sIgM Expression Research Articles

Modulation of B Cell Receptor Signalling By IL-4 In Chronic Lymphocytic Leukaemia

Chronic Lymphocytic leukaemia (CLL) is currently incurable using conventional therapies, although new drugs aimed as signalling pathways are having some therapeutic success. Features of CLL cells reveal a balance between anergy, apoptosis or increased proliferation, with outcome determined by the B cell receptor (BCR). Engagement of the BCR triggers formation of the signalosome leading to calcium [Ca+2]i mobilization and ERK phosphorylation. However other microenvironmental factors affect CLL cell growth and survival and may modulate the BCR response. We have focused on IL-4, produced mainly by T cells and known to rescue CLL cells from apoptosis when cultured in vitro. IL-4 strongly induces phosphorylated STAT6 (STAT6-P) within CLL cells in vitro, therefore we analysed STAT6-P expression within lymph nodes of SLL/CLL patients. We detected STAT6-P expression in SLL/CLL lymph nodes predominantly within proliferation centres. Induction is likely to be via IL-4, since IL-13 and CD40L, which are also capable of inducing STAT6-P, did not produce a STAT6-P signal in CLL cells in vitro.To analyze the effect of IL-4 on BCR signalling, CLL samples were exposed to IL-4 for between 1-72h prior to treatment with anti-IgM. Using calcium flux as a marker of BCR signalling we found that IL-4 significantly increased the calcium flux, as compared to the untreated control, in 63.6% (7/11) of the cases. We showed that the increased calcium flux coincided with an upregulation of sIgM expression observed between 24-72h. Furthermore there was no significant up-regulation of sIgD levels even though calcium flux increased in a proportion of cases (6/9) in response to anti-IgD following IL-4 pre-treatment. We further investigated the effect of IL-4 on known positive and negative regulators of BCR signalling. While no significant changes in CD19 expression were found, CD5 expression was significantly downregulated in all cases which showed IL-4-mediated amplification of signal in response to anti-IgM.Next we evaluated phosphorylated ERK (ERK-P) as a marker of BCR signalling. CLL cells were incubated overnight in the presence or absence of IL-4 and subsequently stimulated with soluble anti-IgM for 15 minutes. We found ERK-P expression was augmented following αIgM activation in the presence of IL-4 compared to the control in a proportion of samples (12/15). When CLL cells were treated with the PI3K inhibitor LY294002 prior to the addition of anti-IgM, ERK-P was reduced due to inhibition of the BCR signalosome upstream of ERK. However pre-treating CLL cells with IL-4 enabled the cells to overcome chemical inhibition of PI3K with LY294002 and restored ERK-P expression. Using densitometry analysis a significantly greater signalling capacity quantified by ERK-P expression (P=0.042) was observed in 15/23 patients. Interestingly, 73.3% of those 15 cases were unmutated CLL (U-CLL), the aggressive form of the disease.Taken together, these data shows that IL-4 is able to enhance conventional BCR mediated signalling and may partially overcome chemical inhibition of the classical BCR pathway in CLL cells. The augmentation of BCR signalling is partly mediated by an increase in sIgM receptor density but may also be partly regulated by modulation of the positive and negative regulators of BCR signalling. Together these data indicate an important role of the microenvironment in BCR signalling in CLL. Disclosures:No relevant conflicts of interest to declare.

Identification in CLL of circulating intraclonal subgroups with varying B-cell receptor expression and function

AbstractChronic lymphocytic leukemia (CLL) is a tumor of circulating B cells, variably stimulated and anergized following exposure to antigen in lymphoid tissues. Down-modulation of surface IgM (sIgM) occurs, but expression and signal capacity can recover in vitro and apparently in vivo during recirculation. We have now dissected individual circulating clones of CLL cases according to sIgM expression level by differential binding to bead-bound anti-IgM. Four clear subgroups (SG1-4) with increasing sIgM were identified in 37/37 cases. Engagement of sIgM induced phosphorylation of PLCγ2 and ERK1/2 at levels ranging from very low in SG1 to high in SG4. Phosphorylation was suppressed by the BTK inhibitor ibrutinib. Expression of CXCR4 also increased from SG1 to SG4, but markers of previous activation and proliferation were dominant in SG1. Incubation of whole CLL populations in vitro led to striking increases in CXCR4 expression as well as recovery of sIgM. Clonal analysis reveals dynamic SGs following presumed antigen stimulation in tissues. SG4 represents a fully recovered, potentially dangerous population equipped to migrate to tissue and receive a proliferative stimulus. SG1 likely represents a postmitotic unresponsive “resting” population. The effect of ibrutinib on the small SG4 population may be the critical factor in therapeutic success.

