Dieter Kube

Mapping of the human IL10 gene and further characterization of the 5' flanking sequence.

Abstract Interleukin-10 (IL-10) is an important regulatory cytokine whose involvement extends into diverse areas of the human immune system. Recent characterization of the promoter and 5’ flanking regions has demonstrated the presence of positive and negative regulatory segments in the promoter. In addition, the characterization of two dinucleotide repeat elements immediately upstream of the gene has shown that there is considerable polymorphism directly associated with the human IL10 gene. In the present report, we describe the mapping of the human IL10 gene to a discrete area of chromosome 1, the definition of a potential cytokine-responsive segment 3 – 4 kilobases upstream of the transcription initiation site, and the identification of two new point mutations in the immediate promoter region with their distribution in a panel of 75 unrelated healthy individuals. These data should further the understanding of how the IL10 gene is controlled in humans and how its function may vary between individuals.

Recurrent mutation of the ID3 gene in Burkitt lymphoma identified by integrated genome, exome and transcriptome sequencing

Burkitt lymphoma is a mature aggressive B-cell lymphoma derived from germinal center B cells. Its cytogenetic hallmark is the Burkitt translocation t(8;14)(q24;q32) and its variants, which juxtapose the MYC oncogene with one of the three immunoglobulin loci. Consequently, MYC is deregulated, resulting in massive perturbation of gene expression. Nevertheless, MYC deregulation alone seems not to be sufficient to drive Burkitt lymphomagenesis. By whole-genome, whole-exome and transcriptome sequencing of four prototypical Burkitt lymphomas with immunoglobulin gene (IG)-MYC translocation, we identified seven recurrently mutated genes. One of these genes, ID3, mapped to a region of focal homozygous loss in Burkitt lymphoma. In an extended cohort, 36 of 53 molecularly defined Burkitt lymphomas (68%) carried potentially damaging mutations of ID3. These were strongly enriched at somatic hypermutation motifs. Only 6 of 47 other B-cell lymphomas with the IG-MYC translocation (13%) carried ID3 mutations. These findings suggest that cooperation between ID3 inactivation and IG-MYC translocation is a hallmark of Burkitt lymphomagenesis.

The Epstein-Barr virus oncoprotein, latent membrane protein-1, reprograms germinal centre B cells towards a Hodgkin's Reed-Sternberg-like phenotype

Although the latent membrane protein‐1 (LMP1) of the Epstein–Barr virus (EBV) is believed to be important for the transformation of germinal centre (GC) B cells, the precise contribution of this viral oncogene to lymphoma development is poorly understood. In this study, we used a non‐viral vector‐based method to express LMP1 in primary human GC B cells. Gene expression profiling revealed that LMP1 induced in GC B cells transcriptional changes characteristic of Hodgkins lymphoma cell lines. Strikingly, LMP1 down‐regulated the expression of B‐cell‐specific genes including B‐cell receptor components such as CD79A, CD79B, CD19, CD20, CD22, and BLNK. LMP1 also induced the expression of ID2, a negative regulator of B‐cell differentiation. Our data suggest that in EBV‐positive cases, LMP1 is likely to be a major contributor to the altered transcriptional pattern characteristic of Hodgkin/Reed–Sternberg cells, including the loss of B‐cell identity. Copyright

Mosaics of gene variations in the Interleukin-10 gene promoter affect interleukin-10 production depending on the stimulation used

Interleukin-10 (IL-10), a cytokine involved in many aspects of the immune response shows interindividual variations in their expression. However, genetic variations of the 5′-flanking region of the IL-10 gene (PIL-10) are poorly characterised with respect to different stimuli. New extended haplo- and genotypes are identified present at differing frequencies in three geographically separated populations. Their influence on IL-10 expression have been assessed in vitro after stimulation of leukocytes with lipopolysaccharide (LPS), dibutyryl-cAMP or following immortalisation with Epstein–Barr virus (lymphoblastoid cell line (LCL)). Interindividual differences of IL-10 production were found to be related to single-nucleotide polymorphisms (SNP) haplotype −6752/–6208 in LCLs (P<0.02), and for haplotypes comprising SNPs –6752/-6208/-3538 after LPS stimulation (P<0.03). Carriers of the IL10.G microsatellite with 22, 24 or 26 dinucleotide repeats linked with the −1087G SNP, exhibited the highest levels of IL-10 expression. Contrasting IL-10 secretion patterns were found for IL10.R microsatellite alleles characterised by 15 dinucleotide repeats: after LPS stimulation this allele was associated with high IL-10 production (P<0.007), but with low IL-10 levels in LCLs (P< 0.038). Thus, the effects of mosaics of genetic elements in the PIL-10 on the capacity of leukocytes to produce IL-10 depend on the agent inducing IL-10 expression.

