Expression of CD1c, CD1d and Presence of Invariant NKT Cells in Hodgkin Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3659-3659
Author(s):  
Chuanhui Xu ◽  
Riemer de Vries ◽  
Lydia Visser ◽  
Arjan Diepstra ◽  
Stephan D Gadola ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Cell Suspensions ◽  
Inkt Cell ◽  
Inkt Cells ◽  
Lipid Antigens ◽  
The Mean

Abstract Abstract 3659 Poster Board III-595 Introduction Hodgkin lymphoma (HL) is a B-cell neoplasm characterized by a minority of neoplastic cells, the so-called Hodgkin and Reed-Sternberg (HRS) cells. The HRS cells are located within an extensive infiltrate of reactive cells, including T cells, B cells, plasma cells, stromal cells, eosinophils and macrophages. CD1 molecules are non-classical MHC I-like molecules that present lipid antigens to T cells, triggering a specific immune response. Of the five CD1 isoforms (CD1a, CD1b, CD1c, CD1d and CD1e) expressed in human tissue, only CD1c and CD1d are expressed in B cells. The T cell receptors (TCRs) of T cells that recognize CD1c are indistinguishable from those that recognize MHC class I or II complexes. In contrast, CD1d presents lipid antigens to invariant Natural Killer T cells (iNKT cells), which are characterized by the expression of a semi-invariant Vα24Jα18 chain plus a limited set of β chains. CD4− and CD4+ iNKT cells were demonstrated as two distinct functional subsets in terms of cytokine production and cytotoxic activation. Immunoregulatory CD1 restricted T cells have been implicated in the pathogenesis of many cancers, but its role in HL is still unknown. In this study, we analyzed the expression of CD1c, CD1d and presence of iNKT cells in HL. Methods Expression of CD1c and CD1d was determined by immunocytochemistry in four HL cell lines KMH2, L1236, L428 and U-HO1. CD1c and CD1d expression in both HRS cells and reactive cells was studied in 46 HL patient tissues by immunohistochemistry. Cell suspensions of ten HL cases were studied for the presence of iNKT cells using monoclonal antibodies against human iNKT cell (clone 6B11) and TCR Vβ11 (clone C21) by flow cytometry. The percentage of CD4+ iNKT cells was determined in the gated iNKT cell population. Results Cytoplasmic CD1d expression was found in four HL cell lines and membranous CD1d expression was found in KMH2 and L1236. CD1c expression was negative in all four HL cell lines. CD1d expression was detected in HRS cells in 46% (21/46) of the HL cases. No correlation was observed between CD1d expression and presence of EBV in HRS cells. In contrast, CD1c expression was negative in HRS cells in all HL cases. Both CD1c and CD1d were detected in reactive cells in all HL cases albeit at varying frequencies. The mean percentage of CD1d restricted iNKT cells was 4% (range 0.8-8%) in HL cell suspensions irrespective of CD1d expression status in HRS cells. In reactive lymph node (RLN) the mean percentage of iNKT cells was also 4% (range 0.4-7%). Approximately half of the iNKT cells were CD4+ in both HL and RLN cell suspensions. Conclusion Expression of CD1d was found in four HL cell lines and in HRS cells of ∼50% of the HL cases. In HL cell suspensions, about 4% (range 0.8-8%) of the reactive background cells were iNKT cells. Disclosures: No relevant conflicts of interest to declare.