B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia

Abstract SCI-25The B-cell receptor (BCR) is a flexible and variable environmental sensor with no fixed ligand. The central component is surface Ig (sIg), which appears to function at several levels, mediating a “tonic” signal essential for survival and also binding to antigens via its variable (V) regions. Expression of sIg and reliance on BCR signals generally persist in neoplastic B cells. Indolent tumors, including chronic lymphocytic leukemia (CLL), allow insight into the pathogenetic role of the BCR prior to therapy as well as revealing key proteins within these pathways for drug targeting. CLL is heterogeneous, arising at two points of differentiation, generating two major subsets, one with unmutated Ig V genes (U-CLL), another with mutated Ig V genes (M-CLL). U-CLL appears to develop from naïve B cells of the natural antibody repertoire aimed against common pathogens. The clinical behavior of the two subsets differs, with U-CLL being of poorer prognosis. Evidence for antigen drive on both subsets comes from detecting “endocytosis in vivo,” whereby sIgM expression and signal capacity in blood cells are variably downmodulated, but can recover in vitro. Mysteriously, sIgD of the same presumed antigen specificity shows no evidence for endocytic downmodulation in vivo. CLL cells apparently engage antigens via sIgM in tissue sites, leading to proliferation and downmodulation, with reexpression gradually occurring during transit through the blood. Expression of CXCR4 closely follows that of sIgM, and clonal analysis reveals subpopulations of potentially dangerous cells with high sIgM/CXCR4 primed for tissue-based proliferative stimulation. In contrast to normal B cells, this is an iterative process exposing the proliferating CLL cells to further genetic changes. Overall higher sIgM levels and increased signal capacity in U-CLL likely account for more aggressive clinical behavior. BCR-induced membrane-proximal events include LYN-mediated phosphorylation of Iga/b followed by recruitment of the tyrosine kinase Syk. Signal propagation then involves Btk and PLCg2. LYN-dependent phosphorylation of CD19 also recruits the p85 subunit of PI3K, a known survival mechanism in CLL. Downstream events include upregulation of MYC proto-oncoprotein expression and induction of MYC-regulated target genes such as cyclin D2, with both proteins detected in proliferation centers. Pathways to increased cell survival include induction of the antiapoptotic MCL1 protein and inactivation of the proapoptotic activity of BIM(EL/L) via enhanced phosphorylation. The ability to phosphorylate BIM(EL) was highly correlated with mutational status and with requirement for treatment. While these events delineate BCR-activated pathways, they provide only the skeleton. sIgM signaling is highly dependent on the polymeric nature of the antigen, with responses to solid-phase stimulus producing a higher and more prolonged signal than the soluble form. Clearly, CLL cells have to integrate BCR signals with those from other receptors for the multitude of microenvironmental factors. This is a two-way process, since BCR signals operate “inside-out” by modulating the expression of molecules involved in migration and adhesion. The fact that the glycan composition of sIgM is also modulated to a mannosylated form, potentially able to bind to mannose-binding lectins, could contribute to the latter. Clinical effects of Syk, Btk and PI3Kd inhibitors, known to affect BCR signaling and potentially other pathways, are both explicable and exciting. Disclosures:No relevant conflicts of interest to declare.

Anergic Responses Characterize a Large Fraction of Human Autoreactive Naive B Cells Expressing Low Levels of Surface IgM

B cell anergy represents an important mechanism of peripheral immunological tolerance for mature autoreactive B cells that escape central tolerance enforced by receptor editing and clonal deletion. Although well documented in mice, the extent of its participation in human B cell tolerance remains to be fully established. In this study, we characterize the functional behavior of strictly defined human naive B cells separated on the basis of their surface IgM (sIgM) expression levels. We demonstrate that cells with lower sIgM levels (IgM(lo)) are impaired in their ability to flux calcium in response to either anti-IgM or anti-IgD cross-linking and contain a significantly increased frequency of autoreactive cells compared with naive B cells with higher levels of sIgM. Phenotypically, in healthy subjects, IgM(lo) cells are characterized by the absence of activation markers, reduction of costimulatory molecules (CD19 and CD21), and increased levels of inhibitory CD22. Functionally, IgM(lo) cells display significantly weaker proliferation, impaired differentiation, and poor Ab production. In aggregate, the data indicate that hyporesponsiveness to BCR cross-linking associated with sIgM downregulation is present in a much larger fraction of all human naive B cells than previously reported and is likely to reflect a state of anergy induced by chronic autoantigen stimulation. Finally, our results indicate that in systemic lupus erythematosus patients, naive IgM(lo) cells display increased levels of CD95 and decreased levels of CD22, a phenotype consistent with enhanced activation of autoreactive naive B cells in this autoimmune disease.

