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.

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 lambda 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 kappa-chain revision, probably due to locus inactivation by the kappa-deleting element. We propose that the lambda 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 V(H)DJ(H) gene sequencing can confirm clonal identity of B cells. Identification of the same IgV(H)4-34 sequence in serial sections in two fetuses confirmed local accumulation of related cells in each case. IgV(H)4-34 was also amplified from an additional two samples, and the D and J repertoire compared with a unique database of unselected V(H)4-34 genes from postnatal gut. Distinguishing characteristics of Ig lambda genes in postnatal gut were also studied in the fetus. According to these parameters, fetal and postnatal B cells are unrelated.

Analysis of strand biased 'G'.C hypermutation in human immunoglobulin V(lambda) gene segments suggests that both DNA strands are targets for deamination by activation-induced cytidine deaminase.

Somatic hypermutation. which diversifies the immunoglobulin repertoire by introducing mutations into rearranged IgV genes, is dependent on the expression of activation-induced cytidine deaminase (AID). It has been proposed that AID deaminates DNA directly, generating mutations at C bases. Mutations from C and G are linked, and it has been suggested that mutations from G arise either during repair of DNA following deamination of C or by deamination of C on both DNA strands. Studies demonstrating that AID deaminates ssDNA on the non-transcribed strand support the former hypothesis. However, analyses of microsequences surrounding mutations suggest that the G.C mutator acts on both DNA strands equivalently. Unusually, in human IgV(lambda) genes, there is G.C strand bias favoring mutation from G. In IgV(lambda), 92% of mutations from G occur in GNW motifs. Hotspots for mutation from G IgV(lambda) are often independent of C nucleotides in the context of local microsequence. This independence of G and C mutation, yet retained dependence on local microsequence suggests that mutations from G arise independent of C on the non-transcribed strand. We suggest that both DNA strands are deaminated and that the transcribed strand is preferentially deaminated in human IgV(lambda) resulting in bias towards mutations from G.

Imprint of somatic hypermutation differs in human immunoglobulin heavy and lambda chain variable gene segments.

Somatic hypermutation (SHM) introduces mutations into immunoglobulin (Ig) variable gene segments, thus diversifying the B cell repertoire prior to positive selection of high affinity variants during maturation of T cell-dependent B cell responses. Somatic hypermutation of Ig heavy chain generates predominantly single base substitutions, favoring transition rather than transversion substitutions, and tends to direct mutations to specific 4-mer target sequences with G in second and C in third position. Here we have analyzed heavily mutated, nonproductively rearranged Ig lambda chain variable gene segments from human intestinal plasma cells, controlling for germline composition of the genes and local sequence variability. We have observed significant G.C strand bias in IgV(lambda), and differences in some di- and trinuleotide target preferences in IgV(lambda) compared to IgV(H). There is also a significant tendency to accumulate adjacent triplet mutations in IgV(lambda), which is not evident in IgV(H) in normal circumstances. These observations suggest that some aspect of the mechanism of somatic hypermutation operates differently in human immunoglobulin heavy and lambda light chain variable gene segments.

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 Crohn's 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.

Characterization of cells of the B lineage in the human adult greater omentum.

Peritoneal B cells and their omental precursors play an important role in the immune response of the peritoneal cavity and mucosal surfaces in mice. We have previously shown that peritoneal and mucosal B lineage cells are unlikely to be significantly linked in humans. However, the status of the omentum remains unknown. Here, using immunohistochemistry, we observed that sparse, quiescent B cells and occasional clusters of B cells were present in the omentum and that plasma cells, predominantly with cytoplasmic immunoglobulin G (IgG), were present. We analysed sequences of immunoglobulin genes amplified using reverse transcriptase-polymerase chain reaction (RT-PCR) from the normal human greater omentum, and describe the characteristics of variable region genes used by IgG, IgA and IgM. We focused on the properties of IgVH4 and IgVH5 families to allow comparisons of like with like between different Ig isotypes and cells from different immune compartments. We observed that the IgM genes were derived from a mixed population with mutated and unmutated immunoglobulin sequences. All IgVH4 and IgVH5 genes used by IgA and IgG from omental cells showed evidence of somatic hypermutation but the load of mutations was not significantly different to that seen in either the systemic or the mucosal compartments. The trends observed, including the dominance of IgG plasma cells, the IgA1/IgA2 ratio being biased towards IgA1, JH1 usage, and a moderate level of somatic mutations, link omental B lineage cells with the systemic compartment. These observations reinforce previous studies highlighting the difference between human and murine B-cell compartments and their relationship to the mucosal immune system.

