CHECKPOINT FOR B CELL SURVIVAL IN HUMAN GUT

B cells are cells of the immune system. B cells fight infections by identifying infectious particles via very specific receptors on the B cell surface, called the B cell receptors. B cell receptors recognise infectious agents by their individual shape and they bind to them in a lock-and-key way. Millions of B cells circulate through the blood and tissues of the body and each B cell has a B cell receptor that is unique to that B cell. As a consequence, no matter what infectious agent finds its way into the body, there will be a B cell with a receptor of the complementary shape to bind it.

If a B cell binds to an infectious particle via its B cell receptor, the B cell may become activated and secrete its B cell receptor that will bind to and fight the infectious agent. When the B cell receptor is secreted it is referred to as antibody.

The process that generates the huge and diverse set of B cells with unique receptors has a major associated hazard. B cells can be produced that bind to the body's own cells and tissues and can attack them. Other cells of the immune system called T cells have the capacity to regulate B cells and they themselves can discriminate well between self and non-self. However, many B cells can make T cell independent responses and these are particularly dangerous if not properly regulated for specificity.

The infectious agents that activate B cells in a T-cell independent way include those that cause some types of pneumonia. B cells have an additional trick to recognise these agents because they have a repeating pattern of shapes on the surface. B cells may be able to recognise the shape through the B cell receptor, but also the regularity with which the shape is presented.

Experiments in our lab suggest that B cell selection that would prevent self-reactivity and promote responsiveness to particles that activate B cells independent of T cells happens in the gut. The gut contains a lot of 'friendly' bacteria that constantly stimulate the immune system it contains. Our experiments suggest that this environment supports stages in B cell development that are largely ignored in models of human B cell immunology or assessment of human disease. We call this a 'checkpoint' because it is a stage of B cell development where only cells that have a required set of properties are allowed to pass.

The gut is involved in the development of B cells in different ways in many species, including chickens, mice, sheep and rabbits. Therefore an influence of the gut on human B cell development is important and highly likely to occur, but as yet totally mysterious.

The aims of experiments described in this application are to understand how B cells mature in the gut and how this is regulated.

Peripheral B cell development and selection occurs after B cells exit the bone marrow and enter the blood as transitional B cells and before maturation. Having observed that transitional type 2 (T2) B cells selectively enter the gut and become activated, we proposed that human gut hosts a selective stage in B cell development. We reasoned that a checkpoint would be evidenced by divergence in cell behaviour in a population: cells would have alternative fates (eg life or death) dependent on a selective process. However whilst funded by MR/L009382/1 we did not observe this in T2 cells in GALT. We therefore analysed a subsequent stage in T2 B cell development implicated by mouse models: marginal zone (MZ) B cells. We identified CD27+IgD+ cells in GALT that are clonally separate from classical memory cells. They are members of the same clones as CD27+IgD+ cells that circulate in the blood and they also have clone members in GALT GCs.

CD27+IgD+ cells from GALT are relatively non-viable in culture and express very high levels of negative B cell regulators TACI and FCRL4. In contrast, classical memory B cells from GALT and CD27+IgD+ cells in blood do not.

We propose a model for MZ B cell development and selection in human gut and wish to interrogate and build the model further by asking the following questions:

What are the key differences between CD27+IgD+ B cells in the gut compared to the blood?

What induces the CD27+IgD+TACIhiFCRL4hi state?

What does survival of CD27+IgD+TACIhiFCRL4hi cells depend on?

The gut is involved in B cell development in many species. We propose that understanding the role played by GALT and the microbiota in human MZ B cell development as planned here will result in a paradigm shift in B cell biology that will support investigation of B cell associated pathologies and underpin novel approaches to establishing human B cell tolerance.

Using data generated during our current period of funding from the MRC (MR/L009382/1) and published data from ourselves and others, we propose a model of B cell development in gut that will change basic perceptions of human B cell biology. The experiments described in this application aim to address key questions in our model. We anticipate major scientific impact as we initiate international discussion of the model we propose. Since this is a study of basic human B cell physiology it will also have clear clinical and pharmaceutical impact in addition to impact through training and mentoring.

Scientific Impact:
The nature of cells with the phenotype of marginal zone B cells that reside in spleen and circulate in blood has been debated fiercely. The community is currently divided into on the one hand, those who think that marginal zone B cells are early memory cells and on the other hand, those who think they are a population that develop independent of memory. Our unpublished data will make a decisive contribution to this debate by identifying that marginal zone B cells and memory B cells develop separately in the gut. We show this in two different ways; by analysis of B cell clone composition and by phenotypic markers of B cell development and function. This proposal, that aims to consolidate and develop this model, is therefore of high scientific importance. We anticipate that when our data is published we will also review this to gain maximum debate of the new concepts we will be introducing into this field.

Clinical Impact:
Our model of B cell development in the gut includes a stage at which B cells with unfavorable B cell receptors are selected against. Failure of such a mechanism could contribute directly to the development of some autoimmune diseases. Indeed, we have already proposed that B cells in SLE access our checkpoint inefficiently. Regulation of B cell tolerance is also important in transplantation and an ability to regulate this to minimize the effect of antibodies to the graft to graft rejection would be highly beneficial. We propose that the marginal zone B cells we identify in the gut that are separate to memory cells are the benign counterparts of MALT lymphomas. These tumours are known in some cases to remain responsive to certain exogenous stimuli. Understanding the physiology of their benign counterparts may identify new therapeutic strategies to achieve remission.

Pharmaceutical Impact:
We anticipate that the project could have pharmaceutical impact through identification of new route to intervene therapeutically in autoimmune diseases, transplantation and MALT lymphoma.

Impact through training and mentoring:
The post doc appointed to the project will have already completed a post doc and her CV will be enhanced by the work we are currently submitting for publication. Therefore, the phase of her career in which this project would be undertaken would be important because it would lead to the point at which she would apply for career development fellowships. This project would have impact on her career by continuing to encourage her independent thinking and ownership of her project should she wish to develop an aspect of it independently in the future.

We have three major areas of strength that will enable us to make the impact as planned

1. We have already made considerable progress allowing us to create our model of B cell development in the gut. We have extensive experience in all aspects of the project and are therefore well placed academically to take it forwards.
2. We have strong infrastructure to support translational research
3. We are located in a large teaching hospital where patients are aware of the research and support this through the generous donation of tissue and blood samples