Higher-order CpG-DNA stimulation reveals distinct activation requirements for marginal zone and follicular B cells in lupus mice

Mouse follicular B cells express TLR9 and respond vigorously to stimulation with single-stranded CpG-oligodeoxynucleotides (ODN). Surprisingly, follicular B cells do not respond to direct stimulation with other TLR9 ligands, such as bacterial DNA or class A(D) CpG-ODN capable of forming higher-order structures, unless other cell types are present. Here, we show that priming with interferons or with B cell-activating factor, or simultaneous co-engagement of the B cell receptor for antigen (BCR), can overcome this unresponsiveness. The effect of interferons occurs at the transcriptional level and is mediated through an autocrine/paracrine loop, which is dependent on IRF-1, IL-6 and IL-12 p40. We hypothesize that the lack of bystander activation of follicular B cells with more complex CpG ligands may be an important safety mechanism for avoiding autoimmunity. This will prevent resting B cells from responding to foreign or self-derived hypomethylated double-stranded CpG ligands unless these ligands are either delivered through the B cell receptor or under conditions where B cells are simultaneously co-engaged by activated plasmacytoid dendritic cells or TH1 cells. A corollary is that the heightened responsiveness of lupus B cells to TLR9-induced stimulation cannot be ascribed to unprimed follicular B cells, but is rather mediated by hypersensitive marginal zone B cells.

Analysis of the effect of different NKT cell subpopulations on the activation of CD4 and CD8 T cells, NK cells, and B cells

Objective. NKT cells have diverse immune regulatory functions including activation of cells involved in Th1- and Th2-type immune activities. Most previous studies have investigated the functions of NKT cells as a single family but more recent evidence indicates the distinct functional properties of NKT cell subpopulation. This study aims to determine whether NKT cell subpopulations have different stimulatory activities on other immune cells that may affect the outcome of NKT cell-based immunotherapy. Methods. NKT cells and NKT cell subpopulations (CD4(+)CD8(-), CD4(-)CD8(+), CD4(-)CD8(+)) were cocultured with PBMC and their activities on immune cells including CD4(+) and CD8(+) T cells, NK cells, and B cells were assessed by flow cytometry. The production of cytokines in culture was measured by enzyme-linked immunsorbent assay. Results. The CD4(+)CD8(-) NKT cells demonstrated substantially greater stimulatory activities on CD4(+) T cells, NK cells, and B cells than other NKT cell subsets. The CD4(-)CD8(+) NKT cells showed the greatest activity on CD8(+) T cells, and were the only NKT cell subset that activated these immune cells. The CD4(-)CD8(-) NKT cells showed moderate stimulatory activity on CD4(+) T cells and the least activity on other immune cells. Conclusion. The results here suggest that NKT cell subpopulations differ in their abilities to stimulate other immune cells. This highlights the potential importance of manipulating specific NKT cell subpopulations for particular therapeutic situations and of evaluating subpopulations, rather than NKT cells as a group, during investigation of a possible role of NKT cells in various disease settings. (c) 2006 International Society for Experimental Hematology. Published by Elsevier Inc.

Host B cells produce IL-10 following TBI and attenuate acute GVHD after allogeneic bone marrow transplantation

Host antigen-presenting cells (APCs) are known to be critical for the induction of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT), but the relative contribution of specific APC subsets remains unclear. We have studied the role of host B cells in GVHD by using B-cell-deficient mu MT mice as BMT recipients in a model of CD4-dependent GVHD to major histocompatlibility complex antigens. We demonstrate that acute GVHD is initially augmented in mu MT recipients relative to wild-type recipients (mortality: 85% vs 44%, P < .01), and this is the result of an increase in donor T-cell proliferation, expansion, and inflammatory cytokine production early after BMT. Recipient B cells were depleted 28-fold at the time of BMT by total body irradiation (TBI) administered 24 hours earlier, and we demonstrate that TBI rapidly induces sustained interleukin-110 (IL-10) generation from B cells but not dendritic cells (DCs) or other cellular populations within the spleen. Finally, recipient mice in which B cells are unable to produce IL-10 due to homologous gene deletion develop more severe acute GVHD than recipient mice in which B cells are wild type. Thus, the induction of IL-10 in host B cells during conditioning attenuates experimental acute GVHD.

