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1.  Pre-B Cell Receptor Signaling Induces Immunoglobulin κ Locus Accessibility by Functional Redistribution of Enhancer-Mediated Chromatin Interactions 
PLoS Biology  2014;12(2):e1001791.
Chromatin conformation analyses provide novel insights into how variable segments in the immunoglobulin light chain gene become accessible for recombination in precursor B lymphocytes.
During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline Vκ transcription. To investigate whether pre-BCR signaling modulates Vκ accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb Vκ region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3′κ enhancer with Vκ genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and Vκ genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used Vκ genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the Vκ region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the Vκ region, whereby the two enhancers play distinct roles.
Author Summary
B lymphocyte development involves the generation of a functional antigen receptor, comprising two heavy chains and two light chains arranged in a characteristic “Y” shape. To do this, the receptor genes must first be assembled by ordered genomic recombination events, starting with the immunoglobulin heavy chain (IgH) gene segments. On successful rearrangement, the resulting IgH μ protein is presented on the cell surface as part of a preliminary version of the B cell receptor—the “pre-BCR.” Pre-BCR signaling then redirects recombination activity to the immunoglobulin κ light chain gene. The activity of two regulatory κ enhancer elements is known to be crucial for opening up the gene, but it remains largely unknown how the hundred or so Variable (V) segments in the κ locus gain access to the recombination system. Here, we studied a panel of pre-B cells from mice lacking specific signaling molecules, reflecting absent, partial, or complete pre-BCR signaling. We identify gene regulatory changes that are dependent on pre-BCR signaling and occur via long-range chromatin interactions between the κ enhancers and the V segments. Surprisingly the light chain gene initially contracts, but the interactions then become more functionally redistributed when pre-BCR signaling occurs. Interestingly, we find that the two enhancers play distinct roles in the process of coordinating chromatin interactions towards the V segments. Our study combines chromatin conformation techniques with data on transcription factor binding to gain unique insights into the functional role of chromatin dynamics.
doi:10.1371/journal.pbio.1001791
PMCID: PMC3928034  PMID: 24558349
2.  Dietary Restriction and Fasting Arrest B and T Cell Development and Increase Mature B and T Cell Numbers in Bone Marrow 
PLoS ONE  2014;9(2):e87772.
Dietary restriction (DR) delays ageing and extends life span. Both long- and short-term DR, as well as short-term fasting provide robust protection against many “neuronal and surgery related damaging phenomena” such as Parkinson’s disease and ischemia-reperfusion injury. The exact mechanism behind this phenomenon has not yet been elucidated. Its anti-inflammatory actions prompted us to thoroughly investigate the consequences of DR and fasting on B and T cell compartments in primary and secondary lymphoid organs of male C57Bl/6 mice. In BM we found that DR and fasting cause a decrease in the total B cell population and arrest early B cell development, while increasing the number of recirculating mature B cells. In the fasting group, a significant reduction in peripheral B cell counts was observed in both spleen and mesenteric lymph nodes (mLN). Thymopoiesis was arrested significantly at double negative DN2 stage due to fasting, whereas DR resulted in a partial arrest of thymocyte development at the DN4 stage. Mature CD3+ T cell populations were increased in BM and decreased in both spleen and mLN. Thus, DR arrests B cell development in the BM but increases the number of recirculating mature B cells. DR also arrests maturation of T cells in thymus, resulting in depletion of mature T cells from spleen and mLN while recruiting them to the BM. The functional relevance in relation to protection against organ damage needs to be determined.
doi:10.1371/journal.pone.0087772
PMCID: PMC3913690  PMID: 24504160
3.  Allelic exclusion of the immunoglobulin heavy chain locus is independent of its nuclear localization in mature B cells 
Nucleic Acids Research  2013;41(14):6905-6916.
