[Show abstract][Hide abstract] ABSTRACT: Clinical studies investigating the impact of natural killer (NK) cells in allogeneic hematopoietic stem cell transplantation settings have yielded promising results. However, NK cells are a functionally and phenotypically heterogeneous population. Therefore, we addressed the functional relevance of specific NK cell subsets distinguished by expression of CD117, CD27 and CD11b surface markers in graft-versus-leukemia (GVL)-reaction and graft-versus-host-disease (GVHD). Our results clearly demonstrate that the subset of c-Kit−CD27−CD11b+ NK cells expressed multiple cytotoxic pathway genes and provided optimal graft-versus-leukemia-effects, while significantly reducing T cell proliferation induced by allogeneic dendritic cells. Furthermore, these NK cells migrated to inflamed intestinal tissues where graft-versus-host-colitis was efficiently mitigated. For the first time, we identified the c-Kit−CD27−CD11b+ NK cell population as the specific effector NK cell subset capable of significantly diminishing GVHD in fully mismatched bone marrow transplantation settings. In conclusion, the subset of c-Kit−CD27−CD11b+ NK cells not only supports GVL, but also plays a unique role in the protection against GVHD by migrating to the peripheral GVHD target organs where they exert efficient immunoregulatory activities. These new insights demonstrate the importance of selecting the optimal NK cell subset for cellular immunotherapy following allogeneic hematopoietic stem cell transplantation.
[Show abstract][Hide abstract] ABSTRACT: Basophils are known to modulate the phenotype of CD4+ T cells and to enhance Th2 responses in vitro and in vivo. In this study, we demonstrate that murine basophils inhibit proliferation of CD4+ T cells in autologous and allogeneic mixed lymphocyte reactions. The inhibition is independent of Fas and MHC class II, but dependent on activation of basophils with subsequent release of interleukin (IL)-4 and IL-6. The inhibitory effect of basophils on T cell proliferation can be blocked with antibodies against IL-4 and IL-6 and is absent in IL-4 / IL-6 double deficient mice. In addition, we show that basophils and IL-4 have beneficial effects on disease activity in a murine model of acute graft-versus-host-disease (GvHD). When basophils were depleted with the antibody MAR-1 before induction of GvHD, weight loss, GvHD score, mortality and plasma TNF levels were increased while injection of IL-4 improved GvHD. Basophil-depleted mice with GvHD also have increased numbers of CD4+ T cells in the mesenteric lymph nodes. Our data show for the first time that basophils suppress autologous and allogeneic CD4+ T cell proliferation in an IL-4 dependent manner.This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Efficient formation of early germinal centers (GCs) depends on the close interaction between GC B cells and antigen-primed CD4+ follicular helper T cells (TFH cells). A tight and stable formation of TFH/B cell-conjugates is required for cytokine-driven immunoglobulin class switching and somatic hypermutation (SHM) of GC B cells. Recently it has been shown that the formation of TFH/B cell-conjugates is crucial for B-cell differentiation and class switch following infection with Leishmania (L.) major parasites. However, the subtype of dendritic cells (DCs) responsible for TFH-cell priming against dermal antigens is thus far unknown. Utilizing a transgenic C57BL/6 mouse model designed to trigger the ablation of Langerin+ DC subsets in vivo, we show that the functionality of TFH/B cell-conjugates is disturbed after depletion of Langerhans cells (LCs): LC-depleted mice show a reduction in SHM in B cells isolated from TFH/B cell-conjugates and markedly reduced GC reactions within skin-draining lymph nodes. In conclusion this study reveals an indispensable role for LCs in promoting GC B-cell differentiation following cutaneous infection with L. major parasites. We propose that LCs are key regulators of GC formation and therefore have broader implications for the development of allergies and autoimmunity as well as for future vaccination strategies.This article is protected by copyright. All rights reserved
European Journal of Immunology 10/2014; 44(10). DOI:10.1002/eji.201344263 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Although advance care planning and the completion of advance directives (ADs) are important tools to avoid unwanted aggressive care once patients have lost their decision-making capacity, only a minority of cancer patients are admitted with completed ADs, and little is known about patients' wishes regarding AD consultations.
For 1 year, every new patient admitted to the hematology/oncology outpatient clinic of the University Hospital Regensburg received a self-administered questionnaire comprising a self-evaluation of AD knowledge and questions about preferences regarding consultation partners and the time of consultation. Disease-related data were collected from medical records. Statistics were calculated with SPSS.
Of the 500 questionnaires handed out, 394 (75%) were evaluable and analyzed. Twenty-eight percent of the participants had completed an AD (living will or health care proxy). Ninety-two percent of the participants without ADs had never received a consultation offer from any professional involved. Only 20% perceived a clear relation between cancer and AD consultations. More than 50% of the participants without ADs were in favor of consultations 'now' or 'in a few weeks', while more than 40% objected to AD consultations.
