Tjasso Blokzijl

BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas.

In a previous study we demonstrated high expression of the non‐coding BIC gene in the vast majority of Hodgkins lymphomas (HLs). Evidence suggesting that BIC is a primary microRNA transcript containing the mature microRNA‐155 (miR‐155) as part of a RNA hairpin is now accumulating. We therefore analysed HL cell lines and tissue samples to determine whether miR‐155 is also expressed in HL. High levels of miR‐155 could be demonstrated, indicating that BIC is processed into a microRNA in HL. Most non‐HL subtypes were negative for BIC as determined by RNA‐ISH. However, in diffuse large B cell lymphoma (DLBCL) and primary mediastinal B cell lymphoma (PMBL), significant percentages of positive tumour cells were observed in 12/18 and 8/8 cases. A higher proportion of tumour cells were positive for BIC in DLBCL with activated B cell‐like phenotype than in DLBCL with germinal centre B cell‐like phenotype. Differential BIC expression was confirmed by qRT‐PCR analysis. Northern blot analysis showed expression of miR‐155 in all DLBCL and PMBL derived cell lines and tissue samples analysed. In summary, we demonstrate expression of primary microRNA BIC and its derivative miR‐155 in HL, PMBL and DLBCL. Copyright

High expression of B-cell receptor inducible gene BIC in all subtypes of Hodgkin lymphoma.

In a search for genes specifically expressed in Reed–Sternberg (RS) cells of Hodgkin lymphoma (HL), we applied the serial analysis of gene expression (SAGE) technique on the HL‐derived cell line DEV. Genes highly expressed in DEV were subjected to an RT‐PCR analysis to confirm the SAGE results. For one of the genes, a high expression was observed in DEV and other HL‐derived cell lines but not in non‐Hodgkin lymphoma (NHL)–derived cell lines and normal controls, suggesting an HL‐specific expression. This gene corresponds to the human BIC gene, a member of the noncoding mRNA‐like molecules. RNA in situ hybridization (ISH) indicated an exclusive nucleolar localization of BIC transcripts in all RS cells in 91% of HL cases, including nodular lymphocyte predominance (NLP) HL and classical HL. Analyses of normal human tissues revealed BIC transcripts in only a small number of CD20‐positive B‐cells in lymph node and tonsil tissue, albeit at a much lower level compared to that of RS cells. BIC RT‐PCR in the Burkitt lymphoma–derived cell line Ramos demonstrated a significant up‐regulation upon cross‐linking of the B‐cell receptor (BcR). IκBα‐mediated blocking of NF‐κB translocation in Ramos did not effect the up‐regulation of BIC expression upon BcR triggering, suggesting that activation of NF‐κB is not involved in regulation of BIC expression. In summary, our data show that expression of BIC is specific for RS cells of HL. In normal tissue, BIC is expressed weakly in a minority of germinal center B cells. Expression of BIC can be modified/influenced by BcR triggering, indicating that BIC might play a role in the selection of B cells.

Lack of BIC and microRNA miR-155 expression in primary cases of Burkitt lymphoma.

We previously demonstrated high expression of primary‐microRNA BIC (pri‐miR‐155) in Hodgkin lymphoma (HL) and lack of expression in most non‐Hodgkin lymphoma subtypes including some Burkitt lymphoma (BL) cases. Recently, high expression of BIC was reported in BL in comparison to pediatric leukemia and normal peripheral‐blood samples. In this study, we extended our series of BL cases and cell lines to examine expression of BIC using RNA in situ hybridization (ISH) and quantitative RT‐PCR (qRT‐PCR) and of miR‐155 using Northern blotting. Both BIC RNA ISH and qRT‐PCR revealed no or low levels of BIC in 25 BL tissue samples [including 7 Epstein–Barr virus (EBV)–positive cases] compared to HL and normal controls. In agreement with these findings, no miR‐155 was observed in BL tissues. EBV‐negative and EBV latency type I BL cell lines also showed very low BIC and miR‐155 expression levels as compared to HL cell lines. Higher levels of BIC and miR‐155 were detected in in vitro transformed lymphoblastoid EBV latency type III BL cell lines. An association of latency type III infection and induction of BIC was supported by consistent expression of BIC in 11 and miR‐155 in 2 posttransplantation lymphoproliferative disorder (PTLD) cases. In summary, we demonstrated that expression of BIC and miR‐155 is not a common finding in BL. Expression of BIC and miR‐155 in 3 latency type III EBV–positive BL cell lines and in all primary PTLD cases suggests a possible role for EBV latency type III specific proteins in the induction of BIC expression.