In developing B cells, the immunoglobulin heavy chain (IgH) locus is thought to move from repressive to permissive chromatin compartments to facilitate its scheduled rearrangement. In mature B cells, maintenance of allelic exclusion has been proposed to involve recruitment of the non-productive IgH allele to pericentromeric heterochromatin. Here, we used an allele-specific chromosome conformation capture combined with sequencing (4C-seq) approach to unambigously follow the individual IgH alleles in mature B lymphocytes. Despite their physical and functional difference, productive and non-productive IgH alleles in B cells and unrearranged IgH alleles in T cells share many chromosomal contacts and largely reside in active chromatin. In brain, however, the locus resides in a different repressive environment. We conclude that IgH adopts a lymphoid-specific nuclear location that is, however, unrelated to maintenance of allelic exclusion. We additionally find that in mature B cells—but not in T cells—the distal VH regions of both IgH alleles position themselves away from active chromatin. This, we speculate, may help to restrict enhancer activity to the productively rearranged VH promoter element.
doi:10.1093/nar/gkt491
PMCID: PMC3737562  PMID: 23748562
4.  Evaluation of Clinical and Immunological Responses: A 2-Year Follow-Up Study in Children with Allergic Rhinitis due to House Dust Mite 
Mediators of Inflammation  2013;2013:345217.
Background. Allergic rhinitis is a disease with polarization towards Th2 and a defect of regulatory T cells. Immunological changes have been reported after immunotherapy treatment. However, there is not much known about the natural course of allergic rhinitis with respect to clinical manifestation and the relation with immunological responses. Objective. To evaluate clinical symptoms of allergic rhinitis, in relation to in vivo allergen-specific skin responses and in vitro allergen-specific effector and regulatory T cells determined at baseline and after two years. Methods. From a large trial, 59 children were randomly selected. The following variables were compared: clinical symptoms, allergen skin tests, specific IgE, T-cell proliferation, IL-5, IL-13, IFN-gamma, IL-10, TGF-beta, CD4+CD25hi cells, and Foxp3 expression. Results. Allergic symptoms had decreased after two years. Whereas skin test reactions correlated between years 0 and 2, there was no change in the size of the reaction. Also, proinflammatory reactions did not change after two years, with a positive correlation between years 0 and 2. No relevant changes were observed with respect to regulatory cells. Conclusion. Whereas, comparable to immunotherapy, allergic complaints decrease, the immunological changes of specific T-cell activity (both effector cells and regulator cells) which are observed after immunotherapy, do not change.
doi:10.1155/2013/345217
PMCID: PMC3655673  PMID: 23737646
5.  The Mucosal Adjuvant Cholera Toxin B Instructs Non-Mucosal Dendritic Cells to Promote IgA Production Via Retinoic Acid and TGF-β 
PLoS ONE  2013;8(3):e59822.
It is currently unknown how mucosal adjuvants cause induction of secretory immunoglobulin A (IgA), and how T cell-dependent (TD) or -independent (TI) pathways might be involved. Mucosal dendritic cells (DCs) are the primary antigen presenting cells driving TI IgA synthesis, by producing a proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), Retinoic Acid (RA), TGF-β or nitric oxide (NO). We hypothesized that the mucosal adjuvant Cholera Toxin subunit B (CTB) could imprint non-mucosal DCs to induce IgA synthesis, and studied the mechanism of its induction. In vitro, CTB-treated bone marrow derived DCs primed for IgA production by B cells without the help of T cells, yet required co-signaling by different Toll-like receptor (TLR) ligands acting via the MyD88 pathway. CTB-DC induced IgA production was blocked in vitro or in vivo when RA receptor antagonist, TGF-β signaling inhibitor or neutralizing anti-TGF-β was added, demonstrating the involvement of RA and TGF-β in promoting IgA responses. There was no major involvement for BAFF, APRIL or NO. This study highlights that synergism between CTB and MyD88-dependent TLR signals selectively imprints a TI IgA-inducing capacity in non-mucosal DCs, explaining how CTB acts as an IgA promoting adjuvant.
