Checkpoint governing B cell fate decisions in human gut-associated lymphoid tissue.

Antibodies are small protein molecules that circulate in the blood and that are also secreted onto the moist body surfaces such as the eyes and intestines. Tears, saliva and mucus for example all contain antibodies. Antibodies fight bugs by binding to them. Antibody binding to bugs can neutralize them to stop them causing harm, or else antibodies can target bugs for attack by other components of the immune system.
Antibodies are made by B cells that display the antibody they can make on their surface. Each B cell is unique and displays its own particular shape of antibody on its surface. In order for each B cell and its antibody to be different to the next one, B cells develop by a process that includes some random events. The genetic material that codes for antibody is shuffled during B cell development so that a massive range of genetic codes for antibodies is generated. As a consequence, no matter what bug or vaccine enters the body there will be a B cell displaying an antibody that fits.
A down-side to a process that generates B cell antibody diversity in a way that includes random events, is that some B cells are produced that display antibodies that could be harmful. To protect the body from harmful antibodies, checkpoints remove B cells with harmful specificities as they develop. No one knows exactly what a checkpoints is - especially in humans where it is difficult to do such investigations.
We know that checkpoints exist because people have looked at the antibodies that B cells make as they develop. B cells develop initially in the bone marrow and are released into the blood as immature B cells (transitional type 1 [T1] cells) that mature into transitional type 2 (T2) cells before becoming mature naïve B cells. Through each stage, the percentage of B cells that have harmful antibody specificities decreases, and this must involve a checkpoint of some kind.
We have shown that B cells at the T2 stage of development preferentially go into the part of the immune system responsible for protecting the intestines (the gut-associated lymphoid tissue [GALT]). GALT is particularly rich in fragments of harmless bugs from the intestine that drive the production of the antibodies that are destined to enter the gut in the mucus. Unlike most of the rest of the immune system, GALT is constantly active from very soon after birth. When the T2 immature B cells are in this setting some become activated. Since most B cells are likely to pass through the gut at some point, this must have consequences because the B cell population would mature without the ones activated in GALT.
To understand this process further we wish to answer the following 4 questions:
1. When B cells become activated in GALT, do they live or die? Are the cells that are activated selective removed and disposed of or actually used in some way?
2. Is there any difference in the antibody reactivities of the B cells that are activated compared to those that are not? This would help us to understand the affect this process has on the developing B cell population and how this process is likely to shape the entire mature B cell population.
3. Are the T2 cells activated in GALT going on to make antibodies that will pass into intestinal mucus?
4. The GALT is constantly activated by bacterial fragments from the gut. Do the T2 subset of cells that are activated have some kind of role in regulating that process?
This project is important because it investigates a phase in human B cell development that we have discovered and that was not known at all before. We have evidence that failure of this phase results production of harmful antibodies. We must understand the basic properties of the system we have discovered to understand how GALT protects from harmful antibody production and to find the best way to develop this field for maximum impact on human health.

We have shown that the T2 subset of transitional B cells enters the gut associated lymphoid tissue where some become activated. We hypothesise that the response of T2 cells to the microbiota in GALT determines their fate, thus constituting a checkpoint in B cell development. We have evidence that this system fails in SLE and could thus protect against autoimmunity. We now wish to ask:

1. Do activated transitional B cells in GALT have the properties of cells that will live or die? We know that cells enter and become activated in GALT, but the tissue does not accumulate them, so we wish to decipher their fate by flow cytometry and cell culture.

2. How do the specificities of the activated versus the non-activated cells differ? Whatever the fate of transitional B cells, it is likely to be dependent on the specificity of the B cell receptor. We propose to determine the specificity of T2 cells in GALT that are activated or not, and compare their specificities.

3. Is there any evidence that activated transitional B cell can enter the IgA response? The intestinal IgA response is the largest and most extensive B cell response in the body. Some of this IgA is known to be polyspecific or autoimmune which are also features of transitional B cells that are removed from the B cell repertoire as it matures. We will therefore ask if GALT transitional B cells can be co-otped into the IgA response.

4. Is there evidence that transitional B cells activated in GALT acquire functional properties? Some transitional cells can be immunomodulatory, a property that would be highly relevant to the mucosal microenvironment.

Extravasation of T2 cells into GALT and activation of a subset of them would alter the repertoire of the T2 cell population that remained naive and unactivated. Understanding the consequences of this for intestinal and systemic immunity is therefore a high priority.

This project will have academic impact by generating published work and data for presentations at scientific meetings.

Probably the major impact will be enhancement of quality of life, health and well being. The applicants work closely with clinical teams and the relevance of data to disease pathogenesis and patient care is constantly under discussion. Once published, findings developed in this way can have broad clinical impact. Several examples of how impact has previously evolved or is currently evolving in this way in our lab are described below:

1. Gastric lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma): We observed in the lab that tumour cells from gastric MALT lymphoma divide in vitro only in the presence of T cells and Helicobacter pylori organisms. This was coupled with clinical and pathological observations of associations between gastric MALT lymphoma and Helicobacter pylori. This justified treating patients with gastric MALT lymphoma with Helicobacter pylori eradication therapy that resulted in tumour regressions in some cases, thus directly impacting patient care globally.

2. Orofacial granulomatosis (OFG): This was previously classified as chronic oral inflammation that was a manifestation of Crohn's disease. We identified that the OFG lesions included IgE producing cells. This supported a clinical study showing effectiveness of dietary modification in treating OFG. These observations together changed national guidelines for treating this condition.

3. Granulomatosis with polyangiitis (GPA): This is an autoimmune disease associated with inflammation of the nasal mucosa and upper airways and the production of pathogenic autoantibodies. Patients can achieve remission following B cell depletion treatment with rituximab that may be repeated regularly for some patients. We have observed that indication for re-treatment may be more easily visualised by quantifying the frequencies of T cell subsets, the profile of which can start to change even before blood borne B cells have re-populated. This is work in progress that illustrates how clinicians and scientists working together can consider direct clinical impact and consider changes in practice alongside the generation of data for peer review publications.

We thus have a strategy for clinical impact and a track record to demonstrate success.