Author: Douwe de Graaf (s1022717)

B-cells, bone marrow-derived lymphocytes, play a crucial role in the humoral immune response, as part of the adaptive immune system. B-cells can perform several functions, like displaying foreign antigens as an antigen presenting cell and after activation becoming antibody-secreting plasma cells or memory cells. There are two types of B-cell activation: T-cell dependent and T-cell independent 1, 2. T-dependent activation takes place in germinal centers (GCs) in peripheral lymph nodes 3. APCs present an antigen, which is recognized by a T-cell, that will proliferate and differentiate to effector and memory T-cells. Helper T-cells then activate B-cells, which eventually will differentiate into plasma cells and start to produce antigen specific antibodies in peripheral tissues. In GCs, B-cells switch from the IgM isotype to a downstream type like IgA, IgD, IgE and IgG, so called class switch recombination (CSR). Somatic hypermutation of V region genes (SHM) also takes place in GCs, by which antibodies acquire a higher affinity to the recognized antigen 4, 5. CSR, SHM and B-cell expansion may also take place outside GCs by T-cell independent B-cell activation. This type of activation takes place without co-stimulation of helper T-cells and is caused directly by antigens or by antigen presenting macrophages and related signalling molecules 6.

Continuously, the human gut is exposed to lots of antigens. Therefore it requires a variety of strategies to protect the body, like humoral and cellular immune responses. Specific intestinal humoral immunity is mediated by IgA en IgM producing B-cells in the lamina propria (LP) of the mucosa 7. 70-80% of all Ig producing cells are found in the intestinal mucosa, which makes it a significant lymphoid organ and the biggest source of IgA 4. IgA+ B-cells are thought to originate from precursors in Peyer's patches and isolated lymphoid follicles of gut associated lymphoid tissue (GALT). Research has shown that CD40 knockout mice still have got normal numbers of intestinal IgA+ B-cells although they can not make GCs 8. In humans T-independent CSR may take place in the LP of the gut, driven by APRIL, a cytokine that is produced by epithelial cells in the colon 9. The VH gene repertoire of these colonic IgA+ B-cells is rather limited, which could be caused by clonal relationships between these cells 10. These data suggest that precursors of IgA+ plasma cells are able to expand in the intestinal LP. Also, as soon as in 1978 research already showed that that local expansion of IgA+ cells may exist 11.

Recently, Yavaraj et al. published their article 'Evidence for Local Expansion of IgA Plasma Cell Precursors in Human Ileum', which deals with the contribution of local expansion of IgA producing B-cells to the total small intestinal IgA plasma cell pool 12. Because there is still a lot unknown about local expansion of B-cells, this may give an answer to this process in the gut.

For this research, biopsies of the ileum of patients, undergoing endoscopy for different problems, where used. These biopsies were treated with an IgA specific immunohistological staining and IgA plasma cells were counted. Of these IgA producing plasma cells, the VH gene usage, clonal relationships between B cells in a sample, mutation patterns in these B cells were determined. With these data, they were able to determine the number of precursor B cells that gave rise to the B cells found in the biopsies.

After molecular cloning and H-chain CDR3 (H-CDR3) spectrotyping, IgA VH gene sequence analysis revealed that there were many clonally related sequences within a biopsy, which could not be found between different biopsies. They estimated that ~100 - 300 precursor B cells were responsible for the 75.000 IgA producing cells per biopsy. This means that these precursor cells, after leaving the GC, must have divided 9-10 times to reach this number of cells. All B-cells contained mutations. In clonally related sequences, most of these mutations were also shared. No clonally related sequences between different samples from the same individual were found. This means that that these cells must have arrived as mutated cells in the LP and must expanded locally. Taken together, these data suggest that after the well known proliferation and expansion of B-cells in GCs, a second wave of IgA producing B-cell expansion takes place in the LP of the small intestine.

The function and location of IgA producing B-cells is not completely known. Although it is clear that CSR and SHM take place in GCs, research has shown that these processes may also occur outside GCs. Still, it is unclear where this T-cell independent CSR and B-cell expansion take place. The authors of this article have tried to answer this question concerning B-cells in the LP of the gut.

