The formation of mutated IgM memory B cells in rat splenic marginal zones is an antigen dependent process.

Previous studies in rodents have indicated that only a minor fraction of the immunoglobulin heavy chain variable region (IGHV-Cµ) transcripts carry somatic mutations and are considered memory B cells. This is in marked contrast to humans where nearly all marginal zone B (MZ-B) cells are mutated. Here we show in rats that the proportion of mutated IgM+ MZ-B cells varies significantly between the various IGHV genes analyzed, ranging from 27% mutated IGHV5 transcripts to 65% mutated IGHV4 transcripts. The observed data on mutated sequences in clonally-related B cells with a MZ-B cell or follicular B (FO-B) cell phenotype indicates that mutated IgM+ MZ-B and FO-B cells have a common origin. To further investigate the origin of mutated IgM+ MZ-B cells we determined whether mutations occurred in rearranged IGHV-Cµ transcripts using IGHV4 and IGHV5 genes from neonatal rat MZ-B cells and FO-B cells. We were not able to detect mutations in any of the IGHV4 and IGHV5 genes expressed by MZ-B cells or FO-B cells obtained from neonatal rat spleens. Germinal centres (GCs) are absent from neonatal rat spleen in the first few weeks of their life, and no mutations were found in any of the neonatal sequences, not even in the IGHV4 gene family which accumulates the highest number of mutated sequences (66%) in the adult rat. Therefore, these data do not support the notion that MZ-B cells in rats mutate their IGHV genes as part of their developmental program, but are consistent with the notion that mutated rat MZ-B cells require GCs for their generation. Our findings support that the splenic MZ of rats harbors a significant number of memory type IgM+ MZ-B cells with mutated IGHV genes and propose that these memory MZ-B cells are probably generated as a result of an antigen driven immune response in GCs, which still remains to be proven.

Class-switched marginal zone B cells in spleen have relatively low numbers of somatic mutations.

The vast majority of rodent splenic marginal zone (MZ)-B cells are naive IgM(+) cells. A small fraction of these MZ-B cells carry mutated V-genes, and represent IgM(+) memory MZ-B cells. Here we reveal further heterogeneity of B cells with a MZ-B cell phenotype, by providing evidence for the existence of class-switched memory MZ-B cells in the rat. In essence, we observed IGHV5 encoded Cγ transcripts, among FACS-purified MZ-B cells, defined as HIS24(low)HIS57(bright) cells. Furthermore, we found that most IgG encoding transcripts are mutated. There is no significant difference in IGHV5 repertoire and subclass usage of these IgG encoding transcripts collected from B cells with a MZ-B cell phenotype and B cells with a follicular (FO) B cell phenotype. However, the IGHV5 genes encoding for IgG antibodies of MZ-B cells exhibited significantly fewer mutations, compared to those with a FO-B cell phenotype. In one rat we found a clonally related set of IgG encoding sequences, of which one was derived from the MZ-B cell fraction and the other from the FO-B cell fraction. We speculate that these two subpopulations of class-switched B cells are both descendants from naive FO-B cells and are generated in germinal centers. Class-switched memory cells with a MZ-B cell phenotype may provide the animal with a population of IgG memory cells that can respond rapidly to blood-borne pathogens.

Organization of the variable region of the immunoglobulin heavy-chain gene locus of the rat.

We have mapped and annotated the variable region of the immunoglobulin heavy (IGH) gene locus of the Brown Norway (BN) rat (assembly V3.4; Rat Genomic Sequence Consortium). In addition to known variable region genes, we found 12 novel previously unidentified functional IGHV genes and 1 novel functional IGHD gene. In total, the variable region of the rat IGH locus is composed of at least 353 unique IGHV genes, 21 IGHD genes, and 5 IGHJ genes, of which 131, 14, and 4 are potentially functional genes, respectively. Of all species studied so far, the rat seems to have the highest number of functional IGHV genes in the genome. Rat IGHV genes can be classified into 13 IGHV families based on nucleotide sequence identity. The variable region of the BN rat spans a total length of approximately 4.9 Mb and is organized in a typical translocon organization. Like the mouse, members of the various IGHV gene families are more or less grouped together on the genome, albeit some members of IGHV gene families are found intermingled with each other. In the rat, the largest IGHV gene families are IGHV1, IGHV2, and IGHV5. The overall conclusion is that the genomic organization of the variable region of the rat IGH locus is strikingly similar to that of the mouse, illustrating the close evolutionary relationship between these two species.

Evidence for local expansion of IgA plasma cell precursors in human ileum.

