B-cell Fractions Research Articles

Crosstalk between Chronic Lymphocytic Leukemia (CLL) B-Cells and Marrow Stromal Cells: Implication for CLL B-Cell Activation and Survival.

It is believed that malignant cells “condition” the microenvironment to facilitate tumor cell survival. We hypothesized that crosstalk between CLL B-cells and marrow stromal cells impacts both cell types bi-directionally and ultimately contributes to leukemic cell apoptotic resistance. To test this hypotheses, bone marrow stromal cells from core bone biopsies from CLL patients were isolated and cultured using methods we have previously described (Leuk Res 2007 31(7):899). Subsequently, we determined the impact of co-culture on CLL B-cell features including apoptosis and CD38 expression. In addition, we evaluated the release of angiogenic cytokines on co-culture and signal events in the stromal cells. Immunophenotyping demonstrated that cultured bone biopsy derived stromal cells were CD73+, CD105+, CD146+, CD14−, CD45−, CD34−, HLA-DR-, suggesting they were mesenchymal stem cells (MSC). Co-culture of these MSC with CLL B-cells protected CLL B-cells from both spontaneous apoptosis (SA) and drug-induced (fludarabine and chlorambucil) apoptosis (DA). For SA, the mean survival of CLL B-cells with or without co-culture of MSC for 5 days were 56.9 ± 10.0 and 7.7 ±3.7 (p<0.05), respectively. When CLL B cells were treated with fludarabine or chlorambucil, the fraction of CLL cells tightly adherent to MSC (TA-CLL) showed higher survival than a less adherent but viable fraction of CLL B-cells. The mean survival of TA-CLL cells treated with 10 μM of fludarabine for 48 hours in the presence of MSC were 67.5 ± 3.6 vs 29.8 ± 11.1 without MSC (P<0.05), respectively. When CLL cells with evidence for CD38 expression were co-cultured with MSC, both the percentage of CD38 positive cells and level of expression of CD38 per cell were up-regulated (mean fold change: CD38 percentage, 2.7, p<0.05; CD38 MFI, 1.9, p<0.05) after 2 weeks. In contrast, the CD38 percentage and expression were not changed in cells with minimal CD38 expression when these CLL B-cells were co-cultured with MSC. In addition, co-culture of MSC with CLL cells induced rapid ERK and AKT phosphorylation (within 30 min) in the MSC on immunoblot analysis. When CLL B cells and MSCs were cultured in transwells, the activation of ERK and AKT in MSC occurred at similar levels, indicating that activation of MSC was mediated by soluble factors. In addition, co-culture led to increased secretion of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) as well as a decrease of thrombospondin-1 (TSP-1) in the culture medium. These findings confirm that co-culture of CLL B-cells and MSC culminates in “angiogenic switch.” Taken together, these results strongly suggest interactions between MSC and CLL B cells are a bi-directional process. In leukemic cells, the interaction not only protects against spontaneous and drug induced apoptosis but also leads to an increase in CD38 expression consistent with an activated status. In MSC, the interaction leads to activation of ERK and AKT. Co-culture also facilitates angiogenic switching. These results underscore the dynamic and complex nature of the interactions between bone marrow stromal cells and CLL B-cells. Further studies are needed to dissect how crosstalk between CLL B-cells and MSC relates to disease progression, and determines whether these interactions can be targeted with therapeutic intent.

The BCL6 Oncogene Drives an Epigenomic Program Linking Stem Cells to the Pathogenesis of Human Diffuse Large B Cell Lymphoma in Mice.

