The Role of Follicular T Cells in the Alloimmune Response and Alloantibody Formation

Kidney failure requiring kidney replacement therapy (end-stage kidney disease) is a problem that affects over 3 million people worldwide and over 30 000 people in the UK. Around 1 in every 2000 people in the UK is at risk of developing end-stage kidney disease over their lifetime, and needing kidney replacement therapy. Kidney replacement therapy can be carried out in a number of ways, but all have side effects and complications. For example machines that do the job of the kidneys by cleaning the blood, can save lives but require the person to spend at least 9 hours a week attached to the machine. One of the best ways of treating end-stage kidney disease is to perform a kidney transplant, an operation that gives a donated kidney, either from someone on the organ donor register or a friend or relative who gives one of their two kidneys, to the patient. This single kidney can do the job of the machine, and clean the blood effectively enough to allow the person to go back to work and lead a nearly normal life. However, kidney transplants do not last forever, and one of the main reasons they fail is called rejection, where the body's immune system recognises the transplant as being different to itself, and therefore attacks it as if it were a bug such a flu virus or meningitis bacterium. One of the ways it does this is to produce antibodies, proteins that stick to the transplant and flag it up to immune cells as something that needs to be attacked.
Currently we use lots of strong medications to dampen down the immune system and prevent this attack; however these medications have side effects by increasing the risk of infection and cancer. Maintaining the balance between keeping the organ alive and preventing severe infection or cancer is difficult. Most transplanted kidneys do very well early on, but by 10 years following a transplant, nearly half will have failed, and most of these will have failed because the immune system has begun to attack them despite the medication. When this happens, patients need to start another form of kidney replacement therapy, or they can have another transplant. However each time someone receives a transplant, it becomes harder to dampen down the immune system because more of these antibodies are produced. The best solution therefore is not to keep giving someone a transplant, but to make the one they have last for as long as possible. Currently our treatments are not very good at this, and so research needs to be done to allow us to improve these treatments so we can prolong the life of the transplant without significantly increasing the risk of cancers or infection. One of the best ways of doing this would be to see what is different between a patient whose immune system starts to attack the transplant, and one whose immune system is controlled by the medication. This can show us new ways of dampening down the immune system, and also help us to find new ways to tell in advance who is at risk of attacking the transplant and hence who needs strong medication to prevent this. This research is designed to look at the immune cells that are involved in attacking the transplant, particularly those that are important for producing antibodies, as these are much harder to dampen down and can live for many years in areas of the body that are protected from the affects of the medication. It is thought that these antibodies are the cause of most kidney transplant failures, and preventing them being made is one of the main ways we can try to prolong the life of transplants. Trying to look in more detail at the cells that interact to produce these antibodies will allow us to see new ways of preventing this interaction, but also hopefully ways of predicting in advance who is at risk of this, so we can begin treatment before these cells have moved to the areas where they are protected from medication effects.

Aims and objectives: To examine the roles of follicular T cells in the formation of alloantibody in humans and mice undergoing transplantation, the development of humoral rejection in humans and mice undergoing transplantation and the development of autoantibodies in humans undergoing transplantation.
Human work:
1.Flow cytometry: Identifying cells of interest using a panel of surface and intracellular markers to compare numbers and phenotype between the lymphoid tissue and peripheral blood. Markers will include: T cells: CD3, CD4, CD8, CD45RA, CCR7, CD25, HLA-DR, CD127, CXCR5, CCR6, CXCR3, IL-23R, CD57, Bcl6, FoxP3. B cells: CD19, CD20, CD24, CD27, CD10, CD38, CD138, IgM, IgD, CD71, Bcl6. NK/NKT cells: CD16, CD56, NKG2D, CD57, Va24, CD3, CD4, CD8. Mononuclear cells: CD14, CD16, CD11c, CD11b, CCR2, CX3CR1, CCR7, HLA-DR, CCR5, CCR1
2.Immunohistology: Using sections of lymph node (fixed and frozen) or biopsy (fixed and paraffin embedded) we will use a more limited panel of antibodies to identify B cell follicles (IgD), germinal centres (CD57) and T cells within these follicles (CD4, FoxP3, Bcl6)
3.Functional analysis: Using flow sorting, we will separate cell subtypes and perform functional studies looking at the B helper and suppressive functions of different cell subtypes.
4.Cytokine analysis: Using serum taken at the time of blood sampling we will assess for antibody levels and proinflammatory cytokines including IL-21
Mouse work:
1.We will use two models of transplantation to explore the roles of follicular CD4 T cells, using a model antigen system to look at responses to OVA and HEL. We will adoptively transfer in cells with the congenic marker CD45.1 to allow us to track antigen specific cells throughout the response to the transplanted tissue.
2.By manipulating this model, we will be able to adjust both inflammatory stimuli and follicular T cell numbers to see the effects of different conditions on the development of antibodies in the model systems.

This research is likely to have a wide range of direct and indirect benefits, including academic, financial and social, in addition to the academic benefits described in the previous section. Firstly from the perspective of renal transplant recipients, whose quality adjusted life years are lower than the general population due to the burden of both their disease and immunosuppression, and become significantly worse if their transplant fails and they return to dialysis. Any intervention that may prolong the life of the transplant without unnecessarily increasing the immunosuppressive burden is likely to have a positive impact on the quality of life of these patients.
In addition, there are a number of social and financial benefits to prolonging the life of a renal transplant. It is estimated that a patient on haemodialysis for end stage renal failure costs the NHS over £35 000 per year. A transplant costs £17 000 per patient plus immunosuppression costs of £5000 per year, so savings in the initial year are only around £10 000, however over the lifespan of a transplant the cost saving is around £25 800 per year compared to dialysis. With over 2700 kidney transplants in 2010-11, these patients are now saving the NHS nearly £70 million every year that the transplant functions.
In addition to this there are wider economic benefits as a functioning transplant will allow patients to lead near-normal lives and contribute to the working population of the country, providing economic benefits and general contributions to society.
With the involvement of the Transplant Research Immunology Group in the ONE study, a European wide organisation funded by the European Union and dedicated to the development of immunotherapy leading to transplant tolerance. This research, which will build on knowledge of alloreactivity, will be of benefit to the study and others investigating the possibility of transplant tolerance and minimal, patient targeted immunosuppression. With links to pharmaceutical companies, any new therapeutic targets could be easily followed up with drug development.
The timescale for transplant tolerance is unclear, trials are ongoing but further research is required. However this research could lead to identification of new therapeutic targets within the timeframe of the fellowship. Translational application of the data to test new therapeutics is likely to take longer, approximately 5-10 years after completion of the fellowship. However we predict that the economic and social benefits of improved transplant longevity are likely to occur within our professional lifetimes.