Somatic mutation in autoantibody-associated VH genes of circulating IgM+IgD+ B cells

Naive B cells expressing IgM and IgD on their surface have no or little somatic mutations in V genes. We have demonstrated that the human IgM⁺IgD⁺B cell clone (0 – 81), which expresses nephritogenic idiotypes, produces IgM anti-DNA antibodies which show monospecificity to DNA. Using a DNA probe which specifically links to the V_H gene of antibody 0 – 81, we identified the counterpart germ-line V gene of 0 – 81, V3-7, which appears to be used by pathogenic autoantibodies in humans. Clone 0 – 81, which may belong to naive B cells in terms of cell phenotype, uses a somatically mutated V3-7 gene. We further studied DNA sequences of V3-7 genes in circulating IgM⁺IgD⁺B cells from normal subjects and patients with systemic lupus erythematosus (SLE). The results revealed that rearranged V3-7 genes in IgM⁺IgD⁺B cells from patients with SLE contained somatically mutated sequences at significantly increased frequencies. These data indicate an abnormal maturation of B cells in autoimmune states that may be associated with an escape of self-reactive B cells from the elimination process in the germinal center.     

IgD expression on B cells is more efficient than IgM but both receptors are functionally equivalent in up-regulation CD80/CD86 co-stimulatory molecules

The expression and function of IgM and IgD antigen receptors were studied in a series of anti-hen egg lysozyme (HEL) immunoglobulin (Ig)-transgenic mice expressing either IgM alone, IgD alone, or both IgM and IgD. B cell surface expression of IgD was found to be more efficient than that of IgM. Thus antigen receptor density on IgD⁺, IgM^? B cells was twofold higher than on IgM⁺, IgD^? B cells despite the presence of sevenfold lower levels of membrane heavy chain mRNA, and coexpression of IgD with IgM led to almost complete inhibition of surface IgM. In addition, less extensive down-regulation of IgD occurred following exposure to antigen in vitro. When regulation of CD80/CD86 co-stimulatory molecules by surface Ig was examined, up-regulation of the former was initiated at lower antigen concentrations on IgM^?, IgD⁺ compared to IgM⁺, IgD^? B cells. On correcting for antigen receptor density, however, induction of CD80/CD86 by IgM and IgD was comparable. Taken together, these results reinforced the functional similarity of IgM and IgD antigen receptors while at the same time revealing differences in expression which may explain their simultaneous presence on mature B cells.     

IgD class switching: Identification of a novel recombination site in neoplastic and normal B cells

IgD on normal B lymphocytes usually is co-expressed with IgM. A minority of normal plasma cells and rare B cell malignancies express exclusively IgD (IgM^?-IgD⁺). The low frequency has been explained by the lack of a recognizable switch region within the Cμ-Cδ intron. We analyzed four cases of IgM^?IgD⁺ hairy cell leukemia (HCL) by Southern (DNA) blot analysis and identified two cases with a recombinatorial event within the Cμ-Cδ intron and deletion of Cμ. DNA sequence analysis of junctional regions showed that Sμ or the immediate upstream region was used as a donor site and that the Cμ-Cδ intronic σδ region was used as acceptor site. Using polymerase chain reaction, we subsequently analyzed whether similar Sμ-σδ recombinations occur in normal tonsils containing IgM^?IgD⁺ plasma cells. Multiple products with a size range of 200–800 base pairs were detected in all four individuals, suggesting clustering of acceptor sites within σδ. Sequence analysis of three cloned products showed Sμ-σδ recombinations similar those observed in HCL. The σδ region contains a relatively high content of pentameric repeats with an extremely G-rich area and appears to function as a vestigial switch recombination site in normal and neoplastic IgM^?-IgD⁺ B cells.     

