Annie Visser

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.

Serum levels of BAFF, but not APRIL, are increased after rituximab treatment in patients with primary Sjögren's syndrome: data from a placebo-controlled clinical trial

B cell depletion therapy with rituximab (RTX; 2 weekly infusions of 1000 mg, premedication: 100 mg prednisolone) in primary Sjogrens syndrome (pSS) patients is effective in reducing subjective and objective symptoms.1 As B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are important cytokines involved in B cell survival and activation, we assessed in pSS patients included in a double-blind, randomised, placebo-controlled trial1 the effects of RTX on serum BAFF and APRIL levels up to 48 weeks after RTX. Serum concentrations of BAFF and APRIL were measured by ELISA using kits from RD median 1277 pg/ml (range 907–3802 pg/ml)) compared with healthy controls (n=10; median 983 pg/ml (range 600–1564 pg/ml)); p<0.01; figure 1A). Also, baseline serum APRIL levels were significantly higher in pSS patients (median 15 098 pg/ml (range 1891–228 591 pg/ml)) than in healthy controls (median 1965 pg/ml (range 889–4567 pg/ml); p<0.05; …

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.

Persistence of immunoglobulin-producing cells in parotid salivary glands of patients with primary Sjogren's syndrome after B cell depletion therapy

Objectives To assess the persistence of immunoglobulin-producing cell populations in the parotid salivary glands of patients with primary Sjögrens syndrome (pSS) after B cell depletion therapy with rituximab. Methods Thirteen patients with pSS and four control patients were included in this study. Patients with pSS were treated with rituximab or placebo. Sequence analysis was carried out on IgA- and IgG-encoding transcripts extracted from parotid salivary gland biopsy specimens taken before treatment and at 12–16 and 36–52 weeks after treatment. Results At baseline, many clonally related sequences were seen in patients with pSS. The number of clonal expansions was significantly higher in patients with pSS than in control patients. Clonal expansions were composed of IgA- and/or IgG-expressing cells. Rituximab did not significantly alter the degree of clonal expansions. Groups of clonally related cells had members which were shared between biopsy specimens taken before and after treatment. Mutation frequencies of immunoglobulin sequences from clonally related cells in patients with pSS were higher after treatment. Conclusions Rituximab treatment does not alter the characteristic features of increased clonal expansions seen in the parotid salivary glands of patients with pSS. The presence of clonally related immunoglobulin-producing cells before and after rituximab treatment strongly suggests that immunoglobulin-producing cells persist in salivary glands of patients with pSS despite B cell depletion. The presence of mixed isotype expression within groups of clonally related cells indicates local class switching in salivary glands of patients with pSS. Persistent immunoglobulin-producing cells may underlie disease relapse after treatment.

Attenuation of Follicular Helper T Cell-Dependent B Cell Hyperactivity by Abatacept Treatment in Primary Sjögren's Syndrome

To assess the effect of abatacept (CTLA‐4Ig), which limits T cell activation, on homeostasis of CD4+ T cell subsets and T cell–dependent B cell hyperactivity in patients with primary Sjögrens syndrome (SS).

Abatacept attenuates T follicular helper-cell-dependent B-cell hyperactivity in primary Sjögren's syndrome.

To assess the effect of abatacept (CTLA‐4Ig), which limits T cell activation, on homeostasis of CD4+ T cell subsets and T cell–dependent B cell hyperactivity in patients with primary Sjögrens syndrome (SS).

Impact of Hibernation on Gut Microbiota and Intestinal Barrier Function in Ground Squirrels

Like many hibernators, the 13-lined ground squirrel fasts during winter. The loss of food intake not only affects the hibernator’s gut structure and function but also has the potential to modify the resident gut microbiota. Here we examined the effect of hibernation on numbers of cecal microbes, microbial production of short chain fatty acids, intestinal macromolecular permeability, and expression of the tight junction protein occludin. Compared with the active season, bacteria detected by probes specific for Lactobacillus–Enterococcus and Eubacteria–Clostridium groups were reduced during hibernation but the Bacteroides–Prevotella group was less affected. Cecal concentrations of short chain fatty acids were similar in active season squirrels, aroused hibernators, and hibernators in early torpor, but lower in late torpor. Gut permeability to a macromolecular marker increased during hibernation, as did tight junction localization and phosphorylation of occludin. The results suggest that hibernation selectively alters the gut microbiota, possibly reflecting differential sensitivity to loss of dietary polysaccharides. Hibernation impairs intestinal barrier function, which may require compensatory mechanisms to enhance tight junctional integrity and thus maintain a healthy relationship of the hibernator host with its resident gut microbiota until food intake resumes in the spring.

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.

Acquisition of N-Glycosylation Sites in Immunoglobulin Heavy Chain Genes During Local Expansion in Parotid Salivary Glands of Primary Sjogren Patients

Previous studies revealed high incidence of acquired N-glycosylation sites acquired N-glycosylation sites in RNA transcripts encoding immunoglobulin heavy variable region (IGHV) 3 genes from parotid glands of primary Sjögren’s syndrome (pSS) patients. In this study, next generation sequencing was used to study the extent of ac-Nglycs among clonally expanded cells from all IGVH families in the salivary glands of pSS patients. RNA was isolated from parotid gland biopsies of five pSS patients and five non-pSS sicca controls. IGHV sequences covering all functional IGHV genes were amplified, sequenced, and analyzed. Each biopsy recovered 1,800–4,000 unique IGHV sequences. No difference in IGHV gene usage was observed between pSS and non-pSS sequences. Clonally related sequences with more than 0.3% of the total number of sequences per patient were referred to as dominant clone. Overall, 70 dominant clones were found in pSS biopsies, compared to 15 in non-pSS. No difference in percentage mutation in dominant clone-derived IGHV sequences was seen between pSS and non-pSS. In pSS, no evidence for antigen-driven selection in dominant clones was found. We observed a significantly higher amount of ac-Nglycs among pSS dominant clone-derived sequences compared to non-pSS. Ac-Nglycs were, however, not restricted to dominant clones or IGHV gene. Most ac-Nglycs were detected in the framework 3 region. No stereotypic rheumatoid factor rearrangements were found in dominant clones. Lineage tree analysis showed in four pSS patients, but not in non-pSS, the presence of the germline sequence from a dominant clone. Presence of germline sequence and mutated IGHV sequences in the same dominant clone provide evidence that this clone originated from a naïve B-cell recruited into the parotid gland to expand and differentiate locally into plasma cells. The increased presence of ac-Nglycs in IGHV sequences, due to somatic hypermutation, might provide B-cells an escape mechanism to survive during immune response. We speculate that glycosylation of the B-cell receptor makes the cell sensitive to environmental lectin signals to contribute to aberrant B-cell selection in pSS parotid glands.