Resistance to the Novel Translation Inhibitor Silvestrol Is Mediated by Elevated Mcl-1 Expression.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1737-1737
Author(s):  
David M. Lucas ◽  
Ellen J. Sass ◽  
Ryan B. Edwards ◽  
Li Pan ◽  
Gerard Lozanski ◽  
...  
Keyword(s):  
Drug Resistance ◽  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Multi Drug Resistance ◽  
Parental Line

Abstract Abstract 1737 Poster Board I-763 We previously reported the efficacy and B-cell selectivity of the natural product silvestrol in acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), using both primary cells and B-cell lines. We also showed that silvestrol inhibits translation, resulting in rapid depletion of the short half-life protein Mcl-1 followed by mitochondrial damage and apoptosis. Cencic et al. reported that silvestrol directly blocks translation initiation by aberrantly promoting interaction of eIF4A with capped mRNA (PLoS One 2009; 4(4):e5223). However, the loss of Mcl-1 in breast and prostate cancer cell lines is delayed relative to what we observe in B-leukemias (48 hr vs. 4-6 hr in CLL and ALL cells). Additionally, silvestrol does not reduce Mcl-1 expression in normal T-cells to the same extent that it does in B-cells, potentially explaining in part the relative resistance of T-cells to this agent. We therefore investigated cell-type differences, as well as the importance of Mcl-1, in silvestrol-mediated cytotoxicity. We incubated the ALL cell line 697 with gradually increasing concentrations of silvestrol to generate a cell line (697-R) with resistance to 30 nM silvestrol (IC50 of parental 697 < 5 nM). No differences between 697-R and the parental line were detected upon detailed immunophenotyping. However, cytogenetic analysis revealed a balanced 7q;9p translocation in 697-R not present in the parental 697 cell line that may be related to the emergence of a resistant clone. We also detected no difference in expression of multi-drug resistance proteins MDR-1 and MRP, which can contribute to resistance to complex amphipathic molecules such as silvestrol. In contrast, we found that baseline Mcl-1 protein expression is strikingly increased in 697-R cells relative to the parental line, although these cells still show similar percent-wise reduction in Mcl-1 upon re-exposure to 80 nM silvestrol. To investigate whether this resistance to silvestrol is reversible, 697-R cells were maintained without silvestrol for 6 weeks (∼18 passages). During this time, viability remained near 99%. Cells were then treated with 30 nM silvestrol. Viability was 94% at 48 hr post-treatment and returned to 99% within a week, while parental 697 cells with the same treatment were completely dead. Baseline Mcl-1 levels remained elevated in 697-R even with prolonged silvestrol-free incubation. These results indicate that the resistance phenotype is not rapidly reversible, as is seen with transient upregulation of multi-drug resistance or stress-response proteins. Additionally, silvestrol moderately induces the transcription of several pro-apoptotic Bcl-2 family members and results in elevated levels of these proteins despite its translation inhibitory activity. Interestingly, no such activity is detected in silvestrol-treated normal T-cells. Together, these results support the hypothesis that in B-cells, silvestrol induces cell death by altering the balance of pro- and anti-apoptotic factors, and that increased Mcl-1 protein can force the balance back toward survival. This work further underscores the importance of Mcl-1 in silvestrol-mediated cytotoxicity. We are now investigating the mechanism of Mcl-1 upregulation in 697-R cells to identify a factor or pathway that can be targeted therapeutically to circumvent resistance. Silvestrol is currently undergoing preclinical pharmacology and toxicology investigation by the U.S. National Cancer Institute Drug Development Group at the Stage IIA level to facilitate its progression to Phase I clinical testing. Disclosures No relevant conflicts of interest to declare.

Contribution of Deregulated miRNAs to the Phenotypic Characteristic of Hodgkin Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 265-265
Author(s):  
Lu Ping Tan ◽  
Bart-Jan Kroesen ◽  
Enrico Tiacci ◽  
Gerben Duns ◽  
Erwin Seinen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Mirna Expression ◽  
Conflicts Of Interest ◽  
System Development ◽  
Cell Phenotype ◽  
Immune System Development

