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1.  IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases 
Nature  2014;507(7492):366-370.
B lymphocytes have critical roles as positive and negative regulators of immunity. Their inhibitory function has so far been associated primarily with interleukin (IL)-10 because B cell-derived IL-10 can protect against autoimmune disease and increase susceptibility to pathogens1,2. Here, we identify IL-35-producing B cells as novel key players in the negative regulation of immunity. Mice in which only B cells did not express IL-35 lost their ability to recover from the T cell-mediated demyelinating autoimmune disease experimental autoimmune encephalomyelitis (EAE). In contrast, these mice displayed a strikingly improved resistance to infection with the intracellular bacterial pathogen Salmonella typhimurium, as shown by their superior containment of the bacterial growth and their prolonged survival both after primary infection, and upon secondary challenge after vaccination, compared to control mice. The increased immunity found in mice lacking IL-35 production by B cells was associated with a higher activation of macrophages and inflammatory T cells, as well as an enhanced stimulatory function of B cells as antigen-presenting cells (APC). During Salmonella infection IL-35- and IL-10-producing B cells corresponded to two largely distinct sets of surface-IgM+CD138hiTACI+CXCR4+CD1dintTim1int plasma cells expressing the transcription factor Blimp1. During EAE CD138+ plasma cells were also the major source of B cell-derived IL-35 and IL-10. Collectively, our data unravel the importance of IL-35-producing B cells in regulation of immunity, and highlight IL-35 production by B cells as a novel therapeutic target for autoimmune and infectious diseases. More generally, this study emphasizes the central role of activated B cells, particularly plasma cells, and their production of cytokines in the regulation of immune responses in health and disease.
PMCID: PMC4260166  PMID: 24572363
2.  Upregulation of Immunoproteasome Subunits in Myositis Indicates Active Inflammation with Involvement of Antigen Presenting Cells, CD8 T-Cells and IFNγ 
PLoS ONE  2014;9(8):e104048.
In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers.
Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses.
Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood.
Immunoproteasomes seem to indicate IIM activity and suggest that dominant involvement of antigen processing and presentation may qualify these diseases exemplarily for the evolving therapeutic concepts of immunoproteasome specific inhibition.
PMCID: PMC4123911  PMID: 25098831
3.  Cell-Specific Type I IFN Signatures in Autoimmunity and Viral Infection: What Makes the Difference? 
PLoS ONE  2013;8(12):e83776.
Gene expression profiling of peripheral blood mononuclear cells (PBMCs) has revealed a crucial role for type I interferon (IFN) in the pathogenesis of systemic lupus erythematosus (SLE). However, it is unclear how particular leucocyte subsets contribute to the overall type I IFN signature of PBMCs and whole blood samples.Furthermore, a detailed analysis describing the differences in the IFN signature in autoimmune diseases from that observed after viral infection has not been performed to date. Therefore, in this study, the transcriptional responses in peripheral T helper cells (CD4+) and monocyte subsets (CD16− inflammatory and CD16+ resident monocytes) isolated from patients with SLE, healthy donors (ND) immunised with the yellow fever vaccine YFV-17Dand untreated controls were compared by global gene expression profiling.It was striking that all of the transcripts that were regulated in response to viral exposure were also found to be differentially regulated in SLE, albeit with markedly lower fold-change values. In addition to this common IFN signature, a pathogenic IFN-associated gene signature was detected in the CD4+ T cells and monocytes from the lupus patients. IL-10, IL-9 and IL-15-mediated JAK/STAT signalling was shown to be involved in the pathological amplification of IFN responses observed in SLE. Type I IFN signatures identified were successfully applied for the monitoring of interferon responses in PBMCs of an independent cohort of SLE patients and virus-infected individuals. Moreover, these cell-type specific gene signatures allowed a correct classification of PBMCs independent from their heterogenic cellular composition. In conclusion, our data show for the first time that monocytes and CD4 cells are sensitive biosensors to monitor type I interferon response signatures in autoimmunity and viral infection and how these transriptional responses are modulated in a cell- and disease-specific manner.