Structural characterisation of the distal 5' flanking region of the human interleukin-10 gene.

Interleukin-10 (IL-10) is an important immunoregulatory cytokine. The recent characterisation of the proximal 5′ flanking region of IL-10 led to the identification of the promoter region. Two polymorphic dinucleotide repeats and 10 single nucleotide polymorphisms (SNPs) have been identified and suggested to be useful genetic markers in several diseases. We have sequenced a further 5275 bp from −9296 to −4021 of the distal part of the 5′ flanking region of the human IL-10 gene from the cosmid clone pWE15-4/11. Our sequence analysis reveals a high density of Alu-repeats within the IL-10 gene locus, including three novel, related structures which we term Alu-IL10 (A-C). Using three overlapping PCR products spanning 5110 bp of this distal part of the IL-10 gene the following single base pair substitutions were identified: at −8571 C/T , −8531 G/A , −6752 A/T , −6208 G/C, −5402 C/G. In addition a heterozygous three base pair deletion at −7400 was observed. The SNPs at −8571 C/T and −8531 G/A are contained within an Alu-repeat. These data should further the understanding of how the IL-10 gene is controlled in man and how its function may vary between individuals.

Down-regulation of BLIMP1α by the EBV oncogene, LMP-1, disrupts the plasma cell differentiation program and prevents viral replication in B cells: implications for the pathogenesis of EBV-associated B-cell lymphomas

An important pathogenic event in Epstein-Barr virus (EBV)-associated lymphomas is the suppression of virus replication, which would otherwise lead to cell death. Because virus replication in B cells is intimately linked to their differentiation toward plasma cells, we asked whether the physiologic signals that drive normal B-cell differentiation are absent in EBV-transformed cells. We focused on BLIMP1α, a transcription factor that is required for plasma cell differentiation and that is inactivated in diffuse large B-cell lymphomas. We show that BLIMP1α expression is down-regulated after EBV infection of primary germinal center B cells and that the EBV oncogene, latent membrane protein-1 (LMP-1), is alone capable of inducing this down-regulation in these cells. Furthermore, the down-regulation of BLIMP1α by LMP-1 was accompanied by a partial disruption of the BLIMP1α transcriptional program, including the aberrant induction of MYC, the repression of which is required for terminal differentiation. Finally, we show that the ectopic expression of BLIMP1α in EBV-transformed cells can induce the viral lytic cycle. Our results suggest that LMP-1 expression in progenitor germinal center B cells could contribute to the pathogenesis of EBV-associated lymphomas by down-regulating BLIMP1α, in turn preventing plasma cell differentiation and induction of the viral lytic cycle.

The Epstein-Barr virus oncoprotein latent membrane protein 1 induces expression of the chemokine IP-10: Importance of mRNA half-life regulation

The latent membrane protein 1 (LMP1) of Epstein‐Barr Virus (EBV) is the main inducer of immuno‐modulatory molecules affecting growth and survival of EBV‐infected cells. However, the network of signalling pathways involved remains to be elucidated. Here we show that LMP1 may regulate cellular genes like IFN‐γ‐inducible protein‐10 kDa (IP‐10) not only through transcriptional but also post‐transcriptional mechanisms. LMP1‐mediated IP‐10 expression is independent from IFN‐γ, TNF‐α or IL‐18. Transcriptional activation of IP‐10 by LMP1 or CD40 stimulation depends on an NF‐κB motif within the proximal 435 bp fragment. Carboxy‐terminal activating regions 1 or 2 of LMP1 are sufficient to direct IP‐10 promoter activation. IP‐10 induction is inhibited by blockade of p38/SAPK2 with SB 202190, which results in decreased IP‐10 mRNA half‐life without affecting IP‐10 promoter activity. Thus, LMP1‐mediated p38/SAPK2 activation regulates transcript stability. This new mechanism of gene regulation demonstrates the potential of the oncoprotein LMP1 to orchestrate a network of signalling pathways at different regulatory levels including mRNA stability.

