Does the phenotype of antigen specific anti-donor memory B cells determine the long-term outcome after renal transplantation?

Renal transplantation is the preferred treatment modality for the majority of patients with kidney failure, offering superior outcomes in terms of length and quality of life, as well as cost effectiveness, when compared to other forms of therapy including dialysis. Despite advances in knowledge and clinical care, most transplanted kidneys do not survive for the natural lifespan of the recipient and fail after an average of 10 years. The most common cause for failure of transplanted kidneys is persistent damage caused by the immune system, which is called chronic rejection. Surprisingly little is known about the precise mechanisms by which the immune system causes kidney damage.
However, we do know that some patients, who have antibodies against the kidney, are at greater risk of suffering from chronic rejection: the presence of the antibody makes it easy to identify these people. However, from what we know about how the immune system works, the antibody alone is insufficient to cause all the damage, so it's likely that other mechanisms involving cells of the immune system are involved.
One of the crucial cells is called the B lymphocyte (or B cell). This is an important cell that captures 'antigens' from the donor kidney and processes them in such a way that they can activate other immune cells, particularly other lymphocytes called CD4+ T cells. We have recently learned a lot about different types of B cells, from both animal and human studies, and it appears as though they can both stimulate and suppress immune-mediated damage through their interactions with CD4+ T cells.
The supervisors of my project have preliminary data to suggest that these two different types of B cells can be found in transplant recipients, and have developed assays to measure their influence on the activation of CD4+ T cells. Importantly, the activity of these cells has been linked to the survival and performance of kidney transplants, so these assays are telling us something important about the future survival of the kidney transplant.
In my project, I will utilize the fact that Guy's Hospital transplants a large number of patients who are already known to have activated B and T cells against their donor kidney, making them the ideal patient group in which to study the nature of the B cells and their effect on T cell activity. In addition, Guy's is performing two large randomized controlled clinical trials in which B cells are being depleted from kidney transplant recipients, making these ideal populations in which the study the effect of depleting the different types of B cell and in particular to ascertain whether depletion of the B cells that suppress T cells is detrimental to the function of the transplant. In all my studies, I will use modern techniques to try to determine the molecular mechanisms by which the B&T cells are interacting, which is likely to lead to ways in which to selectively manipulate the different type of B cells to improve transplant outcomes.

Objective 1: Phenotype of antigen-binding B cells in HLA-sensitised patients: On PBMC from selected patients on the highly sensitised programme at Guy's (and a small number of healthy controls), HLA-binding B cells will be defined by multicolour flow cytometric analysis using biotinylated HLA proteins (corresponding to mismatched antigens against which the recipient has an antibody). To define the role of B cells in IFNgamma production responses to donor antigens (from living donors) and the same purified HLA proteins will be tested using an ELISPOT assay. In all analyses, responses to CMV gpB protein will act as a control for antigen binding, processing and presentation. Soluble inhibitors of B cell receptor activation and anti-HLA class II blocking antibodies will confirm antigen presentation by B cells. Cross-sectional and prospective analyses will allow detailed correlation between results and functional outcomes and study of the evolution of B cell memory responses.
Objective 2: Antigen-binding B cell phenotypes pre- and post-rituximab. ELISPOT patterns and the phenotype of memory B cells (pre- and post- rituximab) will be defined using PBMC from two multicentre RCT's assessing rituximab as induction therapy (REMIND) or as treatment for CR (RituxiCAN-C4). The phenotype of reconstituting HLA-binding B cells will be tested using samples 12 or 24 months post-depletion.
Objective 3: Mechanisms of B cell modulation of IFNgamma production by T cells. RNA profiles from HLA-antigen binding B cells with contrasting immunoregulatory properties will be compared, using Illumina's HT 12v4 beadchip arrays. Differentially expressed genes will be identified by ANOVA analyses using Partek's Genomics suit software and validated by quantitative RT-PCR protein expression. In addition, detailed surface phenotyping of described B cell markers will be performed.

The overarching aim of this research is to maximise the lifespan of solid organ transplants by seeking ways to address the problem of chronic allograft rejection, which is the single biggest cause of allograft failure, causing thousands of patients across the world to return to dialysis each year.

Simply put, the benefits of transplantation are threefold: improved quality of life, improved quantity of life and for positive economic reasons. The most important beneficiaries are therefore transplant recipients themselves. Although focused on kidney transplant recipients, this work has the potential to be translated to recipient of all other solid organs.

Currently 1000 patients per year die waiting for solid organ transplantation, as donor organs are in very short supply. Maximising allograft lifespan reduces the need for re-transplantation, meaning that these organs can instead be transplanted into new patients, offering them greater quality of life and improved survival. From a health economic standpoint, this improves cost-effectiveness by reducing the need for expensive dialysis therapy, and allows patients to remain economically active. This is of clear benefit to not only the UK, but also all other societies offering organ transplantation.

The ultimate goal is the development of tolerance, allowing transplant recipients to maintain stable graft function in the absence of immunosuppression. This benefits patients by relieving the burden of side effects from immunosuppressive drugs, including life-threatening infection, diabetes and malignancy; it also benefits the National Health Service and wider society by eradicating the significant economic cost of funding these expensive medications, freeing resources for alternative areas of expenditure.

Regulatory B cells have been implicated as key players in tolerant patients and it is anticipated that this work will further elucidate the phenotype of B cells capable of regulating T cell activation, in the setting of renal transplant recipients. The study of regulatory B cells is in its relative infancy, much like the study of regulatory T cells in the 1980s. It is hoped that this work will substantially contribute to our knowledge of regulatory B cell phenotype, with repercussions across disciplines interested in B cell biology, notably autoimmunity, infection, vaccination and cancer, with the possibility of positively impacting patients beyond the field of transplantation.