Probing novel modes of molecular recognition by the leukocyte immunoglobulin-like receptor family

This research project focusses on a group of related proteins, the Leukocyte Immunglobulin-like Receptor (LIR) family, which are expressed on the surface of a range of different immune cells. These receptors are thought to play important roles in regulating the immune system, by transmitting signals that either help activate or inactivate cells on which they are expressed. These cells include B cells, which are important for antibody production, T cells, which can kill cells infected with viruses, and Natural Killer (NK) cells that can kill infected or tumour cells. Also, many of the receptors are expressed on specialsed cells called dendritic cells (DCs) that present foreign molecules derived from infecting organisms to T, B and NK cells, and by doing so activate them to eradicate invading viruses, bacteria, or even tumour cells. Therefore, understanding LIR receptor function is important, not only in helping us grasp how the immune system normally regulates itself, but also in the design of new therapies for cancer or viral infection based on the use of particular immune cells such as T cells or DCs. A better understanding of LIR function could also lead to improved strategies for the treatment of autoimmune disorders. The ability of LIR receptors to send stimulating or inhibiting signals to the immune cell on which they are present depends critically on the receptors recognising target molecules on the surface of another cell. Therefore, in order to understand how LIR receptors function, we need to understand what target molecules they recognise, how they recognise them, and what the consequences are. This research proposal will use a range of different techniques to answer these questions, and in doing so will significantly increase our understanding of LIR receptor function. The proposal builds on previous work by Dr Willcox in this area, which involved analysing how a receptor called LIR-1 recognises a target molecule called class I MHC that is involved in presenting parts of virus-derived proteins to T cells. This work led to new ideas about what target molecules other LIR receptors recognise. In particular it suggested that one group of LIR receptors (group 1 LIRs) recognise target molecules similar to class I MHC, while another group (group 2 LIRs) recognise different target molecules. The work outlined in this proposal will involve a series of binding studies to directly test what target molecules are recognised by poorly characterised group 1 LIRs, and will include work on CD1, a molecule similar to class I MHC that has recently been identified as a target molecule recognised by the group 1 receptor LIR-2 by Dr Willcox' group. In addition to binding studies, the structures of group 1 LIR recognition events will be analysed by generating crystals of the proteins involved and exposing them to x-rays. Also, the experiments proposed aim to identify what molecules are recognised by group 2 LIRs, including LIR-5, a receptor that plays an important role in dampening down the function of T cells and the presenting DC cells. These experiments build on recent results from Dr Willcox' laboratory suggesting that LIR-5 recognises a target molecule present on the surface of activated T cells, and will also involve examining the structures of such receptors. Collectively, the experiments outlined in this proposal will help us to understand the function of poorly studied LIR receptors. In doing so, they will significantly increase our understanding of how immune responses are controlled, and could potentially lead to improved strategies for the treatment of cancer, viral infection and autoimmune diseases.

The Leukocyte Immunoglobulin-like Receptor (LIR) family consists of a group of immunoregulatory cell-surface receptors expressed on a range of immune cells, including myeloid and lymphoid lineages. The prototypic receptor, LIR-1, is an inhibitory receptor that recognises a broad range of host class I MHC, but the family also includes other inhibitory (LIR-2, -3, -5, -8) and activatory (LIR-6, -7, ILT8, 10, 11) members, and one soluble receptor (LIR-4). This proposal combines structural, biophysical, and cellular approaches to improve understanding of LIR receptor function in immune regulation. It builds on structural studies on LIR-1/class I MHC recognition by Dr Willcox, which led to the hypothesis that one subset of LIRs (group 1 LIRs, namely LIR-1, -2, -4, -6, -7) binds class I MHC-like molecules associated with B2m while another (group 2 LIRs, namely LIR-3, -5, -8, ILT8, 10, 11) binds novel ligands. The proposal focuses on two key aspects of LIR receptor recognition. Firstly, binding and crystallographic studies will determine which of the poorly characterised receptors LIR-4, LIR-6 and LIR-7 recognise class I MHC ligands, and the molecular properties of these interactions. Also, we have determined the B2m-associated lipid presentation molecule CD1 is a ligand for the group 1 receptor LIR-2, and will characterise LIR/CD1 recognition using similar techniques. Secondly, we will define the structure and ligands of group 2 receptors, focussing mainly on the inhibitory receptor LIR-5, which is upregulated on tolerogenic DCs. This builds on recent results in the Willcox lab, including use of LIR-5 tetramers to stain activated T cells, and preliminary LIR-5 crystallisation. Flow cytometry studies using tetrameric LIR-5 will establish the biochemical nature and expression pattern of the LIR-5 ligand on different cell types. Assuming the ligand is proteinaceous, proteomic approaches will be used to identify candidate ligands for verification in direct binding assays.