Abstract Abstract 265 In Hodgkin Lymphoma (HL), the Hodgkin Reed-Sternberg (HRS) cells are a minority of large mono- or multi-nucleated B cells characterized by a loss of B cell phenotype, constitutive NF-kB activation, a disturbed cell cycle and anti-apoptotic features. In this study we investigated the role of deregulated miRNA expression in the pathogenesis of HL. MiRNA in situ hybridization (ISH) in HL tissue was performed to determine expression of miRNAs previously reported to be highly abundant in HL cell lines, in HRS cells. Next we identified the miRNA-targetome of two HL cell lines by immunoprecipitation of RISC in untransfected and transfected cell lines. miRNA ISH confirmed expression of miR-17-5p, miR-24, miR-106a, miR-146a, miR-150, miR-155, miR-181b and miR-210 in HRS cells. Ago2-immunoprecipitation followed by microarray analysis of the co-immunoprecipitated mRNA revealed that the miRNA-targetome of HL comprises of about 2,500 genes. Inhibition of the anti-miR-17 seed family revealed that about 500 of these genes are regulated by miRNAs of the miR-17 seed family. Gene ontology (GO) analysis for the total miRNA-targetome of HL showed a significant enrichment of genes involved in the regulation of cell cycle, apoptosis, immune system development and NF-kB cascade. The miRNA-targetome of HL contained several genes known to be mutated in HRS cells, including A20, FAS, NFKB1A, NFKB1E, PERP and SOCS1. Also, using previously reported gene expression data, we defined a set of genes downregulated in HL cell lines (L428 and L1236) compared to germinal center B cells (GCB) and compared them to the miRNA-targetome of the same cell lines. This resulted in the identification of 149 genes in L428 and 183 genes in L1236 that were subjected to miRNA mediated repression. Unexpectedly, only a few of all the reported inactivated genes in HRS cells that might contribute to loss of B cell phenotype (MYBL1 and CXCR4) were found to be regulated by miRNAs in HL. In conclusion, we confirmed the expression of miRNAs in the HRS cells of HL tissue and identified miRNA repressed genes in HL. Our data indicated that aberrant miRNA expression contributes to the deregulation of apoptosis, cell cycle, and NF-kB pathways but not loss of B cell phenotype in HL. Disclosures: No relevant conflicts of interest to declare.

CCL3 and CCL4 Plasma Levels Correlate with Established Prognostic Markers in Chronic Lymphocytic Leukemia: Towards a Simple, ELISA-Based Assay for Risk Assessment.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 358-358
Author(s):  
Mariela Sivina ◽  
Elena Hartmann ◽  
Diana Krupnik ◽  
Ruth LaPushin ◽  
Michael Keating ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Plasma Levels ◽  
Prognostic Markers ◽  
Conflicts Of Interest ◽  
High Plasma ◽  
Lymphocytic Leukemia ◽  
White Blood Count ◽  
Mutational Status ◽  
The Mean

Abstract Abstract 358 During the induction of normal immune responses, activated B cells secrete the chemokines CCL3 and CCL4 for recruitment of regulatory T cells (Nat Immunol. 2:1126-32, 2001). This may represent a mechanism enabling cognate interactions between rare antigen-specific T and B cells. We previously reported that CCL3 and CCL4 RNA and protein are induced in CLL cells by co-culture with nurselike cells and after B cell antigen receptor (BCR) cross-linking (Blood 113:3050-8, 2009). We found higher levels of CCL3 and CCL4 in ZAP-70 positive cases, and CCL3 and CCL4 secretion was abrogated by the Syk inhibitor R406. These findings suggest that CCL3/CCL4 secretion by CLL cells correlates with the signaling capacity of the BCR. Also, we previously noticed higher plasma levels of CCL3 and CCL4 in CLL patients, when compared to healthy controls. In this study, we measured CCL3 and CCL4 plasma levels by ELISA in 351 CLL patients, and correlated CCL3 and CCL4 levels with various prognostic markers, including RAI stage, immunoglobulin heavy chain variable region gene (IgVH) mutational status, ZAP-70, β2 microglobulin, CD38, white blood count (WBC), and cytogenetic subgroups. We found that the concentrations of CCL3 and CCL4 were significantly higher in plasma samples from CLL patients who had CLL cells that used unmutated IgVH genes or that expressed ZAP-70 or CD38, or who had an advanced stage of their disease (see Table 1). For example, the mean CCL3 plasma level was 56.4 ± 8.8 pg/ml in patients with CLL cells that used unmutated IgVH (mean ± SEM, n=123) versus 14.5 ± 2.4 in patients with CLL cells that used mutated IgVH (mean ± SEM, n=139, p<0.001). The mean CCL4 level was 171.4 ± 26.3 in patients that had CLL cells that used unmutated IgVH and 92.5 ± 17.5 in patients that had CLL that expressed mutated IgVH. On the other hand, the relative absolute white cell count or presence of chromosomal abnormalities did not correlate with high plasma levels of CCL3 or CCL4. Immunohistochemistry revealed that CCL4 was predominantly expressed in proliferation centers in approximately 50% of the cases. Currently, we are evaluating whether high plasma levels of CCL3 and/or CCL4 are associated with a relatively short time from diagnosis to requiring initial treatment by the IWCLL-working group criteria. Also, we are exploring whether high plasma levels of CCL3 or CCL4 are associated with high proportions of infiltrating T cells in proliferation centers. These studies suggest that patients can be stratified by their relative plasma levels of CCL3 and/or CCL4 and that high plasma-levels of theses chemokines define a characteristic that may be associated with aggressive disease. Disclosures: No relevant conflicts of interest to declare.