Before this article was published, it was thought that T-cell independent B-cell expansion took place in peripheral tissues without being processed in GCs 9. However, Yuvaraj et al. have shown that most of these clonally related B-cells underwent CSR and SHM in GCs. These selected and committed cells went back to gut tissue and had a second wave of expansion in the LP of the gut. This expansion is probably the result of direct interaction between products of intestinal microorganisms and these B-cells. Expansion of these cells is probably necessary for homeostasis of the intestinal flora 12.

This article is important because for a long time it was thought that selected and committed B-cells, could not expand anymore after leaving the GCs. This research shows that this is not the case. A second wave of expansion might be a common feature of B-cells. These new insights might change the general way we look at this subtype of lymphocytes.

In this article a new approach to determine clonal relationship between B-cells was used. To estimate the number of precursor B-cells that gave rise to all IgA producing cells in a biopsy they used 'covering of sampling'. Here for they used I.J. Good's formula 'coverage = (1 -n/N) * 100%', in which 'n' is the number of unique sequences and N is the total number of different sequences. This formula was originally developed by the United Kingdom to break German codes in WO II and later on used in bird watching. 100% coverage means that every new sample is already known and related to another sample. When every new sample is unknown, the coverage is 0%. In this research a sample was a IgA producing B-cell. Good's formula served very well to determine the numbers of precursor B-cells and the number of times these cells must have divided to cause this level of expansion 13.

A number of earlier studies that researched clonal relationships between different biopsies of the same individual only investigated a very restricted population of IgA+ cells, by amplification of a single VH gene (VH 4-34) 14, selection of particular H-CDR3 lengths 10 and detection of antigen-specific IgA production after vaccination 15, which all conclude that clonally related IgA+ B-cells can be found at distant sides in the gut. However, Yavaray et al. looked at potentially all VH genes instead of a selection, by which they didn't observe clonally related sequences 12.

Despite the innovative use of an old formula, this research also has some weak points. Although they have shown that a second wave of expansion occurs after the first one, they did not investigated which signals and mechanisms are responsible for their findings. To fully understand this second wave of B-cell expansion, they should elucidate these points also. The perspective should be broader to be able to determine if their discovered mechanism holds true for all IgA producing B-cells in the human body.

Before this research it was not completely known if plasma cells can expand in the human ileum, outside GCs 6. These data strongly suggest that this is the case. What previously was thought to be T-cell independent B-cell expansion could also be a second wave of expansion 12. With this new information in mind, these researches should be repeated to find out if it really was T-cell independent expansion of 'nave' B-cells or a second wave of expansion of selected and committed B-cells.

By using Good's formula 13 to estimate the 'coverage of sampling' of B-cells in a biopsy it is now possible to cluster clonally related ileal IgA producing cells derived from one precursor cell. Before this research this has not been done. This formula has not been used in a similar way before. Probably it can also be used in other fields of biomedical sciences to get more accurate and better results.

The authors did not mention any therapeutic perspectives, because they only tried to elucidate a natural occurring mechanism of human B-cells. They do point out that somatic mutations outside GCs might result in poly reactive and/or auto reactive Abs 16, which might cause disease. A better understanding of the ways that B-cells expand may be a way to treat immunological diseases in a different way.

Because patient material was used, it is only possible to use a limited amount of gut tissue. Animal models can make it possible to research the phenomenon of second wave expansion more thoroughly. In animal models, the level of local expansion in the gut could be determined more precisely by techniques like single cell picking and analysis of the contribution of Toll-like receptors (TLRs) and cytokines like APRIL and BAFF, which all play a role in T-independent B-cell activation 9, 17-19. Also animal models should be investigated which lack the first or second wave of expansion 8.

Before these new findings are understood completely, signaling molecules, signaling pathways and cellular mechanisms involved in local B-cell expansion, should be investigated thoroughly. Certainly this will change our knowledge about local B-cell expansion.


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