Characterization of intracellular signaling profiles induced by antigen receptor activation in murine B cell subsets (132.30)

Abstract Protein phosphorylation and Ca2+ mobilization are among major signaling parameters characterizing the activation status of B lymphocytes. This study identifies quantitative differences between intracellular signaling profiles in subsets of follicular, marginal zone, transitional T1, T2, T3 murine B cells. Phosphorylation levels of signaling proteins and Ca2+ responses upon BCR engagement with anti-IgM were measured by multicolor flow cytometry. The results revealed previously uncharacterized diversity of signaling profiles in different B cells subsets that correlated with their developmental stage and previous antigen exposure. At least three important trends were identified: 1) B cells at earlier developmental stages had higher activation threshold, baseline Ca2+ and phosphorylation activity, and Ca2+ dynamics that in some instances did not correlate with sIgM expression levels; 2) phosphorylation levels within particular B cell subsets were heterogeneous which suggests the presence of various cell groups with different signaling properties within each subset; 3) analysis of phosphorylation profiles in individual signaling molecules revealed molecule-specific nonlinear characteristics of dose-response curves that are indicative of quantitative signaling differences between B cell subsets and subset-specific balance of BCR signaling cascades. These preliminary findings provide rationale for investigating roles of different B cell subsets in alterations of immune tolerance.

Expression of Surface IgG Is High in Chronic Lymphocytic Leukaemia, and Co-Expression with IgM Confers a Worse Prognosis.

Abstract 4371 IntroductionThe classical immunophenotype of Chronic Lymphocytic Leukaemia (CLL) involves weak or moderate expression of surface immunoglobulin (sIg) M and D. Expression of sIgG has generally been considered to be rare (published estimates 4-28%). The B Cell Receptor is thought to be central to the pathophysiology of CLL. Patients and MethodsWe present data from an analysis of 748 patients with CLL who have had flow cytometry performed at Barts and the London NHS trust. This includes patients from several other clinical centres serving a population of 3.7 million. A cohort of cases (n=26) displaying differing patterns of sIgG and sIgM expression was investigated further by: 4-colour flow cytometry; detection of constant (C) gene mRNA transcripts including Ca, Cm, Cg, or Cd to confirm isotype status; variable heavy chain somatic mutation rate (VH status); and functionality of the sIg using calcium flux and cell proliferation assays ([H3]- thymidine incorporation) after BCR crosslinking using isotype-specific F(ab')2 anti-human Ig. We also analysed whether differing patterns of sIgG and sIgM expression conveyed prognostic impact. Results- Prevalence of sIgG expression399 of the 748 patients with CLL had complete sIgG and sIgM expression data. We found that 33% of all CLL cases express sIgG (19% with co-expression of sIgG and sIgM). 19% appeared to have neither sIgG nor sIgM expression. When tested for, sIgD appeared to be present in most of this IgG-IgM- subset. The expression pattern appeared to be stable over time in the majority of cases. Functionality of sIgGTo explore the signalling capacity of the sIg, a cohort of 26 patients with readily available cells was chosen that displayed a variety of expression patterns of sIgG and sIgM (6 IgG-IgM+, 3 IgG+IgM-, 15 IgG+IgM+, 2 IgG-IgM- ). This cohort underwent further testing:Four-colour flow cytometry confirmed the presence of both sIgG and sIgM on single CLL cells in cases of IgG+IgM+ identified in the diagnostic laboratory, as well as the sIgG and sIgM expression of other cases. In the IgG-IgM+ cases 3 of 6 were VH mutated, one had Cg mRNA transcripts detectable, with all expressing Cm. The IgG+IgM- cases were VH mutated and contained only mRNA for Cg gene transcripts. Of the IgG+IgM+ cases 10/15 were VH mutated. All had detectable Cg and Cm transcripts, with several expressing Ca and Cd. One IgG-IgM- case had no detectable C-gene transcripts but detectable VDJ-genes, the other had Cm and Cd transcripts detectable in the absence of sIgG and sIgM. Both IgG-IgM- cases were VH mutated. Crosslinking of the sIg with appropriate anti sIgG or sIgM F(ab')2 resulted in calcium flux and cell proliferation. These data confirm that the expressed sIgG and sIgM is produced by the CLL cell, and are functionally competent to signal. Prognostic impact: Survival and treatment data were available on 382 patients in our database. Of these, 244 had complete sIgG and sIgM expression determination. Analyses of the sIgG and sIgM expression subgroups suggested that while there is no difference in time to first treatment (median 4.5 years), the IgG+IgM+ group (n=52, median OS 7 years) had a significantly shorter overall survival (OS) compared to the IgG-IgM+ (n=126, median survival not reached), IgG+IgM- (n=29, median OS 11 years), or IgG-IgM- (n=37, median survival not reached) groups. [Display omitted] ConclusionsIn a large cohort of patients with CLL, we show that expression of sIgG is common, with a large number of cases co-expressing sIgG and sIgM simultaneously. When stimulated, both isotypes are capable of signal transduction resulting in cellular activation. These malignant cells appear to have deranged mechanisms of Class Switch Recombination. The IgG+IgM+ group of patients appears to have a more treatment resistant disease, conferring shortened survival. Many different groups have suggested the B Cell Receptor to be an important component of CLL pathophysiology. Our data support these models and provide further insight into the underlying biology of CLL, as well as offering another prognostic marker and possible target for treatment. Disclosures:No relevant conflicts of interest to declare.