B-Cell Receptors Of Follicular Lymphoma Patients Recognize Themselves

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 633-633 ◽  
Author(s):  
Angela Schulz ◽  
Debra K Czerwinski ◽  
Ronald Levy
Keyword(s):  
T Cells ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Lines ◽  
Protein Modification ◽  
Conflicts Of Interest ◽  
Fc Receptors ◽  
Fc Receptor ◽  
Surface Binding

Abstract Follicular Lymphoma (FL) is the most common indolent lymphoma and is characterized by retained surface B-cell receptor (BCR) expression despite ongoing V region somatic mutation. Furthermore, every tumor has a unique BCR. Together, this suggests that the BCR is important for the survival of the malignant B cell. Recognition of a target antigen could lead to a constant stimulation of the malignant cells and serve as a driving force. Recombinant BCRs from a series of FL patients were expressed in the form of assembled proteins and all of IgG3 isotype. Recently we reported that 25% of these BCRs have binding activity against a human epithelial cell line1. In the current study we aimed to broaden the search for putative auto-antigens. As FL cells are in close contact with various peripheral blood cells (PBMC), an epitope on the surface of these cells might serve as a putative auto-antigen. Therefore, we incubated PBMCs from healthy donors with the lymphoma derived BCRs and quantified surface binding by FACS. We gated separately on B-cells, CD4 T-cells, CD8 T-cells, monocytes and natural killer (NK) cells. Thereby, we identified 7 out of 25 tumor-derived BCRs which bound to at least one cell type. Four of these strongly bound to NK, B-cells and monocytes from multiple different donors. None of the tested antibodies bound to T-cells. We tested the four reactive BCRs against B-cell lines (Daudi, Raji, Ramos, DHL-4, FL-18 and Reh) and one T-cell line (MOLT-4). We found that Daudi and to a lesser extent Raji but none of the other cell lines were positive for the same lymphoma derived BCRs as PBMCs. Because of the observed binding pattern we hypothesized that a common protein modification might be the target. We therefore treated the cell lines with tunicamycin, an inhibitor for N-linked glycosylation, in order to test if sugars might be the targets of the lymphoma derived BCRs. Surprisingly, this led to stronger binding of the same BCRs. it is known that de-glycosylation of Fc receptors increases Fc binding, suggesting that Fc receptors might be the target. In line with this hypothesis Daudi cells have Fc receptors but Ramos cells do not. Moreover, T-cells are the only PBMCs which do not express Fc receptors. Therefore, we tested Fc receptors directly as targets. Recombinant Fcγ1 (CD64) and Fcγ3 (CD16) were positive in ELISA tests with the four recombinant lymphoma-derived BCRs. In addition, blocking Fcγ2 (CD32) and Fcγ3 receptors on PBMCs before staining with lymphoma derived BCRs resulted in a signal reduction. In order to define if the Fc part of these BCRs is passively bound by Fc receptors or if the Fc receptor is recognized as specific target by the variable BCR regions we produced F(ab)2 fragments. Staining PBMCs with these abolished binding, suggesting that the Fc region of the lymphoma derived BCRs is bound necessary for cellular binding. As all BCRs have the same IgG3 constant region they should all bind to Fc receptors through their Fc regions with the same affinity. In vivo Fc receptor bearing cells are not activated by the binding of a single antibody but only when an antibody cluster is formed e.g. the antibody-coated surface of a pathogen. We therefore hypothesized that the BCRs which bound Fc receptors exist as clusters. This would be conceivable if the BCRs recognized a part of them-selves as target which subsequently would lead to dimer or oligomerization. Indeed, a Fc binding BCR which was coated on an ELISA plate could be detected with a biotinylated counterpart of itself. This same phenomenon was recently described to be the case for chronic lymphocytic leukemia2. Experiments are ongoing to elucidate which region of the BCRs are recognized as targets and for which percentage of them for which this is true. In conclusion these findings suggest a new target for the BCR of FL cells whose constant presence in its microenvironment might represent a novel mechanism of chronic BCR stimulation. 1Sachen KL et al., Blood 2012, 2Dühren von Minden M et al., Nature 2012 Disclosures: No relevant conflicts of interest to declare.