Subepithelial B cells in the human palatine tonsil. I. Morphologic,cytochemical and phenotypic characterization

This study describes the purification of a subset of tonsillar B cells which share phenotypic, morphologic and cytochemical features with subepithelial (SE) B cells. These cells, which represented the 5–10% of the total tonsillar B cells, were found in the Percoll gradient fraction of highest density, together with resting follicular mantle (FM) B cells. The latter B cells, however, expressed surface CD5 and could be removed by an immune rosetting procedure. The remaining small CD5⁻ B cells had a surface phenotype (IgM⁺, IgD⁺, CD23⁻, CD38^±, CD10⁻, CD44⁺) that was different from that of FM (IgM⁺, IgD⁺, CD23⁺, CD39⁺, CD38⁻, CD10⁻, CD44^±) and of germinal center (GC) (CD23⁻, CD39⁻, CD38⁺, CD10⁺, CD44^±, IgG⁺) B cells isolated from the same cell suspensions. Furthermore, the absence of surface activation markers (CD71 and CD69) and of surface IgG allowed us to distinguish small CD5⁻ B cells from activated and memory cells migrating within Percoll fractions of lower density. In situ immunohistochemical studies revealed that B cells with an identical phenotype as that of small CD5⁻ B cells could be detected predominantly in the SE region (lamina propria) of the tonsil, and also within the epithelium lining the cryptae. This area was also comprised of a relatively minor proportion of activated B cells, not found in the small CD5⁻ B cell fraction owing to the separation procedure used. Consistent with the notion that the SE area could be a site of B cell activation was also the presence of activated macrophages and of plasma cells. Thirty to forty percent of small CD5⁻ B cells isolated in suspension were positive for the endogeneous alkaline phosphatase (ALP) activity. In contrast, only a few FM B cells were ALP⁺, while GC cells were consistently ALP⁻. In situ studies also demonstrated a prevalent expression of ALP activity by the B cells in the SE area. At the ultrastructural level, small CD5⁻ B cells were clearly different from both FM and GC B cells. They displayed a cytoplasm more extended than that of FM B cells with abundant endosomes and plasma membrane projections, and a speckled pattern of nuclear heterochromatin distribution. When fixed tissue sections were examined, cells with identical ultrastructural features could be demonstrated in the tonsillar lamina propria. Collectively, the above data demonstrate an identity of features between the small CD5⁻ B cells isolated in suspension and SE B cells analyzed in situ. Since tonsillar SE B cells are generally thought to represent the homolog of the extrafollicular B cells (including those of the splenic marginal zone), these studies may provide new opportunities for functional studies on this so far incompletely characterized B cell subset.     

The differentiation of human memory B cells into specific antibody-secreting cells is CD40 independent

It is generally accepted that memory B cells can be defined by their ability to produce, upon antigenic challenge, somatically mutated antibody molecules characterized by an increased affinity and by the expression of a downstream heavy chain isotype. However, the inability to isolate this particular B cell compartment has precluded the study of memory B lymphocyte physiology in man. We previously reported on the identification of an IgD^? B cell subset in human tonsils that we defined as CD38^? B cells, whose phenotype is highly reminiscent of that of memory B lymphocytes from the splenic marginal zone of rodents. In the present study, we developed a model of the measles virus (MV)-specific secondary antibody response in vitro to assess the presence of memory B lymphocytes in different B cell subsets isolated from human tonsils and explore the activation requirements of human memory B cells. Our findings show that the memory B cell pool resides in the CD38^? B cell subpopulation and that the differentiation of MV-activated memory B cells into antibody-secreting cells can be achieved upon co-stimulation with interleukin (IL)-2 and IL-10, but does not require engagement of CD40. Interestingly, the CD40-mediated signal was found to synergize with Ig-cross-linking agents for the proliferation of memory B cells, but strongly suppressed their capacity to differentiate along the plasmacytoid pathway. Collectively, our results suggest that the CD40 signaling pathway is instrumental for the clonal expansion of the memory B cell pool, but does not operate in the later phase of the response, which allows their maturation into antibody-secreting cells.     

