is that the intestine is constantly exposed both to billions of harmless and beneficial microorganisms, and also to microbes that can cause infections. The immune cells in the intestine therefore face a formidable challenge. They have to recognise, attack and destroy potentially harmful infectious agents without damaging the body, but they must not mount similar attacks against food, or harmless microorganisms. These harmless materials are not just ignored, active processes are triggered to make sure that immune cells cannot respond to them and cause unnecessary damage to the body. It is important to understand how these immune responses in the intestine are controlled, so that we can find better ways to both activate these responses (e.g. for vaccination, or to control infection), and to reduce their strength when they cause damage to the intestine (e.g. during inflammatory bowel disease).

The two cell types that are most important for controlling immune responses in the intestine are "dendritic cells" and "T cells". These cells interact with each other, and the outcome of this interaction defines the type of immune responses that are generated. Dendritic cells are found in the wall of the intestine, where they are able to acquire samples of proteins from their local environment, from food and microbes. After acquiring these proteins, the dendritic cells leave the intestine and travel to lymph nodes, where the T cells are found. Depending on the source of the protein the dendritic cell has acquired, the dendritic cell will cause the T cell to respond in different ways. For instance, if the dendritic cell has taken up protein from food, the T cell will be de-activated, so that an immune response is not accidentally made against a harmless food protein. On the other hand, the dendritic cell has acquired proteins from a harmful microbe, the dendritic cell will be activated by signals it will have also received from the microbe. This activated dendritic cell will then cause the responding T cell to become activated. The activated T cell will then be able to make a response against the microbe, and kill it. Different types of T cell responses are required to kill different types of microbes, so T cells activated to help kill bacteria will produce different molecules to T cells activated to help kill viruses or large parasitic organisms.

This response is difficult to study because very small numbers of dendritic cell : T cell interactions have very large effects on the type of response that then occurs. It has been very challenging to discover the details of how these small numbers of dendritic cells work. By studying immune responses to parasites, we have recently identified two molecules that appear to have important effects on dendritic cell functions.

Our project has two objectives. The first is to investigate how these two molecules work, and therefore understand the details of how dendritic cells control this type of immune response. Our second objective is to use methods we have developed to identify new molecules that are important for controlling immune responses in the intestine. To achieve this second objective we will take advantage of new technology that is able to identify many of the molecules produced by very small numbers of cells within larger populations.

Understanding the functions of dendritic cells in the way we propose should help us to manipulate the immune response in a wide range of important situations, perhaps by improving the efficiency of vaccines, or preventing the inappropriate and damaging immune responses that cause the symptoms of inflammatory bowel disease.

The molecular control of cDC2 functions is not sufficiently understood. Progress has been hampered by the heterogeneous nature of cDC2s in vivo; the few activated cDC2 needed to prime naïve T cells are outnumbered by non-activated cDCs that also migrate. We have recently shown that, after stimulation with Schistosome Egg Antigen (SEA), cDC2 become able to prime naïve T cells to become Th2 in vivo. But at a molecular level our understanding of cDC2 functions, particularly Th2-priming cDC2s, is still insufficient.

The intestine is a productive tissue for the study of cDC2 functions; it contains more cDCs and T cells than any other organ system. We have established techniques for purification and functional analysis of bona fide migrating intestinal cDC2.

We have performed microarray analyses of both types of cDC2s (CD11b+ single-positive cDC2 and CD11b+CD103+ double-positive cDC2). Both cDC2 populations prime Th2 responses in vivo after stimulation with SEA. We found only three genes that significantly change their expression in both SEA-stimulated cDC2 populations. Our RNA sequencing data confirm that these genes are expressed by cDC2s in the small intestine, lymph, and intestinal lamina propria. Two of the three genes (Rasgrp3 and Il1f9) are known to control cytokine production by myeloid cells, but neither has yet been ascribed a function in cDCs.

Here we have two aims. First, we will investigate the functions of Rasgrp3 and Il1f9, to understand how they contribute to cDC2s ability to prime Th2 responses, both in vitro and in vivo. Second, we will deliver labelled antigens in vivo and generate single-cell sequencing data to identify clusters of genes expressed by the few activated antigen-carrying Th2-competent migratory lymph cDC2s, among the heterogeneous migratory cDC2.

Thus, we aim to uncover novel molecular functions in cDC2, that will enable improved manipulation of the adaptive immune response, both in the intestine and in other tissues.

Application and Exploitation
Given the basic science nature of the project, we do not expect commercially exploitable results in the short term. However, we anticipate that the study will identify cellular and molecular targets that could be exploited for treatment of intestinal inflammation or to improve the efficacy of oral vaccinations. If so, the University has an active Research & Enterprise Office with experience in translating basic science to clinically applicable studies, as do several members of our Institute.
The applicant, SM, manages a number of projects investigating the role of DCs in driving inflammation in patients with spondyloarthropathy, inflammatory bowel disease, and alopecia, and is actively involved with NHS colleagues and colleagues in the Institute in investigating possibilities for therapeutic modulation of the immune system in these diseases. SM also has an extensive network of colleagues through his activities with the British Society for Immunology.
The basic science that underpins the previous work in the Milling lab has generated insights with potential for commercial exploitation. These have generated significant industry funding for work in the laboratory; four members of the research team currently work on projects with substantial components of industrial funding.

Communications and Engagement
Academic Engagement: We will publish the outputs from this project in Open Access Journals to ensure as broad dissemination as possible. We will also deposit manuscripts in the University of Glasgow's established ePrint repository (Enlighten), which is compatible with Google and other search engines. Journals usually demand or allow uploading of complex data sets and when this is not possible, we will make the data available to scientific colleagues through remote access to the University server, or shared by email, upon enquiry. Sequencing data will be made publically available through the GEO database. Our results will be presented at national and international scientific meetings relevant to mucosal immunology and DC biology, which are attended by both clinicians and basic immunologists.

Public Engagement:
The University of Glasgow has excellent facilities and connections for disseminating scientific information to members of the public and media via its Press Office and Research & Enterprise Office. Both Prof Milling and members of his research group are regular contributors to public engagement event, both locally and nationally.
Research in the Milling lab provides excellent projects for undergraduate and postgraduate training, and informs teaching at the Glasgow University Immunology degree. SM, and all members of the Milling lab are actively involved in these knowledge transfer activities. We have contributed to broader educational activities through hosting work experience projects, engaging with the British Society for Immunology, and by contributing to outreach programmes with local school children and adults. These activities will be continued throughout the lifetime of the project.

Capacity and Involvement
Given their personal experience with engagement activities of all kinds, it is envisaged that the PI will be primarily responsible for these activities. The postdoctoral researcher will also present research findings in writing and orally, and will participate in public engagement activities, under the guidance of the PI.

Deliverables and Milestones
The majority of these activities will take place throughout the period of the project, with no formal timetable being in place. However, our experience is that several opportunities for public engagement will arise for the PI each year. Only in the case where a patentable development occurs can we anticipate a delay in releasing data and so we do not anticipate temporal restrictions on data sharing.