scholarly journals Cognate interaction with iNKT cells expands IL-10–producing B regulatory cells

2015 ◽  
Vol 112 (40) ◽  
pp. 12474-12479 ◽  
Author(s):  
Emilie E. Vomhof-DeKrey ◽  
Jennifer Yates ◽  
Thomas Hägglöf ◽  
Paula Lanthier ◽  
Eyal Amiel ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Plasma Cells ◽  
Inkt Cell ◽  
Vaccine Adjuvants ◽  
Regulatory Cells ◽  
Inkt Cells ◽  
Marginal Zone B Cells

Successful induction of B-cell activation and memory depends on help from CD4+ T cells. Invariant natural killer T (iNKT) cells (glycolipid-specific, CD1d-restricted innate lymphocytes) provide both cognate (direct) and noncognate (indirect) helper signals to enhance B-cell responses. Both forms of iNKT-cell help induce primary humoral immune responses, but only noncognate iNKT-cell help drives humoral memory and plasma cells. Here, we show that iNKT cognate help for B cells is fundamentally different from the help provided by conventional CD4+ T cells. Cognate iNKT-cell help drives an early, unsustained germinal center B-cell expansion, less reduction of T follicular regulatory cells, an expansion of marginal zone B cells, and early increases in regulatory IL-10–producing B-cell numbers compared with noncognate activation. These results are consistent with a mechanism whereby iNKT cells preferentially provide an innate form of help that does not generate humoral memory and has important implications for the application of glycolipid molecules as vaccine adjuvants.

scholarly journals Innate and cytokine-driven signals, rather than microbial antigens, dominate in natural killer T cell activation during microbial infection

2011 ◽  
Vol 208 (6) ◽  
pp. 1163-1177 ◽  
Author(s):  
Manfred Brigl ◽  
Raju V.V. Tatituri ◽  
Gerald F.M. Watts ◽  
Veemal Bhowruth ◽  
Elizabeth A. Leadbetter ◽  
...  
Keyword(s):  
T Cells ◽  
Natural Killer ◽  
T Cell Activation ◽  
Cell Activation ◽  
Constitutive Expression ◽  
Microbial Pathogens ◽  
Inkt Cell ◽  
Microbial Infection ◽  
Inkt Cells ◽  
Natural Killer T