Deubiquitination of FANCD2 Is Required for DNA Crosslink Repair

SummaryMonoubiquitination of FANCD2 and PCNA promotes DNA repair. It causes chromatin accumulation of FANCD2 and facilitates PCNA's recruitment of translesion polymerases to stalled replication. USP1, a protease that removes monoubiquitin from FANCD2 and PCNA, was thought to reverse the DNA damage response of these substrates. We disrupted USP1 in chicken cells to dissect its role in a stable genetic system. USP1 ablation increases FANCD2 and PCNA monoubiquitination but unexpectedly results in DNA crosslinker sensitivity. This defective DNA repair is associated with constitutively chromatin-bound, monoubiquitinated FANCD2. In contrast, persistent PCNA monoubiquitination has negligible impact on DNA repair or mutagenesis. USP1 was previously shown to autocleave after DNA damage. In DT40, USP1 autocleavage is not stimulated by DNA damage, and expressing a noncleavable mutant in the USP1 knockout strain partially rescues crosslinker sensitivity. We conclude that efficient DNA crosslink repair requires FANCD2 deubiquitination, whereas FANCD2 monoubiquitination is not dependent on USP1 autocleavage.

High Frequency of Surface Immunoglobulin IgG in B-Cell Chronic Lymphocytic Leukaemia.

Background: A common diagnostic feature of chronic lymphocytic leukaemia (CLL) is the weak surface immunoglobulin (sIg) expression of the IgM/D isotypes. The occurrence of sIgG is believed to be an atypical finding in CLL.Aims: To demonstrate that sIgG expression is a real and not artefactual by molecular and functional assays.Methods: 194 unselected, consecutive cases of CLL were analysed during routine diagnostic testing for sIgG and sIgM. Flow cytometry was performed using fresh whole blood and red cell lysis (Pharmalyse solution, BD). For confirmation of these results and demonstration of functionality, we used 4-colour flow cytometry, PCR for mRNA detection, proliferation assay (3H incorporation), and calcium flux assay.Results: 23% (44/194) of all CLL cases displayed sIgG, with 39 being sIgG+ alone. PCR results showed a distinct band indicating the presence of mRNA for IgG. Four-colour flow cytometry identified single cells that co-expressed CD19/CD160/sIgG and ruled out sIgM expression. In vitro proliferation assays demonstrated that sIgG is functional when triggered by F(Ab')2 fragments specific for sIgG isotype. The strength of the signal in one case after stimulation via sIgG led to a 3.5-fold increase in cellular proliferation. Calcium flux assays also demonstrated that the IgG isotype was functional, one case showing a 50% increase in fluorescence intensity after stimulation via sIgG.Discussion: 23% of CLL cases have sIgG expression. In addition to a high frequency of sIgG expression, this study has shown sIgG to be capable of signalling. If antigen-drive is important in CLL, an IgG B-cell receptor should have higher affinity for a putative antigen, which potentially would be a poor prognostic feature. However, such cases would also be expected to show mutated VH genes, indicating a better prognosis. Further study is required to elucidate the pathological significance of sIgG expression and its role in CLL.

Reversible anergy of sIgM-mediated signaling in the two subsets of CLL defined by VH-gene mutational status

AbstractThe 2 subsets of chronic lymphocytic leukemia (CLL), of worse or better prognosis, likely derive from pre-GC unmutated B cells, or post-GC mutated B cells, respectively. Different clinical behavior could relate to the ability of tumor cells to respond to surface (sIg)–mediated signals. Unmutated cases (U-CLL) have an increased ability to phosphorylate p72Syk in response to sIgM ligation compared to mutated cases (M-CLL). We now confirm and further investigate this differential signaling in a large cohort by [Ca2+]i mobilization. Cases responding to sIgM ligation express higher levels of CD38, ZAP-70, and sIgM. However, CD38 does not influence signaling in vitro or associate with response in bimodal CD38-expressing cases. Similarly, ZAP-70 expression is not required for response in either U-CLL or M-CLL. Strikingly, partially or completely anergized sIgM responses from each subset can recover both sIgM expression and signal capacity spontaneously in vitro or following capping/endocytosis. This provides direct evidence for engagement of putative antigen in vivo. Signaling via sIgD differs markedly being almost universally positive in both U-CLL and M-CLL, with no association with CD38 or ZAP-70 expression. Downstream signaling pathways, therefore, appear intact in CLL, locating anergy to sIgM, mainly in M-CLL. Integration of differential isotype-specific effects mediated by (auto)antigen may determine tumor behavior.