Oncology patients have a large unmet demand for AD consultations. However, a relevant percentage of these patients object to AD consultations. Structured and early AD consultation offers should be made, and early discussions about indications for aggressive treatment should take place.
[Show abstract][Hide abstract] ABSTRACT: Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.
[Show abstract][Hide abstract] ABSTRACT: Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.
[Show abstract][Hide abstract] ABSTRACT: Human blood monocytes comprise at least 3 subpopulations that differ in phenotype and function. Here, we present the first in-depth regulome analysis of human classical (CD14(++)CD16(-)), intermediate (CD14(+)CD16(+)), and nonclassical (CD14(dim)CD16(+)) monocytes. Cap analysis of gene expression adapted to Helicos single-molecule sequencing was used to map transcription start sites throughout the genome in all 3 subsets. In addition, global maps of H3K4me1 and H3K27ac deposition were generated for classical and nonclassical monocytes defining enhanceosomes of the 2 major subsets. We identified differential regulatory elements (including promoters and putative enhancers) that were associated with subset-specific motif signatures corresponding to different transcription factor activities and exemplarily validated novel downstream enhancer elements at the CD14 locus. In addition to known subset-specific features, pathway analysis revealed marked differences in metabolic gene signatures. Whereas classical monocytes expressed higher levels of genes involved in carbohydrate metabolism, priming them for anaerobic energy production, nonclassical monocytes expressed higher levels of oxidative pathway components and showed a higher mitochondrial routine activity. Our findings describe promoter/enhancer landscapes and provide novel insights into the specific biology of human monocyte subsets.
[Show abstract][Hide abstract] ABSTRACT: CD4(+)CD25(+)FOXP3(+) human regulatory T cells (Tregs) are essential for self-tolerance and immune homeostasis. Here, we describe the promoterome of CD4(+)CD25(high)CD45RA(+) naïve and CD4(+)CD25(high)CD45RA(-)memory Tregs and their CD25(-) conventional T-cell (Tconv) counterparts both before and after in vitro expansion by cap analysis of gene expression (CAGE) adapted to single-molecule sequencing (HeliScopeCAGE). We performed comprehensive comparative digital gene expression analyses and revealed novel transcription start sites, of which several were validated as alternative promoters of known genes. For all in vitro expanded subsets, we additionally generated global maps of poised and active enhancer elements marked by histone H3 lysine 4 monomethylation and histone H3 lysine 27 acetylation, describe their cell type-specific motif signatures, and evaluate the role of candidate transcription factors STAT5, FOXP3, RUNX1, and ETS1 in both Treg- and Tconv-specific enhancer architectures. Network analyses of gene expression data revealed additional candidate transcription factors contributing to cell type specificity and a transcription factor network in Tregs that is dominated by FOXP3 interaction partners and targets. In summary, we provide a comprehensive and easily accessible resource of gene expression and gene regulation in human Treg and Tconv subpopulations.
[Show abstract][Hide abstract] ABSTRACT: Next-generation sequencing of the hypervariable V3 region of the 16s rRNA gene isolated from serial stool specimens that had been collected from 31 patients receiving allogeneic stem cell transplantation was performed to elucidate variations in the composition of the intestinal microbiome in the course of allogeneic SCT. Metagenomic analysis was complemented by strain-specific enterococcal PCR and indirect assessment of bacterial load by liquid chromatography-tandem mass spectrometry of urinary indoxyl sulfate. At the time of admission, patients showed a predominance of commensal bacteria. After transplantation, a relative shift toward enterococci was observed, which was more pronounced under antibiotic prophylaxis and treatment of neutropenic infections. The shift was particularly prominent in patients that developed subsequently or suffered from active gastrointestinal GvHD. The mean proportion of enterococci in post-transplant stool specimens was 21 % in patients that did not develop gastrointestinal GvHD as compared to 46% in those that subsequently developed GI GvHD, and 74% at the time of active GvHD. Enterococcal PCR confirmed predominance of E. faecium or both, E. faecium and E. faecalis in these specimens. As a consequence of the loss of bacterial diversity, mean urinary indoxyl sulfate levels dropped from 42.5 (±11 μmol/L) to 11.8 (±2.8 μmol/L) in all post-transplant samples, and 3.5 (±3 μmol/L) in samples from patients with active GvHD. Our study reveals major microbiome shifts in the course of allogeneic stem cell transplantation, which occur in the period of antibiotic treatment but are more prominent in association with gastrointestinal GvHD. Our data indicate early microbiome shifts and a loss of diversity of the intestinal microbiome that may affect intestinal inflammation in the setting of allogeneic stem cell transplantation.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 01/2014; 20(5). DOI:10.1016/j.bbmt.2014.01.030 · 3.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytosine-guanosine dinucleotide (CpG) motifs are immunostimulatory components of bacterial DNA and activators of innate immunity through Toll-like receptor 9 (TLR9). Administration of CpG oligodeoxynucleotides before the onset of experimental colitis prevents intestinal inflammation by enforcement of regulatory mechanisms. It was investigated whether physiologic CpG/TLR9 interactions are critical for the homeostasis of the intestinal immune system.