PMCID: PMC3877094  PMID: 24391825
4.  Ectopic Runx1 Expression Rescues Tal-1-Deficiency in the Generation of Primitive and Definitive Hematopoiesis 
PLoS ONE  2013;8(7):e70116.
The transcription factors SCL/Tal-1 and AML1/Runx1 control the generation of pluripotent hematopoietic stem cells (pHSC) and, thereby, primitive and definitive hematopoiesis, during embryonic development of the mouse from mesoderm. Thus, Runx1-deficient mice generate primitive, but not definitive hematopoiesis, while Tal-1-deficient mice are completely defective. Primitive as well as definitive hematopoiesis can be developed “in vitro” from embryonic stem cells (ESC). We show that wild type, as well as Tal-1−/− and Runx1−/− ESCs, induced to differentiation, all expand within 5 days to comparable numbers of Flk1+ mesodermal cells. While wild type ESCs further differentiate to primitive and definitive erythrocytes, to c-fms+Gr1+Mac1+ myeloid cells, and to B220+CD19+ B- and CD4+/CD8+ T-lymphoid cells, Runx1−/− ESCs, as expected, only develop primitive erythrocytes, and Tal-1−/− ESCs do not generate any hematopoietic cells. Retroviral transduction with Runx1 of Runx1−/− ESCs, differentiated for 4 days to mesoderm, rescues definitive erythropoiesis, myelopoiesis and lymphopoiesis, though only with 1–10% of the efficiencies of wild type ESC hematopoiesis. Surprisingly, Tal-1−/− ESCs can also be rescued at comparably low efficiencies to primitive and definitive erythropoiesis, and to myelopoiesis and lymphopoiesis by retroviral transduction with Runx1. These results suggest that Tal-1 expression is needed to express Runx1 in mesoderm, and that ectopic expression of Runx1 in mesoderm is sufficient to induce primitive as well as definitive hematopoiesis in the absence of Tal-1. Retroviral transduction of “in vitro” differentiating Tal-1−/− and Runx1−/− ESCs should be a useful experimental tool to probe selected genes for activities in the generation of hematopoietic progenitors “in vitro”, and to assess the potential transforming activities in hematopoiesis of mutant forms of Tal-1 and Runx1 from acute myeloid leukemia and related tumors.
PMCID: PMC3726448  PMID: 23922928
5.  Epigenetic quantification of tumor-infiltrating T-lymphocytes 
Epigenetics  2011;6(2):236-246.
The immune system plays a pivotal role in tumor establishment. However, the role of T-lymphocytes within the tumor microenvironment as major cellular component of the adaptive effector immune response and their counterpart, regulatory T cells (Treg), responsible for suppressive immune modulation, is not completely understood. This is partly due to the lack of reliable technical solutions for specific cell quantification in solid tissues. Previous reports indicated that epigenetic marks of immune cells, such as the Treg specifically demethylated region (TSDR) within the FOXP3 gene, may be exploited as robust analytical tool for Treg-quantification. Here, we expand the concept of epigenetic immunophenotyping to overall T-lymphocytes (oTL). This tool allows cell quantification with at least equivalent precision to FACS and is adoptable for analysis of blood and solid tissues. Based on this method, we analyze the frequency of Treg, oTL and their ratio in independent cohorts of healthy and tumorous ovarian, colorectal and bronchial tissues with 616 partly donor-matched samples. We find a shift of the median ratio of Treg-to-oTL from 3–8% in healthy tissue to 18–25% in all tumor entities. Epigenetically determined oTL frequencies correlate with the outcome of colorectal and ovarian cancers. Together, our data show that the composition of immune cells in tumor microenvironments can be quantitatively assessed by epigenetic measurements. This composition is disturbed in solid tumors, indicating a fundamental mechanism of tumor immune evasion. Epigenetic quantification of T-lymphocytes serves as independent clinical parameter for outcome prognosis.
