Influence of human thymocytes on B-lymphocyte differentiation in man.

Human thymocytes from children less than 6 years of age were tested for their influence on differentiation of normal B cells. The addition of either thymocytes or a culture supernatant from thymocytes to normal peripheral blood lymphocytes (PBL) enhanced pokeweed mitogen-induced B-cell differentiation as tested in a plaque-forming assay for antibody to sheep red blood cells. The thymocytes, however, could not substitute for T lymphocytes in cultures of PBL which had been previously depleted of T lymphocytes. Further, prior treatment of thymocytes with concanavalin A did not result in generation of suppressor cells for either B-cell differentiation or for the responses of PBL to mitogens. Thus, although thymocytes were functionally immature by these assays as compared to mature T lymphocytes they exerted an influence on B-cell differentiation in cultures of normal peripheral blood lymphocytes.     

Quantification of the leukocyte common antigen (CD45) in mature B-cell malignancies.

CD45 is a glycoprotein expressed on all lymphohematopoietic cells. Its expression increases during normal B-cell differentiation and remains stable on mature cells. Although it is widely known that CD45 antigen expression is decreased in B-acute lymphocytic leukemia (ALL), only scarce and contradictory information is available on CD45 expression on mature B-cell malignancies. In healthy adults (n = 15), CD45 expression on B lymphocytes was lower than that on T cells. In patients with chronic lymphocytic leukemia (CLL; n = 22), CD45 expression on malignant cells was lower than that on the whole lymphocyte population of healthy adults (n = 28) and on normal B lymphocytes (n = 15). In 6 of the 22 CLL patients, the malignant cell population could be separated from the normal lymphocyte population on the CD45-side scatter (SSC) plot. In 16 CLL patients, there was some degree of overlap between the malignant and normal cells with respect to CD45 expression. For these patients, there was an inverse correlation between CD45 expression on the whole lymphocyte population and the percentage of malignant cells in this population. In two patients with mantle cell lymphoma (MCL), CD45 expression on the malignant cells appeared lower than that on normal B cells and on the whole lymphocyte population. In six patients with hairy cell leukemia (HCL), CD45 expression on hairy cells was comparable to that on the whole lymphocyte population of healthy adults, but slightly higher than that of normal B cells. Evaluation of CD45 expression may help to characterize mature B-cell malignancies.     

SC3 monoclonal antibody defines a novel specific human B-cell surface antigen differentially expressed on B-cell leukaemias and lymphomas and involved in the proliferation of normal and malignant B lymphocytes

The monoclonal antibody SC3 was raised against the NK leukaemia cell line YTindi. It detected a 98-kDa surface antigen with weak expression on a restricted number of leukaemia cell lines under reducing conditions. SC3 mAb labelled 5-10% of normal peripheral blood lymphocytes corresponding almost exclusively to B lymphocytes, and 60-70% of tonsillar B cells. It did not react with erythrocytes, platelets or monocytes whereas it stained granulocytes. The aim of the present study was to examine the expression and functional effects of SC3 mAb reactive epitope on normal and malignant B cells. Most SC3+ B cells from healthy donors were CD23+, some co-expressed CD5 and CD27 and a few were CD38+. SC3 epitope was expressed exclusively by B-lineage malignant proliferations, including B-lineage ALL. Practically, all B-CLL studied expressed SC3 mAb reactive epitope although with variable intensity, while MCL and PLL were negative. Other low grade and high grade B-NHL were variably stained. SC3 mAb alone triggered the proliferation of CD2-depleted PBL and significantly increased the proliferation induced by suboptimal concentrations of LPS. This effect was much weaker with B-CLL cells but was increased after cross-linking with an anti-IgM antibody. The restricted expression pattern combined with molecular weight and functional data indicate that SC3 mAb may detect a novel B-cell antigen mostly expressed by early and naive B cells. Although its expression in B-cell malignancies was not limited to a single differentiation stage, it might confer specific functional characteristics to the positive malignant cells.     

