Signaling through murine CD38 is impaired in antigen receptor-unresponsive B cells

CD38 is a 42-kDa membrane associated enzyme which converts NAD into cyclic ADP-ribose (cADPR), a Ca²⁺-mobilizing second messenger, and ADP-ribose (ADPR). Agonistic antibodies to murine CD38 deliver a potent growth co-stimulus to mature splenic B lymphocytes. In this report we demonstrate a striking relationship between CD38-mediated mitogenesis and the ability of surface IgM to promote B cell proliferation. Tolerized B lymphocytes obtained from a double-transgenic mouse model of B cell tolerance do not proliferate in response to antigen stimulation through the Ig receptor or to agonistic anti-CD38 antibodies. Similarly, B-1 cells isolated from the peritoneal cavity of normal mice, and splenic B cells isolated from newborn mice were also unresponsive to both anti-IgM and anti-CD38 stimulation. All of these CD38-unresponsive B cells expressed normal levels of cell surface CD38 and responded to numerous other stimuli. CD38 immunoprecipitated from these B cell populations was normal in size and effectively hydrolyzed NAD, suggesting that the defect in CD38 signaling likely occurs downstream of CD38 itself. Signaling through CD38 and IgM does not always have identical effects on B cells since anti-CD38 cannot deliver inhibitory growth or differentiation signals to normal B cells or immature B cell lines. Nevertheless, the correlative data with these multiple B cell models of unresponsiveness suggests that the signaling pathway utilized by CD38 and IgM intersect, possibly sharing at least one of the crucial components of the Ig receptor signaling cascade.     

A role for membrane IgD in the tolerance of pathological human rheumatoid factor B cells

Under non-autoimmune conditions, rheumatoid factor (RF) B cells coexist peacefully with their antigen (IgG), or can be transiently activated during secondary immune responses because they can present xenoantigens to specific T cells captured in immune complex form. Such a situation should lead to affinity maturation of RF B cells and potentially dangerous production of high-affinity RF. We used two lines of transgenic mice expressing a somatically mutated pathological human RF in presence (IgM and IgD) or in absence (IgM only) of surface IgD, and confirm that RF B cell tolerance can result from an antigen-induced specific, but incomplete, deletion of naive RF B cells after antigen encounter. This deletion mainly concerns immature, transitional B cells. On the contrary, mature, IgM- and IgD-expressing RF B cells are resistant to such a deletion. These IgM and IgD RF B cells are functional and activable through both B cell receptor dependent (anti-IgM) and independent (LPS) pathways, but they are not fully responsive to human IgG either in vivo or in vitro. Taken together, these results suggest that another mechanism could be involved in the silencing of mature naive IgM and IgD RF B cells.     

The antigen receptor complex on cord B lymphocytes.

The neonatal immune system responds to a restricted range of antigens, producing largely IgM antibody of low affinity. Comparison of the components of the B-cell antigen receptor complex shows significantly elevated membrane levels of IgM in neonatal B cells, compared with adult cells. CD79, which acts as the signal transducer for membrane immunoglobulin, is elevated in parallel with IgM, while IgD is elevated to a lesser degree. CD19, CD21, CD22 and CD81, which are all involved in transmitting activation signals when immunoglobulin is engaged, are not elevated. CD32, which is involved in negative regulation of activation, is present at reduced levels on cord B cells. The elevation of B-cell membrane IgM persists during infancy. Neonatal B cells respond in vitro to interleukin-4 (IL-4) by further elevation of membrane IgM levels. The elevated level of membrane IgM may make neonatal B cells easier to trigger by low concentrations of antigen, but in vitro activation and immunoglobulin modulation experiments did not show significant differences between cord and adult B-cell responses to anti-IgM.     

