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T-cell lymphomas are heterogeneous and rare lymphatic malignancies with unfavorable prognosis. Consequently, new therapeutic strategies are needed. The enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and responsible for lysine 27 trimethylation of histone 3. EZH2 is overexpressed in several tumor entities including T-cell neoplasms leading to epigenetic and consecutive oncogenic dysregulation. Thus, pharmacological EZH2 inhibition is a promising target and its clinical evaluation in T-cell lymphomas shows favorable results. We have investigated EZH2 expression in two cohorts of T-cell lymphomas by mRNA-profiling and immunohistochemistry, both revealing overexpression to have a negative impact on patients’ prognosis. Furthermore, we have evaluated EZH2 inhibition in a panel of leukemia and lymphoma cell lines with a focus on T-cell lymphomas characterized for canonical EZH2 signaling components. The cell lines were treated with the inhibitors GSK126 or EPZ6438 that inhibit EZH2 specifically by competitive binding at the S-adenosylmethionine (SAM) binding site in combination with the common second-line chemotherapeutic oxaliplatin. The change in cytotoxic effects under pharmacological EZH2 inhibition was evaluated revealing a drastic increase in oxaliplatin resistance after 72 h and longer periods of combinational incubation. This outcome was independent of cell type but associated to reduced intracellular platinum. Pharmacological EZH2 inhibition revealed increased expression in SRE binding proteins, SREBP1/2 and ATP binding cassette subfamily G transporters ABCG1/2. The latter are associated with chemotherapy resistance due to increased platinum efflux. Knockdown experiments revealed that this was independent of the EZH2 functional state. The EZH2 inhibition effect on oxaliplatin resistance and efflux was reduced by additional inhibition of the regulated target proteins. In conclusion, pharmacological EZH2 inhibition is not suitable in combination with the common chemotherapeutic oxaliplatin in T-cell lymphomas revealing an EZH2-independent off-target effect.

Chronic hepatitis C virus (HCV) infection leads to a complex interplay with adaptive immune cells that may result in cryoglobulinemia or lymphoma. Although direct-acting antiviral (DAA) therapy has decreased the incidence of severe liver damage, its effect on extrahepatic HCV manifestations such as B cell dyscrasias is still unclear. Here, we used immunosequencing to analyze HCV imprinting on B cell receptor (BCR) repertoires in patients with chronic HCV mono-infection or a sustained virological response (SVR) after DAA therapy. The majority of patients had a B cell signature with high somatic hypermutation and richness. Convergence to specific immunoglobulin genes produced high-connectivity complementarity-determining region 3 (CDR3) networks. Interestingly, IGHV1-69 CDR1 and CDR3 mutations characterizing highly neutralizing HCV antibodies corresponded to recurrent point mutations found in clonotypic BCRs of high-grade lymphomas. These BCRs did not show autonomous signaling but a lower activation threshold. B cells carrying these point mutations showed a persisting oncogenic transcriptome signature with dysregulation in signaling nodes such as CARD11, MALT1, RelB, MAPK, and NFAT. Collectively, this study provides evidence that lymphoma-like cells may result from the anti-HCV immune response and may persist for years after SVR, leading to HCV-related B cell dyscrasias and increased lymphoma risk beyond viral elimination.

Engraftment and differentiation of donor hematopoietic stem cells is decisive for the clinical success of allogeneic stem cell transplantation (alloSCT) and depends on the recipient's bone marrow (BM) niche. A damaged niche contributes to poor graft function post-alloSCT, but the underlying mechanisms and the role of BM multipotent mesenchymal stromal cells (MSC) are ill defined. Upon multivariate analysis in 732 individuals, we observed a reduced presence of proliferation-capable MSC in BM aspirates from patients (N=196) who had undergone alloSCT (OR 0.61, p=.028). This was confirmed by paired analysis in 30 patients showing a higher frequency of samples with a lack of MSC presence post-alloSCT compared to pre-alloSCT. This reduced MSC presence was associated with reduced survival of patients following alloSCT and specifically with impaired graft function. Post-alloSCT MSC showed diminished in vitro proliferation along with a transcriptional antiproliferative signature, upregulation of epithelial-mesenchymal transition and extracellular matrix pathways and altered impact on cytokine release upon contact with hematopoietic cells. In order to avoid in vitro culture bias, we isolated the CD146+/CD45-/HLA-DR- BM cell fraction, which comprised the entire MSC population. The post-alloSCT isolated native CD146+MSC showed a similar reduction in proliferation capacity and shared the same antiproliferative transcriptomic signature as for post-alloSCT CFU-F-derived MSC. Taken together, our data show that alloSCT confers damage to the proliferative capacity of native MSC, which is associated with reduced patient survival after alloSCT and impaired engraftment of allogeneic hematopoiesis. These data represent the basis to elucidate mechanisms of BM niche reconstitution post-alloSCT and its therapeutic manipulation.

