Peter M. Dammers

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 (VH) genes by H-CDR3 spectrotyping, cloning, and sequencing of VH genes from murine intestinal IgA-producing plasma cells reveals a very restricted usage of VH genes and multiple clonally related sequences. The restricted usage of VH 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 VH 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.

Most Marginal Zone B Cells in Rat Express Germline Encoded Ig VH Genes and Are Ligand Selected

The present study was performed to analyze whether marginal zone B (MZ-B) cells in nondeliberately immunized adult rats are selected on basis of the specificity of their B cell receptor, and to determine to what extent memory B cells contribute to the MZ-B cell subset. To this end, the Ig PC7183 VH gene repertoire was studied among VHDJH-μ transcripts expressed in four sequential stages of B cell development, of two individual untreated adult rats. B cell subsets, i.e., pro/pre-B cells and newly formed B (NF-B) cells from bone marrow, and recirculating follicular B cells and MZ-B cells from spleen were sorted by flow cytometry. In addition, from one these rats, cells were microdissected from follicular and MZ areas of the spleen and productive PC7183 VH gene rearrangements were analyzed for the presence of somatic mutations. Sequence analysis reveals that most MZ-B cells in the adult rat, either defined by flow cytometry or by their anatomical location in the spleen, express germline encoded VH genes (naive MZ-B cells) and a minor fraction (about 20%) of the MZ-B cells carry somatic mutations (memory MZ-B cells). In addition, we show that naive MZ-B cells are a selected population of cells, both based on PC7183 VH gene repertoire and on the length of the Ig heavy (H) chain complementarity-determining region 3 (H-CDR3) region, i.e., PC7183 VHDJH-μ transcripts of MZ-B cells carry significantly shorter H-CDR3 regions than other B cell subsets.

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 VH 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 ∼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.

Presence of Germline and Full-Length IgA RNA Transcripts Among Peritoneal B-1 Cells

Next to conventional B cells (or B-2 cells), peritoneal B-1 cells have been shown to contribute significantly to the production of IgA-secreting plasma cells in the gut. Evidence for this was mainly based on studies comprising manipulated animals, including lethally X-irradiated and transgenic mice. To examine the ability of peritoneal B-1 cells from untreated mice to switch actively to IgA in vivo, we performed RT-PCR analysis on FACS-sorted peritoneal B-cell subsets from untreated BALB/c mice in order to examine the presence of germline Cα mRNA and mature Cα mRNA transcripts. Germline Cα and mature Cα transcripts were readily detectable in peritoneal B-1 cells (defined as IgMbright/IgDdull), but not, or very little, in peritoneal B-2 cells (defined as IgMdull/IgDbright). Moreover, by subdividing the B-l-cell population in CD5+ B-1a cells and CD5- B-1b cells, it was shown that in vivo expression of germline Cα and mature Cα transcripts was largely restricted to the B-1b-cell lineage. These results indicate that peritoneal B-1 cells indeed are capable to switch to IgA under normal physiological conditions and hereby further support the view that B-1 cells contribute significantly to the mucosal IgA response, albeit this function appears to be restricted to the B-1b-cell subset.

Slow recovery of follicular B cells and marginal zone B cells after chemotherapy: implications for humoral immunity.

Although most chemotherapeutic agents are known to cause primarily reduction or suppression of immune responses, surprisingly little is known about the influence of cytostatic agents on lymphoid tissue compartments such as the splenic marginal zone. The marginal zone plays an important role in the defence against encapsulated bacteria, which are potential candidates for postchemotherapeutic infections. We studied the effect of three different cytostatic agents (cisplatin, methotrexate, and cyclophosphamide) on B cell subpopulations in a rat model. Rats received a single dose of a single cytostatic agent and were sacrificed at different time points after treatment. Bone marrow, blood, mesenteric lymph nodes and spleens were analysed by flow‐cytometry and immunohistochemistry. All three cytostatic agents showed severe bone marrow depression. CP and MTX showed only mild reduction of cell populations in the spleen. CyPh showed a severe reduction of recirculating follicular B (RF‐B) cells and marginal zone B (MZ‐B) cells. At day 24 most populations were already recovered, but RF‐B cells and MZ‐B cells were still reduced. The reduction of the marginal zone and late recovery may imply that, beside the overall increased infection risk due to neutropenia, patients treated with chemotherapy are at risk for developing infections from encapsulated bacteria for a considerable period of time after treatment, extending beyond the period of bone marrow depression.

Immunoglobulin VH‐gene usage of autoantibodies in mercuric chloride‐induced membranous glomerulopathy in the rat

Brown‐Norway (BN) and Dorus Zadel Black (DZB) rats develop a T‐cell‐dependent membranous glomerulopathy (MGP) with high proteinuria and antiglomerular basement membrane (GBM) autoreactive antibodies (Abs), upon exposure to mercuric chloride (HgCl2). Laminin is an important autoantigenic target of the anti‐GBM Abs, absorbing ≈ 30% of the anti‐GBM reactivity. Although many anti‐GBM Abs have undergone isotype switching, it is currently unclear whether affinity maturation occurs during the HgCl2‐induced autoimmune response. To address this question we analysed the rearranged immunoglobulin heavy chain variable‐region genes (VHDJH regions) of 15 mAbs that were previously obtained from HgCl2‐treated rats. Seven of these mAbs exhibit reactivity towards laminin. Our study showed that the VH‐gene usage of antilaminin mAbs is largely restricted to the PC7183 VH‐gene family (six out of seven). In addition, we demonstrated that at least three out of six laminin reactive and five out of six non‐laminin‐binding mAbs are encoded by germline VH genes (a total of eight out of 12 mAbs). Of the eight mAbs that are encoded by germline VH genes, seven are of a non‐immunoglobulin M (IgM) isotype, indicating that isotype switching has occurred in these mAbs in the absence of somatic mutations. The mutations observed in the VH genes of the four remaining mAbs do not provide strong evidence for antigenic selection. The data support the notion that B cells in this model of MGP are not subjected to affinity maturation and probably result from polyclonal B‐cell activation.

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.

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.