Reconstitution of EBV-directed T cell immunity by adoptive transfer of peptide-stimulated T cells in a patient after allogeneic stem cell transplantation for AITL.

Reconstitution of the T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by stimulation with peptides representing defined epitopes covering multiple HLA restrictions. T cells were adoptively transferred to a patient who had developed persisting high titers of EBV after allogeneic stem cell transplantation for angioimmunoblastic T-cell lymphoma (AITL). T cell receptor beta (TCRß) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring of T cell clonotypes at a molecular level after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.

A novel approach to detect resistance mechanisms reveals FGR as a factor mediating HDAC inhibitor SAHA resistance in B-cell lymphoma.

Histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) are not commonly used in clinical practice for treatment of B-cell lymphomas, although a subset of patients with refractory or relapsed B-cell lymphoma achieved partial or complete remissions. Therefore, the purpose of this study was to identify molecular features that predict the response of B-cell lymphomas to SAHA treatment. We designed an integrative approach combining drug efficacy testing with exome and captured target analysis (DETECT). In this study, we tested SAHA sensitivity in 26 B-cell lymphoma cell lines and determined SAHA-interacting proteins in SAHA resistant and sensitive cell lines employing a SAHA capture compound (CC) and mass spectrometry (CCMS). In addition, we performed exome mutation analysis. Candidate validation was done by expression analysis and knock-out experiments. An integrated network analysis revealed that the Src tyrosine kinase Gardner-Rasheed feline sarcoma viral (v-fgr) oncogene homolog (FGR) is associated with SAHA resistance. FGR was specifically captured by the SAHA-CC in resistant cells. In line with this observation, we found that FGR expression was significantly higher in SAHA resistant cell lines. As functional proof, CRISPR/Cas9 mediated FGR knock-out in resistant cells increased SAHA sensitivity. In silico analysis of B-cell lymphoma samples (n = 1200) showed a wide range of FGR expression indicating that FGR expression might help to stratify patients, which clinically benefit from SAHA therapy. In conclusion, our comprehensive analysis of SAHA-interacting proteins highlights FGR as a factor involved in SAHA resistance in B-cell lymphoma.

A new method to prevent carry-over contaminations in two-step PCR NGS library preparations.

Two-step PCR procedures are an efficient and well established way to generate amplicon libraries for NGS sequencing. However, there is a high risk of cross-contamination by carry-over of amplicons from first to second amplification rounds, potentially leading to severe misinterpretation of results. Here we describe a new method able to prevent and/or to identify carry-over contaminations by introducing the K-box, a series of three synergistically acting short sequence elements. Our K-boxes are composed of (i) K1 sequences for suppression of contaminations, (ii) K2 sequences for detection of possible residual contaminations and (iii) S sequences acting as separators to avoid amplification bias. In order to demonstrate the effectiveness of our method we analyzed two-step PCR NGS libraries derived from a multiplex PCR system for detection of T-cell receptor beta gene rearrangements. We used this system since it is of high clinical relevance and may be affected by very low amounts of contaminations. Spike-in contaminations are effectively blocked by the K-box even at high rates as demonstrated by ultra-deep sequencing of the amplicons. Thus, we recommend implementation of the K-box in two-step PCR-based NGS systems for research and diagnostic applications demanding high sensitivity and accuracy.

IκB-ζ controls the constitutive NF-κB target gene network and survival of ABC DLBCL.

Constitutive activation of the nuclear factor-κ B (NF-κB) pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL). Recurrent mutations of NF-κB regulators that cause constitutive activity of this oncogenic pathway have been identified. However, it remains unclear how specific target genes are regulated. We identified the atypical nuclear IκB protein IκB-ζ to be upregulated in ABC compared with germinal center B-cell-like (GCB) DLBCL primary patient samples. Knockdown of IκB-ζ by RNA interference was toxic to ABC but not to GCB DLBCL cell lines. Gene expression profiling after IκB-ζ knockdown demonstrated a significant downregulation of a large number of known NF-κB target genes, indicating an essential role of IκB-ζ in regulating a specific set of NF-κB target genes. To further investigate how IκB-ζ mediates NF-κB activity, we performed immunoprecipitations and detected a physical interaction of IκB-ζ with both p50 and p52 NF-κB subunits, indicating that IκB-ζ interacts with components of both the canonical and the noncanonical NF-κB pathway in ABC DLBCL. Collectively, our data demonstrate that IκB-ζ is essential for nuclear NF-κB activity in ABC DLBCL, and thus might represent a promising molecular target for future therapies.

Histone acetylation and DNA demethylation of T cells result in an anaplastic large cell lymphoma-like phenotype.