Invariant natural killer T cells (iNKT cells) are critical for host defense against a variety of microbial pathogens. However, the central question of how iNKT cells are activated by microbes has not been fully explained. The example of adaptive MHC-restricted T cells, studies using synthetic pharmacological α-galactosylceramides, and the recent discovery of microbial iNKT cell ligands have all suggested that recognition of foreign lipid antigens is the main driver for iNKT cell activation during infection. However, when we compared the role of microbial antigens versus innate cytokine-driven mechanisms, we found that iNKT cell interferon-γ production after in vitro stimulation or infection with diverse bacteria overwhelmingly depended on toll-like receptor–driven IL-12. Importantly, activation of iNKT cells in vivo during infection with Sphingomonas yanoikuyae or Streptococcus pneumoniae, pathogens which are known to express iNKT cell antigens and which require iNKT cells for effective protection, also predominantly depended on IL-12. Constitutive expression of high levels of IL-12 receptor by iNKT cells enabled instant IL-12–induced STAT4 activation, demonstrating that among T cells, iNKT cells are uniquely equipped for immediate, cytokine-driven activation. These findings reveal that innate and cytokine-driven signals, rather than cognate microbial antigen, dominate in iNKT cell activation during microbial infections.

scholarly journals HDAC11 plays an essential role in regulating OX40 ligand expression in Hodgkin lymphoma

Blood ◽  
2011 ◽  
Vol 117 (10) ◽  
pp. 2910-2917 ◽  
Author(s):  
Daniela Buglio ◽  
Noor M. Khaskhely ◽  
Kui Shin Voo ◽  
Hector Martinez-Valdez ◽  
Yong-Jun Liu ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Essential Role ◽  
Histone Deacetylase Inhibitors ◽  
Interleukin 10 ◽  
Antitumor Immune Response ◽  
Dependent Manner ◽  
Small Interfering Rnas

AbstractIn Hodgkin lymphoma (HL), the malignant cells are surrounded by a large number of reactive infiltrating inflammatory cells, including OX40-expressing T cells and interleukin 10 (IL-10)–producing regulatory T (T-reg) cells. These T-reg cells can suppress the immune response and thus contribute to the maintenance of immune tolerance and to insufficient antitumor response. The engagement of OX40L with the OX40 receptor is essential for the generation of antigen-specific memory T cells and for the induction of host antitumor immunity. In the present study, we investigated whether histone deacetylase inhibitors (HDACis) may induce a favorable antitumor immune response by regulating the expression of OX40L in HL. We found that HDACis up-regulated OX40L surface expression in HL cell lines in a dose-dependent manner. Small interfering RNAs (siRNAs) that selectively inhibited HDAC11 expression, significantly up-regulated OX40L and induced apoptosis in HL cell lines, and silencing HDAC11 transcripts increased the production of tumor necrosis-α (TNF-α) and IL-17 in the supernatants of HL cells. Furthermore, HDACI-induced OX40L inhibited the generation of IL-10–producing type 1 T-reg cells. These results demonstrate for the first time that HDAC11 plays an essential role in regulating OX40L expression. Pharmacologic inhibition of HDAC11 may produce a favorable antitumor immune response in patients with HL.

scholarly journals MicroRNA High Throughput Loss-of-Function Screening Reveals an Oncogenic Role for miR-21-5p in Hodgkin Lymphoma

2018 ◽  
Vol 49 (1) ◽  
pp. 144-159 ◽  
Author(s):  
Ye Yuan ◽  
Fubiao Niu ◽  
Ilja M. Nolte ◽  
Jasper Koerts ◽  
Debora de Jong ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Growth ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Western Blotting ◽  
High Throughput ◽  
Annexin V ◽  
Loss Of Function ◽  
Empty Vector ◽  
Competition Assays