doi:10.1371/journal.pone.0059822
PMCID: PMC3603891  PMID: 23527272
6.  Bruton’s tyrosine kinase mediated signaling enhances leukemogenesis in a mouse model for chronic lymphocytic leukemia 
In chronic lymphocytic leukemia (CLL) signals from the B cell receptor (BCR) play a major role in disease development and progression. In this light, new therapies that specifically target signaling molecules downstream of the BCR continue to be developed. While first studies on the selective small molecule inhibitor of Bruton’s tyrosine kinase (Btk), Ibrutinib (PCI-32765), demonstrated that Btk inhibition sensitizes CLL cells to apoptosis and alters their migratory behavior, these studies however did not address whether Btk-mediated signaling is involved in the process of CLL leukemogenesis. To investigate the requirement of Btk signaling for CLL development, we modulated Btk expression in the IgH.ETμ CLL mouse model, which is based on sporadic expression of the simian oncovirus SV40 T-antigen in mature B cells. To this end, we crossed IgH.ETμ mice on a Btk-deficient background or introduced a human Btk transgene (CD19-hBtk). Here we show that Btk deficiency fully abrogates CLL formation in IgH.ETμ mice, and that leukemias formed in Btk haplo-insufficient mice selectively expressed the wild-type Btk allele on their active X chromosome. Conversely, Btk overexpression accelerated CLL onset, increased mortality, and was associated with selection of non-stereotypical BCRs into CLL clones. Taken together, these data show that Btk expression represents an absolute prerequisite for CLL development and that Btk mediated signaling enhances leukemogenesis in mice. We therefore conclude that in CLL Btk expression levels set the threshold for malignant transformation.
PMCID: PMC3555194  PMID: 23359016
Chronic lymphocytic leukemia (CLL); bruton’s tyrosine kinase (Btk); B cell receptor signaling
7.  Granuloma Formation in Pulmonary Sarcoidosis 
Sarcoidosis is a granulomatous disorder of unknown cause, affecting multiple organs, but mainly the lungs. The exact order of immunological events remains obscure. Reviewing current literature, combined with careful clinical observations, we propose a model for granuloma formation in pulmonary sarcoidosis. A tight collaboration between macrophages, dendritic cells, and lymphocyte subsets, initiates the first steps toward granuloma formation, orchestrated by cytokines and chemokines. In a substantial part of pulmonary sarcoidosis patients, granuloma formation becomes an on-going process, leading to debilitating disease, and sometimes death. The immunological response, determining granuloma sustainment is not well understood. An impaired immunosuppressive function of regulatory T cells has been suggested to contribute to the exaggerated response. Interestingly, therapeutical agents commonly used in sarcoidosis, such as glucocorticosteroids and anti-TNF agents, interfere with granuloma integrity and restore the immune homeostasis in autoimmune disorders. Increasing insight into their mechanisms of action may contribute to the search for new therapeutical targets in pulmonary sarcoidosis.
doi:10.3389/fimmu.2013.00437
PMCID: PMC3857538  PMID: 24339826
pulmonary sarcoidosis; granuloma; formation; integrity; dendritic cells; T helper 1 cells; T helper 17 cells; regulatory T cells
8.  Evidence for local dendritic cell activation in pulmonary sarcoidosis 
Respiratory Research  2012;13(1):33.
Background
Sarcoidosis is a granulomatous disease characterized by a seemingly exaggerated immune response against a difficult to discern antigen. Dendritic cells (DCs) are pivotal antigen presenting cells thought to play an important role in the pathogenesis. Paradoxically, decreased DC immune reactivity was reported in blood samples from pulmonary sarcoidosis patients. However, functional data on lung DCs in sarcoidosis are lacking. We hypothesized that at the site of disease DCs are mature, immunocompetent and involved in granuloma formation.
Methods
We analyzed myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in broncho-alveolar lavage (BAL) and blood from newly diagnosed, untreated pulmonary sarcoidosis patients and healthy controls using 9-color flowcytometry. DCs, isolated from BAL using flowcytometric sorting (mDCs) or cultured from monocytes (mo-DCs), were functionally assessed in a mixed leukocyte reaction with naïve allogeneic CD4+ T cells. Using Immunohistochemistry, location and activation status of CD11c+DCs was assessed in mucosal airway biopsies.