Regulation of the natural killer activity of lymphocytes from healthy donors and patients with chronic lympholeukemia through the interaction of T- and non-T cells

The role of T- and B-cell cooperative interaction in the regulation of natural killer (NK) activity in human peripheral blood lymphocytes was studied. It has been shown that preincubation of normal donor mononuclear cells (MNC) for 48 h is followed by the loss of NK activity, while the incubation of the isolated T- and non-T-cell subsets does not lead to an analogous fall in the killer lymphocyte function. NK activity of MNC and isolated lymphocyte subsets in normal donors is shown to exceed that of CLL patients. The absence of preincubation effect on NK activity level of MNC, T- and non-T-cells in CLL patients has been also found. The findings obtained suggest that as a result of T- and B-cell interaction during preincubation differentiation of young T lymphocytes with NK cellular properties takes place. It is followed by the loss in NK activity. B-cell defect in CLL patients might cause the absence of preincubation effect on NK activity of T cells.     

Genome-wide DNA methylation analysis in precursor B-cells

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182–200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.     

Phenotypic heterogeneity in three cases of lymphoid malignancy with chromosomal translocations in 14q11

Lymphoid malignancies bearing translocations involving the region 14q11 are thought to have a T-cell phenotype because their pathogenesis is postulated to involve the aberrant juxtaposition of DNA sequences active in T-cell differentiation (T-cell antigen receptor genes) and proto-oncogenes. We present here three lymphoid malignancies with translocations involving 14q11. Although one had a T-cell phenotype, the other two had B-cell lineage phenotypes. Our findings confirm that not all lymphoid malignancies with translocations involving 14q11 represent T-cell clonal expansions.     

Regulatory role of CD19 molecules in B-cell activation and differentiation

Cluster of differentiation ([CD]) 19 antigens are B-cell-specific molecules expressed on virtually all human cells of the B-lymphocyte lineage except plasma cells. We produced a new anti-CD19 monoclonal antibody (McAb), CLB-CD19, that was used to study the role of CD19 molecules in B-cell activation. Anti-CD19 McAb induced mobilization of free intracellular calcium ([Ca2+]i) in Daudi cells, but not in normal spleen or tonsillar B cells, for which crosslinking with a second anti-mouse Ig antibody was not required. Anti-CD19 McAb inhibited B-cell proliferation induced by anti-IgM coupled to Sepharose beads. This inhibitory effect was overcome by the addition of nonmitogenic concentrations of phorbol myristate acetate. Anti-CD19 McAb did not interfere with Staphylococcus aureus- or B-cell growth factor-induced B-cell proliferation. Anti-CD19 McAb inhibited T-cell-dependent polyclonal B-cell differentiation in pokeweed mitogen-, interleukin 2-, or anti-CD3-driven culture systems. Delayed addition studies showed that once differentiation of B cells was induced, CD19 molecules lost their regulating function. Taken together, our results indicate that CD19 molecules play a regulatory role in B-cell proliferation and differentiation.     

Genome-wide DNA methylation analysis in precursor B-cells

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182–200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.     

Lysophosphatidic acid (LPA) protects primary chronic lymphocytic leukemia cells from apoptosis through LPA receptor activation of the anti-apoptotic protein AKT/PKB

Lysophosphatidic acid (LPA) protects epithelial and fibroblast cell lines from apoptosis. In B-cells, LPA acts as a growth factor promoting cell proliferation. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD19+/CD5+ B-lymphocytes primarily through a block in apoptosis. The mechanisms underlying this defect are not fully understood. We investigated whether LPA could be a survival factor in CLL cells. Herein, we demonstrate that LPA protects B-cell lines BJAB and I-83 and primary CLL cells but not normal B-cells from fludarabine- and etoposide-induced apoptosis. Furthermore, LPA prevented spontaneous apoptosis in primary CLL cells. The LPA1 expression was found to be increased in primary CLL cells compared with normal B-cells correlating with LPA prevention of apoptosis. Treatment of primary CLL cells with the LPA receptor antagonist, diacylglycerol pyrophosphate, reverses the protective effect of LPA against apoptosis, and down-regulation of the LPA1 by siRNA blocked LPA-mediated protection against spontaneous apoptosis in primary CLL cells. The protective effect of LPA was inhibited by blocking activation of the phosphatidylinositol 3-kinase/AKT signaling pathway. These results indicate that LPA is a survival factor in B-cell lines and primary CLL cells but not normal B-cells. Thus, drugs targeting the LPA receptors might be an effective therapy against B-cell-derived malignancies such as CLL.     