Diffuse large-B-cell lymphoma (DLBCL) is the most common lymphoid malignancy in adults. The BCL6 oncogene, a transcriptional repressor, is frequently involved in DLBCL pathogenesis. Constitutive expression of BCL6 in GC cells leads to larger GCs and development of DLBCL in mice, although constitutive expression of BCL6 earlier in the B-cell lineage has the opposite effect and abrogates B-cell development. Limitations in our knowledge of how BCL6 mediates transcriptional programming to impose the malignant phenotype presents a barrier towards improved understanding and therapeutic targeting of B-cell lymphomas. Recent advances in stem cell biology suggest that many tumors are sustained by a pool of biological distinct cancer stem cells (CSCs), which may be uniquely susceptible to epigenomic programming by oncogenes and require specific therapeutic targeting. Although CSCs have not yet been identified in DLBCL, we hypothesized that such cells may exist in these tumors and their characterization could be critical for studies of lymphomagenesis and therapeutics. In order to test this hypothesis our group developed a mouse model where BCL6 is expressed transiently in the stem cell compartment at levels comparable to normal GC cells. These animals undergo completely normal hematopoiesis, except that 100% develop lymphomas within 20 to 28 weeks. Extensive histopathologic, molecular and expression array phenotyping demonstrate that these tumors are most similar to human DLBCLs. However, these tumor cells do not express BCL6, and so must be transcriptionally programmed at the stem cell level to behave in a malignant fashion. Moreover, transplantation of DLBCL cells does not propagate the tumor whereas transplantation of the CSCs rapidly re-establishes the DLBCL in recipient mice. In-depth microarray and QPCR transcriptional profiling demonstrate that BCL6 imposes a B cell lymphoma program in the stem cell compartment. This transcriptional programming subsequently becomes latent during hematopoiesis and reappears only in the mature B-cell fraction. We reasoned that this effect must be dependent on epigenetic programming imposed by BCL6 in CSCs. To determine whether this is the case we compared and contrasted the epigenetic signature of CSCs, and DLCBL tumors in the transgenic animals vs. those of normal stem cells and mature B-cells in control animals. This was performed using the HELP (HpaII tiny fragment enrichment by ligation mediated PCR) method with a custom designed high-density oligonucleotide microarray that allows quantitative detection of DNA methylation at 24,000 murine gene promoters. The data show that BCL6 imposes a unique DNA hypermethylation signature in CSCs that persists in the DLBCL cells. Taken together, our results suggest that BCL6 mediates transcriptional and epigenomic programming in stem cells that can latently persist during differentiation (thus allowing early B-cell development to proceed) and impose the DLBCL phenotype in their mature progeny. Since human lymphomas exhibit similar transcriptional programming, therapeutic targeting of the BCL6 epigenomic program might enhance the power of anti-lymphoma regimens to eradicate the disease.

T-Cell Function Is Critical for Murine Cholesterol Gallstone Formation

The formation of cholesterol gallstones is a complex process involving contributions from genes and environmental factors. Although gallbladder inflammation is believed to be common during cholelithogenesis, the role of immunologic factors is unknown. The role of adaptive immunity in cholesterol cholelithogenesis was analyzed utilizing immunocompetent Helicobacter spp.-infected and -uninfected BALB/c and congenic immunodeficient Rag2(-/-) (Rag) mice. Lymphocyte transfer studies were performed to determine which cellular subset was responsible for cholesterol gallstone formation. Also, gallbladder inflammation was quantified to determine the nature of the inflammatory response associated with cholelilithogenesis. When fed a lithogenic diet for 8 weeks, wild-type mice developed significantly more cholesterol gallstones (27%-80% prevalence) than Rag mice ( approximately 5%, P < .05). Helicobacter spp.-infected BALB/cJ mice displayed statistically significant increases in cholesterol gallstone prevalence compared with uninfected mice (81% vs. 39%; P < .05). Transfer of splenocytes or T lymphocytes to Rag2(-/-) mice increased stone prevalence markedly (26% and 40% respectively; P < .05), whereas transfer of B cells was not appreciably cholelithogenic (13%). The adaptive immune response increased the expression of gallbladder Muc genes and accumulation of mucin gel. In addition, T cells and cholesterol monohydrate crystals induced proinflammatory gene expression in the gallbladder, which likely contributes to gallbladder dysfunction. These studies indicate that T cells are critical in murine cholesterol cholelithogenesis. Furthermore, cholesterol monohydrate crystals induce expression of proinflammatory cytokines in a T-cell-dependent fashion. Acquired immunity and inflammation are likely to be crucial factors in cholesterol gallstone pathogenesis, rather then merely the result of cholelithogenesis.

Effects of long-term continuous use of immune-enhancing enteral formula on nutritional and immunologic status in non-surgical patients

Objective Although the perioperative use of immune-enhancing enteral formula (IEEF) effectively reduces the rate of infectious complications, whether chronic use of IEEF is beneficial is unknown. A prospective randomized clinical trial was performed to examine the safety and effectiveness of long-term IEEF on nutritional and immunologic status in non-surgical patients receiving total enteral nutrition through the gastrostomy access route. Methods A total of 30 patients were randomly assigned to two groups in which they received total enteral nutrition, an IEEF (n = 15) or a regular polymeric enteral formula (control group; n = 15) for 12 wk. Nutritional and immunologic variables were periodically examined. Results Serum concentrations of insulin-like growth factor-I increased significantly for the IEEF group throughout the study. Although serum concentrations of dihomo-γ-linoleic acid decreased significantly in the IEEF group, serum concentrations of eicosapentaenoic acid and docosahexaenoic acid increased significantly, as did concentrations of serum arginine and ornithine. The CD4/CD8 ratio and natural killer cell activity also increased for the IEEF group, but the differences were not significant. The B-cell fraction increased and the T-cell fraction of peripheral lymphocytes decreased for the IEEF group. Neither infectious nor non-infectious complications occurred during the study period in either group, except for a significant increase in serum urea nitrogen and uric acid concentrations for the IEEF group. Conclusion Long-term use of IEEF is safe in non-surgical patients and results in a significant increase in serum insulin-like growth factor-I concentrations in association with increased humoral immunity.

