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.     

Human IgM+IgD+ B cells,the major B cell subset in the peripheral blood,express Vϰ genes with no or little somatic mutation throughout life

Peripheral blood B cells of a 67-year-old person were separated into IgM⁺IgD⁺, IgM⁺IgD^?, and IgM^?IgD^? subsets, and nucleotide sequences of expressed immunoglobulin light chain variable (V) regions encoded by V?3 and V?4 gene family members were determined from amplified cDNA. V region sequences from IgM⁺IgD⁺ cells (the major B cell population in the blood) showed no or little somatic mutation (0.3%), in contrast to V? sequences from IgM⁺IgD^? and IgM^?IgD^? B cells (2.0% and 3.9%, respectively). This suggests that in the human like in the mouse, and independently of age, somatically mutated memory B cells accumulate in the compartment of IgM^?IgD^? cells, whereas the IgM⁺IgD⁺ subpopulation consists of cells whose antibody repertoire is mainly determined by V region gene rearrangements and N-region insertion, at the molecular level. The somatically mutated IgM+IgD^? cells may represent early descendants of IgM⁺IgD⁺ cells recruited into the memory cell compartment.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

B cell subpopulations separated by CD27 and crucial collaboration of CD27+ B cells and helper T cells in immunoglobulin production

B cell immunoglobulin production is regulated by helper T cells through direct interaction and secreted cytokines. In the present study, we functionally analyzed CD27 in cord and peripheral blood B cells. Adult peripheral blood B cells were separated into CD27⁺ and CD27^? cells, which differed in their morphology. Cord blood B cells did not express CD27, and CD27 expression on peripheral blood B cells increased with age. Only CD27⁺ B cells had the ability to produce immunoglobulin, which was increased by contact with a tumor necrosis factor-related transmembrane ligand, CD70. Adult peripheral blood CD27⁺ B cells can be further subdivided into two discrete subtypes: IgD^?CD27⁺ and IgD⁺ CD27⁺ B cells. IgD^? CD27⁺ B cells produce IgG, IgM and IgA, whereas IgD⁺ CD27⁺ B cells predominantly produce IgM. The addition of activated CD4⁺ CD45RO T cells expressing CD70 caused down-regulation of CD27 expression on activated B cells, and this down-modulation was completely blocked by anti-CD70 monoclonal antibody, indicating direct T-B cell contact via CD27/CD70. The triggering via CD27 and CD40 additively increased the immunoglobulin production under Staphylococcus aureus Cowan strain plus interleukin-2 stimulation. Taken together, our findings demonstrate that peripheral blood B cells are separated into subpopulations by CD27 and IgD expression and that CD27⁺ B cells produce large amounts of immunoglobulin by interaction with the CD70 molecule.     

Toll-like receptor 4-mediated signaling regulates IL-7-driven proliferation and differentiation of B-cell precursors

Lipopolysaccharide （LPS） is known to be a potent activator of mature B cells by signaling through Toll-like receptor 4 （TLR4）. Its impact on early B-cell development, however, is not well defined. When comparing to C3H/HeN mice, TLR4-mutant C3H/HeJ mice showed an increase in the number of pro-B and pre-B cells in the bone marrow. When cultured in the presence of IL-7, the proliferation of pro-B and large pre-B cells was significantly inhibited by LPS, possibly due to reduced IL-7 receptor-a （IL-7Ra） expression. Meanwhile, the generation of IgM＋/IgD＋ B cells was greatly enhanced in IL-7 cultures of pro-B and pre-B cells. Consistent with these results, treatment with LPS facilitated the progression of adoptively transferred B220＋IgM-IgD- precursors into IgD＋ cells. Overall, these data suggest that LPS has a profound influence on early B-cell development, which may contribute to the deregulated B-cell development under physiological and pathological conditions such as bacterial infections.     

Lymphocyte membrane immunoglobulins: similarities between human IgD and mouse IgD-like molecules.