PMCID: PMC3278789  PMID: 20962591
tumor immunology; regulatory T cells; T-lymphocytes; epigenetic immunophenotyping; DNA methylation
6.  SiPaGene: A new repository for instant online retrieval, sharing and meta-analyses of GeneChip® expression data 
BMC Genomics  2009;10:98.
Microarray expression profiling is becoming a routine technology for medical research and generates enormous amounts of data. However, reanalysis of public data and comparison with own results is laborious. Although many different tools exist, there is a need for more convenience and online analysis with restriction of access and user specific sharing options. Furthermore, most of the currently existing tools do not use the whole range of statistical power provided by the MAS5.0/GCOS algorithms.
With a current focus on immunology, infection, inflammation, tissue regeneration and cancer we developed a database platform that can load preprocessed Affymetrix GeneChip expression data for immediate access. Group or subgroup comparisons can be calculated online, retrieved for candidate genes, transcriptional activity in various biological conditions and compared with different experiments. The system is based on Oracle 9i with algorithms in java and graphical user interfaces implemented as java servlets. Signals, detection calls, signal log ratios, change calls and corresponding p-values were calculated with MAS5.0/GCOS algorithms. MIAME information and gene annotations are provided via links to GEO and EntrezGene. Users access via https protocol their own, shared or public data. Sharing is comparison- and user-specific with different levels of rights. Arrays for group comparisons can be selected individually. Twenty-two different group comparison parameters can be applied in user-defined combinations on single or multiple group comparisons. Identified genes can be reviewed online or downloaded. Optimized selection criteria were developed and reliability was demonstrated with the "Latin Square" data set. Currently more than 1,000 arrays, 10,000 pairwise comparisons and 500 group comparisons are presented with public or restricted access by different research networks or individual users.
SiPaGene is a repository and a high quality tool for primary analysis of GeneChips. It exploits the MAS5.0/GCOS pairwise comparison algorithm, enables restricted access and user specific sharing. It does not aim for a complete representation of all public arrays but for high quality analysis with stepwise integration of reference signatures for detailed meta-analyses. Development of additional tools like functional annotation networks based on expression information will be future steps towards a systematic biological analysis of expression profiles.
PMCID: PMC2657156  PMID: 19265543
7.  Synthesis, Storage, and Release of Vascular Endothelial Growth Factor/Vascular Permeability Factor (VEGF/VPF) by Human Mast Cells: Implications for the Biological Significance of VEGF206 
Molecular Biology of the Cell  1998;9(4):875-884.
Mast cells have been implicated in various diseases that are accompanied by neovascularization. The exact mechanisms by which mast cells might mediate an angiogenic response, however, are unclear and therefore, we have investigated the possible expression of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in the human mast cell line HMC-1 and in human skin mast cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that mast cells constitutively express VEGF121, VEGF165, and VEGF189. After a prolonged stimulation of cells for 24 h with phorbol 12-myristate 13-acetate (PMA) and the ionophore A23187, an additional transcript representing VEGF206 was detectable, as could be verified by sequence analysis. These results were confirmed at the protein level by Western blot analysis. When the amounts of VEGF released under unstimulated and stimulated conditions were compared, a significant increase was detectable after stimulation of cells. Human microvascular endothelial cells (HMVEC) responded to the supernatant of unstimulated HMC-1 cells with a dose-dependent mitogenic effect, neutralizable up to 90% in the presence of a VEGF-specific monoclonal antibody. Flow cytometry and postembedding immunoelectron microscopy were used to detect VEGF in its cell-associated form. VEGF was exclusively detectable in the secretory granules of isolated human skin mast cells. These results show that both normal and leukemic human mast cells constitutively express bioactive VEGF. Furthermore, this study contributes to the understanding of the physiological role of the strongly heparin-binding VEGF isoforms, since these were found for the first time to be expressed in an activation-dependent manner in HMC-1 cells.
PMCID: PMC25314  PMID: 9529385

Results 1-7 (7)