A Soluble BAFF Antagonist, BR3-Fc, Decreases Peripheral Blood B Cells and Lymphoid Tissue Marginal Zone and Follicular B Cells in Cynomolgus Monkeys

BAFF (also known as BLyS), a member of the tumor necrosis factor superfamily, plays a critical role in the maturation and development of B cells. BAFF has three receptors on B cells, the most crucial of which is BR3. In this study, we demonstrate the biological outcome of BAFF blockade in cynomolgus monkeys using a soluble fusion protein consisting of human BR3 and human IgG1 Fc. In vitro, BR3-Fc blocked BAFF-mediated survival and proliferation of cynomolgus monkey B cells. Weekly treatment of cynomolgus monkeys with BR3-Fc for 13 to 18 weeks resulted in significant B-cell reduction in the peripheral blood and in lymphoid organs. CD21(high) B cells in lymphoid tissues, a subset analogous to human marginal zone B cells, expressed nearly twofold higher BR3 levels than did CD21(med) B cells. Lymphoid tissue flow cytometric analysis showed that BR3-Fc reduced this CD21(high) B-cell subset to a greater extent than it reduced CD21(med) B cells. Dual-label immunohistochemistry and morphometric image analysis supported these results by demonstrating that BR3-Fc reduced a significant proportion of the B cells within the splenic inner and outer marginal zones. These findings should prove very useful in guiding the desired therapeutic use of BR3-Fc for autoimmune diseases in the clinic.

IRF-4/MUM-1 Expression Is a Critical Switch in the Generation of Plasma Cells Versus Memory B-Cells.

Most types of human B-cell lymphomas harbor somatically mutated Ig variable region genes, reflecting their origin from cells that have undergone the germinal center (GC) reaction of T-dependent immune responses. The lymphomas exhibit diverse phenotypes and clinical behaviors, likely as a consequence of differences both in the mechanisms of transformation and in the specific target cell. We have recently identified two distinct subsets of GC B-cells that may represent late stages of the GC-reaction and may be the precursors of plasma cells and memory B-cells. The corresponding subsets are characterized by downregulation of the GC-marker BCL6 and the alternative expression of IRF-4/MUM-1 or nuclear c-Rel. These two subsets seem to reflect distinct cellular programs which are altered during B-lymphomagenesis in various tumor subtypes which co-express BCL6, IRF-4 and nuclear c-Rel, an event never observed in normal B-cells. In order to gain insights into the physiologic role of IRF-4/MUM-1 and nuclear c-Rel in GC-development, we are ablating their expression specifically in mouse GC B-cells. Transgenic mice were generated that carry an IRF-4/MUM-1 null allele and a conditional IRF-4/MUM-1 allele which, following Cre-mediated deletion of the loxP-flanked promotor region and exons 1 and 2, expresses eGFP, thus allowing to track the development of the IRF-4/MUM-1 deficient cells at the single cell level. IRF-4/MUM-1fl/- mice were crossed with transgenic mice that express the Cre-recombinase specifically in B-cells undergoing class-switch to IgG1, an event occurring in a large fraction of GC B-cells. Upon immunization with sheep red blood cells or nitrophenyl-(NP)-KLH, mice unable to express IRF-4/MUM-1 in late GC B-cells (IRF-4/MUM-1fl/-/Cγ 1Cre/+), in contrast to control mice (IRF-4/MUM-1fl/+/Cγ 1Cre/+), did not develop plasma cells (IgG1+CD138+) in the peripheral lymphoid organs, blood, and bone marrow. On the other hand, the generation of memory B-cells appears normal since antigen-specific B-cells were present in the spleen (eGFP+B220+PNA-IgG1+) and blood (eGFP+B220+CD38+IgG1+). These results suggest that the IRF-4/MUM-1 gene product is required for the development of antigen-selected GC B-cells into plasma cells. We suggest that the expression of IRF-4/MUM-1 in a GC centrocyte is the critical event in the commitment of B-cells to differentiate into a plasma cell versus a memory B-cell, and are currently testing the role of nuclear c-Rel in the same process.