Post-acute sequelae of COVID-19 (PASC) are long-term consequences of SARS-CoV-2 infection that can substantially impair the quality of life. Underlying mechanisms ranging from persistent viruses to innate and adaptive immune dysregulation have been discussed. Here, we profiled the plasma of 181 individuals from the cohort study for digital health research in Germany (DigiHero), including individuals after mild to moderate COVID-19 with or without PASC and uninfected controls. We focused on soluble factors related to monocyte/macrophage biology and on circulating SARS-CoV-2 spike (S1) protein as a potential biomarker for persistent viral reservoirs. At a median time of 8 months after infection, we found pronounced dysregulation in almost all tested soluble factors, including both pro-inflammatory and pro-fibrotic cytokines. These immunological perturbations were remarkably independent of ongoing PASC symptoms per se, but further correlation and regression analyses suggested PASC-specific patterns involving CCL2/MCP-1 and IL-8 that either correlated with sCD162, sCD206/MMR, IFN-α2, IL-17A and IL-33, or IL-18 and IL-23. None of the analyzed factors correlated with the detectability or levels of circulating S1, indicating that this represents an independent subset of patients with PASC. These data confirm prior evidence of immune dysregulation and persistence of viral protein in PASC and illustrate its biological heterogeneity that still awaits correlation with clinically defined PASC subtypes.

The B-cell architecture of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is complex since it is composed of malignant lymphocyte-predominant cells along with a rich B-cell bystander environment. To gain insight into molecular determinants of disease transformation, we studied B-cell evolutionary trajectories in lymphoma tissue from diagnosis to relapse or transformation to non-Hodgkin lymphoma by next-generation sequencing of immunoglobulin heavy chains. Patients with NLPHL that later transformed were older and showed IgD negativity, absence of the characteristic IGHV3/IGHD3/IGHJ6 lymphocyte-predominant rearrangement and high repertoire clonality. We constructed phylogenetic trees within the compartment of the malignant clone to investigate clonal evolution. In all relapsing cases, the lymphocyte-predominant rearrangement was identical at diagnosis and relapse. NLPHL cases with transformation showed more complex trajectories with strong intraclonal diversification. The dominant founder clone in transformations showed clonal evolution if derived from the same cell of origin, or arose from a different cell of origin. Together, our data point to a significant role of antigenic drive in the transformation of NLHPL and identify high B-cell repertoire clonality with dominant intraclonal lymphocyte-predominant cell diversification as a hallmark of transformation. Sequencing of initial paraffin-embedded tissue may therefore be applied diagnostically to identify NLPHL cases with high risk of transformation. publishedVersion

Background and aims Autoimmune hepatitis (AIH) is a chronic liver disease that regularly relapses when immunosuppression is tapered. It is thought to be driven by T-cells, whereas the etiologic impact of an apparently deregulated B lineage system, as evidenced by hypergammaglobulinemia and autoantibodies, remains elusive. We set out to investigate T and B cell repertoires supporting chronic inflammation in AIH. Approach and results T and B cell receptor (TCR/BCR) and human leukocyte antigen (HLA) next-generation immunosequencing were used to record immune signatures from a cohort of 60 patients with AIH and disease controls. Blood and liver B lineage immune metrics were not indicative of a dominant directional antigen selection apart from a slight skewing of IGHV-J genes. More importantly, we found strong AIH-specific TRBV-J skewing not attributable to the HLA-DRB1 specificities of the cohort. This TCR repertoire bias was generated as a result of peripheral T cell (de)selection and persisted in disease remission. Using a clustering algorithm according to antigenic specificity, we identified liver TCR clusters that were shared between patients with AIH but were absent or deselected in patients with other liver pathologies. Conclusions Patients with AIH show profound and persisting T-cell architectural changes that may explain high relapse rates after tapering immunosuppression. Liver T-cell clusters shared between patients may mediate liver damage and warrant further study.

