Laurent Boursier

Characteristics of Human IgA and IgM Genes Used by Plasma Cells in the Salivary Gland Resemble Those Used in Duodenum But Not Those Used in the Spleen

Immunologically, the parotid salivary gland is an effector site that secretes large quantities of polyspecific Abs into the saliva, mainly of the IgA isotype. It is considered to be part of the common mucosal immune system but the inductive site for the Ab-producing cells of the salivary gland has not yet been clearly identified. The origin and diversity of cells of B lineage can be investigated by analyzing their Ig heavy chain genes (IgH). We have obtained sequences of IgM and IgA VH4–34 genes from plasma cells in human salivary gland, duodenal lamina propria, and splenic red pulp. Related sequences were found in different areas sampled within each tissue studied, indicating that the plasma cells carrying these genes are widespread with limited diversity. Examples of related IgH genes that are isotype switched were also seen in the salivary gland. The genes from plasma cells of the salivary gland were highly mutated, as were duodenal plasma cell sequences. The level of mutation was significantly higher than that seen in splenic plasma cell sequences. Analysis of CDR3 regions showed that the sequences from salivary gland had significantly smaller CDR3 regions than sequences from spleen, due to differences in number and type of DH regions used. Sequences from duodenum also had smaller CDR3 regions. Therefore, plasma cells from human duodenum and salivary gland showed characteristics that differed from those of human splenic plasma cells.

Analysis of immunoglobulin genes in splenic marginal zone lymphoma suggests ongoing mutation

Splenic marginal zone lymphoma (SMZL) is a low-grade primary splenic B cell lymphoma, originally thought to be related to splenic marginal zone B cells. Later studies showed that SMZL sometimes may be accompanied by villous lymphocytes in the peripheral blood, a condition previously characterized as splenic lymphoma with villous lymphocytes (SLVL). The relationship between SMZL and splenic marginal zone B cells has recently been called into question. We report four further cases of SMZL, two of which were associated with villous lymphocytes in the peripheral blood. In addition to immunophenotypical analysis, we have studied the IgV(H) genes in each case, because the extent and patterns of their mutation can indicate the normal B cell counterpart of lymphomas. The IgV(H) genes in the four cases of SMZL studied are mutated, which is consistent with their origin from postfollicular marginal zone B cells. Evidence of ongoing mutation was also observed. This contrasts with a study showing that blood-borne tumor cells in SLVL show no sign of ongoing mutation. It is possible that the ongoing mutations in the cases studied here are acquired in a splenic microenvironment, such as that found in the follicle center.

Effect of somatic hypermutation on potential N-glycosylation sites in human immunoglobulin heavy chain variable regions

Immunoglobulins are known to be variably glycosylated. Although most carbohydrate is likely to be associated with the constant region, the variable region may also be glycosylated. Variable region glycosylation is known to be associated with antigen binding; in one model, the presence of carbohydrate on the external surface of CDR2 was shown to increase the affinity by up to ten-fold. In this study we have studied the effect of somatic hypermutation on potential sites of N-glycosylation in IgV(H) genes used by mucosal plasma cells secreting IgM, IgA and IgG in adults and children. Mucosal plasma cells secreting IgM, IgA and IgG are all heavily mutated from childhood. We have observed a tendency to lose the germline encoded N-glycosylation sites in all populations studied (a range of 43-67% of genes showing loss of the site). The tendency to lose the site was associated with a higher frequency of somatic hypermutation. We have also analysed the tendency to create potential N-glycosylation sites, and observed that this was greater in IgA and IgG than IgM and was again associated with the high frequency of somatic hypermutation. We observed no evidence of selection for either loss or gain of potential N-glycosylation sites. The changes in potential glycosylation status associated with a high frequency of somatic hypermutation is likely to increase the diversity of mucosal immunoglobulins, but is not as likely to affect the peripheral immune system where the frequency of somatic hypermutation is generally lower.