Coexpression of BMI-1 and EZH2 Polycomb Group Genes in Reed-Sternberg Cells of Hodgkin’s Disease

The human BMI-1 and EZH2 polycomb group (PcG) proteins are constituents of two distinct complexes of PcG proteins with gene regulatory activity. PcG proteins ensure correct embryonic development by suppressing homeobox genes, and they also contribute to regulation of lymphopoiesis. The two PcG complexes are thought to regulate different target genes and probably have different tissue distributions. Altered expression of PcG genes is linked to transformation in cell lines and induction of tumors in mutant mice, but the role of PcG genes in human cancers is relatively unexplored. Using antisera specific for human PcG proteins, we used immunohistochemistry and immunofluorescence to detect BMI-1 and EZH2 PcG proteins in Reed-Sternberg cells of Hodgkins disease (HRS). The expression patterns were compared to those in follicular lymphocytes of the lymph node, the normal counterparts of HRS cells. In the germinal center, expression of BMI-1 is restricted to resting Mib-1/Ki-67(-) centrocytes, whereas EZH2 expression is associated with dividing Mib-1/Ki-67(+) centroblasts. By contrast, HRS cells coexpress BMI-1, EZH2, and Mib-1/Ki-67. Because HRS cells are thought to originate from germinal center lymphocytes, these observations suggests that Hodgkins disease is associated with coexpression of BMI-1 and EZH2 in HRS cells.

miRNA analysis in B-cell chronic lymphocytic leukaemia: proliferation centres characterized by low miR-150 and high BIC/miR-155 expression.

Several miRNAs have been reported to be associated with immunoglobulin heavy chain (IgH) mutation and ZAP‐70 expression status in blood samples of B‐cell chronic lymphocytic leukaemia/small lymphocytic lymphoma (B‐CLL/SLL). In the bone marrow and lymphoid tissues, proliferation centres (PCs) represent an important site of activation and proliferation of the neoplastic cells, suggesting that these tissues better reflect the biology of CLL than circulating blood cells. We collected 33 lymph nodes and 37 blood CLL samples and analysed IgH mutation status and ZAP‐70 expression status. Expression of 15 miRNAs was analysed by qRT‐PCR and RNA‐ISH. Sixty‐three per cent of the lymph node cases contained mutated IgH genes and 49% of the lymph node cases were ZAP‐70‐positive, and a significant correlation was observed between ZAP‐70 expression and IgH mutation status. Of the blood CLL samples, 49% contained mutated IgH sequences. The miRNA expression pattern in CLL lymph node and blood samples was very similar. Three of 15 miRNAs (miR‐16, miR‐21, and miR‐150) showed a high expression level in both blood and lymph node samples. No difference was observed between ZAP‐70‐positive or ‐negative and between IgH‐mutated or unmutated cases. No correlation was found between miR‐15a and miR‐16 expression levels and 13q14 deletion in the blood CLL samples. RNA in situ hybridization (ISH) revealed strong homogeneous staining of miR‐150 in the tumour cells outside the PCs. In reverse BIC/pri‐miR‐155 expression was observed mainly in individual cells including prolymphocytes of the PCs. This reciprocal pattern likely reflects the different functions and targets of miR‐150 and miR‐155. Copyright

Dimeric galectin-1 induces IL-10 production in T-lymphocytes: an important tool in the regulation of the immune response

Galectin‐1, a β‐galactoside binding protein that can occur as both a monomer and a homodimer, binds to leucocyte membrane antigens such as CD7, CD43, and CD45, and has immune‐regulatory functions in several animal models of autoimmune disease. However, its mechanism of action is only partially understood. In this study, a marked increase in IL‐10 mRNA and protein levels was demonstrated in non‐activated and activated CD4+ and CD8+ T‐cells, following treatment with a high concentration (dimeric form), but not a low concentration (monomeric form), of recombinant galectin‐1 protein. IL‐10 is known to suppress TH1 type immune responses and upregulation of IL‐10 may thus contribute to the immune‐regulatory function of galectin‐1. Galectin‐1 was strongly expressed on the endothelial cells of human kidney allografts, suggesting a role in the regulation of immune responses in transplantation. Administration of high concentrations of galectin‐1 may be a useful tool in the treatment of T‐cell‐mediated diseases. Copyright

Distinct BMI-1 and EZH2 Expression Patterns in Thymocytes and Mature T Cells Suggest a Role for Polycomb Genes in Human T Cell Differentiation

BMI-1 and EZH2 Polycomb-group (PcG) proteins belong to two distinct protein complexes involved in the regulation of hematopoiesis. Using unique PcG-specific antisera and triple immunofluorescence, we found that mature resting peripheral T cells expressed BMI-1, whereas dividing blasts were EZH2+. By contrast, subcapsular immature double-negative (DN) (CD4−/CD8−) T cells in the thymus coexpressed BMI-1 and EZH2 or were BMI-1 single positive. Their descendants, double-positive (DP; CD4+/CD8+) cortical thymocytes, expressed EZH2 without BMI-1. Most EZH2+ DN and DP thymocytes were dividing, while DN BMI-1+/EZH2− thymocytes were resting and proliferation was occasionally noted in DN BMI-1+/EZH2+ cells. Maturation of DP cortical thymocytes to single-positive (CD4+/CD8− or CD8+/CD4−) medullar thymocytes correlated with decreased detectability of EZH2 and continued relative absence of BMI-1. Our data show that BMI-1 and EZH2 expression in mature peripheral T cells is mutually exclusive and linked to proliferation status, and that this pattern is not yet established in thymocytes of the cortex and medulla. T cell stage-specific PcG expression profiles suggest that PcG genes contribute to regulation of T cell differentiation. They probably reflect stabilization of cell type-specific gene expression and irreversibility of lineage choice. The difference in PcG expression between medullar thymocytes and mature interfollicular T cells indicates that additional maturation processes occur after thymocyte transportation from the thymus.