Significance A hyperinflammatory syndrome reminiscent of toxic shock syndrome (TSS) is observed in severe COVID-19 patients, including children with Multisystem Inflammatory Syndrome in Children (MIS-C). TSS is typically caused by pathogenic superantigens stimulating excessive activation of the adaptive immune system. We show that SARS-CoV-2 spike contains sequence and structure motifs highly similar to those of a bacterial superantigen and may directly bind T cell receptors. We further report a skewed T cell receptor repertoire in COVID-19 patients with severe hyperinflammation, in support of such a superantigenic effect. Notably, the superantigen-like motif is not present in other SARS family coronaviruses, which may explain the unique potential for SARS-CoV-2 to cause both MIS-C and the cytokine storm observed in adult COVID-19., Multisystem Inflammatory Syndrome in Children (MIS-C) associated with COVID-19 is a newly recognized condition in children with recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. These children and adult patients with severe hyperinflammation present with a constellation of symptoms that strongly resemble toxic shock syndrome, an escalation of the cytotoxic adaptive immune response triggered upon the binding of pathogenic superantigens to T cell receptors (TCRs) and/or major histocompatibility complex class II (MHCII) molecules. Here, using structure-based computational models, we demonstrate that the SARS-CoV-2 spike (S) glycoprotein exhibits a high-affinity motif for binding TCRs, and may form a ternary complex with MHCII. The binding epitope on S harbors a sequence motif unique to SARS-CoV-2 (not present in other SARS-related coronaviruses), which is highly similar in both sequence and structure to the bacterial superantigen staphylococcal enterotoxin B. This interaction between the virus and human T cells could be strengthened by a rare mutation (D839Y/N/E) from a European strain of SARS-CoV-2. Furthermore, the interfacial region includes selected residues from an intercellular adhesion molecule (ICAM)-like motif shared between the SARS viruses from the 2003 and 2019 pandemics. A neurotoxin-like sequence motif on the receptor-binding domain also exhibits a high tendency to bind TCRs. Analysis of the TCR repertoire in adult COVID-19 patients demonstrates that those with severe hyperinflammatory disease exhibit TCR skewing consistent with superantigen activation. These data suggest that SARS-CoV-2 S may act as a superantigen to trigger the development of MIS-C as well as cytokine storm in adult COVID-19 patients, with important implications for the development of therapeutic approaches.

We identified a subset of Chronic Lymphocytic Leukemia (CLL) patients with high Signaling Lymphocytic Activation Molecule Family (SLAMF) receptor-related signaling that showed an indolent clinical course. Since SLAMF receptors play a role in NK cell biology, we reasoned that these receptors may impact NK cell-mediated CLL immunity. Indeed, our experiments showed significantly decreased degranulation capacity of primary NK cells from CLL patients expressing low levels of SLAMF1 and SLAMF7. Since the SLAMFlow signature was strongly associated with an unmutated CLL immunoglobulin heavy chain (IGHV) status in large datasets, we investigated the impact of SLAMF1 and SLAMF7 on the B cell receptor (BCR) signaling axis. Overexpression of SLAMF1 or SLAMF7 in IGHV mutated CLL cell models resulted in reduced proliferation and impaired responses to BCR ligation, whereas the knockout of both receptors showed opposing effects and increased sensitivity toward inhibition of components of the BCR pathway. Detailed molecular analyzes showed that SLAMF1 and SLAMF7 receptors mediate their BCR pathway antagonistic effects via recruitment of prohibitin-2 (PHB2) thereby impairing its role in signal transduction downstream the IGHV-mutant IgM-BCR. Together, our data indicate that SLAMF receptors are important modulators of the BCR signaling axis and may improve immune control in CLL by interference with NK cells.

Post-acute sequelae of COVID-19 (PASC) is emerging as global problem with unknown molecular drivers. Using a digital epidemiology approach, we recruited 8,077 individuals to the cohort study for digital health research in Germany (DigiHero) to respond to a basic questionnaire followed by a PASC-focused survey and blood sampling. We report the first 318 participants, the majority thereof after mild infections. Of those, 67.8% report PASC, predominantly consisting of fatigue, dyspnea, and concentration deficit, which persists in 60% over the mean 8-month follow-up period and resolves independently of post-infection vaccination. PASC is not associated with autoantibodies, but with elevated IL-1β, IL-6, and TNF plasma levels, which we confirm in a validation cohort with 333 additional participants and a longer time from infection of 10 months. Blood profiling and single-cell data from early infection suggest the induction of these cytokines in COVID-19 lung pro-inflammatory macrophages creating a self-sustaining feedback loop.