CD27+ B cells in human lymphatic organs: re-evaluating the splenic marginal zone

The marginal zone of human spleens is regarded as an organ‐specific region harbouring sessile memory B cells. This opinion has arisen by extrapolating from results obtained in mice and rats. Detection of CD27⁺ B cells in situ now revealed similarities among the most superficial region of B‐cell follicles in human spleens, reactive lymph nodes, inflamed appendices, tonsils and terminal ilea. The follicular surface in these organs consists of small naïve immunoglobulin D (IgD)⁺ CD27^– B cells predominating in an inner area and larger IgD^+/– CD27⁺ B cells prevailing in a more superficial position. CD27⁺ B cells may, however, also occupy the entire follicular periphery around the germinal centre. Together with additional peculiarities this distribution indicates a fundamental microanatomical difference among the human and rodent splenic white pulp. We hypothesize that the follicular periphery represents a recirculation compartment both for naïve and memory/natural reactive B cells in all human secondary lymphatic organs. This assumption implies a difference in recirculation behaviour among human and rodent B memory cells.     

Unique properties of memory B cells of different isotypes

Summary: Memory antibody responses are typically seen to T-cell-dependent antigens and are characterized by the rapid production of high titers of high-affinity antigen-specific antibody. The hallmark of T-cell-dependent memory B cells is their expression of a somatically mutated, isotype-switched B-cell antigen receptor, features that are mainly generated in germinal centers. Classical studies have focused on isotype-switched memory B cells (mainly IgG isotype) and demonstrated their unique intrinsic properties in terms of localization and responsiveness to antigen re-exposure. However, recent advances in monitoring antigen-experienced B cells have revealed the considerable heterogeneity of memory B cells, which include unswitched IgM⁺ and/or unmutated memory B cells. The IgM and IgG type memory B cells reside in distinct locations and appear to possess distinct origins and effector functions, together orchestrating humoral memory responses.     

B-lymphocyte Subpopulations in Patients with Selective IgA Deficiency

Introduction

Selective deficiency IgA (IgAD) is the most common primary abnormality of immunoglobulin production with unknown pathophysiology. It is genetically related to common variable immunodeficiency (CVID), where besides IgA also IgG and frequently IgM serum levels are decreased. In this study we focused on determination of B-lymphocyte developmental stages and searching for similarities between CVID and IgAD.

Materials and Methods

Using flow cytometry we determined major lymphocyte subpopulations and B-lymphocyte subsets: na?ve (CD27^-IgD⁺), marginal zone cells (CD27⁺IgD⁺), class-switched memory cells (CD27⁺IgD^-), ??double-negative?? B cells (CD27^-IgD^-), transitional cells (IgM⁺⁺CD38⁺⁺), plasmablasts (CD38⁺⁺⁺IgM⁺ or IgM^-), and CD21^lowCD38^low cells in 80 patients with IgAD, 48 patients with CVID, and 80 control persons.

Results

Compared to healthy controls, a decrease in the absolute number and frequency of CD4+ cells (both?P?P?=?0.035) as well as plasmablasts (P?lowCD38^low subset (P?=?0.007) was observed in IgAD patients compared to control persons. No significant differences were observed in the remaining B-cell developmental subsets. A decrease in CD27⁺IgD^- (<0.4% of peripheral blood lymphocytes), frequently observed in CVID patients and also previously reported in IgAD, was found in only five patients (6%) with IgAD, two of them being first-degree relatives of CVID patients.

Conclusion

Our results show a decrease of terminally differentiated B-lymphocyte subsets in patients with IgAD, similar as previously found in patients with CVID, but these results are less expressed than in CVID patients.     

Identification of a novel subpopulation of germinal center B cells characterized by expression of IgD and CD70

CD27, which belongs to the tumor necrosis-factor receptor family, is expressed on germinal center (GC) but not on naive B cells, suggesting an important function of this molecule in the regulation of the GC reaction. We described here the expression of CD70, which is the ligand for CD27. We observed that in most tonsils, CD70 is only expressed on part of the IgD^?, CD38^? B cell population, which have been described as memory B cells. However, in 10 % of the tonsils tested, CD70⁺ IgD⁺ GC were found. The CD70⁺ GC B cells were small cells that also expressed CD44 and CD39, but were CD10^? and CD38^?, suggesting that they represent very recent immigrants that are in the process of forming a GC. The concordant expression of CD27 and its ligand CD70 on this primordial subset of GC B cells suggests an important role for CD27/CD70 interaction at this stage of GC formation.     