Establishment and Maintenance of Kaposi's Sarcoma-Associated Herpesvirus Latency in B Cells

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is the infectious cause of Kaposi's sarcoma and is also associated with two B-cell lymphoproliferative diseases, primary effusion lymphoma and the plasmablastic form of multicentric Castleman's disease. KSHV is also found in the B-cell fraction of peripheral blood mononucleocytes of some KS patients. Despite in vivo infection of B cells and the ability of KSHV to infect many cell types in culture, to date B cells in culture have been resistant to KSHV infection. However, as shown here, the lack of infection is not due to the inability of B cells to support latent KSHV infection. When KSHV DNA is introduced into B cells, the virus is maintained as an episome and can establish and maintain latency over the course of months. As in all primary effusion lymphoma cell lines, there is a low level of spontaneous lytic replication in latently infected BJAB cells. Importantly, viral gene expression is similar to that of primary effusion lymphoma cell lines. Furthermore, the virus can be reactivated to higher levels with specific stimuli and transmitted to other cells, indicating that this is a productive infection. Thus B cells in culture are capable of establishing, maintaining, and reactivating from latency. These studies provide a controlled system to analyze how KSHV alters B cells during KSHV latency and reactivation.

Emmprin, a cell surface inducer of matrix metalloproteinases (MMPs), is expressed in T-cell lymphomas.

Degradation of the extracellular matrix by matrix metalloproteinases (MMPs) is a crucial step in tumour invasion and metastasis. In human carcinomas, tumour cell-fibroblast interactions (TFIs) have been demonstrated to play a role in the up-regulation of MMP levels in tumours, and emmprin is a surface molecule on tumour cells that stimulates nearby fibroblasts to produce MMP-1, 2, and 3. T-cell lymphomas frequently show extranodal organ involvement and skin invasion, but a role for TFIs in their invasion has not been examined in detail. This study investigated TFIs in T-cell lymphomas with special reference to emmprin expression and MMP production. Immunohistochemically, only germinal centre cells and some histiocytes expressed emmprin in non-neoplastic lymph nodes (ten cases), while all T-cell lymphomas [14 cases of adult T-cell leukaemia/lymphoma (ATLL), six cases of lymphoblastic lymphoma, seven cases of anaplastic large cell lymphoma, and nine cases of angio-immunoblastic T-cell lymphoma] expressed emmprin strongly and diffusely. FACS analysis of peripheral blood from normal individuals revealed that small fractions of B-cells, T-cells, and monocytes expressed emmprin, whereas emmprin-expressing T-cells were much increased in number, and expressed this protein to a higher level, in ATLL patients. In vitro co-cultures of emmprin-positive HTLV-1-transformed lymphocytes (MT-2) and emmprin-negative human fibroblasts enhanced the production of pro-MMP-2 (gelatinase A) and active MMP-2, compared with cultures of either cell type alone. This stimulation was inhibited by an activity-blocking peptide against emmprin. Moreover, in histopathological sections from patients with ATL skin involvement, MMP-2 was demonstrated in fibroblasts around infiltrating ATL cells, but not in fibroblasts in non-diseased areas. In conclusion, emmprin is overexpressed by T-lymphoma cells, when compared with normal counterparts, and facilitates MMP-2 production via interactions with fibroblasts, which could play a role in stromal invasion by lymphoma cells.

In vitro and in vivo evidence of PNH cell sensitivity to immune attack after nonmyeloablative allogeneic hematopoietic cell transplantation

AbstractIt has been proposed that paroxysmal nocturnal hemoglobinuria (PNH) cells may proliferate through their intrinsic resistance to immune attack. To evaluate this hypothesis, we examined the impact of alloimmune pressure on PNH and normal cells in the clinical setting of nonmyeloablative allogeneic hematopoietic cell transplantation (HCT). Five patients with severe PNH underwent HCT from an HLA-matched family donor after conditioning with cyclophosphamide and fludarabine. PNH neutrophils (CD15+/CD66b–/CD16–) were detected in all patients at engraftment, but they subsequently declined to undetectable levels in all cases by 4 months after transplantation. To test for differences in susceptibility to immune pressure, minor histocompatibility antigen (mHa)–specific T-cell lines or clones were targeted against glycosylphosphatidylinositol (GPI)–negative and GPI-positive monocyte and B-cell fractions purified by flow cytometry sorting. Equivalent amounts of interferon-γ (IFN-γ) were secreted following coculture with GPI-negative and GPI-positive targets. Furthermore, mHa-specific T-cell lines and CD8+ T-cell clones showed similar cytotoxicity against both GPI-positive and GPI-negative B cells. Presently, all 5 patients survive without evidence of PNH 5 to 39 months after transplantation. These in vitro and in vivo studies show PNH cells can be immunologically eradicated following nonmyeloablative HCT. Relative to normal cells, no evidence for a decreased sensitivity of PNH cells to T-cell–mediated immunity was observed.