¹³¹I-labeled IgD-like molecules extracted from the membrane of mouse spleen, and lymph node cells and their constitutive chains have been characterized by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis (SDS-PAGE) followed by radioautography. Unreduced mouse IgD-like molecules concentrated in two definite bands (IgD₁ and IgD₂). IgD, migrated slightly ahead of ¹³¹I-labeled mouse membrane IgM and displayed a mobility very similar to that of ¹³¹I-labeled IgD extracted from the membrane of human tonsil cells. IgD₂ migrated faster than both IgD₁ and human membrane IgD (mIgD), but slower than ¹⁴C-labeled mouse IgG2 ^b used as marker. The different mobility of IgD₁ and IgD₂ was due to difference in size of their respective H-chains (δ₁ and δ₂), as shown by SDS-PAGE analysis of the two molecules after reduction. Mol.wt. determination of IgD₁, IgD₂ and of the corresponding constitutive chains are consistent with an H₂L₂ structure. IgD₁ and IgD₂ were partially replaced following prolonged dialysis by molecules with a SDS-PAGE mobility consistent with an HL structure, IgD₃. In contrast to that found for spleen and lymph node, IgD₁ only was detected on the surface of bone marrow cells. Immunofluorescence experiments have shown that while the majority of mouse lymph node or spleen cells bear IgM and IgD, IgM was found to be the predominant Ig class on the surface of bone marrow cells, although a certain number of IgD-IgM and a few IgD-positive cells were found. Experiments where mouse lymphocytes have been treated with pronase have indicated that, in analogy with mlgD of primates, mouse IgD-like molecules are very labile and can be removed from the cell surface by very mild proteolytic treatment.     

Molecular cloning of human RP105

RP105 is a 105-kDa type I membrane protein of the leucine-rich repeat (LRR) family. Anti-RP105 sensitizes B cells to antigen-receptor-mediated apoptosis, but protects B cells from radiation-induced apoptosis and stimulates B cell proliferation. The sequence of the mouse RP105 has been reported. Here, we report the characterization of the human RP105. The 2.6-kb cDNA encodes a protein of 661 amino acids which displays 78% homology with mouse RP105. The 22 LRR and the 9 potential N-linked glycosylation sites within the extracellular region are conserved. While previous studies have shown that RP105 is expressed on surface IgM⁺IgD⁺⁺ B cells in mice, human RP105 was shown to be expressed on all subsets of mature B cells and dendritic cells. Human RP105 gene was mapped to the long arm of chromosome 5, where numerous cytokines and receptors have been localized.     

Identification of two distinct CD5- B cell subsets from human tonsils with different responses to CD40 monoclonal antibody

This study investigated the response of different CD5^? B cell subsets to CD40 monoclonal antibody (mAb) in various combinations with interleukin (IL)-4 or rabbit anti-human μ chain antibody (a-μ-Ab). The different CD5 B cell subsets were isolated from tonsillar B cell suspensions depleted of CD5⁺ B cells and subsequently fractionated on Percoll density gradients. While resting CD5⁺ B cells proliferated and produced IgM molecules in response to a-μ-Ab, IL-4 and CD40 mAb as well as to Staphylococcus aureus Cowan strain I (SAC) and IL-2, resting CD5^? B cells, which were co-purified in the same 60% Percoll fractions, consistently failed to respond. These cells were, however, activated by the stimuli employed, as demonstrated by their capacity to express the surface activation markers CD69, CD25 and CD71. Resting CD5⁺ B cells had the typical phenotype of mantle zone B cells (IgM⁺ IgD⁺ CD39+ CD38^? CD10^? CDw75^dim), whereas resting CD5^? B cells were CD38^? CD39^? CD 10^? CDw75 intermediate and expressed surface IgM but relatively little surface IgD and could not be classified as mantle zone or germinal center cells. The finding that purified germinal center cells (CD38⁺ CD10⁺ CD39^? CDw75^bright, IgG⁺) responded to CD40 mAb and IL-4 and also to SAC plus IL-2 further underlined the differences to resting CD5^? B cells. However, some of the data collected suggest possible relationships between CD5^? B cells and germinal center cells. The CD5^? B cells isolated from the 50 % Percoll fraction proliferated in response to a-μ-Ab, CD40 mAb and IL-4 as well as to SAC and IL-2. These cells had the same mantle zone B cell phenotype as the CD5⁺ B cells, but their capacity to respond to the stimuli in vitro was unrelated to a possible contamination with CD5⁺ B cells, as documented by the appropriate controls. Furthermore, upon exposure to SAC or phorbol esters, the large majority of CD5^? B cells from the 50 % Percoll fraction did not express surface CD5 and there was very little if any accumulation of CD5 mRNA. Finally, most of the cycling cells in the stimulated CD5^? B cells did not express CD5. The CD5^? B cells from the 50 % Percoll fraction were comprised of a consistent proportion of cells that expressed surface activation markers. The removal of these cells abrogated the capacity of the suspensions to respond to the stimuli in vitro, possibly suggesting that these cells additional activation signals in vivo which were essential to acquire the capacity to respond and that could not be reproduced in vitro. The present study underlines the phenotypic and functional heterogeneity of CD5^? B cells and contributes to the identification of two subsets of these cells which differ in phenotype, tissue distribution and in vitro responses to different stimuli.