Neural progenitor number is regulated by nuclear factor-kappa B p65 and p50 subunit-dependent proliferation rather than cell survival

The number of cells generated by a proliferating stem or precursor cell can be influenced both by proliferation and by the degree of cell death/survival of the progeny generated. In this study, the extent to which cell survival controls progenitor number was examined by comparing the growth characteristics of neurosphere cultures derived from mice lacking genes for the death inducing Bcl-2 homologue Hara Kiri (Hrk), apoptosis-associated protein 1 (Apaf1), or the prosurvival nuclear factor-kappa B (NF kappa B) subunits p65, p50, or c-rel. We found no evidence that Hrk or Apaf1, and by inference the mitochondrial cell death pathway, are involved in regulating the number of neurosphere-derived progeny. However, we identified the p65p50 NF kappa B dimer as being required for the normal growth and expansion of neurosphere cultures. Genetic loss of both p65 and p50 NF kappa B subunits resulted in a reduced number of progeny but an increased proportion of neurons. No effect on cell survival was observed. This suggests that the number and fate of neural progenitor cells are more strongly regulated by cell cycle control than survival. (c) 2005 Wiley-Liss, Inc.

Adenocarcinoma of colon differentiating as dome epithelium of gut-associated lymphoid tissue

Aims: An early adenocarcinoma of the ascending colon was confined to a mass of gut-associated lymphoid tissue (GALT). The first description of an adenocarcinoma of colon differentiating as dome epithelium is presented. Methods and results: A plaque-like carcinoma was identified opposite the ileocaecal valve in an asymptomatic 56-year-old man with a family history of colorectal cancer. Malignant epithelium was confined to a mass of GALT filling but limited to the submucosa, Characterization of the neoplasm was undertaken by means of mucin histochemistry, immunohistochemistry, electron microscopy and assessment of DNA microsatellite instability status. The malignant epithelium comprised well differentiated columnar cells with a microvillous brush border and expressing MUC1, but no goblet cells or expression of MUC2. The demonstration of focal clusters of intraepithelial B-lymphocytes supported the presence of functioning M-cells within the malignant neoplasm. The cancer was DNA microsatellite stable despite the finding of tumour infiltrating lymphocytes. Conclusions: There is evidence for the origin of colorectal neoplasia from dome epithelium in both experimental models and microreconstruction studies of early adenomas in nonpolypotic human colorectal mucose, It is suggested that the lymphocyte-rich subset of colorectal cancer that expresses MUC1 but not MUC2 may be differentiating as dome epithelium of gut-associated lymphoid tissue.

Orexin-stimulated MAP kinase cascades are activated through multiple G-protein signalling pathways in human H295R adrenocortical cells: diverse roles for orexins A and B

Orexins A and B (ORA and ORB) are neuropeptide hormones found throughout the central nervous system and periphery. They are required for a host of physiological processes including mitogen-activated protein kinase (MAPK) regulation, steroidogenesis, appetite control and energy regulation. While some signalling mechanisms have been proposed for individual recombinant orexin receptors in generic mammalian cell types, it is clear that the peripheral effects of orexin are spatially and temporally complex. This study dissects the different G-protein signalling and MAPK pathways activated in a pluripotent human adrenal H295R cell line capable of all the physiological steps involved in steroidogenesis. Both extracellular receptor kinase 1/2 (ERK1/2) and p38 were phosphorylated rapidly with a subsequent decline, in a time- and dose-dependent manner, in response to both ORA and ORB. Conversely, there was little or no direct activation of the ERK5 or JNK pathway. Analysis using signalling and MAPK inhibitors as well as receptor-specific antagonists determined the precise mediators of the orexin response in these cells. Both ERK1/2 and p38 activation were predominantly Gq- and to a lesser extent Gs-mediated; p38 activation even had a small Gi-component. Effects were broadly comparable for both orexin sub-types ORA and ORB and although most of the effects were transmitted through the orexin receptor-1 subtype, we did observe a role for orexin receptor-2-mediated activation of both ERK1/2 and p38. Cortisol secretion also differed in response to ORA and ORB. These data suggest multiple roles for orexin-mediated MAPK activation in an adrenal cell-line, this complexity may help to explain the diverse biological actions of orexins with wide-ranging consequences for our understanding of the mechanisms initiated by these steroidogenic molecules.