Intra- and extrathymic B cells in physiologic and pathologic conditions

Summary Normal thymuses and thymuses with lymphofollicular hyperplasia have been examined immunohistologically using immunoenzymatic single and double labelling methods and a panel of monoclonal antibodies against B lymphocyte differentiation antigens (CD19-, CD20-, CD21-, CD22-, CD23- and CD37ag) and human immunoglobulins (IgM, IgD) for the presence and localisation of B lymphocytes and cells expressing B cell differentiation antigens. The numerous hyperplastic lymph follicles which occur in the pathological condition of lymphofollicular hyperplasia of the thymus were found to originate in the extrathymic compartment of the interlobular septal space. This area was found to be blown up by the growing lymph follicles with exactly the same cellular composition as their counterparts in the peripheral lymphatic tissue. Some of the B lymphocytes expressing the immunophenotype of follicular mantle zone lymphocytes which were detected in the thymic medulla probably infiltrated through discontinuities of the border between the perivascular space and the thymic medulla. Apart from this primarily extrathymic B cell compartment, B lymphocytes and cells expressing B cell antigens were found within the thymus medulla of normal control thymuses of different ages from fetal to adult life. These cells were detected as a small subpopulation in normal fetal, juvenile and adult thymuses. Morphologically they could be subdivided into small, round lymphoid cells accounting for less than 1% of medullary lymphoid cells, and into a larger variant, asteroidally shaped because of short cytoplasmic processes. These asteroid cells were even more infrequent than the lymphoid variant. Immunophenotype (CD19ag+, CD20ag+, CD22ag+, CD37ag+, IgM+, IgD+) and morphology of the first cell type led to the conclusion that the lymphoid cells were in fact B lymphocytes. They were scattered throughout the medulla of fetal and juvenile and adult thymuses alike. The second, the asteroid cell type, constantly expressed CD20ag and inconstantly IgM, CD22ag and CD37ag; furthermore, CD23ag was detected in a subset of the asteroid cells either restricted to the perinuclear zone or expressed in the entire cytoplasma and on the plasma membrane. The asteroid cells were located in the corticomedullary region of the fetal thymuses but were randomly distributed with a tendency to Hassall's corpuscles in juvenile and adult thymuses. They often formed rosettes with non-B lymphocytes. It can be concluded that a small number of B cells and asteroid cells of still uncertain origin, but expressing B cell antigens, are constitutive elements of the fetal and adult thymic medulla. It can be assumed that the asteroid cell might represent a novel type of thymic accessory cell and that the rosetting of non-B lymphocytes around this asteroid cell might simulate or in fact be the earliest B cell interaction of maturing T cells.Abbreviation mAb(s) monoclonal antibody(-ies) - CDxxag antigen defined by the mAb cluster xx - CDxx(mAb) mAb of the cluster xx This study was supported by the Land Baden-Württemberg (Förderung der AIDS Forschung)Dedicated to Prof. Dr. V. Becker on the occasion of his 65th birthday     

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)

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.     

不同体外刺激对小鼠腹腔产生IL-10的B细胞的影响

目的:探索不同体外刺激对小鼠腹膜腔B细胞产生IL-10的影响.方法:在不同刺激剂作用下,体外培养小鼠腹腔细胞5或48小时,流式细胞仪分析CD19+IL-10+ B细胞比例.结果:PIM刺激5小时后,小鼠腹腔CD19+IL-10+ B细胞比例明显升高至14.69%(P<0.01),在PIM刺激的基础上加入anti-IgM F(ab′)2或anti-CD40均不能进一步提高腹腔B细胞IL-10的表达(P>0.05),而加入IPS后,CD19+IL-10+ B细胞比例升高至平均26.53%(P< 0.01).LPS、anti-CD40刺激48小时可明显升高CD19+IL-10+ B细胞比例,但能够产生更强BCR信号的coated anti-IgM刺激抑制小鼠腹腔B细胞IL-10的表达.结论:体外刺激5小时,LPS+PIM是腹腔产生IL-10的B细胞活化的最佳刺激,刺激48小时,LPS和anti-CD40可进一步促进腹腔B细胞IL-10的表达,这一作用可能是通过诱导腹腔B10前体细胞成熟实现的.而anti-IgM刺激对腹腔B细胞产生IL-10的影响依赖于刺激时间和信号强度.     