Background: The COVID-19 pandemic called for a fast conduct of studies to establish vaccines and therapies, but also to identify the natural history and drivers of post-acute sequelae of COVID-19 (PASC). Digital epidemiology may serve this aim by rapidly generating large sample sizes allowing dedicated analyses of biomaterial in a subsample of interest. Methods: Of 129,733 households in Halle (Saale) invited to the cohort study for digital health research in Germany (DigiHero), 8,077 individuals participated, among these 919 that reported prior positive SARS-CoV-2 testing in their households. These were invited to respond to a PASC-focused questionnaire and to undergo blood sampling for cytokine and autoantibody profiling. Cytokine profiles were validated in a second cohort with early infections and single-cell transcriptomics datasets. Results: The analysis is based on the first 318 DigiHero participants, 258 thereof on average eight months after mostly mild infection. PASC were reported in 67.8% of cases and consisted predominantly in fatigue, dyspnea and concentration deficit. The recovery from PASC was not associated with post-infection vaccination suggesting that it may not be driven by a cryptic SARS-CoV-2 reservoir. We confirmed the high percentage of individuals with autoantibodies after COVID-19, but found no association with PASC. While our data show that a broad range of cytokines remain deregulated long after infection, IL-1s, IL-6 and TNF-α represented a triad that was associated with PASC. Blood profiling and single-cell data from early infection indicated that these cytokines are induced in COVID-19 lung pro-inflammatory macrophages creating a feedback loop that may trigger their long-term activation. Conclusion: We provide evidence for a long-lasting cytokine signature potentially underlying many of the clinical symptoms of PASC that may be driven by macrophage primed during acute infection. This study demonstrates how the combination of digital epidemiology with selective biobanking can rapidly generate hints towards disease mechanisms.

In parasite and viral infections, aberrant B cell responses can suppress germinal center reactions thereby blunting long-lived memory and may provoke immunopathology including autoimmunity. Using COVID-19 as model, we set out to identify serological, cellular and transcriptomic imprints of pathological responses linked to autoreactive B cells at single-cell resolution. We show that excessive plasmablast expansions are prognostically adverse, correlate with auto-antibody production, but do not hinder the formation of neutralizing antibodies. While plasmablasts followed IL-4 and BAFF driven developmental trajectories, were polyclonal and not enriched in autoreactive B cells, we identified two memory populations (CD80+/ISG15+ and CD11c+/SOX5+/T-bet+/-) with immunogenetic and transcriptional signs of autoreactivity that may be the cellular source of auto-antibodies in COVID-19 and that may persist beyond recovery. Immunomodulatory interventions discouraging such adverse responses may be useful in selected patients to shift the balance from autoreactivity towards long-term memory., Graphical Abstract

A considerable fraction of B cells recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with germline-encoded elements of their B cell receptor, resulting in the production of neutralizing and nonneutralizing antibodies. We found that antibody sequences from different discovery cohorts shared biochemical properties and could be retrieved across validation cohorts, confirming the stereotyped character of this naive response in coronavirus disease 2019 (COVID-19). While neutralizing antibody sequences were found independently of disease severity, in line with serological data, individual nonneutralizing antibody sequences were associated with fatal clinical courses, suggesting detrimental effects of these antibodies. We mined 200 immune repertoires from healthy individuals and 500 repertoires from patients with blood or solid cancers - all acquired prior to the pandemic - for SARS-CoV-2 antibody sequences. While the largely unmutated B cell rearrangements occurred in a substantial fraction of immune repertoires from young and healthy individuals, these sequences were less likely to be found in individuals over 60 years of age and in those with cancer. This reflects B cell repertoire restriction in aging and cancer, and may to a certain extent explain the different clinical courses of COVID-19 observed in these risk groups. Future studies will have to address if this stereotyped B cell response to SARS-CoV-2 emerging from unmutated antibody rearrangements will create long-lived memory.

Summary We profiled adaptive immunity in COVID-19 patients with active infection or after recovery and created a repository of currently >14 million B and T cell receptor (BCR, TCR) sequences from blood of these patients. The B cell response showed converging IGHV3-driven BCR clusters closely associated with SARS-CoV-2 antibodies. Clonality and skewing of TCR repertoires was associated with interferon type I and III responses, early CD4+ and CD8+ T cell activation and counterregulation by the coreceptors BTLA, Tim-3, PD-1, TIGIT and CD73. Tfh, Th17-like and nonconventional (but not classical anti-viral) Th1 cell polarizations were induced. SARS-CoV-2-specific T cell responses were driven by TCR clusters shared between patients with a characteristic trajectory of clonotypes and traceability over the disease course. Our data provide fundamental insight into adaptive immunity to SARS-CoV-2 with the actively updated repository providing a resource for the scientific community urgently needed to inform therapeutic concepts and vaccine development., Highlights • Convergent B cell responses to SARS-CoV-2 epitopes are predominantly naïve • Shared T cell clusters emerge over disease course in recovering COVID-19 patients • A receptor sequence repository from COVID-19 patients is established for public use, It is unclear how immune responses to Sars-CoV-2 differ in patients with mild versus severe COVID-19 disease. Schultheiß et al. define specific immune receptor sequences associated with different disease courses and established an actively updated sequence repository for public use.