Insulin release from cultured B-cells

Established RlNm5F and lN111 R1 and newly available HlT-T15 and UMR 407/3 B-cell lines have been successfully maintained in vitro. With the exclusion of UMR 407/3 cells, all lines were continuously propagable. Doubling times and plating efficiencies for HlT-T15, RlNm5F, lN111 R1 and UMR 407/3 cells were 20 hours and 85%, 31 hours and 76%, 24 hours and 80% and 38 hours and 94% respectively. All the cell lines were anchorage dependent, but only UMR 407/3 cells grew to confluence. Only HlT-T15 and UMR 407/3 cells produced a true insulin response to glucose but glucose markedly increased the rate of D-[U14C]glucose oxidation by all the cell lines. Glucose induced insulin release from HlT-T15 cells was biphasic with an exaggerated first phase. Insulin release from HlT-T15, RlNm5F and IN111 R1 cells was stimulated by amino acids and sulphonylureas. Glucagon stimulated insulin release from HlT-T15 and RlNm5F cells while somatostatin and pancreatic polypeptide inhibited release. These observations suggest that net insulin release from the whole islet may be the result of significant paracrine interaction. HlT-T15 and RlNm5F cell insulin release was stimulated by forskolin and inhibited by imidazole. Ca2+ channel blockade and calmodulin inhibition suppressed insulin release from HlT-T15, RlNm5F and IN111 R1 cells. In addition phorbol esters stimulated insulin release from RlNm5F cells. These data implicate cAMP, Ca2+ and protein kinase-C in the regulation of insulin release from cultured B-cells. Acetylcholine increased insulin release from HlT-T15 and RlNm5F cells. Inhibition of the response by atropine confirmed the involvement of muscarinic receptors. HlT-T15 cell insulin release was also inhibited by adrenaline. These observations suggest a possible role for the autonomic nervous system in the modulation of insulin release. Preliminary studies with a human insulinoma maintained in monolayer culture have demonstrated a limited life span of some seven weeks, a continuous low level of insulin release but no insulin response to glucose challenge.

Lymphoid aggregates that resemble tertiary lymphoid organs define a specific pathological subset in metal-on-metal hip replacements

Aseptic lymphocyte-dominated vasculitis-associated lesion (ALVAL) has been used to describe the histological lesion associated with metal-on-metal (M-M) bearings. We tested the hypothesis that the lymphoid aggregates, associated with ALVAL lesions resemble tertiary lymphoid organs (TLOs). Histopathological changes were examined in the periprosthetic tissue of 62 M-M hip replacements requiring revision surgery, with particular emphasis on the characteristics and pattern of the lymphocytic infiltrate. Immunofluorescence and immunohistochemistry were used to study the classical features of TLOs in cases where large organized lymphoid follicles were present. Synchrotron X-ray fluorescence (XRF) measurements were undertaken to detect localisation of implant derived ions/particles within the samples. Based on type of lymphocytic infiltrates, three different categories were recognised; diffuse aggregates (51%), T cell aggregates (20%), and organised lymphoid aggregates (29%). Further investigation of tissues with organised lymphoid aggregates showed that these tissues recapitulate many of the features of TLOs with T cells and B cells organised into discrete areas, the presence of follicular dendritic cells, acquisition of high endothelial venule like phenotype by blood vessels, expression of lymphoid chemokines and the presence of plasma cells. Co-localisation of implant-derived metals with lymphoid aggregates was observed. These findings suggest that in addition to the well described general foreign body reaction mediated by macrophages and a T cell mediated type IV hypersensitivity response, an under-recognized immunological reaction to metal wear debris involving B cells and the formation of tertiary lymphoid organs occurs in a distinct subset of patients with M-M implants. © 2013 Mittal et al.

Distinct Differentiation Programs Triggered by IL-6 And LPS in Teleost IgM+ B Cells in the Absence of Germinal Centers

We would like to thank Lucia Gonzalez and Maria Sanz for technical assistance. Professor Øystein Evensen is also acknowledged for providing us with the inactivated IPNV. This work was supported by the European Research Council (ERC Starting Grant 2011 280469) and by the European Commission under the 7th Framework Programme for Research and Technological Development (FP7) of the European Union (Grant Agreement 311993 TARGETFISH). T. W. received funding from the MASTS pooling initiative (The Marine Alliance for Science and Technology for Scotland). MASTS is funded by the Scottish Funding Council (grant reference HR09011).