A characteristic feature of anaplastic large cell lymphoma is the significant repression of the T-cell expression program despite its T-cell origin. The reasons for this down-regulation of T-cell phenotype are still unknown. To elucidate whether epigenetic mechanisms are responsible for the loss of the T-cell phenotype, we treated anaplastic large cell lymphoma and T-cell lymphoma/leukemia cell lines (n=4, each) with epigenetic modifiers to evoke DNA demethylation and histone acetylation. Global gene expression data from treated and untreated cell lines were generated and selected, and differentially expressed genes were evaluated by real-time reverse transcriptase polymerase chain reaction and western blot analysis. Additionally, histone H3 lysine 27 trimethylation was analyzed by chromatin immunoprecipitation. Combined DNA demethylation and histone acetylation of anaplastic large cell lymphoma cells was not able to reconstitute their T-cell phenotype. Instead, the same treatment induced in T cells: (i) an up-regulation of anaplastic large cell lymphoma-characteristic genes (e.g. ID2, LGALS1, c-JUN), and (ii) an almost complete extinction of their T-cell phenotype including CD3, LCK and ZAP70. In addition, suppressive trimethylation of histone H3 lysine 27 of important T-cell transcription factor genes (GATA3, LEF1, TCF1) was present in anaplastic large cell lymphoma cells, which is in line with their absence in primary tumor specimens as demonstrated by immunohistochemistry. Our data suggest that epigenetically activated suppressors (e.g. ID2) contribute to the down-regulation of the T-cell expression program in anaplastic large cell lymphoma, which is maintained by trimethylation of histone H3 lysine 27.

Deep sequencing of MYC DNA-binding sites in Burkitt lymphoma.

BACKGROUND: MYC is a key transcription factor involved in central cellular processes such as regulation of the cell cycle, histone acetylation and ribosomal biogenesis. It is overexpressed in the majority of human tumors including aggressive B-cell lymphoma. Especially Burkitt lymphoma (BL) is a highlight example for MYC overexpression due to a chromosomal translocation involving the c-MYC gene. However, no genome-wide analysis of MYC-binding sites by chromatin immunoprecipitation (ChIP) followed by next generation sequencing (ChIP-Seq) has been conducted in BL so far. METHODOLOGY/PRINCIPAL FINDINGS: ChIP-Seq was performed on 5 BL cell lines with a MYC-specific antibody giving rise to 7,054 MYC-binding sites after bioinformatics analysis of a total of approx. 19 million sequence reads. In line with previous findings, binding sites accumulate in gene sets known to be involved in the cell cycle, ribosomal biogenesis, histone acetyltransferase and methyltransferase complexes demonstrating a regulatory role of MYC in these processes. Unexpectedly, MYC-binding sites also accumulate in many B-cell relevant genes. To assess the functional consequences of MYC binding, the ChIP-Seq data were supplemented with siRNA- mediated knock-downs of MYC in BL cell lines followed by gene expression profiling. Interestingly, amongst others, genes involved in the B-cell function were up-regulated in response to MYC silencing. CONCLUSION/SIGNIFICANCE: The 7,054 MYC-binding sites identified by our ChIP-Seq approach greatly extend the knowledge regarding MYC binding in BL and shed further light on the enormous complexity of the MYC regulatory network. Especially our observations that (i) many B-cell relevant genes are targeted by MYC and (ii) that MYC down-regulation leads to an up-regulation of B-cell genes highlight an interesting aspect of BL biology.

Classical Hodgkin's lymphoma shows epigenetic features of abortive plasma cell differentiation.

BACKGROUND: Epigenetic changes are involved in the extinction of the B-cell gene expression program of classical Hodgkin's lymphoma. However, little is known regarding epigenetic similarities between cells of classical Hodgkin's lymphoma and plasma cell myeloma, both of which share extinction of the gene expression program of mature B cells. DESIGN AND METHODS: Global histone H3 acetylation patterns were determined in cell lines derived from classical Hodgkin's lymphoma, plasma cell myeloma and B-cell lymphoma by chromatin immunoprecipitation and subsequent hybridization onto promoter tiling arrays. H3K27 trimethylation was analyzed by chromatin immunoprecipitation and real-time DNA polymerase chain reaction for selected genes. Epigenetic modifications were compared to gene expression data. RESULTS: Characteristic B-cell genes were hypoacetylated in classical Hodgkin's lymphoma and plasma cell myeloma cell lines as demonstrated by comparison of their histone H3 acetylation patterns to those of B-cell lines. However, the number of genes jointly hyperacetylated and expressed in classical Hodgkin' lymphoma and plasma cell myeloma cell lines, such as IRF4/MUM1 and RYBP, is limited. Moreover, H3K27 trimethylation for selected characteristic B-cell genes revealed that this additional epigenetic silencing is much more prevalent in classical Hodgkin's lymphoma than in plasma cell myeloma. CONCLUSIONS: Our epigenetic data support the view that classical Hodgkin's lymphoma is characterized by abortive plasma cell differentiation with a down-regulation of characteristic B-cell genes but without activation of most genes typical of plasma cells.