ALVAC-mediated gene transfer is efficient in lymphoid malignancies of T-and early B-cell origin, but not in tumors arising from mature B-cells

Natural attenuation of ALVAC virus in mammals makes it an attractive vector for cancer vaccine therapy of immunocompromised hosts, such as patients with lymphoid malignancies. However, the transduction efficiency of ALVAC constructs in lymphoid tumors has not yet been characterized. We studied a wide spectrum of human T- and B-cell leukemia and lymphomas and found significant heterogeneity of the ALVAC-mediated gene product expression in these tumors. While ALVAC-B7.1, ALVAC-B7.2, or ALVAC-luciferase vectors effectively expressed recombinant genes in malignancies arising from T- or early B-cell precursors, negative or low expression of ALVAC recombinant genes occurred in tumors arising from mature B-cells. We showed that ALVAC-encoded B7.1 or B7.2 was continuously expressed on the infected, and subsequently irradiated, leukemia cells, and only cells with ALVAC-mediated expression of costimulatory molecules (but not unmodified leukemia cells or those infected with the ALVAC-parental vector) induced significant proliferation and IFN-gamma production by alloreactive T-cells. These data provide the rationale for clinical studies using the ALVAC vector system for gene transfer into lymphoid tumors of T- and early B-cell origin to render them more immunogenic, while alternative strategies should be considered for immunotherapy of mature B-cell malignancies.     

Manipulation of cellular redox parameters for improving therapeutic responses in B-cell lymphoma and multiple myeloma

Developing novel combined-modality therapeutic approaches based on understanding of the involvement of redox biology in apoptosis of malignant cells is a promising approach for improving clinical responses in B-cell lymphoma and multiple myeloma. Therapeutic modalities that generate reactive oxygen species (i.e., radiation, photodynamic therapy, and specific chemotherapeutic drugs) have been shown to be selectively cytotoxic to malignant B-cells. In this review, we will discuss agents that induce apoptosis in B-cell tumors by oxidative stress. Subsequently, a novel biochemical rationale (based on fundamental differences in cancer vs. normal cell oxidative metabolism) for combining oxidative stressors with radiotherapy and chemotherapy, that may lead to designing of more effective treatment strategies for B-cell malignancies, will be discussed. Besides providing potential curative benefit, such novel therapies could also selectively target and inhibit the emergence of drug-resistance in tumor cells, which is a major determinant of treatment failure in many B-cell malignancies.     

Expression of the CD31 antigen in normal B-cells and non Hodgkin's lymphomas

The role cell adhesion molecules play in the biological and clinical behaviour of non Hodgkin's lymphomas (NHL) has been reported in several studies. This study reports the findings on B-cells taken from various healthy control tissues and compared them to B-cells from 83 malignant B-lymphomas, that had been classified according to the WHO classification. Flow cytometry was used to investigate the surface expression of CD31, an adhesion molecule involved in B-cell development and vascular adhesion mechanisms. Quantification of the fluorescence signals showed specific patterns of CD31 expression on normal B-cell subpopulations and different NHL groups. Our results demonstrate that CD31 expression is modulated during the differentiation process in normal B-cells, high in pre-B-I cells, low in pre-B-II precursors, intermediate in the mature B-cell subpopulations or, depending on the functional state absent in activated follicular centre cells, present in pre- and post- germinal centre cells. When the CD31 expression is evaluated as fluorescence intensity in NHL, it reveals a heterogeneous pattern related to histogenetic derivation (high in small lymphocytic lymphoma, low in follicular lymphoma, intermediate in marginal zone and large cell lymphomas). These observations suggest that CD31 might well play a critical role in the ontogeny and physiology of B-lymphocytes. Therefore, on the basis of these observations we propose the CD31 molecule as an interesting additional useful parameter to be used for the differential diagnosis of NHL and hypothese that it has a pathophysiologic role in NHL evolution.     

Umbilical cord mesenchymal stem cells suppress B-cell proliferation and differentiation

Mesenchymal stem cells (MSCs) may be obtained from umbilical cord as an abundant and noninvasive source. However, the immunomodulatory properties of umbilical cord-MSCs (UC-MSCs) were poorly studied. In this study, we aimed to investigate the effects of UC-MSCs on B-cell proliferation and differentiation. UC-MSCs were found to suppress the proliferation of B cells isolated from murine spleen. Moreover, UC-MSCs markedly suppressed B-cell differentiation as shown by the decreased number of CD138+cells and reduced levels of IgM and IgG production in coculture. As revealed by transwell experiments, soluble factors produced by UC-MSCs might be involved in mediating B-cell suppression. The Blimp-1 mRNA expression was suppressed whereas the PAX-5 mRNA expression was induced in coculture. Finally, UC-MSCs modified the phosphorylation pattern of Akt and p38 pathways, which were involved in B-cell proliferation and differentiation. These results may further support the potential therapeutic use of UC-MSCs in treating autoimmune disorders.     