Differential contribution of Wiskott-Aldrich syndrome protein to selective advantage in T- and B-cell lineages

AbstractSomatic mosaicism because of in vivo reversion has been recently reported in a small number of patients affected with Wiskott-Aldrich syndrome (WAS). Flow cytometry analysis of WAS protein (WASP) expression has shown that these patients carried revertant cells only among T lymphocytes. Here, we have used high-resolution capillary electrophoresis to analyze genomic DNA from highly purified cells of one of these patients and detected revertant sequences also within the B-cell fraction. The demonstration of revertant cells among both T and B lymphocytes in this patient is consistent with the reversion event having occurred in a common lymphoid progenitor. However, although WASP-expressing T cells showed selective advantage and were readily detectable in the periphery of the mosaic patient, revertant B lymphocytes remained below the detection threshold of flow cytometry. These findings suggest that, contrary to T cells, differentiation and survival of B lymphocytes is minimally dependent on WASP.

Tetrocarcin-A—induced ER stress mediates apoptosis in B-CLL cells via a Bcl-2—independent pathway

AbstractTetrocarcin-A (TC-A), an antibiotic agent isolated from actinomycetes, has recently been described to antagonize Bcl-2 functions, thereby sensitizing tumor cells to cell death signals under control of Bcl-2. In this study, we analyzed the direct proapoptotic effect of TC-A in the B-chronic lymphocytic leukemia (B-CLL) model. We focused on the signal cascade triggered by TC-A in B-CLL cells and identified activated mitochondrial as well as endoplasmatic reticulum (ER) stress signals. The expression levels of known effector molecules mediating mitochondrial signaling, such as Bax and Bid, and the antagonistic molecule Bcl-2 did not influence sensitivity of B-CLL cells to TC-A. Furthermore, the molecular chaperone and sensor of ER stress, HSP70, though significantly up-regulated in B-CLL cells undergoing TC-A—triggered apoptosis, was ineffective to exert its anti-apoptotic function described in multiple cell death pathways. Autologous T cells of B-CLL patients were significantly less sensitive to TC-A as were also T cells from healthy donors when compared with their normal B-cell fraction. Furthermore, sensitivity of B-CLL cells to TC-A treatment in vitro was dependent neither on the expression levels of CD38—a prognostic factor for survival of B-CLL patients as well as for their response to therapy—nor on the clinical stage or pretreatment status of patients. From our data showing that TC-A induced a cell death pathway via ER stress preferentially in B cells and that it acted independently of important markers of drug sensitivity and of clinical markers, we conclude that TC-A might represent an attractive candidate drug for further evaluation in preclinical trials.

Leucocyte populations and responses to immunization and photoperiod manipulation in Atlantic salmon ( Salmo salar L.) 0+ smolt

The immune status of season-independent Atlantic salmon smolt may be affected by changes in photoperiod and high rearing temperatures. Variations in different leucocyte subpopulations were measured in photo-manipulated out-of-season (0+) Atlantic salmon smolt by use of flow cytometry and specific monoclonal antibodies. 0+ smolts were produced by exposing parr to continuous light (LD 24:0) until June 10 followed by a “winter” photoperiod (LD 12:12) for 6 weeks, and then continuous light. Three different groups were studied; untreated smolt (UT), smolt injected with a commercial multivalent oil-adjuvanted vaccine (VA), and smolt injected with the saline/oil-adjuvant used in the vaccine (AD). The fish were injected in the “winter” photoperiod. From introduction of “winter” photoperiod until 2 weeks after seawater transfer, fractions of B-cells (identified by G2H3), neutrophiles (identified by E3D9) as well as polymorphonuclear and B-cells (identified by C4B6) were monitored. From introducing continuous photoperiod and until seawater transfer, there were variations for all groups within the leucocyte subpopulations identified by the monoclonal antibodies (MAb) used. Neutrophiles had a minor increase, while the number of B-cells decreased. C4B6, identifying both types of cells recognised by E3D9 and G2H3 remained stable, indicating that the leucocyte fraction identified by C4B6 was not changed, but there was redistribution with respect to the numbers of B-cells and neutrophiles. After seawater transfer, another redistribution of leucocytes took place. A minor reduction for the neutrophiles was observed, while at the same time, both B-cells and the polymorphonuclear cells increased. It was not possible to identify any effect of vaccine and/or adjuvant on the leucocytes studied. The specific serum antibody levels measured by enzyme-linked immunosorbent assay (ELISA) were low during the experimental period. A low immune response was observed measured as antibody-producing cells (APC) by ELISPOT. The low levels of specific antibodies supported the findings of low APC numbers. There were indications that UT had lower numbers of APC compared to AD and VA. The results show that changes with respect to fractions of leucocyte populations take place during parr–smolt transformation, and these changes may affect the cellular and humoral immune response to vaccination. Different photoperiod regimes may have different effects on leucocyte populations and general conclusions should not be made from the single photoperiod studied.