Follicular Lymphoma-Like B-Cells in Healthy Individuals: A Novel Intermediate Step in Early Lymphomagenesis.

Follicular lymphoma (FL) is one of the most common B-cell lymphoma, and remains virtually incurable despite its relatively indolent nature. T(14;18)(q32;q21), the genetic hallmark and early initiating event of FL pathogenesis, is also present at low frequency (10−5–10−7) in blood from healthy individuals (HI), indicating that t(14;18) and the ensuing BCL2 overexpression is necessary but not sufficient for malignant transformation. It has long been assumed that in HI, t(14;18) is carried by circulating quiescent naïve B-cells, where its oncogenic potential would be restrained. Yet, several reports, including long-term persistence and immunomodulation of t(14;18)+ cells in lymphoma-free individuals, led us to question this model and investigate the status of circulating t(14;18)+ cells in HI. We first determined if t(14;18)+ cells are naïve B-cells by assessing class-switch recombination (CSR) on the translocated allele. Using 2 long-range PCR assays designed to amplify unswitched BCL2/Sμ and switched BCL2/Sg regions, DNA samples from 6 HI with t(14;18) were tested. Contrary to previous assumptions, our data clearly show that most peripheral t(14;18)+ cells already underwent CSR (n=5/6) and therefore that most t(14;18)+ cells are not naïve B-cells. Are they then memory B cells? Naïve and memory B cell subsets from 9 HI were isolated by cell sorting according to IgD and CD27 markers, and the rate of t(14;18) analyzed in each subset relatively to that of the total B cells. Strikingly, while the level of naïve t(14;18)+ cells remained at baseline for all individuals, memory B-cells tightly accounted for the wide modulation of t(14;18) frequencies observed between individuals. In addition, sequence analysis of t(14;18) clones revealed that this wide modulation was not due to the accumulation of clonally unrelated t(14;18) naïve B-cells, but rather to the clonal expansion of t(14;18)-bearing memory B-cells. To further define the t(14;18)+ cells, we next examined the repartition of the translocation in the IgD−/CD27+ and IgD+/CD27+ memory B-cell subsets. Unexpectedly, we found that the IgD+/CD27+ subset contained significantly higher rates of translocation than the IgD−/CD27+, both in terms of prevalence and frequency. Thus, while CSR is found in the majority of translocated alleles (~75%), most t(14;18)+ memory B cells have not switched their productive allele (~70%) and express an IgM/D. Most importantly, although atypical among physiological peripheral B-cells, this “allelic paradox” is a specific hallmark of FL, and suggests the presence of the same selective pressure in favor of sIgM expression on a B-cell population that is at the same time permanently driven to switch. In line with B-cell hyperplasia in BCL2 transgenic mice slowly progressing to low grade lymphoma, it is likely that “FL-like” cells in HI are rescued by BCL2 from apoptosis, and “frozen” at a differentiation stage in which constitutive AID expression drives continuous somatic hypermutation and CSR activity, two mechanisms conferring a high propensity for genomic instability. Altogether, our findings identify a novel intermediate step in early lymphomagenesis, and strongly impact both on the current understanding of disease progression from potent pre-malignant niches, and on the proper handling of t(14;18) frequency in blood as a potential early biomarker for lymphoma.

Gene Expression Profiling of Diffuse Large B-Cell Lymphomas Supervised by CD21 Expression.

