A multi-disciplinary approach to understanding the immunological basis and potential prevention of graft versus host disease.

The immune system contains different types of white blood cell that work together to fight off disease. These cells grow from stem cells in the bone marrow of healthy people. However, in some people, some of the stem cells and white blood cells become cancerous and this results in leukaemia. In order to treat this, the patient's stem cells and white cells are removed with irradiation and/or chemotherapy. This kills the cancerous cells but it also kills the other healthy white cells that are important for fighting off infections. To remedy this, the patient receives a stem cell transplant from a healthy volunteer. The stem cells go to the patient's bone marrow and differentiate into white blood cells, giving the patient a new immune system. The quicker the stem cells turn into white blood cells, the faster the number of white cells increases. This is called immune reconstitution. The more 'useful' white cells (e.g. ones that can kill cancer cells) that are produced, the less chance there is of the leukaemia recurring and the less likely the patient is to become sick due to other infections. This process of cancer killing is called Graft versus Leukaemia (GVL).
Graft-versus-host disease (GVHD) is an important clinical complication following stem cell transplantation (SCT), caused by the same donor white cells attacking the patient whom they recognise as 'foreign'. This results in significant patient morbidity and mortality and significantly affects their quality of life. However as the process of GVHD is the same as that which mediates GVL, current treatments which stop white cells attacking the patient tissues also stop the same cells from killing cancerous cells. This puts patients at an increased risk of relapse from the original disease. It is now clear that treatments need to be developed which prevent GVHD, but which do not stop GVL.

GVHD has a characteristic clinical picture affecting the skin, gut and liver and our work shows that specific molecules guide patient white cells to these tissues where they cause damage. Therefore one strategy to prevent GVHD is to prevent the white cells getting to the tissues where they can cause damage. Key to this is that cancerous cells will be present in the blood and bone marrow and not in the tissues and therefore this strategy should not prevent GVL.

In our recent studies we have been monitoring immune reconstitution prior to disease development and our data indicate that this is key to predicting which patients will develop disease. Here we will combine our studies of immune reconstitution with our studies of white cell movement to the tissue to a) predict in which patients GVHD will develop, b) in which tissues they will develop disease and c) develop strategies to block movement of the white cells to the tissues.

The development of white cells which will kill tissues and tumour will be monitored during the first 2 weeks following transplant. We will specifically analyse reconstitution of donor white cells, assessing how they move to the tissues and what they do once they have arrived. Importantly for this programme we have access to both donor and patient blood and tissues including skin, gut, liver and bone marrow. Once we have determined how white cells get to the tissues we will test out a number of methods to prevent this.
We are in an excellent position to translate this work to the clinic. The School of Cancer Sciences encompasses one of the largest cancer trials units within the UK bringing together clinical trial expertise from across the University and, in particular, the three large, well-established, UKCRC fully-registered Clinical Trials Units. Therefore we envisage that this work can be successfully and rapidly translated. This will significantly enhance patient outcome and welfare following stem cell transplant.

Our programme seeks to understand the regulation of immune function in the early post-transplant period in order to separate the Graft versus leukaemia response (GVL) and Graft versus Host disease (GVHD). To achieve this, our Programme is based on 3 main areas 1) immune reconstitution during the period of very early immune reconstitution, (2) Regulation of migration of antigen-specific T cells to target tissues during GVHD and (3) development of chemokine blockade as a mechanism to prevent GVHD.
We have shown that functional donor lymphocytes are detectable within the first two weeks after transplant and that kinetics of recovery predict clinical outcome. Here we seek to extend studies of VEIR to specifically monitor development of the alloimmune response utilising flow FISH for detection of donor T cells, multimer technology and High throughput sequencing. These techniques will allow us to identify both GVL and GVHD responses, and to assess the ability of these developing cells to migrate towards, and kill patient tissue. The patient microenvironment will be key to T cell homing during immune reconstitution and therefore we will monitor changes in chemotactic cytokines during VEIR. A major part of this section of work will be to study the influence of the local tissue microenvironment on the development of T-cell responses incorporating a novel skin blister model, tissue explants and fluorescent microscopy. As GVHD is a tissue specific disease, this will be key to understanding disease pathogenesis.
We have shown that chemokines guide T cells towards the tissues in GVHD. Here we will combine our studies of VEIR with our migration work to predict which patients will develop disease, and in which tissues. Once established we will develop pre clinical assays to block T cell migration to classical GVHD sites, using both neutralizing antibodies and siRNA. Development of these represents an opportunity to prevent GVHD without abrogating GVL.

The proposed work is centred around the development of GVH responses, with a key focus being on very early immune reconstitution to separate between development of GVL and GVHD responses. As such, the primary impacts will be clinicians and patients. The study addresses fundamental biological mechanisms and relates them to clinical outcome after HSCT. The work should therefore benefit both clinicians and patients in the longer-term. We have already generated some data that links the expansion of a CD8 T cell population in the first 14 days after transplantation with the development of Graft versus Host Disease two or more weeks later. This would allow clinicians to predict and treat GvHD before clinical symptoms, leading to a reduction in morbidity in HSCT patients. We expect this work to bridge the gap in understanding between pathogenesis of GVHD in the periphery and clinical manifestation of the disease. This will be achieved through incorporation of a skin blister model which will allow us to track development of alloimmune responses, and their movement into the tissues over time. This has not previously been investigated and would be a powerful addition to understanding this disease. Early prediction will allow intervention before clinical symptoms, improving patient welfare and clinical outcome and reducing post transplant complications including infection, GVHD and relapse.
This Programme of work also aims to explore in vitro mechanisms to block T cell homing to tissues incorporating two methods; neutralization antibodies and siRNA knockdown. Thus, the work may have some commercial impact. Depending on the findings of the study, we will approach a UK company to develop reagents that show promise for use in pre-clinical and clinical trials. This process would be facilitated by the world-class clinical trials team at Birmingham. We also plan to study serum free light chains, in collaboration with Binding Site Ltd as a tool for GVHD prediction. This would also provide commercial impact as this assay could be utilised clinically as a simple prognostic test.
As this Programme of work involves study of development of the immune system, this will benefit a number of groups of academics. These will include developmental and neonatal immunologists and stem cell and solid organ transplant biologists. Human stem cell transplant provides a novel in vivo model for study of immune development in a lymphopenic environment and will provide insight into the mechanisms of homeostatic proliferation, which is currently poorly characterised in humans.