Thymic medullary cells expressing B lymphocyte antigens

A small subpopulation of human thymic medullary cells was found to express B cell-restricted and -associated antigens in various combinations. The cells were detected in fetal, juvenile, and adult thymi using indirect immunoenzymatic methods and monoclonal antibodies (MoAbs). Morphologically, they could be subdivided into small, round lymphoid cells, accounting for less than 1% of medullary lymphoid cells, and into a larger variant, even more infrequent in number and asteroid in shape because of short cytoplasmic processes. Immunophenotype (CD19+, CD20+, CD22+, CD37+, IgM+, IgD+) and morphology of the first cell type led to the conclusion that the lymphoid cells were B lymphocytes. The second, asteroid cell type constantly expressed CD20 and inconstantly expressed IgM, CD19, CD22, and CD37; they were often found to form rosettes with non-B lymphocytes. It can be concluded that a small number of B cells and asteroid cells of still uncertain origin, but expressing B cell-restricted antigens, are constitutive elements of the fetal and adult thymic medulla. It can be hypothesized that the asteroid cell might represent a novel type of thymic accessory cell and that the rosetting of non-B lymphocytes around this asteroid cell might simulate or in fact be the earliest B cell interaction of maturing T cells.     

Differential expression of the B cell-restricted molecule CD22 on neonatal B lymphocytes depending upon antigen stimulation

Newborns respond poorly to certain antigens and produce mainly IgM antibodies. By flow cytometry we analyzed on neonatal and adult B cells the expression of CD22, a B cell receptor (BCR)-associated membrane molecule, known as negative modulator of BCR signaling. After T cell-independent (TI-)stimulation with anti-mu F(ab')(2) fragments we found a dramatic decrease in the percentage of neonatal CD22(+) B cells and CD22 mean fluorescence intensity (MFI) shift, whereas adult B cells remained unaffected. Survival and proliferation rates of neonatal B cells were higher compared to adult B cells whereas the degrees of apoptosis and necrosis were comparable. Surprisingly, after stimulation with lower doses of anti-mu apoptosis as well as proliferation increased significantly in contrast to adult B cells. T cell-dependent (TD)-stimulation with anti-CD40 monoclonal antibody and IL-4 resulted in a dramatic increase in the percentage of CD22(+) neonatal B cells in contrast to unaffected adult B cells. CD22 MFI shifts showed no significant changes, respectively. The survival rate was higher for adult B cells, whereas apoptosis and cell death were comparable. These results suggest that TI antigens lower the neonatal BCR signaling threshold via down-regulation of CD22, resulting in hyperresponsive B cells apt to premature apoptosis. On the other hand, up-regulation of CD22 after TD stimulation may allow increased inhibiting influence of CD22 on neonatal BCR signaling, impairing B cell activation and differentiation.     

The follicular versus marginal zone B lymphocyte cell fate decision is regulated by Aiolos, Btk, and CD21

Most splenic B cells in mice that lack Aiolos are mature IgD(hi)IgM(lo) follicular lymphocytes, suggesting that maturation signals delivered via the BCR are enhanced in the absence of Aiolos. The enhanced maturation of follicular B cells is accompanied by the absence of MZ B lymphocytes and the downregulation of CD21 expression in follicular B cells, all of which depend on the generation of signals via Btk, which is in epistasis to Aiolos. The inverse relationship between the strength of BCR signaling and MZ B cell development is supported by an examination of MZ B cells in CD21 null mice. These data support the view that antigens (in contrast to "tonic" signals) drive the development of naive B cells.     