We have reported that CD21 expression in tumor cells is a favorable prognostic factor in diffuse large B-cell lymphomas (DLBCLs)(Ogawa S, et al. Br J Haematol , 2004; 125: 180–86). In the present study, we investigated the relationship between CD21 expression and the groups of germinal center B-cell-like (GCB) DLBCLs and activated B-cell-like (ABC) DLBCLs. The diagnosis of DLBCL was made according to the WHO Classification. CD21 in tumor cells was examined by means of immunohistochemistry using frozen sections. Forty cases of DLBCLs were investigated using Agilent 44K human oligo-microarrays (Agilent Technologies, Palo Alto, CA). GCB and ABC DLBCLs were identified based on Rosenwald's gene set (Rosenwald A, et al. N Engl J Med , 2002; 346: 1937–47). Four cases (10 %) of 40 DLBCLs were found to be GCB DLBCLs, and 36 cases ABC DLBCLs. Incidence of CD21-positive cases was 50% (2/4) in GCB DLBCLs and 39% (14/36) in ABC DLBCLs. Overall survival of 36 patient with ABC DLBCLs were shown in Fig.1. [Display omitted] Signature genes to distinguish between CD21-positive and CD21-negative DLBCLs were as follows; IgM (P=0.00017), CCR6 (P=0.0011), IL7 (P=0.0017), IgK (P=0.0037), EBI2 (P=0.0066), CD24 (P=0.0099) etc. were overexpressed in CD21-negative DLBCLs, and CR2 (CD21) (P=0.0010), MKNK2 (P=0.0028), LMO2 (P=0.00478), CDKN2A (P=0.0059), PDE8B (P=0.0066) etc. were overexpressed in CD21-positive DLBCLs. In 40 cases of DLBCLs, overall survival of patients with sIgM-positive DLBCL was significantly worse than that of patients with sIgM-negative DLBCL (P=0.023). Furthermore, in 216 consecutive cases of DLBCLs analyzed by immunohistochemistry using frozen sections from 1987 to 2004, overall survival according to sIgM expression was significantly different (P=0.013).In conclusion,CD21 expression was a favorable prognostic factor in ABC DLBCLs.A top feature gene to distinguish between CD21-positive and CD21-negative DLBCLs was IgM. CD21 expression seemed to be related to the differentiation level of lymphoma cells.sIgM expression itself was a poor prognostic factor in ABC DLBCLs and in whole DLBCLs.

Effect of Dexamethasone on the Surface Expression of Marker Molecules and Differentiation of Murine B Cells

Background: There are at least two different subsets of B cells, B-1 and B-2. The characteristic features and function of B-2 cells in addition to the effect of steroids on B-2 cells are well-known. Although B-1 cells have different features and functions from B-2 cells, the effect of steroids on B-1 cells is not completely understood. Therefore, this study examined the effects of dexamethasone on peritoneal (or B-1 cells) and splenic B cells (or B-2 cells). Methods: Purified B cells were obtained from the peritoneal fluid and the spleens of mice. The isolated B cells were cultured in a medium and after adding different concentrations of dexamaethasone. The cell survival rate was measured by flow cytometry using propidium iodide. The expression level of the B cell surface marker was analyzed by flow cytometry. During the culture of these cells, immunoglobulin secreted into the culture supernatants was evaluated by an enzyme-linked immunosorbent assay. Results: The survival rate of peritoneal and splenic B cells decreased with increasing dexamethasone concentration. However, the rate of peritofieal B cell apoptosis was lower than that of splenic B cells. CDS and B7.1 expression in peritoneal B cells and CD23 and sIgM expression in splenic B cells after the dexamethasone treatment were reduced. When B cells were treated with dexamethasone, the spontaneous IgM secretion decreased with increasing dexamethasone concentration. Conclusion: Dexamethasone induces apoptosis in peritoneal and splenic B cells. However, peritoneal B cells are less sensitive to dexamethasone. The dexamethasone suppressed expression of the surface markers in peritoneal B cells is different from those in splenic B cells.

Cell surface immunoglobulin regulated checkpoints in chicken B cell development

The bursa of Fabricius is critical for the normal development of B lymphocytes in avian species. Productive colonization of bursal follicles by B cell precursors requires surface immunoglobulin expression. We have shown using retroviral gene transfer that expression of chimeric receptors containing the extracellular and transmembrane domains of murine CD8α and CD8β fused to the cytoplasmic domains of chicken Igα and Igβ can support productive bursal colonization in the chicken embryo in bursal cells lacking the expression of endogenous sIgM. We show here that chimeric receptor expression does not support continued bursal cell development after hatch. However intrabursal administration of anti-CD8 antibodies that ligate the CD8α:Igα chimeric receptor results in maintained numbers of bursal cells that express the chimeric receptor in the absence of endogenous sIgM. These results support a model in which sIgM receptor expression is required for productive bursal colonization in the chick embryo but sIgM receptor ligation is required to support later B cell development after hatch.

Differential Expression of CD180 and sIgM by B Chronic Lymphocytic Leukaemia Cells (B-CLL) Using Mutated and Unmutated IgVh Genes.