Transitional B cell subsets in human bone marrow

B cells originate from precursors in the bone marrow, and the first cells which migrate to the peripheral blood have been classified as ‘transitional B cells’. Transitional B cells have been characterized in human blood with stage 1 (T1) and stage 2 (T2) subsets being proposed. In the present study, 27 normal human bone marrow samples were analysed for transitional B cell markers by eight‐colour flow cytometry. T1 transitional B cells (CD45⁺CD19⁺CD10⁺IgM⁺IgD^lo) and T2 transitional B cells (CD45⁺CD19⁺CD10⁺IgM⁺IgD⁺) were identified in normal bone marrow samples at a mean frequency of 3·2 and 3·1% of total B lineage cells, respectively. A majority of the bone marrow transitional B cells were CD24^hiCD38^hi, the phenotype of blood transitional B cells. Consistent with recent peripheral blood data, T2 B cells had a significantly higher CD21 expression compared with T1 B cells (72·4 versus 40·9%) in the bone marrow. These data raise the possibility that transitional B cells are capable of differentiating from T1 to T2 B cells within the bone marrow. Furthermore, transitional cells at either stages 1 or 2 might be capable of migrating out of the bone marrow.     

Memory B cells at successive stages of differentiation: expression of surface IgD and capacity for self renewal

In recent studies, we have characterized two memory B cell populations capable of giving rise to IgG antibody-producing cells in adoptive recipients. One population carries surface IgD and gives rise to predominantly low-affinity antibody responses; the other lacks detectable surface IgD and gives rise to predominantly high-affinity responses. These memory populations often coexist in individual donors for long periods of time; however, in strongly stimulated donors, the IgD⁺ population is lost after several weeks, and nearly all detectable B cell memory is IgD^? thereafter. In this publication, we show that the IgD⁺ and IgD^? memory populations represent B cells at two successive stages of antigen-dependent differentiation. We used the fluorescence-activated cell sorter (FACS) in a double isolation and transfer protocol to show directly that FACS-isolated IgD⁺ memory cells transferred to adoptive recipients give rise both to IgG antibody-producing cells and to an expanded memory population that is predominantly IgD^?. We also show that FACS-isolated IgD^? memory populations from the original donor “self-renew” (i.e. give rise to more IgD-memory) in adoptive recipients and that these events require supplementation of the isolated memory cells with carrier-primed T cells and antigen. In discussing these findings, we integrate our data with previous evidence on the expression of surface IgG on memory B cells to create an updated view of surface Ig expression during memory development. We also consider these findings in the light of our recent suggestion that the loss of IgD receptors facilitates affinity maturation in the more mature (IgD^?) memory population.     

Identification of a tonsil IgD+ B cell subset with phenotypical and functional characteristics of germinal center B cells

We have identified and isolated a subpopulation of IgD⁺ B cells (IgD⁺ CD38⁺ B cells) from human tonsils which expresses the germinal center (GC)-associated surface markers CD10, CD38, CD75, CD77 and CD95/Fas. The heterogeneity of expression of several markers on IgD⁺ CD38⁺ B cells suggests that this population can be further subdivided into two discrete subtypes. On a functional basis, IgD⁺ CD38⁺ B cells behave as GC B cells as they rapidly and spontaneously undergo apoptosis in vitro and cannot be stimulated to synthesize DNA upon cross-linking of the antigen receptor. However, in contrast with most GC B cells, IgD⁺ CD38⁺ B cells have not completed Ig class switching since they predominantly secrete IgM following stimulation in vitro and lack surface expression of secondary isotypes. Immunoenzymatic staining performed on tonsil tissue sections revealed that IgD⁺ CD38⁺ B cells are located in two distinct histological structures: within the GC of a few classical secondary follicles, in which they appear as scattered cells, and within rare atypical GC, homogeneously constituted of IgD⁺ B cells. Taken together, our findings indicate that IgD⁺ CD38⁺ B cells constitute a novel subset of GC B cells. The possibility that these cells could represent an early stage of the follicular reaction or be generated in response to certain bacterial carbohydrate antigens is discussed.     