Recent studies revealed trajectories of mutational events in early melanomagenesis, but the accompanying changes in gene expression are far less understood. Therefore, we performed a comprehensive RNA-seq analysis of laser-microdissected melanocytic nevi (n = 23) and primary melanoma samples (n = 57) and characterized the molecular mechanisms of early melanoma development. Using self-organizing maps, unsupervised clustering, and analysis of pseudotime (PT) dynamics to identify evolutionary trajectories, we describe here two transcriptomic types of melanocytic nevi (N1 and N2) and primary melanomas (M1 and M2). N1/M1 lesions are characterized by pigmentation-type and MITF gene signatures, and a high prevalence of NRAS mutations in M1 melanomas. N2/M2 lesions are characterized by inflammatory-type and AXL gene signatures with an equal distribution of wild-type and mutated BRAF and low prevalence of NRAS mutations in M2 melanomas. Interestingly, N1 nevi and M1 melanomas and N2 nevi and M2 melanomas, respectively, cluster together, but there is no clustering in a stage-dependent manner. Transcriptional signatures of M1 melanomas harbor signatures of BRAF/MEK inhibitor resistance and M2 melanomas harbor signatures of anti-PD-1 antibody treatment resistance. Pseudotime dynamics of nevus and melanoma samples are suggestive for a switch-like immune-escape mechanism in melanoma development with downregulation of immune genes paralleled by an increasing expression of a cell cycle signature in late-stage melanomas. Taken together, the transcriptome analysis identifies gene signatures and mechanisms underlying development of melanoma in early and late stages with relevance for diagnostics and therapy.

// Tobias Gerber 1, * , Edith Willscher 2, * , Henry Loeffler-Wirth 2 , Lydia Hopp 2 , Dirk Schadendorf 3 , Manfred Schartl 4, 5, 6 , Ulf Anderegg 7 , Gray Camp 1 , Barbara Treutlein 1 , Hans Binder 2 , Manfred Kunz 7 1 Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology Leipzig, 04103 Leipzig, Germany 2 Interdisciplinary Center for Bioinformatics, University of Leipzig, 04107 Leipzig, Germany 3 Department of Dermatology, Venereology and Allergology, University Hospital Essen, 45147 Essen, Germany 4 Department of Physiological Chemistry, University of Wurzburg, Biozentrum, Am Hubland, 97074 Wurzburg, Germany 5 Comprehensive Cancer Center Mainfranken, University Clinic Wurzburg, 97080 Wurzburg, Germany 6 Institute for Advanced Study, 3572 Texas A&M University, College Station, Texas 77843-3572, USA 7 Department of Dermatology, Venereology and Allergology, University of Leipzig, 04103 Leipzig, Germany * These authors contributed equally to this work Correspondence to: Manfred Kunz, email: manfred.kunz@medizin.uni-leipzig.de Keywords: melanoma, single cell transcriptome sequencing, stem cells Received: May 18, 2016 Accepted: November 12, 2016 Published: November 26, 2016 ABSTRACT Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples. Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/ NRAS wild type and BRAF wild type/ NRAS mutant melanoma metastasis, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, pigmentation and cellular stroma. Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data. We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells, suggesting that these kinases might be involved in melanoma progression. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. Finally, we identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs). Patient-derived cultures of the BRAF mutant/ NRAS wild type and BRAF wild type/ NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters. Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma therapy.