Introduction. B cell chronic lymphocytic leukaemia (B-CLL) is a heterogeneous disease as shown by differential expression of a variety of surface and cytoplasmic markers. In a search for markers that could define biological activity of different B-CLL subsets, we have studied the surface expression of the Toll-like receptor (TLR) family member CD180 in relation to other surface markers and mutation status of IgVh genes.Methods. Seventy eight B-CLL patients (68 untreated and 10 treated) and 15 age-matched controls were studied in three different clinics. CD19+ B cells were stained using indirect immuno fluorescence for CD180, surface IgM (sIgM), CD79b and CD38, analysed by flow cytometry and data expressed as the relative antibody binding sites (RBS)/cell for each marker. Monoclonal anti-CD5 antibodies were also used with anti CD180 to determine levels of expression of CD180 in control CD5+ cells. IgVh mutation was determined for 47 patients.Results B-CLL cells had variable levels of CD180 expression, but this was always less (1036 ± 935 RBS/cell) than that expressed by normal blood B cells (5548 ± 2271 RBS/cell) and was stable for up to 18 months. Significantly higher levels of CD180 were expressed by B-CLL cells with mutated (M) compared with those using unmutated (UM) IgVh genes. This was in contrast to the higher levels of expression of sIgM by B-CLL cells using UM than M IgVh genes (Figure).Conclusions. CD180 is expressed at higher levels on B-CLL cells using M than those using UM IgVh genes and is in contrast to the level of expression of sIgM which is higher on B-CLL cells using UM versus M genes.This differential expression of CD180 supports the notion that B-CLL cells using UM IgVh genes represent a population of cells actively responding to signals (perhaps to self antigens) via their surface IgM. [Display omitted]

IL-7 does not prevent pro-B/pre-B cell maturation to the immature/sIgM(+) stage.

IL-7 plays many fundamental roles during murine B lineage development. One reported function is to maintain progenitors in a developmentally immature state by preventing differentiation to the surface IgM (sIgM)(+) stage. Withdrawal of IL-7 from cultures has been shown to lead to increases in mature traits such as RAG expression, IgL rearrangements and expression of sIgM. These observations have been interpreted as an inductive event promoting the differentiation of progenitor cells. In contrast to this, we reproducibly observe sIgM(+) cells that have differentiated in cultures containing IL-7. We find that sIgM(+) cells arise as a normal consequence when B lineage cells are cultured in the presence of IL-7. However, these cells are short-lived and are quickly replaced by newly emerging sIgM(+) cells that differentiate from sIgM(-) progenitors. Withdrawal of IL-7 from these cultures only prevents the survival and proliferation of CD2(-)sIgM(-) pro-B cells but does not change the number of cells that differentiate to the sIgM(+) stage. This changes the ratio of sIgM(-):sIgM(+) cells and results only in an apparent maturation of the culture as a whole. Therefore withdrawal of IL-7 from these cultures acts as a selection event, not an induction event, for populations that are normally present.

In Bacillus subtilis W23, the duet sigmaXsigmaM, two sigma factors of the extracytoplasmic function subfamily, are required for septum and wall synthesis under batch culture conditions.

The synthesis of poly(RboP), the main Bacillus subtilis W23 teichoic acid, is encoded by tarDF-tarABIJKL operons, the latter being controlled by two promoters designated PtarA-int and PtarA-ext. Analysis by lacZ fusions reveals that PtarA-int activity exhibits sharp increases at the beginning and end of the transition between exponential and stationary growth phase. As confirmed by mRNA quantification, these increases are mediated by ECF sigma factors sigmaX and sigmaM respectively. In liquid media, strain W23 sigX sigM double mutants experience serious difficulties in the transition and stationary growth phases. Inactivation of sigmaX- and sigmaM-controlled regulons, which precludes transcription from PtarA-int, leads to (i) delays in chromosome segregation and septation and (ii) a transient loss of up to 30% of the culture OD or lysis. However, specific inactivation of PtarA-int, leading mainly to a shortage of poly(RboP), does not affect growth while, nevertheless, interfering with normal septation, as revealed by electron microscopy. The different sigM transcription in strains W23 and 168 is discussed. In W23, expression of tarA and sigM, which is shown to control divIC, is inversely correlated with growth rate, suggesting that the sigM regulon is involved in the control of cell division.

Clustering of the B cell receptor is not required for the apoptotic response.

We examined the role of BCR cell membrane redistribution in anti-IgM-induced apoptosis in three human B cell lines, RA#1, 2G6, and MC116, that differ in their relative levels of sIgM expression. The apoptotic response was found to be dependent on the nature of the anti-IgM and the cell line. In the cell lines, RA#1 and MC116, sIgM aggregated into patches that were insensitive to the disruption of cholesterol-rich membrane microdomains by nystatin or beta-MCD. The B cell line 2G6 was able to reorganize sIgM into a tight coalescent cap upon anti-IgM treatment. However, in this case, the lipid raft inhibitors nystatin and beta-MCD disrupted the patching. In 2G6 cells, BCR-mediated apoptosis was not affected by nystatin treatment, whereas it increased in beta-MCD pretreated cells. Thus, no evident correlation was found between apoptosis and BCR cell membrane redistribution or lipid raft formation in either of the three cell lines. The data indicate that the apoptotic signal transduction pathway is independent of BCR translocation into lipid rafts and/or aggregation.

Selective enhancement of B cell antigen receptor-mediated antigen presentation by treatment with transforming growth factor-beta.