A Therapeutic Antibody for Cancer, Derived from Single Human B Cells.

Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery.

On the Origin of Natural Antibody

Natural IgM (nIgM) is constitutively present in the serum, where it aids in the early control of viral and bacterial expansions. nIgM also plays a significant role in the prevention of autoimmune disease by promoting the clearance of cellular debris. However, the cells that maintain high titers of nIgM in the circulation had not yet been identified. Several studies have linked serum nIgM with the presence of fetal-lineage B cells, and others have detected IgM secretion directly by B1a cells in various tissues. Nevertheless, a substantial contribution of undifferentiated B1 cells to nIgM titers is doubtful, as the ability to produce large quantities of antibody (Ab) is a function of the phenotype and morphology of differentiated plasma cells (PCs). No direct evidence exists to support the claim that a B1-cell population directly produces the bulk of circulating nIgM. The source of nIgM thus remained uncertain and unstudied.

In the first part of this study, I identified the primary source of nIgM. Using enzyme-linked immunosorbent spot (ELISPOT) assay, I determined that the majority of IgM Ab-secreting cells (ASCs) in naïve mice reside in the bone marrow (BM). Flow cytometric analysis of BM cells stained for intracellular IgM revealed that nIgM ASCs express IgM and the PC marker CD138 on their surface, but not the B1a cell marker CD5. By spinning these cells onto slides and staining them, following isolation by fluorescence-activated cell sorting (FACS), I found that they exhibit the typical morphological characteristics of terminally differentiated PCs. Transfer experiments demonstrated that BM nIgM PCs arise from a progenitor in the peritoneal cavity (PerC), but not isolated PerC B1a, B1b, or B2 cells. Immunoglobulin (Ig) gene sequence analysis and examination of B1-8i mice, which carry an Ig knockin that prohibits fetal B-cell development, indicated that nIgM PCs differentiate from fetal-lineage B cells. BrdU uptake experiments showed that the nIgM ASC compartment contains a substantial fraction of long-lived plasma cells (LLPCs). Finally, I demonstrated that nIgM PCs occupy a survival niche distinct from that used by IgG PCs.

In the second part of this dissertation, I characterized the unique survival niche of nIgM LLPCs, which maintain constitutive high titers of nIgM in the serum. By using genetically deficient or Ab-depleted mice, I found that neither T cells, type 2 innate lymphoid cells, nor mast cells, the three major hematopoietic producers of IL-5, were required for nIgM PC survival in the BM. However, IgM PCs associate strongly with IL-5-expressing BM stromal cells, which support their survival in vitro when stimulated. In vivo neutralization of IL-5 revealed that, like individual survival factors for IgG PCs, IL-5 is not the sole supporter of IgM PCs, but is likely one of several redundant molecules that together ensure uninterrupted signaling. Thus, the long-lived nIgM PC niche is not composed of hematopoietic sources of IL-5, but a stromal cell microenvironment that provides multiple redundant survival signals.

In the final part of my study, I identified and characterized the precursor of nIgM PCs, which I found in the first project to be resident in the PerC, but not a B1a, B1b, or B2 cell. By transferring PerC cells sorted based on expression of CD19, CD5, and CD11b, I found that only the CD19+CD5+CD11b- population contained cells capable of differentiating into nIgM PCs. Transfer of decreasing numbers of unfractionated PerC cells into Rag1 knockouts revealed an order-of-magnitude drop in the rate of serum IgM reconstitution between stochastically sampled pools of 106 and 3x105 PerC cells, suggesting that the CD19+CD5+CD11b- compartment comprises two cell types, and that interaction between the two necessary for nIgM-PC differentiation. By transferring neonatal liver, I determined that the early hematopoietic environment is required for nIgM PC precursors to develop. Using mice carrying a mutation that disturbs cKit expression, I also found that cKit appears to be required at a critical point near birth for the proper development of nIgM PC precursors.

The collective results of these studies demonstrate that nIgM is the product of BM-resident PCs, which differentiate from a PerC B cell precursor distinct from B1a cells, and survive long-term in a unique survival niche created by stromal cells. My work creates a new paradigm by which to understand nIgM, B1 cell, and PC biology.

Expression of high amounts of the CD117 molecule in a case of B-cell non-Hodgkin's lymphoma carrying the t(14:18) translocation.