Functional Maturation of Immature b Cells Accumulated in the Periphery by an Intraperitoneal Administration of a Traditional Chinese Medicine,Xiao-Chai-Hu-Tang (Japanese Name: Shosaiko-To)

Abstract

We found that an intraperitoneal (ip) injection of a traditional Chinese herbal medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to), induced accumulation of B lymphocytes (sIgM⁺) in the peritoneal cavity and spleen. 1) Cell surface marker analysis by a fluorescence-activated cell sorter (FACS) demonstrated that the accumulated B cells on day 4 or 7 after shosaiko-to administration (early phase) were composed mainly of sIgM⁺ IgD^- cells and suggested that these B cells maturated into sIgM⁺ IgD⁺ cells on days 10 or 14 (late phase). Relative decrease of IgM⁺ IgD⁺cells at early phase was more profound in peritoneal cells (PC) than in spleen cells. 2) With respect to spleen lymphocytes, antibody responses to a thymus-independent (TI) antigen of type 2 (trinitrophenylated Ficoll) and a thymus-dependent (TD) antigen (sheep erythrocyte) were enhanced at late phase but not at early phase. In contrast, responses to trinitrophenylated lipopolysaccharide (TNP-LPS) as a TI-1 antigen and LPS as a B cell mitogen or a polyclonal B cell activator were enhanced markedly at early phase but declined at late phase. 3) With respect to peritoneal lymphocytes, responses to LPS were suppressed at early phase but recovered at late phase. Enhanced responses to TI and TD antigen at late phase in spleen lymphocytes and suppressed response to LPS at early phase in peritoneal lymphocytes may be explained by increases of IgM⁺ IgD⁺ mature B cells and IgM⁺ IgD^- immature B cells, respectively, at those times. Enhanced responses to TI-1 or LPS in spleen lymphocytes at early phase may be explained by elevated sensitivity of IgM⁺ IgD⁺ cells which reside in the spleen before shosaiko-to administration and receive the direct stimulation by shosaiko-to, or by acquired responsiveness of IgM⁺ IgD^- cells which migrate after stimulation with shosaiko-to.     

CD73 expression identifies a subset of IgM+ antigen‐experienced cells with memory attributes that is T cell and CD40 signalling dependent

B‐cell memory was long characterized as isotype‐switched, somatically mutated and germinal centre (GC)‐derived. However, it is now clear that the memory pool is a complex mixture that includes unswitched and unmutated cells. Further, expression of CD73, CD80 and CD273 has allowed the categorization of B‐cell memory into multiple subsets, with combinatorial expression of the markers increasing with GC progression, isotype‐switching and acquisition of somatic mutations. We have extended these findings to determine whether these markers can be used to identify IgM memory phenotypically as arising from T‐dependent versus T‐independent responses. We report that CD73 expression identifies a subset of antigen‐experienced IgM⁺ cells that share attributes of functional B‐cell memory. This subset is reduced in the spleens of T‐cell‐deficient and CD40‐deficient mice and in mixed marrow chimeras made with mutant and wild‐type marrow, the proportion of CD73⁺ IgM memory is restored in the T‐cell‐deficient donor compartment but not in the CD40‐deficient donor compartment, indicating that CD40 ligation is involved in its generation. We also report that CD40 signalling supports optimal expression of CD73 on splenic T cells and age‐associated B cells (ABCs), but not on other immune cells such as neutrophils, marginal zone B cells, peritoneal cavity B‐1 B cells and regulatory T and B cells. Our data indicate that in addition to promoting GC‐associated memory generation during B‐cell differentiation, CD40‐signalling can influence the composition of the unswitched memory B‐cell pool. They also raise the possibility that a fraction of ABCs may represent T‐cell‐dependent IgM memory.     