Background Germinal center-derived B cell lymphomas are tumors of the lymphoid tissues representing one of the most heterogeneous malignancies. Here we characterize the variety of transcriptomic phenotypes of this disease based on 873 biopsy specimens collected in the German Cancer Aid MMML (Molecular Mechanisms in Malignant Lymphoma) consortium. They include diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt’s lymphoma, mixed FL/DLBCL lymphomas, primary mediastinal large B cell lymphoma, multiple myeloma, IRF4-rearranged large cell lymphoma, MYC-negative Burkitt-like lymphoma with chr. 11q aberration and mantle cell lymphoma. Methods We apply self-organizing map (SOM) machine learning to microarray-derived expression data to generate a holistic view on the transcriptome landscape of lymphomas, to describe the multidimensional nature of gene regulation and to pursue a modular view on co-expression. Expression data were complemented by pathological, genetic and clinical characteristics. Results We present a transcriptome map of B cell lymphomas that allows visual comparison between the SOM portraits of different lymphoma strata and individual cases. It decomposes into one dozen modules of co-expressed genes related to different functional categories, to genetic defects and to the pathogenesis of lymphomas. On a molecular level, this disease rather forms a continuum of expression states than clearly separated phenotypes. We introduced the concept of combinatorial pattern types (PATs) that stratifies the lymphomas into nine PAT groups and, on a coarser level, into five prominent cancer hallmark types with proliferation, inflammation and stroma signatures. Inflammation signatures in combination with healthy B cell and tonsil characteristics associate with better overall survival rates, while proliferation in combination with inflammation and plasma cell characteristics worsens it. A phenotypic similarity tree is presented that reveals possible progression paths along the transcriptional dimensions. Our analysis provided a novel look on the transition range between FL and DLBCL, on DLBCL with poor prognosis showing expression patterns resembling that of Burkitt’s lymphoma and particularly on ‘double-hit’ MYC and BCL2 transformed lymphomas. Conclusions The transcriptome map provides a tool that aggregates, refines and visualizes the data collected in the MMML study and interprets them in the light of previous knowledge to provide orientation and support in current and future studies on lymphomas and on other cancer entities. Electronic supplementary material The online version of this article (10.1186/s13073-019-0637-7) contains supplementary material, which is available to authorized users.

The vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)-β are key target molecules in glioblastoma that are interdependently regulated. Both pathways drive key malignant features in glioma cells such as angiogenesis, invasiveness and immunosuppression. Here we explore whether TGF-β acts as an escape pathway from VEGF inhibition using gene expression profiling and in vitro and in vivo studies in a panel of syngeneic mouse glioma models. In vivo, single agent activity was observed for the VEGF antibody B20-4.1.1 in three (SMA-540, SMA-560 and GL-261), and for the TGF-β receptor I antagonist LY2157299 in two (SMA-540 and SMA-560) of four mouse glioma models. Transcriptomic profiling of the four mouse glioma models revealed that SMA-497 and GL-261, while unresponsive to LY2157299, were the most immunogenic tumors as defined by Gene Ontology; specifically, up-regulation of chemokine/chemoreceptors genes involved in immune cell recruitment and interferon-related genes were found. Co-targeting of VEGF and TGF-β was ineffective in one refractory model (SMA-497), in which no major changes in tumor immune-infiltrating cells were detected upon mono- or co-treatment settings. Significant prolongation of survival in the GL-261 model was associated with early stage increased infiltration of CD8+ cells, lower numbers of CD11b+ macrophages/microglia and sustained suppression of angiogenesis compared to anti-VEGF treatment alone. Phosphorylated SMAD2 was increased in both tumor cells and CD45+ leukocytes during anti-angiogenic treatment pointing to the possibility of a TGF-β-induced immunosuppressive micro-milieu that is efficiently counteracted by concomitant administration of LY2157299. In conclusion, our study highlights the biological heterogeneity of glioblastoma even among simple mouse models and illustrates that co-targeting of the VEGF and TGF-β pathways might lead to improved tumor control in subsets of glioblastoma linked with angiogenesis impairment and general reduction of the immune-suppressive phenotype.This study was supported by a grant from Oncosuisse (KFS-02694-08-2010)