Specific expression of miR-17-5p and miR-127 in testicular and central nervous system diffuse large B-cell lymphoma.

Recent studies have shown that certain non-coding short RNAs, called miRNAs, play an important role in diffuse large B-cell lymphomas. Patients with diffuse large B-cell lymphoma have great diversity in both clinical characteristics, site of presentation and outcome. The aim of our study is to validate the differential expression in germinal center and non-germinal center diffuse large B-cell lymphoma,s and to study to the extent to which the primary site of differentiation is associated with the miRNA expression profile. We studied 50 cases of de novo diffuse large B-cell lymphoma for the expression of 15 miRNAs (miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-92, miR-127, miR-155, miR-181a and miR-221). Apart from 19 nodal cases without extranodal dissemination (stages I and II), we selected two groups with unambiguous stages I and II extranodal presentation; 9 cases of primary central nervous system, 11 cases of primary testicular and 11 cases of other primary extranodal diffuse large B-cell lymphomas. All cases were analyzed with qRT-PCR. In situ hybridization for the most differentially expressed miRNAs was performed to show miRNA expression in tumor cells, but not in background cells. MiR-21 and miR-19b showed the highest expression levels. No significant differences were seen between germinal center and non-germinal center diffuse large B-cell lymphomas in either the total or the nodal group for any of the 15 miRNAs. Two miRNAs showed significant differences in expression levels for diffuse large B-cell lymphoma subgroups according to the site of presentation. MiR-17-5p showed a significant higher expression level in the central nervous system compared with testicular and nodal diffuse large B-cell lymphomas (P<0.05). MiR-127 levels were significantly higher in testicular than in central nervous system and in nodal diffuse large B-cell lymphomas (P<0.05). We conclude that the location of diffuse large B-cell lymphoma is an important factor in determining the differential expression of miRNAs.

Expression of the T-cell transcription factors, GATA-3 and T-bet, in the neoplastic cells of Hodgkin lymphomas

Since Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL) generally have immunoglobulin gene rearrangements, they are considered to be of B-cell origin. One of the characteristics of HRS cells is a prominent production of various cytokines and chemokines. Cytokine production is generally driven by expression of T-cell transcription factors (TFs). Only limited information is available on the expression of T-cell TFs in HL. Expression of four T-cell TFs and the target cytokine spectrum of these TFs were analyzed in six HL and three large B-cell lymphoma (LBCL) cell lines using quantitative PCR. ERM expression was observed in all HL and LBCL cell lines. Out of HL cell lines, T-bet was expressed in five, GATA-3 in four, and c-Maf in two cell lines. Immunohistochemistry in HL tissues revealed that in 11 of 12 (92%) of the classical HL cases HRS cells were GATA-3 and/or T-bet positive. In three of six cases of nodular lymphocyte predominance type of HL, the neoplastic cells were T-bet positive. Overall, the T-cell TF and cytokine profiles of the HL cell lines showed a considerable degree of consistency. The expression of T-cell TFs may explain the production of various cytokines by HL cell lines and HRS cells.

The CD4+CD26-T-cell population in classical Hodgkin's lymphoma displays a distinctive regulatory T-cell profile

Little is known about the gene expression profile and significance of the rosetting CD4+CD26− T cells in classical Hodgkins lymphoma (cHL). To characterize these T cells, CD4+CD26− and CD4+CD26+ T-cell populations were sorted from lymph node (LN) cell suspensions from nodular sclerosis HL (NSHL) and reactive LNs. mRNA profiles of stimulated and resting cell subsets were evaluated with quantitative RT-PCR for 46 genes. We observed a higher percentage of CD4+CD26− T cells in NSHL than in reactive LNs. The resting CD4+CD26− T cells in NSHL showed higher mRNA levels of CD25, CTLA4, OX40 and CCR4 compared with in LNs, supporting a regulatory T-cell (Treg) type, and this was validated by immunohistochemistry. Moreover, these cells showed low or no expression of the Th1- or Th2-related cytokines IL-2, IFN-γ, IL-13, IL-12B, IL-4, and IL-5, and the chemoattractant receptor CRTH2. Besides Tregs, Th17 cells may exist in NSHL based on the significantly higher IL-17 mRNA level for both T-cell populations in NSHL. Upon stimulation in vitro, lack of upregulation of mRNA levels of most cytokine genes indicated an anergic character for the CD4+CD26− T-cell subset. Anergy fits with the Treg profile of these cells, probably explaining the immunosuppressive mechanism involved in NSHL.