Evolution of a core gene network for skeletogenesis in chordates.

The skeleton is one of the most important features for the reconstruction of vertebrate phylogeny but few data are available to understand its molecular origin. In mammals the Runt genes are central regulators of skeletogenesis. Runx2 was shown to be essential for osteoblast differentiation, tooth development, and bone formation. Both Runx2 and Runx3 are essential for chondrocyte maturation. Furthermore, Runx2 directly regulates Indian hedgehog expression, a master coordinator of skeletal development. To clarify the correlation of Runt gene evolution and the emergence of cartilage and bone in vertebrates, we cloned the Runt genes from hagfish as representative of jawless fish (MgRunxA, MgRunxB) and from dogfish as representative of jawed cartilaginous fish (ScRunx1-3). According to our phylogenetic reconstruction the stem species of chordates harboured a single Runt gene and thereafter Runt locus duplications occurred during early vertebrate evolution. All newly isolated Runt genes were expressed in cartilage according to quantitative PCR. In situ hybridisation confirmed high MgRunxA expression in hard cartilage of hagfish. In dogfish ScRunx2 and ScRunx3 were expressed in embryonal cartilage whereas all three Runt genes were detected in teeth and placoid scales. In cephalochordates (lancelets) Runt, Hedgehog and SoxE were strongly expressed in the gill bars and expression of Runt and Hedgehog was found in endo- as well as ectodermal cells. Furthermore we demonstrate that the lancelet Runt protein binds to Runt binding sites in the lancelet Hedgehog promoter and regulates its activity. Together, these results suggest that Runt and Hedgehog were part of a core gene network for cartilage formation, which was already active in the gill bars of the common ancestor of cephalochordates and vertebrates and diversified after Runt duplications had occurred during vertebrate evolution. The similarities in expression patterns of Runt genes support the view that teeth and placoid scales evolved from a homologous developmental module.

Evidence of haematopoiesis within the developing human diencephalon. Correlations with vertebrate phylogeny.

AIM: The sites of haematopoiesis during human ontogeny can be correlated to the sites where haematopoiesis occurs in vertebrate phylogeny. As haematopoiesis has been described in the diencephalon and pituitary gland of water-inhabiting vertebrates we wanted to find out whether such a phenomenon also occurs in human embryos. MATERIAL AND METHODS: Paraffin-embedded specimens from the diencephalon and pituitary gland of human embryos at the 7th to 22nd gestational week and from adults were investigated by conventional histology and immunohistology for the presence of haematopoietic cells. RESULTS: Cellular accumulations predominantly of erythroid and megakaryocytic lineage were identified in the floor of the developing diencephalon of the 7th/8th gestational week. At the older developmental stages of the 18th to 22nd gestational week loose aggregates of haematopoietic cells within the leptomeningeal spaces adjacent to the hypophyseal infundibulum were detected in 2 out of 7 cases analyzed. CONCLUSIONS: As it has been proposed that lymphohaematopoietic clusters occasionally occur within the brain in bone marrow-less vertebrates as a response to noxious agents, we speculate that this temporal appearance of haematopoietic cell clusters within the diencephalon floor in early human ontogeny could also be due to fetal immunomodulations.

Gene identification and analysis of transcripts differentially regulated in fracture healing by EST sequencing in the domestic sheep.

BACKGROUND: The sheep is an important model animal for testing novel fracture treatments and other medical applications. Despite these medical uses and the well known economic and cultural importance of the sheep, relatively little research has been performed into sheep genetics, and DNA sequences are available for only a small number of sheep genes. RESULTS: In this work we have sequenced over 47 thousand expressed sequence tags (ESTs) from libraries developed from healing bone in a sheep model of fracture healing. These ESTs were clustered with the previously available 10 thousand sheep ESTs to a total of 19087 contigs with an average length of 603 nucleotides. We used the newly identified sequences to develop RT-PCR assays for 78 sheep genes and measured differential expression during the course of fracture healing between days 7 and 42 postfracture. All genes showed significant shifts at one or more time points. 23 of the genes were differentially expressed between postfracture days 7 and 10, which could reflect an important role for these genes for the initiation of osteogenesis. CONCLUSION: The sequences we have identified in this work are a valuable resource for future studies on musculoskeletal healing and regeneration using sheep and represent an important head-start for genomic sequencing projects for Ovis aries, with partial or complete sequences being made available for over 5,800 previously unsequenced sheep genes.

An inversion involving the mouse Shh locus results in brachydactyly through dysregulation of Shh expression.