IgA-Producing Plasma Cells Originate From Germinal Centers That Are Induced by B-Cell Receptor Engagement in Humans

BACKGROUND & AIMS IgA contributes to homeostatic balance between host and intestinal microbiota. Mechanisms that initiate the IgA response are unclear and likely to differ between humans and animal models. We used multiple experimental approaches to investigate the origin of human intestinal plasma cells that produce IgA in the gastrointestinal tract. METHODS Complexity of IgA-producing plasma cell populations in human gastrointestinal mucosa and bone marrow and the specific response to oral cholera vaccine were compared by analysis of immunoglobulin genes. Flow cytometry, gene expression analysis, and immunohistochemistry were used to analyze signaling pathways induced by B-cell receptor engagement in human gut-associated lymphoid tissue (GALT) and involvement of innate immunity in B-cell activation in GALT compared with nonintestinal sites. RESULTS Human intestinal IgA-producing plasma cells appeared to be of germinal center origin; there was no evidence for the population complexity that accompanies multiple pathways of derivation observed in bone marrow. In germinal center B cells of human GALT, Btk and Erk are phosphorylated, CD22 is down-regulated, Lyn is translocated to the cell membrane, and Fos and Jun are up-regulated; these features indicate B-cell receptor ligation during germinal center evolution. No differences in innate activation of B cells were observed in GALT, compared with peripheral immune compartments. CONCLUSIONS IgA-producing plasma cells appear to be derived from GALT germinal centers in humans. B-cell receptor engagement promotes formation of germinal centers of GALT, with no more evidence for innate immune receptor activation in the mucosa than nonintestinal immune compartments. Germinal centers in GALT should be targets of mucosal vaccinations because they are the source of human intestinal IgA response.

IgVH gene analysis suggests that peritoneal B cells do not contribute to the gut immune system in man

The contribution of peritoneal B cells to the intestinal lamina propria plasma cell population is well documented in mice, but unknown in humans. We have analyzed immunoglobulin (Ig) genes of human peritoneal B cells, because such genes show distinctive characteristics in mucosal B cells, particularly highly mutated variable regions. Here, we report the characteristics of variable regiongenes used by IgM, IgA and IgG in peritoneal cells. We focused on the properties of IgVH4–34 to allow comparisons of like‐with‐like between different isotypes and cells from different immune compartments. We observed that the IgM genes were mostly unmutated, and that the mutated subset had less mutations than would be expected in a mucosal B cell population. Likewise, the IgVH4–34 genes used by IgA and IgG from peritoneal B cells had significantly lower numbers of mutations than observed in the mucosal counterparts. Other trends observed, while not reaching statistical significance, followed the trend of peripheral B cells. The peritoneal B cell population had more IgA1 than IgA2 sequences, and there was no dominance of JH4 in the IgA from peritoneum or spleen, in contrast to the mucosal sequences. Overall, this study suggested that human peritoneal B cell are either peripheral or mixed in origin; they are unlikely to represent an inductive compartment for the mucosal B cell system.

Subepithelial dendritic B cells in orofacial granulomatosis

Background: Orofacial granulomatosis (OFG) is a chronic, disfiguring, granulomatous inflammation of the lips and oral mucosa. The pathogenesis is unknown, but it has been linked previously to Crohns disease (CD) and more recently to dietary sensitivity. The oral mucosa is an immunologically responsive site associated with the generation of protective mucosal and systemic immune responses to vaccination and also hyperresponsiveness to allergens in some individuals. Classically, immune responses in oral mucosa are considered to be mediated by mucosa‐associated lymphoid tissues (MALT), secondary lymphoid follicles that are intimately associated with epithelia. Methods: Immunohistochemistry was used to investigate the inflammatory infiltrate in OFG and control tissue samples. Polymerase chain reaction (PCR), cloning of PCR products, and sequencing were used to characterize the local immunoglobulin gene profile in OFG. Results: We describe large, active, dendritic B cells in oral mucosa that were not associated with any organized lymphoid tissues in the local subepithelial microenvironment. They express activation induced cytidine deaminase, which is essential for immunoglobulin gene diversification by somatic hypermutation and class switch recombination. IgE is also expressed by these B cells. They do not align with any other previously described B‐cell subset in secondary lymphoid tissues in terms of morphology, proliferative activity, or phenotype. Conclusions: These subepithelial dendritic B cells may contribute to the immune responsiveness of the oral mucosa, including IgE‐mediated allergic responses. In patients with OFG, further understanding of the role these cells play in oral immunity may lead to novel therapeutic possibilities. (Inflamm Bowel Dis 2010)