The tyrosine kinase Lyn is required for B cell development beyond the T1 stage in the spleen: rescue by over-expression of Bcl-2

We have analyzed the effects of deficiency in the tyrosine kinase Lyn on B cell development using transgenic mice that express a B cell antigen receptor (BCR) of defined specificity (3-83,anti-H-2K(k or b)). In the absence of Lyn, immature B cells are abundant in the bone marrow and spleen up until the T1 stage (IgM(hi) IgD(-) CD21(-)CD23(-)), after which B cell development is severely impaired. The small number of mature B cells that do develop in Lyn-deficient mice express normal levels of the transgenic BCR and lack expression of CD80 and CD86, suggesting they are not activated. In Lyn-deficient animals the presence of a Bcl-2 transgene leads to a dramatic increase in B cell numbers and restores T2 stage (IgM(hi) IgD(hi) CD21(hi) CD23(int)) and mature populations. In 3-83 lyn-/- Bcl-2 Tg mice, a population of lambda-positive cells that also express the 383 idiotype is evident, suggesting that in the absence of lyn isotype exclusion by the transgenic BCR is less efficient. The results indicate that Lyn plays a positive role in the selection and survival of mature B cells in addition to its previously documented negative role in tolerance and B cell activation.     

Age dependency and mutual relations in T and B lymphocyte abnormalities in common variable immunodeficiency patients

Common variable immunodeficiency (CVID) is primary hypogammaglobulinaemia with an unknown aetiopathogenesis. Although various abnormalities of T and B cells have been described, their pathogenetic roles are unclear. We determined T and B lymphocyte subsets known to be abnormal in CVID in order to disclose possible relations between numerical abnormalities in those cells. Markers associated with B cell development (CD21, CD27, IgM, IgD) were determined on B lymphocytes (CD19+); T lymphocyte development (CD45RA, CD45RO, CD62L) and activation markers (CD25, CD27, CD28, CD29, CD38, CD57, HLA-DR) were determined on CD4+ and CD8+ T lymphocytes in 42 CVID patients and in 33 healthy controls. Abnormalities in CD4+ T lymphocyte activation markers (increase in CD29, HLA-DR, CD45RO, decrease in CD27, CD62L, CD45RA) were observed particularly in patients with a decreased number of memory (CD27+) and mature (CD21+) B cells (group Ia according to the Freiburg group's classification), while abnormalities observed in CD8+ cells (increase in CD27 and CD28 and decrease in HLA-DR, CD57 and CD38) did not depend upon grouping patients together according to B lymphocyte developmental subpopulations. We observed correlations between immature B cells (IgM+ CD21-) and expression of CD27, CD62L, CD45RA, CD45RO and HLA-DR on CD4+ T cells in CVID patients but not in the control group. The expression of CD27 and CD45RA on CD4+ T lymphocytes, such as the percentage of IgD+ CD27- and IgD+ CD27+ cells in B lymphocytes, showed age dependency to be more significant than in the control group. Our study demonstrates that T and B lymphocyte abnormalities in CVID are partially related to each other. Some of those abnormalities are not definite, but may evolve with age of the patient.     

Fetal and neonatal development of human spleen: an immunohistological study.

Localization and immunophenotype of lymphocyte subsets in fetal human spleens were studied by employing a panel of monoclonal antibodies (McAb) in an immunoperoxidase staining procedure on frozen tissue sections. Spleens varied from 15 weeks of gestational age (gestational weeks, gw) to newborn. The white pulp consisted of intermediate-sized lymphocytes; no separate compartments could be discerned. Germinal centre development was not observed. Dendritic cells stained for B2, HB5, aC3bR, anti-DRC and OKIa, but in most cases not for immunoglobulin. Although low cellular immunity is observed in neonates, T cells showed adult phenotypes in proportions comparable to the adult situation; immature OKT6(+) lymphocytes were rarely seen. Very few cells stained with anti-NK cell antibody Leu7. B cells all expressed B1, Leu14, aC3bR, T10 and OKIa, were strongly positive for BA1, and mostly stained very weakly for B2 and HB5. Almost all B cells expressed IgM and IgD simultaneously, and very few expressed IgG. IgA-positive cells were absent. At 15 gw a considerable number of IgM(+) B cells showed Leu1 staining, but this decreased during development. These cells may represent the normal counterpart of Leu1(+) IgM(+) cells observed in B-CLL and immunocytic and centrocytic malignant lymphomas. After 25 gw only very few Leu1(+) IgM(+) cells were seen. Altogether, the morphology and immunophenotype of white pulp B cells were different from the predominating adult B-cell subsets, at least until birth. These 'immature' splenic B cells may be precursors for adult splenic B-cell subsets. Considering the presumed role of the marginal zone in the immunity against TI-2 antigens, the absence of a marginal zone at birth may be a main factor in the defective immunity against these antigens in neonates.     