BackgroundThe vascular endothelial growth factor (VEGF) and transforming growth factor (TGF)–β pathways regulate key biological features of glioblastoma. Here we explore whether the TGF-β pathway, which promotes angiogenesis, invasiveness, and immunosuppression, acts as an escape pathway from VEGF inhibition.MethodsThe role of the TGF-β pathway in escape from VEGF inhibition was assessed in vitro and in vivo and by gene expression profiling in syngeneic mouse glioma models.ResultsWe found that TGF-β is an upstream regulator of VEGF, whereas VEGF pathway activity does not alter the TGF-β pathway in vitro. In vivo, single-agent activity was observed for the VEGF antibody B20-4.1.1 in 3 and for the TGF-β receptor 1 antagonist LY2157299 in 2 of 4 models. Reduction of tumor volume and blood vessel density, but not induction of hypoxia, correlated with benefit from B20-4.1.1. Reduction of phosphorylated (p)SMAD2 by LY2157299 was seen in all models but did not predict survival. Resistance to B20 was associated with anti-angiogenesis escape pathway gene expression, whereas resistance to LY2157299 was associated with different immune response gene signatures in SMA-497 and GL-261 on transcriptomic profiling. The combination of B20 with LY2157299 was ineffective in SMA-497 but provided prolongation of survival in GL-261, associated with early suppression of pSMAD2 in tumor and host immune cells, prolonged suppression of angiogenesis, and delayed accumulation of tumor infiltrating microglia/macrophages.ConclusionsOur study highlights the biological heterogeneity of murine glioma models and illustrates that cotargeting of the VEGF and TGF-β pathways might lead to improved tumor control only in subsets of glioblastoma.

Alteration in the expression level of peripheral myelin protein 22 (PMP22) is the most frequent cause for demyelinating neuropathies of Charcot-Marie-Tooth type. Here, we demonstrate a loss of motoneurons (MNs) in the spinal cords from transgenic mice over-expressing Pmp22 (Pmp22(tg)) while mice lacking Pmp22 [Pmp22(ko); knockout (ko)] exhibited normal MN numbers at the symptomatic age of 60 days. In order to describe the molecular changes in affected MNs, these cells were isolated from lumbar spinal cords by laser-capture microdissection. Remarkably, the MNs of the Pmp22(ko) and Pmp22(tg) mice showed different expression profiles because of the altered Pmp22 expression. The changes in the expression profile of MNs from Pmp22(ko) mice resemble those described in MNs from mice after nerve injury and included genes that had been described in neuronal growth and regeneration like Gap43 and Sprr11a. The changes detected in the expression pattern of MNs from Pmp22(tg) mice exhibited fewer similarities to other expression patterns. The specific expression pattern in the MNs of the Pmp22(ko) mice might contribute to the better survival of the MNs. Our study also revealed induction of genes like brain-expressed X-linked 1 (Bex1) and desmoplakin (Dsp) that had recently been found up-regulated in MNs of human amyotrophic lateral sclerosis patients.

Cerebral gliomas of World Health Organization (WHO) grade II and III represent a major challenge in terms of histological classification and clinical management. Here, we asked whether large-scale genomic and transcriptomic profiling improves the definition of prognostically distinct entities. We performed microarray-based genome- and transcriptome-wide analyses of primary tumor samples from a prospective German Glioma Network cohort of 137 patients with cerebral gliomas, including 61 WHO grade II and 76 WHO grade III tumors. Integrative bioinformatic analyses were employed to define molecular subgroups, which were then related to histology, molecular biomarkers, including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q co-deletion and telomerase reverse transcriptase (TERT) promoter mutations, and patient outcome. Genomic profiling identified five distinct glioma groups, including three IDH1/2 mutant and two IDH1/2 wild-type groups. Expression profiling revealed evidence for eight transcriptionally different groups (five IDH1/2 mutant, three IDH1/2 wild type), which were only partially linked to the genomic groups. Correlation of DNA-based molecular stratification with clinical outcome allowed to define three major prognostic groups with characteristic genomic aberrations. The best prognosis was found in patients with IDH1/2 mutant and 1p/19q co-deleted tumors. Patients with IDH1/2 wild-type gliomas and glioblastoma-like genomic alterations, including gain on chromosome arm 7q (+7q), loss on chromosome arm 10q (-10q), TERT promoter mutation and oncogene amplification, displayed the worst outcome. Intermediate survival was seen in patients with IDH1/2 mutant, but 1p/19q intact, mostly astrocytic gliomas, and in patients with IDH1/2 wild-type gliomas lacking the +7q/-10q genotype and TERT promoter mutation. This molecular subgrouping stratified patients into prognostically distinct groups better than histological classification. Addition of gene expression data to this genomic classifier did not further improve prognostic stratification. In summary, DNA-based molecular profiling of WHO grade II and III gliomas distinguishes biologically distinct tumor groups and provides prognostically relevant information beyond histological classification as well as IDH1/2 mutation and 1p/19q co-deletion status.