Mesenteric lymph node cell and lamina propria mononuclear cell (LPMC) populations from BALB/c wild-type (wt) or TLR9 mice were assessed by flow cytometry and proteome profiling. Cytokine secretion was determined and nuclear extracts were analyzed for nuclear factor kappa B (NF-κB) and cAMP response-element binding protein activity. To assess the colitogenic potential of intestinal T cells, CD4-enriched cells from LPMC of wt or TLR9 donor mice were injected intraperitoneally in recipient CB-17 SCID mice.
TLR9 deficiency was accompanied by slight changes in cellular composition and phosphorylation of signaling proteins of mesenteric lymph node cell and LPMC. LPMC from TLR9 mice displayed an increased proinflammatory phenotype compared with wt LPMC. NF-κB activity in cells from TLR9 mice was enhanced, whereas cAMP response-element binding activity was reduced compared with wt. Transfer of lamina propria CD4-enriched T cells from TLR9 mice induced severe colitis, whereas wt lamina propria CD4-enriched T cells displayed an attenuated phenotype.
Lack of physiologic CpG/TLR9 interaction impairs the function of the intestinal immune system indicated by enhanced proinflammatory properties. Thus, physiologic CpG/TLR interaction is essential for homeostasis of the intestinal immune system as it is required for the induction of counterregulating anti-inflammatory mechanisms.
[Show abstract][Hide abstract] ABSTRACT: Single nucleotide polymorphisms (SNPs) have been associated with an increased incidence of invasive aspergillosis (IA) after
allogeneic stem cell transplantation (allo-SCT). We analyzed 41 patients with proven/probable IA after allo-SCT for an association
of SNPs, within the TLR2, TLR4, TLR5, TLR9, and NOD2/CARD15 genes, with susceptibility to IA. The control group consisted
of 130 patients who had allo-SCT but did not develop IA. While no association was found for donor SNPs and the recipients'
risk of IA, analysis of recipient SNPs showed a significant association between the presence of recipient TLR5-Stop SNP (1174C>
T) and the incidence of IA (P = 0.004). Multivariate analysis demonstrated that the recipient TLR5-Stop SNP appeared as an independent risk factor for
IA after allo-SCT. Our study suggests that TLR5 is involved in host defense against Aspergillus fumigatus, and that the recipient TLR5-Stop SNP represents a risk factor for the development of IA after allo-SCT.
Medical mycology: official publication of the International Society for Human and Animal Mycology 07/2013; 51(8). DOI:10.3109/13693786.2013.809630 · 2.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to assess current clinical practice in diagnosis and treatment of acute graft-versus-host disease (aGVHD) we performed a survey among German, Austrian and Swiss allogeneic hematopoietic stem cell transplant (alloHSCT) centers. Thirty-four (47%) of 72 contacted centers completed both the diagnostic and the therapeutic section of the survey representing 65% of alloHSCT activity within the participating countries in 2011. Three pediatric centers answered as requested only the diagnostic part of the survey. In the presence of diarrhea and decreased oral intake after engraftment only 4 centers (12%) do not perform any endoscopy prior to start of immunosuppressive treatment. In case of a skin rash with the differential diagnosis of drug reaction only 12 centers (35%) perform a skin biopsy upfront whereas 19 do so after failure of systemic steroids. In the presence of rapidly increasing cholestasis occurring without any other signs of aGVHD 11 centers (32%) perform a liver biopsy upfront and 14 only after failure of steroid treatment while 9 centers do not perform a liver biopsy at all. Twenty centers (59%) use a percutaneous, 12 a transvenous approach and 1 mini-laparoscopy for liver biopsies. First-line treatment of cutaneous aGVHD stage 1 consists of topical treatment alone in 17 of 31 responding centers (61%), while isolated cutaneous aGVHD stage 3 is treated with systemic steroids (prednisolone below 0.5 mg/kg/day n=2, 0.5-1.0 mg/kg/day n=10, above 1.0-2.5 mg/kg/day n=19) without or with topical agents (steroids n=10, calcineurin inhibitors n=3). In gastrointestinal (GI) manifestations of aGVHD 9 centers (29%) add topical to systemic steroids and 3 consider topical steroids as the only treatment for mild GI and cutaneous aGVHD. The choice of agent for second-line treatment as well as the sequence of administration are extremely heterogeneous most likely due to a lack of convincing data published. Most frequently used are mycophenolate mofetil (n=14), and extracorporeal photopheresis (n=10). Our survey also demonstrates that clinicians chose salvage therapies for steroid-refractory aGVHD based on their centers' own clinical experience.