Epstein-Barr Virus Infection of Naïve B Cells In Vitro Frequently Selects Clones with Mutated Immunoglobulin Genotypes: Implications for Virus Biology

Epstein-Barr virus (EBV), a lymphomagenic human herpesvirus, colonises the host through polyclonal B cell-growth-transforming infections yet establishes persistence only in IgD+ CD27+ non-switched memory (NSM) and IgD− CD27+ switched memory (SM) B cells, not in IgD+ CD27− naïve (N) cells. How this selectivity is achieved remains poorly understood. Here we show that purified N, NSM and SM cell preparations are equally transformable in vitro to lymphoblastoid cells lines (LCLs) that, despite upregulating the activation-induced cytidine deaminase (AID) enzyme necessary for Ig isotype switching and Ig gene hypermutation, still retain the surface Ig phenotype of their parental cells. However, both N- and NSM-derived lines remain inducible to Ig isotype switching by surrogate T cell signals. More importantly, IgH gene analysis of N cell infections revealed two features quite distinct from parallel mitogen-activated cultures. Firstly, following 4 weeks of EBV-driven polyclonal proliferation, individual clonotypes then become increasingly dominant; secondly, in around 35% cases these clonotypes carry Ig gene mutations which both resemble AID products and, when analysed in prospectively-harvested cultures, appear to have arisen by sequence diversification in vitro. Thus EBV infection per se can drive at least some naïve B cells to acquire Ig memory genotypes; furthermore, such cells are often favoured during an LCLs evolution to monoclonality. Extrapolating to viral infections in vivo, these findings could help to explain how EBV-infected cells become restricted to memory B cell subsets and why EBV-driven lymphoproliferative lesions, in primary infection and/or immunocompromised settings, so frequently involve clones with memory genotypes.

Effect of Interleukin-10 Gene Polymorphisms on Clinical Outcome of Patients with Aggressive Non-Hodgkin's Lymphoma: An Exploratory Study

Purpose: Current chemotherapy can achieve high response rates in aggressive non-Hodgkins lymphoma (NHL), but the factors that influence regression and survival remain unknown. The present exploratory study tested the hypothesis whether interleukin-10 (IL-10) polymorphisms predict clinical outcome, leukocytopenia, or infectivity during therapy. IL-10 was chosen because immune alterations are a major risk factor for NHL, and IL-10 is a cytokine involved in inflammatory processes associated with clinical outcome. Experimental Design: Five hundred patients with aggressive NHL treated with CHOP/CHOEP were analyzed for IL-10 gene polymorphisms, including distal loci -7400InDel, -6752AT (rs6676671), and -6208CG (rs10494879) in comparison with proximal loci -3538AT (rs1800890), -1087AG (rs1800896), and -597AC (rs1800872) according to the incidence and outcome of the lymphoma. Results: No differences in allele frequencies or haplotypes were found comparing a cohort of patients with aggressive NHL/diffuse large B-cell lymphoma with a healthy control group. Patients with aggressive NHL characterized by IL-10-7400DelDel had shorter overall survival periods compared with the other genotypes (P = 0.004). The 3-year rate is 43.4% for IL-10-7400DelDel and 73.4% for IL-10-7400InIn and IL-10-7400InDel together. A significant increased risk for event-free survival is found for carriers of the genotype IL-10-6752TT-6208CC-3538AA (P = 0.047). Multivariate analysis of IL-10-7400 gene variation in relation to overall survival adjusted to international prognostic index revealed a relative risk of 1.9 for carriers of IL-10-7400DelDel (P = 0.037). No associations were found analyzing diffuse large B-cell lymphoma patients separately. Conclusion: Our results indicate that IL-10 gene variations could be associated to the clinical course of aggressive NHL, which points out the importance of host factors and respective genetic elements for treatment response.

The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation

The MHC class I chain‐related molecule A (MICA) is a highly polymorphic ligand for the activating natural killer (NK)‐cell receptor NKG2D. A single nucleotide polymorphism causes a valine to methionine exchange at position 129. Presence of a MICA‐129Met allele in patients (n = 452) undergoing hematopoietic stem cell transplantation (HSCT) increased the chance of overall survival (hazard ratio [HR] = 0.77, P = 0.0445) and reduced the risk to die due to acute graft‐versus‐host disease (aGVHD) (odds ratio [OR] = 0.57, P = 0.0400) although homozygous carriers had an increased risk to experience this complication (OR = 1.92, P = 0.0371). Overall survival of MICA‐129Val/Val genotype carriers was improved when treated with anti‐thymocyte globulin (HR = 0.54, P = 0.0166). Functionally, the MICA‐129Met isoform was characterized by stronger NKG2D signaling, triggering more NK‐cell cytotoxicity and interferon‐γ release, and faster co‐stimulation of CD8+ T cells. The MICA‐129Met variant also induced a faster and stronger down‐regulation of NKG2D on NK and CD8+ T cells than the MICA‐129Val isoform. The reduced cell surface expression of NKG2D in response to engagement by MICA‐129Met variants appeared to reduce the severity of aGVHD.