Lambda Light Chain Revision in the Human Intestinal IgA Response

Revision of Ab L chains by secondary rearrangement in mature B cells has the potential to change the specific target of the immune response. In this study, we show for the first time that L chain revision is normal and widespread in the largest Ab producing population in man: intestinal IgA plasma cells (PC). Biases in the productive and non-productive repertoire of λ L chains, identification of the circular products of rearrangement that have the characteristic biases of revision, and identification of RAG genes and protein all reflect revision during normal intestinal IgA PC development. We saw no evidence of IgH revision, probably due to inappropriately orientated recombination signal sequences, and little evidence of κ-chain revision, probably due to locus inactivation by the κ-deleting element. We propose that the λ L chain locus is available and a principal modifier and diversifier of Ab specificity in intestinal IgA PCs.

B cell development and proliferation of mature B cells in human fetal intestine.

B cells are present in human fetal intestine from approximately 14 weeks of gestation. Here we show that this population includes mature, dividing B cells. These are large cells with dendritic processes, resembling human thymic B cells. In addition, we observed IgM+, light chain−, and CD20− cells and local expression of V pre‐B, demonstrating that the human fetal intestine is a site of B cell development. Ig VHDJH gene sequencing can confirm clonal identity of B cells. Identification of the same IgVH4–34 sequence in serial sections in two fetuses confirmed local accumulation of related cells in each case. IgVH4–34 was also amplified from an additional two samples, and the D and J repertoire compared with a unique database of unselected VH4–34 genes from postnatal gut. Distinguishing characteristics of Ig λ genes in postnatal gut were also studied in the fetus. According to these parameters, fetal and postnatal B cells are unrelated.

Mathematical analysis of antigen selection in somatically mutated immunoglobulin genes associated with autoimmunity

Affinity maturation is a process by which low-affinity antibodies are transformed into highly specific antibodies in germinal centres. This process occurs by hypermutation of immunoglobulin heavy chain variable (IgH V) region genes followed by selection for high-affinity variants. It has been proposed that statistical tests can identify affinity maturation and antigen selection by analysing the frequency of replacement and silent mutations in the complementarity determining regions (CDRs) that contact antigen and the framework regions (FRs) that encode structural integrity. In this study three different methods that have been proposed for detecting selection: the binomial test, the multinomial test and the focused binomial test, have been assessed for their reliability and ability to detect selection in human IgH V genes. We observe first that no statistical test is able to identify selection in the CDR antigen-binding sites, second that tests can reliably detect selection in the FR and third that antibodies from nasal biopsies from patients with Wegener’s granulomatosis and pathogenic antibodies from systemic lupus erythematosus do not appear to be as stringently selected for structural integrity as other groups of functional sequences.

Biased JH usage in plasma cell immunoglobulin gene sequences from colonic mucosa in ulcerative colitis but not in Crohn’s disease

BACKGROUND Ulcerative colitis is an inflammatory disease of the colonic and rectal mucosa. Autoantibodies have been observed in ulcerative colitis which may have a role in the pathogenesis of the disease. Evidence also suggests that there is an hereditary predisposition towards the disease, although no individual genes have been identified. AIMS This is a pilot study of immunoglobulin heavy chain genes (IgH) in ulcerative colitis to determine whether they have any particular genetic characteristics which may lead to a better understanding of the disease aetiology. SUBJECTS Colonic or rectal tissue was obtained from five children with ulcerative colitis. Tissue was also obtained from five children with Crohn’s disease and five children who did not have inflammatory bowel disease as controls. METHODS B cells and IgD+ B cells were identified by immunohistochemistry on frozen sections. Areas of lamina propria containing plasma cells, and areas of IgD+ B cells were microdissected. The immunoglobulin genes were PCR amplified, cloned, and sequenced. Sequences were analysed for content of somatic mutations and composition of heavy chain. RESULTS An increase in the use of JH6 and DXP′1, and a decrease in the use of JH4, gene segments in immunoglobulin genes from lamina propria plasma cells, and from virgin IgD+ B cells, was found in patients with ulcerative colitis. These biases were not present in the control groups. CONCLUSIONS There is a fundamental difference in the immunoglobulin genes from patients with ulcerative colitis. Whether this is caused by a difference in content of immunoglobulin gene segments in the germline or a difference in the recombination mechanism is not known.