Background/Aims: Classical Hodgkin lymphoma (cHL) is among the most frequent lymphoma subtypes. The tumor cells originate from crippled germinal center (GC)-B cells that escaped from apoptosis. MicroRNAs (miRNAs) play important roles in B-cell maturation and aberrant expression of miRNAs contributes to the pathogenesis of cHL. Our aim was to identify oncogenic miRNAs relevant for growth of cHL using a high-throughput screening approach. Methods: A lentiviral pool of 63 miRNA inhibition constructs was used to identify miRNAs essential to cell growth in three cHL cell lines in duplicate. As a negative control we also infected cHL cell lines with a lentiviral barcoded empty vector pool consisting of 222 constructs. The abundance of individual constructs was followed over time by a next generation sequencing approach. The effect on growth was confirmed using individual GFP competition assays and on apoptosis using Annexin-V staining. Our previously published Argonaute 2 (Ago2) immunoprecipitation (IP) data were used to identify target genes relevant for cell growth / apoptosis. Luciferase assays and western blotting were performed to confirm targeting by miRNAs. Results: Four miRNA inhibition constructs, i.e. miR-449a-5p, miR-625-5p, let-7f-2-3p and miR-21-5p, showed a significant decrease in abundance in at least 4 of 6 infections. In contrast, none of the empty vector constructs showed a significant decrease in abundance in 3 or more of the 6 infections. The most abundantly expressed miRNA, i.e. miR-21-5p, showed significantly higher expression levels in cHL compared to GC-B cells. GFP competition assays confirmed the negative effect of miR-21-5p inhibition on HL cell growth. Annexin-V staining of cells infected with miR-21-5p inhibitor indicated a significant increase in apoptosis at day 7 and 9 after viral infection, consistent with the decrease in growth. Four miR-21-5p cell growth- and apoptosis-associated targets were AGO2-IP enriched in cHL cell lines and showed a significant decrease in expression in cHL cell lines in comparison to normal GC-B cells. For the two most abundantly expressed, i.e. BTG2 and PELI1, we confirmed targeting by miR-21-5p using luciferase assays and for PELI1 we also confirmed this at the protein level by western blotting. Conclusion: Using a miRNA loss-of-function high-throughput screen we identified four miRNAs with oncogenic effects in cHL and validated the results for the in cHL abundantly expressed miR-21-5p. MiR-21-5p is upregulated in cHL compared to GC-B cells and protects cHL cells from apoptosis possibly via targeting BTG2 and PELI1.

scholarly journals New insights in the biology of Hodgkin lymphoma

Hematology ◽  
2012 ◽  
Vol 2012 (1) ◽  
pp. 328-334 ◽  
Author(s):  
Ralf Küppers
Keyword(s):  
T Cells ◽  
B Cells ◽  
Signaling Pathways ◽  
Hodgkin Lymphoma ◽  
Tumor Cells ◽  
Cytotoxic T Cells ◽  
Cell Gene Expression ◽  
Expression Program ◽  
Multiple Signaling ◽  
Cell Gene

Abstract The Hodgkin and Reed/Sternberg (HRS) tumor cells of classical Hodgkin lymphoma (HL) and the lymphocyte-predominant tumor cells of nodular lymphocyte–predominant HL are both derived from germinal center B cells. HRS cells, however, have largely lost their B-cell gene-expression program and coexpress genes typical of various types of hematopoietic cells. Multiple signaling pathways show a deregulated activity in HRS cells. The genetic lesions involved in the pathogenesis of HL are only partly known, but numerous members and regulators of the NF-κB and JAK/STAT signaling pathways are affected, suggesting an important role for these pathways in HL pathogenesis. Some genetic lesions involve epigenetic regulators, and there is emerging evidence that HRS cells have undergone extensive epigenetic alterations compared with normal B cells. HRS and lymphocyte-predominant cells are usually rare in the lymphoma tissue, and interactions with other cells in the microenvironment are likely critical for HL pathophysiology. T cells represent a main population of infiltrating cells, and it appears that HRS cells both inhibit cytotoxic T cells efficiently and also receive survival signals from Th cells in direct contact with them.

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