Proteomic analysis of B-cell malignancies

The identification of proteins aberrantly expressed in malignant B-cells can potentially be used to develop new diagnostic, prognostic or therapeutic targets. Proteomic studies of B-cell malignancies have made significant progress, but further studies are needed to increase our coverage of the B-cell malignant proteome. To achieve this goal we stress the advantages of using sub-cellular fractionation, protein separation, quantitation and affinity purification techniques to identify hitherto unidentified signalling and regulatory proteins. For example, proteomic analysis of B-cell plasma membranes isolated from patients with mantle cell lymphoma (MCL) identified the voltage-gated proton channel (HVCN1,[1]). This protein has now been characterised as a key modulator of B-cell receptor (BCR) signalling and abrogation of HVCN1 function could have a role in the treatment of B-cell malignancies dependent on maintained BCR signalling [2]. Similarly, proteomic studies on cell lysates from prognostic subtypes of CLL, distinguished by the absence (UM-CLL) or presence (M-CLL) of somatic hypermutation of the immunoglobulin heavy chain locus identified nucleophosmin 1 (NMP1) as a potential prognostic marker [3,4]. Thus, targeted proteomic analysis on selected organelles or sub-cellular compartments can identify novel proteins with unexpected localisation or function in malignant B-cells that could be developed for clinical purposes.     

Enhanced targeting specificity to tumor cells by simultaneous recognition of two antigens

Radioimmunotherapy recently afforded convincing results for B-cell non-Hodgkin's lymphoma treatment with antibody specific for B-cell differentiation antigens. High doses of unlabeled or labeled antibodies are necessary to saturate specific sites on normal B-cells. We thus developed a new targeting strategy, taking advantage of dual binding cooperativity, to enhance the specificity of the radioactive uptake by tumor cells. This approach was evaluated using human Burkitt lymphoma cells (Ramos) which express both CD10 and CD20 antigens. Most normal cells express at most one of these two differentiation antigens but many hematological tumors, including most human B type acute lymphoblastic leukemia cells, express both. Cells pretargeted with two bispecific antibodies, one recognizing CD10 and a histamine derivative (HSG), the other recognizing CD20 and the DTPA-indium complex, bind cooperatively radiolabeled mixed-haptens (DTPA-HSG). Increased binding (about 5-fold compared to binding to only one of CD10 or CD20 antigens) is observed at 37 degrees C, demonstrating the feasibility of the technique. This binding enhancement is a slow process, not observed at 4 degrees C. Such a binding enhancement will increase specificity for targeting isotopes to double antigen positive tumor cells compared to nontumor tissue cells bearing only one of them. This approach might be used to increase tumor irradiation with minimal irradiation of normal cells.     

Lymphokine-induction of memory B-cell differentiation: differential stimulation of large virgin and memory B-cell differentiation

In order to compare and contrast the requirements of virgin and memory B cells for B-cell differentiation factors, a model system was developed in which low-density rat B cells isolated from 4-week primed antigen-draining lymph nodes were cultured in vitro. This large low-density cell population contained B cells which were 90% surface IgM positive and 60% IgD positive and showed moderately elevated Ia staining. When the cell population was stimulated with antigen plus lymphokines or lymphokines alone, antigen-specific IgG antibody was secreted; this was used as a measure of memory cell differentiation. When the cell population was stimulated with mitogen (lipopolysaccharide plus dextran sulfate) plus lymphokines, polyclonal IgG and IgM secretion was seen and was used as a measure of virgin B-cell differentiation. Using this system, we found that lymphokines contained in a Con A-induced rat spleen cell supernatant (CSN) were sufficient to drive both memory and virgin B-cell differentiation. In contrast, lymphokines contained in the supernatant from the murine T-cell hybridoma B151K12 (B151CFS) were able to induce large amounts of polyclonal IgM and IgG secretion but did not support memory B-cell differentiation. When recombinant human IL-2 was added to these cultures, it acted synergistically to augment virgin B-cell differentiation, but this combination of lymphokines was still not able to support memory B-cell differentiation. Furthermore, recombinant rat interferon-gamma and a commercial source of human BCGF, with or without IL-2, were unable to promote significant virgin or memory B-cell differentiation. These data support the hypothesis that memory B cells and virgin B cells differ in their lymphokine requirements for differentiation into antibody-secreting cells.     