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 01/2013; 19(5). DOI:10.1016/j.bbmt.2013.01.018 · 3.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this retrospective analysis, 30 patients with acute GVHD (aGVHD) and 32 patients with chronic GVHD (cGVHD) treated with extracorporeal photopheresis (ECP) performed by the COBE Spectra System were evaluated. After 3 months of ECP treatment, a CR and PR were observed in 9 (30%) and 6 (20%) patients with aGVHD and in 2 (6%) and 12 (38%) patients with cGVHD. In 16 (53%) patients with aGVHD and 9 (28%) with cGVHD ECP treatment was already stopped after 3 months. One (3%) patient with aGVHD and 7 (22%) patients with cGVHD received new additional immunosuppressive therapy started during the first 3 months of ECP treatment and were classified as 'nonresponder' with regard to ECP. Of these patients a PR was achieved in one patient with aGVHD and in three patients with cGVHD. Steroids could be tapered by 50 in 83% of patients with aGVHD and in 29% of patients with cGVHD after 3 months of ECP treatment. Patients with aGVHD achieving a CR or PR showed a significant improved OS after allo-SCT (P=0.019). ECP is associated with significant response rates and successful reduction of steroids in patients with GVHD.Bone Marrow Transplantation advance online publication, 27 August 2012; doi:10.1038/bmt.2012.156.
Bone marrow transplantation 08/2012; 48(3). DOI:10.1038/bmt.2012.156 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve--especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4(high)/CD25(high)/CD45RA(high) 'regulatory T cells' and CD8(high)/CD62L(high)/CD45RA(neg) 'central memory T cells', have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.
PLoS ONE 04/2012; 7(4):e35798. DOI:10.1371/journal.pone.0035798 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg) are pivotal for peripheral self-tolerance. They prevent immune responses to auto- and alloantigens and are thus under close scrutiny as cellular therapeutics for autoimmune diseases and the prevention or treatment of alloresponses after organ or stem cell transplantation. We previously showed that human Treg with a memory cell phenotype, but not those with a naive phenotype, rapidly downregulate expression of the lineage-defining transcription factor FOXP3 upon in vitro expansion. We now compared the transcriptomes of stable FOXP3(+) Treg and converted FOXP3(-) ex-Treg by applying a newly developed intranuclear staining protocol that permits the isolation of intact mRNA from fixed, permeabilized, and FACS-purified cell populations. Whole-genome microarray analysis revealed strong and selective upregulation of Th2 signature genes, including GATA-3, IL-4, IL-5, and IL-13, upon downregulation of FOXP3. Th2 differentiation of converted FOXP3(-) ex-Treg occurred even under nonpolarizing conditions and could not be prevented by IL-4 signaling blockade. Thus, our studies identify Th2 differentiation as the default developmental program of human Treg after downregulation of FOXP3.
The Journal of Immunology 12/2011; 188(3):1275-82. DOI:10.4049/jimmunol.1102288 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Induction of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation along the kynurenine pathway, acts as a potent immunoregulatory loop. To address its role in human allogeneic stem cell transplantation, we measured major tryptophan metabolites, such as quinolinic acid and kynurenine, in serial urine specimens from 51 patients by liquid chromatography-tandem mass spectrometry. Samples were collected between admission and day 90 after transplantation, and metabolite levels were correlated with early clinical events and outcome. In selected patients, IDO gene expression was assessed by quantitative RT-PCR in intestinal biopsies. Surviving patients had significantly lower metabolite levels on days 28, 42, and 90, respectively, compared with patients dying of GVHD and associated complications (n = 10). Kynurenine levels were directly correlated with severity and clinical course of GVHD: Mean urinary quinolinic acid levels were 4.5 ± 0.3 μmol/mmol creatinine in the absence of acute GVHD, 8.0 ± 1.1 μmol/mmol creatinine for GVHD grade 1 or 2, and 13.5 ± 2.7 μmol/mmol creatinine for GVHD grade 3 or 4 (P < .001), respectively. GVHD-dependent induction of IDO was further suggested by increased expression of IDO mRNA in intestinal biopsies from patients with severe GVHD. Our data indicate reactive release of kynurenines in GVHD-associated inflammation.