Acute wrist and foot drop associated with hepatitis C virus related mixed cryoglobulinemia: Rapid response to treatment with rituximab

The nature of the B-cell subsets associated with chronic hepatitis C virus related type II mixed cryoglobulinemia (HCV-MC) is unclear.We report the case of a 64-year-male with acute onset wrist drop and foot drop, secondary to HCV-MC related mononeuritis multiplex, who was treated with rituximab, an anti-CD20⁺ antibody directed against B cells. We monitored the frequency of B-cell subsets in peripheral blood before and after rituximab, and correlated B-cell subset changes with clinical response.Significant improvements in his wrist and foot drop, as well as his vasculitic rash, depression and erectile dysfunction were evident within six days of starting rituximab and have persisted several months after B-cell recovery.More than 95% of CD20⁺ B cells had disappeared from peripheral blood within 1 week, returning to baseline by week 21. CD20⁺CXCR3⁺ frequency at baseline was similar to that at week 21. CD20⁺CD5⁺, the human equivalent of B1 B cells and CD20⁺IgM⁺IgD⁺, naïve B cells were increased. By contrast, CD20⁺CD27⁺ memory cell frequency was reduced.These data suggest that CD27⁺ memory B cells, but not CD5⁺ and IgM⁺IgD⁺ B cells may play a role in the clinical manifestations of cryoglobulinemia.     

IgM- and IgD-bearing peripheral blood lymphocytes differentiate to IgM but not IgG or IgA immunoglobulin-secreting cells

Human peripheral blood mononuclear cells were depleted of surface IgM⁺ or IgD⁺ cells and assayed for mitogen-induced differentiation to immunoglobulin-secreting cells (ISC) of IgM, IgG and IgA classes. Stimulatory agents included T cell-dependent poke weed mitogen, B cell mitogen Staphylococcus aureus bacteria strain Cowan I, and a combination of the two which gives uniform, high levels of ISC from all normal donors. Depletion of either IgM- or IgD-bearing B lymphocytes resulted in loss of cells bearing the other Ig class and blocked most of the mitogenic reactivity to anti-IgM and anti-IgD. Proliferative responses to Cowan I in these depleted populations were about 20% that of unfractionated mononuclear cells. Depletion of T cells increased the mitogenic response to Cowan I and to the two antibody preparations, showing that they are T-independent mitogens. Depletion of IgD⁺ cells caused partial loss of mitogen-induced IgM ISC (22%-60% of unseparated controls) but no loss of IgG or IgA ISC. Depletion of IgM-bearing cells caused complete loss of IgM ISC, but no loss of IgG or IgA ISC. We previously demonstrated that anti-IgM antibody blocked mitogen induction of Ig secretion of these three classes in spleen cells, but only IgM secretion in blood mononuclear cells. Together, the results suggest that the majority of cells in normal blood responding to mitogens to mature to IgG or IgA production belong to IgM^?, IgD^? B cell subsets, in contrast to precursors of secreting cells for these isotypes in the spleen. Thus, these blood precursors appear to be more mature than the corresponding spleen cells.     

Abnormal B cell differentiation in primary Sjögren's syndrome results in a depressed percentage of circulating memory B cells and elevated levels of soluble CD27 that correlate with Serum IgG concentration

The percentage of CD27⁺ B cells in peripheral blood (PB) of patients with primary Sjögren's syndrome (pSS) is significantly decreased compared to normals. In contrast, serum levels of the soluble form of CD27 (sCD27) are significantly higher in pSS patients, with a strong positive correlation between sCD27 and serum IgG levels. In vitro experiments demonstrate that normal B cells cultured under conditions driving plasma cell differentiation result in the production of substantial amounts of sCD27. Analyses of V_H-region genes from sorted CD27⁺ and CD27^? B cells from pSS patients confirm that the CD27⁺ population corresponds to the somatically mutated memory compartment, as in healthy individuals. Together our data indicate that in pSS, there is an abnormal differentiation of B cells to plasma cells resulting in a depression of the circulating memory B-cell pool and the release of significant amounts of sCD27 and IgG.