Systemic analysis of the molecular mechanisms of leukocyte entry into the pancreas

Lymphocytes, also known as white blood cells, defend the body against microbes, viruses and cancer by circulating throughout the body. Aside from lymphoid tissues (spleen, lymph nodes, bone marrow, etc), a small number of lymphocytes can be found in all other tissues, including the pancreas. Entry of lymphocytes in tissues such as the brain, the eyes, the muscles or the pancreas is regulated by specific proteins and processes. Aberrant or facilitated entry of lymphocytes into these non-lymphoid tissues can lead to severe immune pathologies. Notably, lymphocyte infiltrates in the pancreas have been documented in both type 1 and type 2 diabetes. Blocking immune cell entry in the pancreas would certainly provide therapeutic benefit to diabetes patients. Our overall objective is to identify the proteins that allow both normal and aberrant lymphocyte infiltration in the pancreas. We will use two complementary non-targeted approach to identify key pathways facilitating lymphocyte entry into pancreatic tissue. First, by a novel molecular biology approach, namedly ProCode, we will test the role of over 200 proteins in inhibiting or facilitating lymphocyte entry in the pancreas. Second, we will screen over 200 strains of mice from the Collaborative Cross and determine which of these mice present with more or less lymphocytes in the pancreas. As the genome of the Collaborative Cross mice is known, this unbiased approach will reveal genes that influence lymphocyte entry in the pancreas. Altogether, these complementary approaches will unravel the specific molecular pathways that need to be targeted to prevent lymphocyte entry in pancreatic tissue.

Migration of leukocytes into the pancreas is a key pathogenic step in the development of diabetes. In order to extravasate into tissues, circulating leukocytes must first make rolling contacts with the vasculature before forming firm adhesions and subsequently transmigrating through the endothelium and basement membrane. These processes depend on the expression of integrins, which facilitate intercellular and cell-matrix interactions, and chemokines, which guide cell migration. Current studies are predominantly candidate-based and limited to broad neutralisation or gene deletion models, which prevents analysis of the intrinsic requirements of different cell types to infiltrate the diabetic milieu. Our approach will use genetic screening and natural genetic variants to unravel the specific molecular pathways that need to be targeted to prevent diabetogenic immune cell entry in pancreatic tissue. We will use two complementary systematic approaches to identify key pathways facilitating immune cell entry into pancreatic tissue: ProCode CrispR screening and Collaborative Cross natural variant screening. The ProcCode CrispR approach uses transfection of gRNA-lentiviruses, each with a unique protein barcode, to generate a mouse that has a mosaic population of leukocytes with representations of multiple different knockout lines. We can use this system to simultaneously screen for the functional role of all known integrins, adhesion molecules, chemokine receptors and matrix remodelling enzymes in each defined leukocyte population entering the tissues. Collaborative Cross studies compare >200 inbred mouse strains with defined parentage from eight diverse founder strains. By qualitatively and quantitatively assessing pancreatic infiltrate in these strains, we can use the predefined genetics to identify the molecular mediators down to the gene level.

In addition to its immediate impact for basic, pre-clinical and clinical academic research (see Academic Beneficiaries), this work will be relevant for the following groups:

A) The biomedical industry: The proposed work will establish a critical molecular pathways that regulate migration of immune cells into pancreas. Identification of pathways that involve the migration of immune cells is of high interest to the industrial sector in their attempts to develop new treatments for patients with diabetes. Several major biologic treatments for Multiple Sclerosis are based on this process: fingolimod and Natalizumab. Superior treatments are likely to be possible, as both existing drugs have broader than desirable inhibition profiles, impacting both inflammatory and anti-inflammatory cell types. Our project would provide the most comprehensive analysis of migration kinetics, spanning all leukocyte subtypes, creating a key resource for the industry.

B) The Babraham Institute and affiliates: Where relevant, we will directly pursue commercial opportunities generated from this research by developing projects with new and existing industrial collaborators. We will engage in industrial collaborations with the assistance of Babraham Institute Enterprise (BIE). BIE is the wholly owned trading arm of the Babraham Institute (BI) and manages, develops and commercialises the Institute's intellectual property portfolio, as well as facilitating collaborations between the Institute and industry. Successful commercial exploitation of the results will have impact by directly fostering UK and global economic growth. BIE has arrangements for protection and development of intellectual property and a track record in exploitation of the Institute's science. The commercialisation of data will directly benefit the Institute and affiliated parties by generating new revenue.

C) The UK skilled workforce: The project will have immediate impact by enhancing skills within the UK workforce. Undertaking the proposed research will result in recruitment and training of a post-doctoral researcher at a world-class UK research institute in the fields of diabetes immunology, immune cells trafficking and gene regulation and will enable the postdoctoral researcher to advance their career, potentially towards scientific independence. We encourage researchers to gain skills in bioinformatics, flowcytometry and statistics through attendance of regularly held training sessions at the Babraham Institute. These research skills are relevant for both a career in academic or industrial science. The postdoctoral researcher will also gain presentation skills and the opportunity to expand their network of scientific and industrial contacts through the opportunity to attend International conferences, for which funding has been requested. By being involved in this research, a variety of technical and scientific staff at the Babraham Institute, including Animal Facility technicians and managers, will gain new skills required to perform diabetes immunology and CrspR biology experiments. These technical skills will be relevant to a variety of academic and industrial research sectors.