The relationship between monoclonal myeloma precursor B cells in the peripheral blood stem cell harvests and the clinical response of multiple myeloma patients.

The aim of this study was to determine the presence of monoclonal myeloma precursor B cells in peripheral blood stem cell harvests and to investigate their role in the clinical outcome of multiple myeloma patients. A total of 39 multiple myeloma patients were treated with a sequential therapy including double high-dose melphalan therapy followed by a double transplant procedure. The apheresis products for the second transplant were purged using a panel of four or five different mouse monoclonal antibodies against B-cell antigens (CD10, CD19, CD20, CD22 and CD37). In 19/39 patients a tumour-specific CDR III signal was identified in the diagnostic bone marrow. Gene scan analysis after CDR III PCR of the magnetic bead isolated B-cell fraction from the apheresis products in these 19 patients revealed three different patterns: 32% of patients had a predominantly monoclonal B-cell population; 63% of patients had an identifiable monoclonal signal within an oligoclonal B-cell population. In only 1/19 patients were no monoclonal B cells identified in the B-cell population of the apheresis product. A correlation between the clonal pattern and the clinical response after sequential chemotherapy was found. Patients with a predominance of monoclonal myeloma or myeloma precursor B cells had an early relapse or achieved a minimal response or a partial remission. Patients with an oligo- and/or polyclonal pattern achieved a high percentage of partial as well as complete remissions.

Protein kinase C-delta is a target of B-cell antigen receptor signaling

Protein kinase C (PKC) enzymes have been implicated as key intermediates in B-cell antigen receptor (BCR) signaling. Each of the 11 PKC isoforms may phosphorylate different substrates and regulate different cellular processes. In this report we show that PKC-delta (PKC-δ) is a target of BCR signaling. BCR engagement increased the amount of PKC-δ in the membrane-enriched particulate fraction of B-cells, suggesting that BCR activates PKC-δ. BCR ligation also caused substantial tyrosine phosphorylation of PKC-δ. We show that activation of phospholipase C by BCR is necessary for both PKC-δ membrane localization and tyrosine phosphorylation. In contrast, phorbol esters which mimic the action of diacylglycerol could recruit PKC-δ to cellular membranes but did not induce tyrosine phosphorylation of PKC-δ. These data suggest a model in which phospholipase C-dependent production of diacylglycerol recruits PKC-δ to cellular membranes where it is then phosphorylated by BCR-activated tyrosine kinases.

CD5+ B lymphocytes are the main source of antibodies reactive with non‐parasite antigens in Trypanosoma congolense‐infected cattle

Mice infected with African trypanosomes produce exceptionally large amounts of serum IgM, a major part of which binds to non-trypanosome antigens such as trinitrophenol and single-strand DNA. In this paper, we describe that in cattle infected with Trypanosoma congolense and T. vivax, similar antibodies are found, although they bind mainly to protein antigens, such as beta-galactosidase, ovalbumin and ferritin. The parasite non-specific IgM antibodies appear around the same time as the parasite-specific antibodies, but their origin and function are not clear. We tested the hypothesis that CD5+ B cells (or B-1 cells), which increase during trypanosome infections in cattle, are responsible for production of antibodies to non-trypanosome antigens. Splenic CD5+ and CD5- B cells from infected cattle were sorted and tested in a single cell blot assay. The numbers of immunoglobulin-secreting cells were similar in both B-cell populations. However, antibodies with reactivity for non-trypanosome antigens were significantly more prevalent in the CD5+ B-cell fraction and were exclusively IgM. The preference for production of these antibodies by CD5+ B cells and the expansion of this subpopulation during infections in cattle, strongly suggest that CD5+ B cells are the main source of trypanosome non-specific antibodies. We propose that these antibodies are natural, polyreactive antibodies that are predominantly secreted by CD5+ B cells. Since B-1 cells are up-regulated in many states of immune insufficiency, the immunosuppression associated with trypanosome infections may be responsible for the increase of this subset and the concomitant increase in trypanosome non-specific antibodies.