GL7 defines the cycling stage of pre-B cells in murine bone marrow.

We have identified a novel subset of early B lineage cells in the mouse bone marrow (BM) by GL7 expression on cell surface. GL7(+)B220(low) BM cells have a large cell size and are CD43(-to low), CD95(-), Sca-1(-), I-A(low), IgM(-) and IgD(-), suggesting that they are large pre-B cells. These BM cells express lambda5 and VpreB but not terminal deoxytransferase (TdT) and Bcl-2, and approximately 50 % of them are in cell cycle. This fraction was not detected in BM cells of Rag-1-deficient and Scid mice, supporting that GL7(+)B220(low) BM cells belong to fraction C' and D according to Hardy's criteria or to an early large pre-B-II fraction according to Melchers-Rolink's criteria. Furthermore, GL7(+)B220(low) BM cells can differentiate into IgM(+) immature B cells in co-culture with stromal cells. These results suggest that B lymphocytes pass through the GL7(+) pre-B cell stage during differentiation in the BM. Thus, GL7 is the critical marker to define the proliferation stage of large pre-B cells.     

CD11b-mediated migratory property of peripheral blood B cells

BACKGROUND: CD11b belongs to the integrin family and is expressed on neutrophils, monocytes, natural killer cells, and a subset of lymphocytes. Although CD11b expressed on neutrophils and monocytes has been extensively investigated and has been reported to play an important role in the migration of these subsets of leukocytes, the function of CD11b expressed on a subset of B cells has not yet been clarified. OBJECTIVE: To elucidate the functional activity of CD11b expressed on B cells, we characterized the CD11b-expressing cells among the B-cell population and investigated their migratory ability. METHODS: Isolated peripheral blood CD19 + B cells were analyzed by flow cytometry. The migratory ability of B cells was evaluated by the transwell assay, and the contribution of CD11b to this ability was investigated by using an anti-CD11b blocking mAb. RESULTS: The majority of CD27 - IgD + naive B cells were CD11b - , whereas most CD27 + memory cells were CD11b +. Among the CD27 + memory cells, expression of CD11b was stronger on the IgD - cells than on the IgD + cells. In the transwell assay, the migrating cells were predominantly CD27 + IgD - cells, most of which expressed CD11b. The addition of an anti-CD11b blocking mAb resulted in the significant reduction of the number of migrating B cells. CONCLUSION: Memory B cells express CD11b and, in contrast with naive B cells, have high migratory ability. CD11b plays an essential role in the homing process of memory cells.     

Differential effects of manipulating signaling in early T cell development in intestinal intraepithelial lymphocytes and thymocytes