IgA plays a crucial role in establishment and maintenance of mucosal homeostasis between host cells and commensal bacteria. To this end, numerous IgA plasma cells are located in the intestinal lamina propria. Whether the (immediate) precursor cells for these plasma cells can expand locally is not completely known and was studied here. The total number of IgA plasma cells in human ileal biopsies was counted. Sequence analysis of IgA V(H) genes from human ileal biopsies revealed the occurrence of many clonally related sequences within a biopsy, but not between different biopsies. This observation strongly argues for local expansion of IgA precursor cells. By comparing the number of unique sequences with the number of clonally related sequences within a biopsy, we estimated that approximately 100-300 precursors were responsible for the 75,000 IgA-producing cells that were present per biopsy. These precursor cells must therefore have divided locally 9-10 times. Since all sequences contained mutations and most of the mutations present in clonally related sequences were shared, the IgA precursor cells must have arrived initially as mutated cells in the lamina propria. Our data show evidence for the existence of two waves of expansion for IgA-producing cells in human ileum. The first wave occurs during initial stimulation in germinal centers as evidenced by somatic hypermutations. A second wave of expansion of IgA-committed cells occurs locally within the lamina propria as evidenced by the high frequency of clonally related cells.

Recruitment and selection of marginal zone B cells is independent of exogenous antigens.

Marginal zone B (MZ-B) cells of the spleen contribute significantly to the immunity against invasive infections with polysaccharide-encapsulated bacteria. Recent evidence indicates that recruitment and selection of MZ-B cells occurs on the basis of positive selection constraints that likely operate via B cell receptor (BCR) signaling. Previous studies have shown that MZ-B cells carry relatively shorter immunoglobulin (Ig) heavy (H) chain complementarity-determining region 3 (H-CDR3) sequences and express BCR which are thought to be polyreactive. In this scenario, MZ-B cell selection proceeds via engagement of the BCR with exogenous (i.e. microbial gut flora-derived) and/or endogenous (self) antigens. Here, we studied the influence of exogenous antigens on the selection process of MZ-B cells using non-genetically manipulated adult germ-free and conventionally reared infant rats. This study was carried out by H-CDR3 spectratype analysis of V(H)(PC7183)-encoded Ig V(H)DJ(H)-mu transcripts expressed by purified splenic MZ-B cells and other B cell subsets. We show that MZ-B cells in both adult germ-free and conventionally reared infant (14-day-old) rats are H-CDR3-selected cells, providing strong evidence that recruitment and selection of MZ-B cells is driven by self antigens.

Restricted IgA repertoire in both B-1 and B-2 cell-derived gut plasmablasts.

Mucosal IgA is the most abundantly produced Ig upon colonization of the intestinal tract with commensal organisms in the majority of mammals. The repertoire of these IgA molecules is still largely unknown; a large amount of the mucosal IgA cannot be shown to react with the inducing microorganisms. Analysis of the repertoire of used H chain Ig (V(H)) genes by H-CDR3 spectrotyping, cloning, and sequencing of V(H) genes from murine intestinal IgA-producing plasma cells reveals a very restricted usage of V(H) genes and multiple clonally related sequences. The restricted usage of V(H) genes is a very consistent observation, and is observed for IgA plasma cells derived from B-1 or conventional B-2 cells from different mouse strains. Clonal patterns from all analyzed V(H) gene sequences show mainly independently acquired somatic mutations in contrast to the clonal evolution patterns often observed as a consequence of affinity maturation in germinal center reactions in peripheral lymphoid organs and Peyer's patches. Our data suggest a model of clonal expansion in which many mucosal IgA-producing B cells develop in the absence of affinity maturation. The affinity of most produced IgA might not be the most critical factor for its possible function to control the commensal organisms, but simply the abundance of large amounts of IgA that can bind with relatively unselected affinity to redundant epitopes on such organisms.

Marginal zone B cells in neonatal rats express intermediate levels of CD90 (Thy-1).

Here we show that marginal zone (MZ)-B cells in rats can already be detected in neonatal spleen from two days after birth. At this time point, morphologically distinct MZs are not present yet and the vast majority of B cells in spleen are located in a concentric area surrounding the T cell zones (PALS). Before MZs are obviously detectable in spleen (14 days after birth), MZ-B cells seem to be enriched at the outer zones of the concentric B cell areas. Similar to adult rats, neonatal MZ-B cells are intermediate-sized cells that express high levels of surface (s)IgM and HIS57 antigen, and low levels of sIgD and CD45R (HIS24). We show here, however, that in contrast to adult MZ-B cells, MZ-B cells (and also recirculating follicular (RF)-B cells) in neonatal rats express higher levels of CD90 (Thy-1). In adult rats, expression of CD90 on the B cell lineage is confined to immature B cells. We speculate that the expression of CD90 on neonatal MZ-B cells may have implications for their responsiveness to polysaccharide (T cell-independent type 2) antigens.