The c-kit proto-oncogen (CD117) has been described to be present in normal and neoplastic hemopoietic cells including both myeloid and lymphoid lineages. Among the normal lymphoid cells CD117 expression would be restricted to a small subset of NK-cells, and to early T-cell precursors and it is not expressed by normal B-cells. Regarding chronic lymphoproliferative disorders the only data provided up to now suggests that CD117 expression is restricted to cases of Hodgkin's disease and anaplastic large-cell lymphoma. In the present paper we describe a case of a B-cell chronic lymphoproliferative disorder carrying the t(14:18) translocation as demonstrated by molecular studies, in which the flow cytometric immunophenotypic analysis of both peripheral blood and bone marrow samples revealed the expression of high amounts of the CD117 antigen in the surface of the clonal B-cell population. Further studies are necessary to explore both the functional role of c-kit expression in the neoplastic B-cells from this patient and its potential utility for the diagnosis and follow-up of patients with B-cell non-Hodgkin's lymphoma.

NK Cells of Kidney Transplant Recipients Display an Activated Phenotype that Is Influenced by Immunosuppression and Pathological Staging

To explore phenotype and function of NK cells in kidney transplant recipients, we investigated the peripheral NK cell repertoire, capacity to respond to various stimuli and impact of immunosuppressive drugs on NK cell activity in kidney transplant recipients. CD56(dim) NK cells of kidney transplanted patients displayed an activated phenotype characterized by significantly decreased surface expression of CD16 (p=0.0003), CD226 (p<0.0001), CD161 (p=0.0139) and simultaneously increased expression of activation markers like HLA-DR (p=0.0011) and CD25 (p=0.0015). Upon in vitro stimulation via Ca++-dependent signals, down-modulation of CD16 was associated with induction of interferon (IFN)-gamma expression. CD16 modulation and secretion of NFAT-dependent cytokines such as IFN-gamma, TNF-alpha, IL-10 and IL-31 were significantly suppressed by treatment of isolated NK cells with calcineurin inhibitors but not with mTOR inhibitors. In kidney transplant recipients, IFN-gamma production was retained in response to HLA class I-negative target cells and to non-specific stimuli, respectively. However, secretion of other cytokines like IL-13, IL-17, IL-22 and IL-31 was significantly reduced compared to healthy donors. In contrast to suppression of cytokine expression at the transcriptional level, cytotoxin release, i.e. perforin, granzyme A/B, was not affected by immunosuppression in vitro and in vivo in patients as well as in healthy donors. Thus, immunosuppressive treatment affects NK cell function at the level of NFAT-dependent gene expression whereby calcineurin inhibitors primarily impair cytokine secretion while mTOR inhibitors have only marginal effects. Taken together, NK cells may serve as indicators for immunosuppression and may facilitate a personalized adjustment of immunosuppressive medication in kidney transplant recipients.

Expression of tumor-related Rac1b antagonizes B-Raf-induced senescence in colorectal cells

Mutations in the BRAF oncogene have been identified as a tumor-initiating genetic event in mainly melanoma, thyroid and colon cancer, resulting in an initial proliferative stimulus that is followed by a growth arrest period known as oncogene-induced senescence (OIS). It remains unknown what triggers subsequent escape from OIS to allow further tumor progression. A previous analysis revealed that overexpression of splice variant Rac1b occurs in around 80% of colorectal tumors carrying a mutation in BRAF. Using both BRaf-V600E-directed RNAi and overexpression we demonstrate that this mutation does not directly lead to Rac1b overexpression, indicating the latter as an independent event during tumor progression. Nonetheless, we observed that expression of oncogenic BRaf-V600E in non-transformed colonocytes (NCM460 cell line) increased both the transcript and protein levels of p14ARF, p15INK4b and p21CIP1 and led to increased expression of β-galactosidase, all indicators of OIS induction. Interestingly, whereas the protein levels of these markers were reduced upon Rac1b overexpression, the levels of their respective transcripts remained unchanged. Importantly, the co-expression of Rac1b with B-Raf-V600E reverted the OIS phenotype, reducing the expression levels of the cell-cycle inhibitors and β-galactosidase to those of control cells. These data identify increased Rac1b expression as one potential mechanism by which colorectal tumor cells can escape from B-Raf-induced OIS.