A pre-TCR-CD3 signal is required for the efficient maturation of CD4- CD8- thymocytes to the CD4+ CD8+ stage. This study addressed whether a similar signal is required for maturation of intestinal intraepithelial lymphocytes (IEL) that may develop extrathymically. We have shown previously that IEL from mice deficient for CD3- associated zeta chains include an immature population of CD3- CD8alphaalpha+ cells expressing cytoplasmic TCR beta chains but lacking detectable surface TCRalphabeta, CD16 and B220. Here we stimulated the appearance of such IEL in epsilon+/- zeta-/- mice by expression of an activated Lck transgene or in vivo treatment with anti-CD3epsilon. Anti-CD3epsilon treatment of RAG-deficient animals also yielded CD16- B220- IEL. In contrast, expression of a TCRbeta transgene in rag-1(-/-) mice did not stimulate the appearance of CD3- CD8alphaalpha+ CD16- B220- cells. Taken together these data indicate that although anti-CD3epsilon treatment and LckF505 assist in catalyzing a CD16+ B220+ --> CD16- B220- transition, these manipulations are not equivalent to a pre-TCR signal in IEL lymphocytes.     

In vivo effects of LPS on B lymphocyte subpopulations. Migration of marginal zone-lymphocytes and IgD-blast formation in the mouse spleen

The aim of the present study was to analyze the effect of LPS on the localization and differentiation of splenic B lymphocytes. Therefore, we used a double immunoperoxidase technique which enabled us to detect both the IgM+ IgD- marginal zone lymphocytes and the IgM+ IgD+ follicular lymphocytes in the same tissue section. Next to a dramatic disappearance of the predominantly IgM+ IgD- lymphocytes in the marginal zone shortly after an intravenous injection of LPS, an increased number of these cells could be found in the splenic follicles. The present results strongly suggest that the IgM+ IgD- cells in the splenic follicles represent immigrating marginal zone lymphocytes, and not differentiating follicular B cells, because no IgM+ IgD- cells could be observed in the follicles of draining lymph nodes shortly after a subcutaneous injection of a similar amount of LPS. These observations support the suggestion that LPS induces a migration of marginal zone lymphocytes into the follicles. The present results also showed the formation of IgD plasmablasts in the inner PALS and around the terminal arterioles of the spleen after LPS administration. The induction of IgD plasmablasts appeared to be a specific effect of LPS which may be related to its toxic properties.     

Cell autonomous expression of IgD is not essential for the maturation of conventional B cells.

To analyse the function of IgD in vivo, we generated a 'loss of function' mouse model utilizing gene targeting technology. By homologous recombination in a (C57BL/6 x CBA)F1 mouse embryonic stem cell (ES) line one allele of the delta heavy chain gene was rendered non-functional. In chimeric mice obtained after injection of the targeted ES cells into blastocysts derived from severe combined immunodeficient mice we analysed ES cell derived B lymphocytes expressing the targeted or the wild-type allele by using allotype specific reagents. We show that B cells expressing the targeted allele appear in the periphery as IgM+ D- cells at normal frequency. They express the CD23 marker and respond to a T cell dependent antigen. Thus, cell autonomous expression of IgD is neither essential for B cell maturation into an antigen responsive state nor for antigen dependent triggering of the cells into an immune response.     

IgM plays an important role in induction of collagen-induced arthritis

IgM is one major type of B cell receptor (BCR) expressed on most of the B cells from immature to mature stages. During normal B cell ontogeny, signals transduced through the IgM BCR play an important role in regulating B cell maturation and survival at multiple checkpoints. In addition, IgM BCR is also required for antigen-dependent differentiation and activation of B cells. However, whether IgM BCR-mediated signalling is important for the pathogenesis of autoimmune diseases remains elusive. Using IgM-deficient mice, we examined the effect of absence of IgM on the development of collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis (RA). Compared to their wild-type littermates, IgM-deficient mice were either resistant to arthritis induction or developed significantly less severe arthritis. There was a significant decrease of autoantibody production in IgM-deficient mice, particularly IgG2a antibodies, which is believed to be pathogenic in CIA. Thus, although IgM(-/-) mice have relatively normal B cell development with IgD BCR replacing IgM BCR, the absence of IgM-mediated signals has a profound impact on the development of CIA, indicating that IgM plays an important role in the development and pathogenesis of autoimmune arthritis and IgM-mediated signalling is critical in the generation of pathogenic autoreactive antibodies.