Gap-junction communication pathways in germinal center reactions.

Intercellular channels called gap junctions enable multicellular organisms to exchange information rapidly between cells. Though gap junctions are held to be ubiquitous in solid tissues, we have only recently found them in the lymphoid organs. Functional direct cell-cell communication has now been confirmed by us and other groups in bone marrow, thymus, and in secondary lymphoid tissues. What functions do they serve in the lymphoreticular system where, so far, only cytokines/growth factors and adhesion molecules have been considered as regulators? Here we show evidence for and refer to published work about functional direct cellcell communication through gap junctions in germinal center reactions and make proposals for their role in the immune response. We found a large amount of the connexin43 (Cx43) gap junctions in the germinal centers of secondary lymphoid follicles. Ultrastructurally and immunohistologically, most of the junctions were detected on the processes of follicular dendritic cells (FDC) enveloping nondividing centrocytes in the light zone of germinal centers where B-cell selection is thought to take place. Further support for this finding came by revealing the Cx43 mRNA in situ at the same location as the protein. On antigen challenge, gap junctions appeared on the FDC as they formed meshworks in germinal centers. In order to find out which germinal center cells communicate directly, we separated FDC-rich, low-density, B-cell fractions from human tonsil. In culture, we injected single FDC with the low-molecular-weight fluorescent dye, Lucifer Yellow (Mr 457 Da), which passed between adjacent FDC and sometimes from FDC to B cells. Based on these findings and their assigned functions in other tissues, gap junctions may contribute to germinal center reactions in the following ways: (1) they may regulate follicle pattern formation by controlling FDC growth, (2) they may be involved in FDC-B-cell signaling contributing to the final rescue of selected B cells from apoptosis, and (3) they may enable FDC to work as a functional syncytium providing a cellular internet for integrating germinal center events. Data supporting these interpretations are briefly discussed.

Modulation of B-Cell Immunoglobulin Synthesis by Retinoic Acid

Retinoic acid (RA) and its parent compound retinol (ROH, vitamin A) have been recognized as important immunopotentiating agents since the early 1900s. We have focused our studies on the effects of retinoids on B-cell immune function in the newborn infant. The response of cord blood mononuclear cells (CBMC) to formalinized Cowan I strainStaphylococcus aureus(SAC), a T-cell-dependent factor for inducing the differentiation of B cells into immunoglobulin (Ig)-secreting cells, was used as a model system for studying whether RA could alter the immunoglobulin synthesis of newborn B lymphocytes. The addition of RA to SAC-stimulated CBMC cultures produced a 2- to 47-fold increase in IgM synthesis. An ELISA-spot assay showed that the RA-induced enhancement in Ig synthesis was due to the recruitment of more B cells to differentiate into Ig-secreting cells. Whereas RA enhanced IgM production of CBMC stimulated with SAC, RA augmented only IgG production of SAC-stimulated adult peripheral blood mononuclear cells (PBMC). To determine if the differences in dose–response characteristics between CBMC and adult PBMC resided within the target cell, i.e., the B cell, T-cell-enriched and T-cell-depleted (B-cell) fractions from CBMC and adult PBMC were cocultured in various combinations. The isotype, i.e., IgM vs IgG, and the dose–response curve characteristics were intrinsic to the responding B-cell source, i.e., newborn vs adult. Highly purified T cells from CBMC, when preincubated for 36 hr with RA, enhanced IgM synthesis of cord blood B cells. Supernatants from purified T cells generated a factor which could enhance B-cell synthesis. Although interleukin (IL)-2, IL-4, and IL-6 could not be detected by ELISA in the T-cell-derived supernatants, RA probably generates a cytokine/interleukin from T cells which modulates B-cell Ig secretion. RA can also act directly on B cells as evidenced by the augmentation in Ig synthesis of Epstein–Barr virus (EBV)-transformed B-cell lines. These data suggest that RA can have a direct effect on B cells. Since increased proliferation (numbers) of lymphoblastoid B cells was not responsible for the increased amounts of Ig in the supernatant fluids, we examined whether cytokines secreted by EBV-transformed B cells could be acting as an autocrine factor in increasing Ig synthesis. EBV-transformed B-cell clones incubated with RA for 6 days produced a 20- to 45-fold increase in IL-6. An understanding of the mechanisms by which RA enhances B-cell immune function may lead to the use of RA or its derivatives in patients with immune deficiencies and in preterm infants with immature immune systems.