Short digits (Dsh) is a radiation-induced mouse mutant. Homozygous mice are characterized by multiple defects strongly resembling those resulting from Sonic hedgehog (Shh) inactivation. Heterozygous mice show a limb reduction phenotype with fusion and shortening of the proximal and middle phalanges in all digits, similar to human brachydactyly type A1, a condition caused by mutations in Indian hedgehog (IHH). We mapped Dsh to chromosome 5 in a region containing Shh and were able to demonstrate an inversion comprising 11.7 Mb. The distal breakpoint is 13.298 kb upstream of Shh, separating the coding sequence from several putative regulatory elements identified by interspecies comparison. The inversion results in almost complete downregulation of Shh expression during E9.5-E12.5, explaining the homozygous phenotype. At E13.5 and E14.5, however, Shh is upregulated in the phalangeal anlagen of Dsh/+ mice, at a time point and in a region where WT Shh is never expressed. The dysregulation of Shh expression causes the local upregulation of hedgehog target genes such as Gli1-3, patched, and Pthlh, as well as the downregulation of Ihh and Gdf5. This results in shortening of the digits through an arrest of chondrocyte differentiation and the disruption of joint development.

Cloning and expression of an inhibitor of microbial metalloproteinases from insects contributing to innate immunity.

The first IMPI (inhibitor of metalloproteinases from insects) was identified in the greater wax moth, Galleria mellonella [Wedde, Weise, Kopacek, Franke and Vilcinskas (1998) Eur. J. Biochem. 255, 535-543]. Here we report cloning and expression of a cDNA coding for this IMPI. The IMPI mRNA was identified among the induced transcripts from a subtractive and suppressive PCR analysis after bacterial challenge of G. mellonella larvae. Induced expression of the IMPI during a humoral immune response was confirmed by real-time PCR, which documented up to 500 times higher amounts of IMPI mRNA in immunized larvae in comparison with untreated ones. The IMPI sequence shares no similarity with those of tissue inhibitors of metalloproteinases or other natural inhibitors of metalloproteinases, and the recombinant IMPI specifically inhibits thermolysin-like metalloproteinases, but not matrix metalloproteinases. These results support the hypothesis that the IMPI represents a novel type of immune-related protein which is induced and processed during the G. mellonella humoral immune response to inactivate pathogen-associated thermolysin-like metalloproteinases.

Cloning and expression of gallerimycin, an antifungal peptide expressed in immune response of greater wax moth larvae, Galleria mellonella.

A novel defensin-like peptide was identified in the greater wax moth, Galleria mellonella. It was discovered in a haemocyte cDNA bank enriched with transcripts upregulated after immune challenge via subtractive hybridisation and suppressive PCR. The deduced amino acid sequence of the defensin-like peptide exhibits similarities to the antifungal peptides drosomycin from Drosophila melanogaster and heliomicin from Heliothis virescens. Therefore, it has been termed gallerimycin. Upregulation of gallerimycin after stimulation of the immune system by LPS-injection was demonstrated by quantitative real-time PCR. A full-size cDNA was cloned and overexpressed in Escherichia coli Origami cells in order to obtain a functional peptide with disulfide bridges. The recombinant peptide was active against the entomopathogenic fungus Metarhizium anisopliae, but not against yeast, gram-negative and gram-positive bacteria.

Single-cell analysis of CD30+ cells in lymphomatoid papulosis demonstrates a common clonal T-cell origin.

Lymphomatoid papulosis (LyP) represents an intriguing cutaneous T-cell lymphoproliferative disorder with a histologic appearance resembling malignant lymphoma. This finding strongly contrasts with the benign clinical course of the disease. However, in 10% to 20% of cases, LyP can precede, coexist with, or follow malignant lymphoma. In these cases, the same T-cell population has been shown to be present in the LyP as well as in the associated lymphoma. In most LyP cases, there is-despite the sometimes extremely long course of the disease-no evolution of a secondary lymphoma. The investigation of these uncomplicated LyP cases for the presence of clonal T-cell receptor rearrangements has produced heterogeneous results. This might be explained by biologic or technical reasons arising from analyzing whole tissue DNA extracts. To definitively clarify whether the large atypical CD30(+) cells in LyP without associated lymphoma all belong to the same clone or represent individually rearranged T cells, we analyzed the T-cell receptor-gamma rearrangements of single CD30+ as well as of single CD30- cells isolated from 14 LyP lesions of 11 patients. By using this approach we could demonstrate that the CD30+ cells represent members of a single T-cell clone in all LyP cases. Moreover, in 3 patients the same CD30+ cell clone was found in anatomically and temporally separate lesions. In contrast, with only a few exceptions, the CD30- cells were polyclonal in all instances and unrelated to the CD30+ cell clone. Our results demonstrate that LyP unequivocally represents a monoclonal T-cell disorder of CD30+ cells in all instances.