Human intestinal IgA response is generated in the organized gut-associated lymphoid tissue but not in the lamina propria.

BACKGROUND & AIMS: The intestinal lamina propria has traditionally been viewed as the effector site of mucosal immune responses. However, this view has been challenged with the identification, in the murine lamina propria, of an in situ class switch DNA recombination pathway to IgA. In this study, we tested the hypothesis that in situ class switching occurs in the human lamina propria. METHODS: Immunohistochemistry was used to analyze tissue microenvironments and RT-PCR to look for molecular evidence of Ig class switching and to track clonally related cells of B lineage. RESULTS: We found no evidence of proliferation of either lamina propria CD20+ or CD19+ cells or evidence of activation-induced cytidine deaminase mRNA expression outside the organized gut-associated lymphoid tissue, although I alpha-C alpha immunoglobulin germ-line gene transcript expression could be identified in the lamina propria. We identified clonally related cells, including IgA and IgM isotype-switched variants, in multiple samples known to be free of activation-induced cytidine deaminase, organized lymphoid tissue, or cellular proliferation. For 4 groups of cells, the patterns of somatic mutations on the rearranged IgV(H)5 gene segment were more similar between cells from distant sites than from their immediate neighbors, implying dissemination of cells from a common set of precursors. CONCLUSIONS: Our data are inconsistent with a model in which precursors of human IgA-secreting plasma cells are induced or expanded in the lamina propria. The human lamina propria is therefore likely to solely be an effector site of intestinal secretory IgA responses that originate from the organized gut-associated lymphoid tissues.

Biases in Ig lambda light chain rearrangements in human intestinal plasma cells.

Human intestinal lamina propria plasma cells are considered to be the progeny of chronically stimulated germinal centers located in organized gut-associated lymphoid tissues such as Peyer's patches and isolated lymphoid follicles. We have sampled human colonic lamina propria plasma cells and naive and memory B cell subsets from human Peyer's patches by microdissection of immunohistochemically stained tissue sections and used PCR methods and sequence analysis to compare IgVlambdaJlambda rearrangements in the plasma cell and B cell populations. Rearrangements that were either in-frame or out-of-frame between V and J were compared. Usage of IgVlambda families in the in-frame rearrangements from the plasma cells resembled that observed in the mantle cells, suggesting that antigenic selection for cellular specificity does not dramatically favor any particular Vlambda segment. However, in marked contrast, out-of-frame rearrangements involving Vlambda1 and Vlambda2 families are rarely observed in intestinal plasma cells, whereas rearrangements involving Vlambda5 are increased. This resulted in significantly biased ratios of in-frame:out-of-frame rearrangements in these Vlambda families. Out-of-frame rearrangements of IgVlambdaJlambda from plasma cells, including those involving the Vlambda5 family, have a significant tendency not to involve Jlambda1, consistent with the hypothesis that this population includes rearrangements generated by secondary recombination events. We propose that modification of out-of-frame rearrangements of IgVlambdaJlambda exists, probably a consequence of secondary rearrangements. This may be a mechanism to avoid translocations to susceptible out-of-frame IgVlambdaJlambda rearrangements during somatic hypermutation.

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 region genes used by IgM, IgA and IgG in peritoneal cells. We focused on the properties of IgV(H)4-34 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 IgV(H)4-34 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 J(H)4 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.