Bcl-x Rather Than Bcl-2 Mediates CD40-Dependent Centrocyte Survival in the Germinal Center

Both rapid B-cell proliferation and programmed cell death (PCD) occur during the differentiation and selection of B cells within the germinal center. To help elucidate the role of Bcl-x in B-cell antigen selection and PCD within the germinal center, we examined its expression in defined B-cell populations and by immunochemistry of tonsil tissue. Purified B-cell fractions enriched for centrocytes express high amounts of Bcl-x and relatively low amounts of Bcl-2, whereas fractions enriched for centroblasts lack significant levels of both proteins. Consistent with this observation, immunocytochemistry localized Bcl-x within cells scattered throughout the germinal center. Stimulation of tonsil B cells with either CD40 or Staphylococcus aureus Cowan increase bcl-x mRNA and protein levels. Treatment of a cell line with a germinal center phenotype (RAMOS) or the tonsillar B-cell centroblast fraction with CD40 rapidly increased Bcl-x levels and partially rescued B cells from PCD. These data suggest that Bcl-x rather than Bcl-2 may rescue centrocytes during selection in the germinal center.

Expression of granulocyte colony-stimulating factor and granulocyte colony-stimulating factor receptor genes in partially overlapping monoclonal B-cell populations from chronic lymphocytic leukemia patients

B lymphocytes were purified from the peripheral blood of 30 B-cell chronic lymphocytic leukemia (B-CLL) patients and tested for the ability to produce granulocyte colony-stimulating factor (G-CSF) in vitro. Fifteen Staphylococcus aureus Cowan I (SAC)-stimulated, but not unstimulated, B-cell suspensions produced G-CSF in short-term cultures. Accordingly, G-CSF mRNA was detected only in SAC-stimulated B cells. Five CLL B-cell fractions that released G-CSF following exposure to SAC were also incubated with CD40 or anti-mu antibodies in the presence or absence of recombinant (r) interleukin-2 (IL-2) or IL-4. The 5 cell suspensions produced G-CSF only on culture with CD40 monoclonal antibody in combination with rIL-2 or rIL-4. CD5+ B lymphocytes, which represent the normal counterparts of most B-CLL proliferations, did not produce G-CSF under any of the above culture conditions. G-CSF produced by leukemic B lymphocytes was biologically active, because conditioned media of SAC-stimulated cells supported the in vitro growth of myeloid colonies from normal bone marrow progenitors. The colony stimulating activity of CLL B-cell supernatants was ascribed to both G-CSF and granulocyte-macrophage colony stimulating factor. G-CSF receptors (G-CSFRs) were detected on freshly isolated B lymphocytes from 7 of 11 B-CLL patients; 5 of these cell suspensions produced G-CSF in culture, whereas 2 did not. rG-CSF rescued 3 of the 7 G-CSFR+ cell fractions from spontaneous apoptosis but had no effect on their in vitro proliferation.

Detection of Clonai CD34+19+ Progenitors in Bone Marrow of BCL2-IgH–Positive Follicular Lymphoma Patients

AbstractThe frequent occurrence of BCL2-lgH rearrangements in follicular lymphoma (FL) makes detection of low numbers of tumor cells possible by polymerase chain reaction (PCR). The presence of BCL2-lgH in the bone marrow (BM) and peripheral blood of many FL patients at the time of autografting has led to the suggestion that selection of the CD34-enriched fraction may lead to reinfusion of lower numbers of tumor cells. To address this issue, we PCR-amplified BCL2-IgH from fluorescence-activated cell sorting (FACS)-purified BM CD34+ and CD34– fractions in seven FL patients showing a PCR-detectable translocation in the major breakpoint region of BCL2, five of which showed morphological BM involvement. The total CD34+ fraction showed diminished but residual positivity in the first two cases tested. Therefore, BM cells from the remaining five patients were sorted for the CD34+19– immature population, the CD34+19+ B-cell precursors, and the CD34–19+ mature B-cell fraction. The CD34+19– subpopulation was negative in four of five, despite evident BM infiltration in three cases. In contrast, the CD34+19+ fraction was positive in all three cases tested. These cells represented 0% to 50% (mean, 18%) of the total CD34+ population, suggesting that, if reinfusion of BCL2-IgH–positive cells plays a role in postautograft relapse in FL, therapeutic C034 selection procedures should include additional purging of the CD34+19+ B-cell precursors or, at least, assessment of the proportion of CD19+ cells in the CD34+ fraction and its correlation with clinical outcome postreinfusion.