Results
mDCs in BAL, but not in blood, from sarcoidosis patients were increased in number when compared with mDCs from healthy controls. mDCs purified from BAL of sarcoidosis patients induced T cell proliferation and differentiation and did not show diminished immune reactivity. Mo-DCs from patients induced increased TNFα release in co-cultures with naïve allogeneic CD4+ T cells. Finally, immunohistochemical analyses revealed increased numbers of mature CD86+ DCs in granuloma-containing airway mucosal biopsies from sarcoidosis patients.
Conclusion
Taken together, these finding implicate increased local DC activation in granuloma formation or maintenance in pulmonary sarcoidosis.
doi:10.1186/1465-9921-13-33
PMCID: PMC3352267  PMID: 22513006
Sarcoidosis; Dendritic cells; Bronchoalveolar lavage; Granuloma; TNFα
9.  COX-2 inhibition improves immunotherapy and is associated with decreased numbers of myeloid-derived suppressor cells in mesothelioma. Celecoxib influences MDSC function 
BMC Cancer  2010;10:464.
Background
Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature cells that accumulates in tumour-bearing hosts. These cells are induced by tumour-derived factors (e.g. prostaglandins) and have a critical role in immune suppression. MDSC suppress T and NK cell function via increased expression of arginase I and production of reactive oxygen species (ROS) and nitric oxide (NO). Immune suppression by MDSC was found to be one of the main factors for immunotherapy insufficiency. Here we investigate if the in vivo immunoregulatory function of MDSC can be reversed by inhibiting prostaglandin synthesis by specific COX-2 inhibition focussing on ROS production by MDSC subtypes. In addition, we determined if dietary celecoxib treatment leads to refinement of immunotherapeutic strategies.
Methods
MDSC numbers and function were analysed during tumour progression in a murine model for mesothelioma. Mice were inoculated with mesothelioma tumour cells and treated with cyclooxygenase-2 (COX-2) inhibitor celecoxib, either as single agent or in combination with dendritic cell-based immunotherapy.
Results
We found that large numbers of infiltrating MDSC co-localise with COX-2 expression in those areas where tumour growth takes place. Celecoxib reduced prostaglandin E2 levels in vitro and in vivo. Treatment of tumour-bearing mice with dietary celecoxib prevented the local and systemic expansion of all MDSC subtypes. The function of MDSC was impaired as was noticed by reduced levels of ROS and NO and reversal of T cell tolerance; resulting in refinement of immunotherapy.
Conclusions
We conclude that celecoxib is a powerful tool to improve dendritic cell-based immunotherapy and is associated with a reduction in the numbers and suppressive function of MDSC. These data suggest that immunotherapy approaches benefit from simultaneously blocking cyclooxygenase-2 activity.
doi:10.1186/1471-2407-10-464
PMCID: PMC2939552  PMID: 20804550
10.  Low-Dose Cyclophosphamide Synergizes with Dendritic Cell-Based Immunotherapy in Antitumor Activity 
Clinical immunotherapy trials like dendritic cell-based vaccinations are hampered by the tumor's offensive repertoire that suppresses the incoming effector cells. Regulatory T cells are instrumental in suppressing the function of cytotoxic T cells. We studied the effect of low-dose cyclophosphamide on the suppressive function of regulatory T cells and investigated if the success rate of dendritic cell immunotherapy could be improved. For this, mesothelioma tumor-bearing mice were treated with dendritic cell-based immunotherapy alone or in combination with low-dose of cyclophosphamide. Proportions of regulatory T cells and the cytotoxic T cell functions at different stages of disease were analyzed. We found that low-dose cyclophosphamide induced beneficial immunomodulatory effects by preventing the induction of Tregs, and as a consequence, cytotoxic T cell function was no longer affected. Addition of cyclophosphamide improved immunotherapy leading to an increased median and overall survival. Future studies are needed to address the usefulness of this combination treatment for mesothelioma patients.
doi:10.1155/2010/798467
PMCID: PMC2874992  PMID: 20508851
11.  Dendritic cells are crucial for maintenance of tertiary lymphoid structures in the lung of influenza virus–infected mice 
The Journal of Experimental Medicine  2009;206(11):2339-2349.