TGF-beta1 was examined for the ability to regulate Ag-presentation by B cells, using A20-HL B lymphoma cells bearing TNP-specific IgM receptors. Treatment of A20-HL cells with TGF-beta1 at 1 ng/ml, a concentration that inhibited proliferation, enhanced presentation of Ag internalized via surface IgM (sIgM), but not via fluid-phase pinocytosis. TGF-beta1-treatment slightly enhanced surface expression of sIgM, but not of MHC class II molecules. The treatment accelerated recovery of sIgM expression after its removal by ligation with TNP-OVA, and induced prolonged intracellular residence of TNP-OVA internalized via sIgM, which co-localized with intracellular MHC class II molecules. TGF-beta1-treatment increased accumulation of newly synthesized intracellular MHC class II molecules that were localized in compartments positive for lysosome-associated membrane protein 1, although cellular protein synthesis was decreased by the treatment. The accumulated intracellular MHC class II molecules were triggered to the cell surface by ligation of sIgM. Finally, TGF-beta1-treatment induced Igalpha-phosphorylation in response to lower concentrations of TNP-OVA. On the basis of these findings, we conclude that TGF-beta1-treatment of A20-HL cells selectively enhances the ability to present Ag internalized via sIgM, not via fluid-phase pinocytosis, through accelerating sIgM recovery, increasing accumulation of intracellular MHC class II molecules and enhancing the ability of sIgM ligation to induce Igalpha-phosphorylation.

B-Cell Suppression in Adult Mice Injected with Anti-δ Followed by Anti-μ mAb

Cross-linking of surface IgM or IgD B-cell receptors (BCR) with appropriate anti-Ig antibodies induces IgMhigh or IgDhigh B-cell depletion, respectively. The aim of this paper is to analyze how injections of anti-δ followed by anti-μ monoclonal antibodies (mAb) can deplete and suppress B cells and then induce T-independent type 2 antigen tolerance in adult mice even after treatment is stopped. The experimental protocol consisted of three daily injections of anti-δ mAb followed by repeated injections of anti-μ mAb. It shows that a sequential injection of anti-δ and anti-μ mAb induces B-cell depletion and T-independent type 2 response downregulation. Morever, the T-dependent response is maintained, except for the IgG3 isotype. After clearance of the anti-δ mAb from the circulation, B cells reappear as an IgD+ IgM− B-cell population in the bone marrow (BM) and spleen. The origin of IgD+ IgM− cells was studied in scid mouse transfer models. We show that IgD+ IgM− B cells are not mature cells reexpressing sIgD but BM-derived cells that require a T-cell presence to be developed. The lack of sIgM expression by posttranscriptional regulation and the need of T-cell help for escaping anti-μ negative selection suggest strongly that this population had properties similar to those of anergized B cells. These results support the potential use of sequential injections of anti-δ and anti-μ in the prevention of xenograft rejection.

In Vitro Characterization of chB6 Positive and Negative Cells from Early Avian Embryos

Density gradient-separated embryonic spleen and bone marrow cells were enriched for chB6+ cells with positive and negative selection procedures and magnetic cell sorting. The majority of chB6+ cells, consisting of small, dense, strongly chB6+ cells, were prone to apoptosis, which was further accentuated after exposure to monoclonal antibodies directed against chB6 alloantigen, but was largely inhibited by PDBU, leading to maintenance and frequently numerical increases of chB6+ cells after an initial decline. sIgM+ cells within that population followed a very similar pattern, suggesting a PDBU-induced upregulation of sIgM expression on a proportion of chB6+ cells. The protective effect of PDBU on anti-chB6-exposed cells was confirmed with bursal lymphocytes and shown to be entirely PDBU concentration dependent. It was calculated that each 14-day embryonic spleen contained a minimum of 250,000 chB6+ and 3000–4000 sIgM+ cells, respectively. Endogenous apoptosis appeared to be increased with embryonic age, reaching a peak with bursal cells in the posthatching period. A second population of larger, less dense, and weak chB6+ cells, often with vacuoles in a more abundant cytoplasm, differed functionally, expanding numerically in unstimulated cultures and being inhibited by PDBU. No sIgM+ cells developed within this population. It is proposed that this chB6+ fraction may represent progenitors of a previously suggested chB6+ subset of macrophages, in contrast to the dense chB6+, small cells, viewed as B cell progenitors. chB6− cells, consisting predominantly of granulocytes, proliferated vigorously in unstimulated cultures, but were consistently inhibited by PDBU. Coculture of age-matched embryonic bursal stroma with positively and negatively enriched chB6+ cells revealed enhanced protection from apoptosis for chB6+ cells and a PDBU-induced upregulation of sIgM expressing cells. Bursal stroma also had a pronounced positive effect on the proliferation of chB6− cells.