Cancer stem cells may mediate therapy resistance and recurrence in various types of cancer, including glioblastoma. Cancer stemlike cells can be isolated from long-term cancer cell lines, including glioma lines. Using sphere formation as a model for cancer cell stemness in vitro, we derived sphere cultures from SMA-497, SMA-540, SMA-560, and GL-261 glioma cells. Gene expression and proteomics profiling demonstrated that sphere cultures uniformly showed an elevated expression of stemness-associated genes, notably including CD44. Differences in neural lineage marker expression between nonsphere and sphere cultures were heterogeneous except for a uniform reduction of β-III-tubulin in sphere cultures. All sphere cultures showed slower growth. Self-renewal capacity was influenced by medium conditions but not nonsphere versus sphere culture phenotype. Sphere cultures were more resistant to irradiation, whereas both nonsphere and sphere cultures were highly resistant to temozolomide. Nonsphere cells formed more aggressive tumors in syngeneic mice than sphere cells in all models except SMA-560. There were no major differences in vascularization or infiltration by T cells or microglia/macrophages between nonsphere and sphere cell-derived tumors implanted in syngeneic hosts. Together, these data indicate that mouse glioma cell lines may be induced in vitro to form spheres that acquire features of stemness, but they do not exhibit a uniform biologic phenotype, thereby challenging the view that they represent a superior model system.

BACKGROUND: The prognosis of glioblastoma, the most malignant type of glioma, is still poor, with only a minority of patients showing long-term survival of more than three years after diagnosis. We aimed to elucidate the poorly characterized molecular aberrations in glioblastomas of longterm survivors. METHODS: We performed genome- and/or transcriptome-wide molecular profiling of primary tumor samples from 94 glioblastoma patients of the German Glioma Network, including 28 longterm survivors with >36 months overall survival (OS), 20 short-term survivors with

Although the activation of immune cells is the first and thereby pivotal step in the immunological cascade, the current knowledge about the details of this process is quite limited. Recent studies have shown that aromatic compounds, such as B[ a ]P, influence the immune system even at low concentrations. We investigated the effect of a subtoxic B[ a ]P concentration (50 nM) on the proteome and the metabolome of non-activated and activated Jurkat T cells. The GeLC-MS/MS analysis yielded 2624 unambiguously identified proteins. In addition to typical regulatory pathways associated with T cell activation, pathway analysis by Ingenuity IPA revealed several metabolic processes, for instance purine and pyruvate metabolism. The activation process seems to be influenced by B[ a ]P suggesting an important role of the mTOR pathway in the cellular adaptation. B[ a ]P exposure of non-activated Jurkat cells induced signaling pathways such as protein ubiquitination and NRF2 mediated oxidative stress response as well as metabolic adaptation involving pyruvate, purine and fatty acid metabolism. Thus, we validated the proteome results by determining the concentrations of 183 metabolites with FIA-MS/MS and IC-MS/MS. Furthermore, we were able to show that Jurkat cells metabolize B[ a ]P to B[ a ]P-1,6-dione. The combined evaluation of proteome and metabolome data with an integrated, genome-scale metabolic model provided novel systems biological insights into the complex relation between metabolic and proteomic processes in Jurkat T cells during activation and subtoxic chemical exposure.

The most prominent brain region evaluating the significance of external stimuli immediately after their onset is the amygdala. Stimuli evaluated as being stressful actuate a number of physiological processes as an immediate stress response. Variation in the serotonin transporter gene has been associated with increased anxiety- and depression-like behavior, altered stress reactivity and adaptation, and pathophysiology of stress-related disorders. In this study the instant reactions to an acute stressor were measured in a serotonin transporter knockout mouse model. Mice lacking the serotonin transporter were verified to be more anxious than their wild-type conspecifics. Genome-wide gene expression changes in the amygdala were measured after the mice were subjected to control condition or to an acute stressor of one minute exposure to water. The dissection of amygdalae and stabilization of RNA was conducted within nine minutes after the onset of the stressor. This extremely short protocol allowed for analysis of first wave primary response genes, typically induced within five to ten minutes of stimulation, and was performed using Affymetrix GeneChip Mouse Gene 1.0 ST Arrays. RNA profiling revealed a largely new set of differentially expressed primary response genes between the conditions acute stress and control that differed distinctly between wild-type and knockout mice. Consequently, functional categorization and pathway analysis indicated genes related to neuroplasticity and adaptation in wild-types whereas knockouts were characterized by impaired plasticity and genes more related to chronic stress and pathophysiology. Our study therefore disclosed different coping styles dependent on serotonin transporter genotype even directly after the onset of stress and accentuates the role of the serotonergic system in processing stressors and threat in the amygdala. Moreover, several of the first wave primary response genes that we found might provide promising targets for future therapeutic interventions of stress-related disorders also in humans.