Self-renewal of acute lymphocytic leukemia cells is limited by the Hedgehog pathway inhibitors cyclopamine and IPI-926

Conserved embryonic signaling pathways such as Hedgehog (Hh), Wingless and Notch have been implicated in the pathogenesis of several malignancies. Recent data suggests that Hh signaling plays a role in normal B-cell development, and we hypothesized that Hh signaling may be important in precursor B-cell acute lymphocytic leukemia (B-ALL). We found that the expression of Hh pathway components was common in human B-ALL cell lines and clinical samples. Moreover, pathway activity could be modulated by Hh ligand or several pathway inhibitors including cyclopamine and the novel SMOOTHENED (SMO) inhibitor IPI-926. The inhibition of pathway activity primarily impacted highly clonogenic B-ALL cells expressing aldehyde dehydrogenase (ALDH) by limiting their self-renewal potential both in vitro and in vivo. These data demonstrate that Hh pathway activation is common in B-ALL and represents a novel therapeutic target regulating self-renewal and persistence of the malignant clone.     

Difference in genesis between antigen-induced IgM antibody-forming cells and B-cell mitogen-induced IgM antibody-forming cells in mouse spleen cell cultures.

To determine the mechanisms in the triggering of thymus-independent lymphocytes (B cells) for development into antibody-forming cells (AFC), genesis of IgM AFC elicited polyclonally by nonspecific stimulation with B-cell mitogen, such as nystatin and bacterial lipopolysaccharide, was compared with that of IgM AFC specifically elicited by antigenic stimulation, using mouse spleen cell cultures as an experimental system and sheep erythrocytes (SRBC) as a test antigen. Considering that differentiation and proliferation are necessary cellular events for precursor B cells to develop into AFC, the effect of different antimetabolic agents on the generation of each type of AFC in spleen cell cultures was examined. The generation of anti-SRBC IgM hemolysin plaque-forming cells (PFC) in B-cell mitogen-stimulated spleen cell cultures was found to be less susceptible to X-irradiation or mitomycin C than that in the SRBC-stimulated cultures. These apparently paradoxical results were affiirmed using colcemid as an inhibitor of cell mitosis and hydroxyurea (HU) as an inhibitor of cellular DNA synthesis. Thus, when spleen cell cultures responding to either SRBC or B-cell mitogen were exposed to colcemid or HU during a period from 2 days to 3 days after the stimulation, the exponential generation of anti-SRBC IgM PFC in the cultures responding to SRBC was completely halted, whereas that in the cultures responding to B-cell mitogen was not. Furthermore, N⁶, O^2′ -dibutyryl adenosine 3′, 5′ -cyclic monophosphoric acid was found to halt the exponential generation of antigen-induced anti-SRBC IgM PFC but not that of the B-cell mitogen-induced anti-SRBC IgM PFC. From these results it was suggested that B-cell mitogen might stimulate precursor Bμ cells at a late stage in the differentiative pathway to develop into AFC without cell division, and that antigenic stimulation might stimulate relatively primitive precursor Bμ cells to proliferate and then differentiate into AFC. Based on this idea, mechanisms in the triggering of B-cell activation are discussed.     

Suppression of B-cell and T-cell responses by the prostaglandin-induced T-cell-derived suppressor (PITS). I. Analysis of the PITS beta factor

Within populations of mitogenically (PWM) stimulated normal human lymphocytes, the proliferation of B lymphocytes is terminated by T cells. In contrast, T cells limit their own proliferation. T cells thus apparently measure and terminate the proliferation of B cells as well as themselves, suggesting an important role for them in limiting amplification during immune response. Under the culture conditions employed, PWM-induced B- and T-cell proliferation was uncoupled from B-cell differentiation into plasmacytes. Termination of B-cell proliferation in this in vitro model of humoral immune response is independent of B-cell differentiation.