Tertiary lymphoid organs (TLOs) are organized aggregates of B and T cells formed in postembryonic life in response to chronic immune responses to infectious agents or self-antigens. Although CD11c+ dendritic cells (DCs) are consistently found in regions of TLO, their contribution to TLO organization has not been studied in detail. We found that CD11chi DCs are essential for the maintenance of inducible bronchus-associated lymphoid tissue (iBALT), a form of TLO induced in the lungs after influenza virus infection. Elimination of DCs after the virus had been cleared from the lung resulted in iBALT disintegration and reduction in germinal center (GC) reactions, which led to significantly reduced numbers of class-switched plasma cells in the lung and bone marrow and reduction in protective antiviral serum immunoglobulins. Mechanistically, DCs isolated from the lungs of mice with iBALT no longer presented viral antigens to T cells but were a source of lymphotoxin (LT) β and homeostatic chemokines (CXCL-12 and -13 and CCL-19 and -21) known to contribute to TLO organization. Like depletion of DCs, blockade of LTβ receptor signaling after virus clearance led to disintegration of iBALT and GC reactions. Together, our data reveal a previously unappreciated function of lung DCs in iBALT homeostasis and humoral immunity to influenza virus.
doi:10.1084/jem.20090410
PMCID: PMC2768850  PMID: 19808255
12.  GATA-2 Plays Two Functionally Distinct Roles during the Ontogeny of Hematopoietic Stem Cells 
GATA-2 is an essential transcription factor in the hematopoietic system that is expressed in hematopoietic stem cells (HSCs) and progenitors. Complete deficiency of GATA-2 in the mouse leads to severe anemia and embryonic lethality. The role of GATA-2 and dosage effects of this transcription factor in HSC development within the embryo and adult are largely unexplored. Here we examined the effects of GATA-2 gene dosage on the generation and expansion of HSCs in several hematopoietic sites throughout mouse development. We show that a haploid dose of GATA-2 severely reduces production and expansion of HSCs specifically in the aorta-gonad-mesonephros region (which autonomously generates the first HSCs), whereas quantitative reduction of HSCs is minimal or unchanged in yolk sac, fetal liver, and adult bone marrow. However, HSCs in all these ontogenically distinct anatomical sites are qualitatively defective in serial or competitive transplantation assays. Also, cytotoxic drug-induced regeneration studies show a clear GATA-2 dose–related proliferation defect in adult bone marrow. Thus, GATA-2 plays at least two functionally distinct roles during ontogeny of HSCs: the production and expansion of HSCs in the aorta-gonad-mesonephros and the proliferation of HSCs in the adult bone marrow.
doi:10.1084/jem.20031556
PMCID: PMC2213282  PMID: 15466621
GATA-2; hematopoietic stem cells; AGM; haploinsufficiency; gene dosage
14.  Btk Is Required for an Efficient Response to Erythropoietin and for SCF-controlled Protection against TRAIL in Erythroid Progenitors 
Regulation of survival, expansion, and differentiation of erythroid progenitors requires the well-controlled activity of signaling pathways induced by erythropoietin (Epo) and stem cell factor (SCF). In addition to qualitative regulation of signaling pathways, quantitative control may be essential to control appropriate cell numbers in peripheral blood. We demonstrate that Bruton's tyrosine kinase (Btk) is able to associate with the Epo receptor (EpoR) and Jak2, and is a substrate of Jak2. Deficiency of Btk results in reduced and delayed phosphorylation of the EpoR, Jak2, and downstream signaling molecules such as Stat5 and PLCγ1 as well as in decreased responsiveness to Epo. As a result, expansion of erythroid progenitors lacking Btk is impaired at limiting concentrations of Epo and SCF. In addition, we show that SCF induces Btk to interact with TNF-related apoptosis-inducing ligand (TRAIL)–receptor 1 and that lack of Btk results in increased sensitivity to TRAIL-induced apoptosis. Together, our results indicate that Btk is a novel, quantitative regulator of Epo/SCF-dependent expansion and survival in erythropoiesis.
doi:10.1084/jem.20031109
PMCID: PMC2212722  PMID: 15007095
Jak2; Stat5; hematopoiesis; signal transduction

Results 1-14 (14)