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T lymphocytes accumulate in inflamed tissues of patients with chronic inflammatory diseases (CIDs) and express pro-inflammatory cytokines upon re-stimulation in vitro. Further, a significant genetic linkage to MHC genes suggests that T lymphocytes play an important role in the pathogenesis of CIDs including juvenile idiopathic arthritis (JIA). However, the functions of T lymphocytes in established disease remain elusive. Here we dissect the transcriptional and the clonal heterogeneity of synovial T lymphocytes in JIA patients by single-cell RNA sequencing combined with T cell receptor profiling on the same cells. We identify clonally expanded subpopulations of T lymphocytes expressing genes reflecting recent activation by antigen in situ. A PD-1+ TOX+ EOMES+ population of CD4+ T lymphocytes expressed immune regulatory genes and chemoattractant genes for myeloid cells. A PD-1+ TOX+ BHLHE40+ population of CD4+ , and a mirror population of CD8+ T lymphocytes expressed genes driving inflammation, and genes supporting B lymphocyte activation in situ. This analysis points out that multiple types of T lymphocytes have to be targeted for therapeutic regeneration of tolerance in arthritis.

It is a matter of current debate whether the bone marrow is a hub for circulating memory T lymphocytes and/or the home of resident memory T lymphocytes. Here we demonstrate for CD69+ murine CD8+ , and CD69+ murine and human CD4+ memory T lymphocytes of the bone marrow, making up between 30 and 60% of bone marrow memory T lymphocytes, that they express the gene expression signature of tissue-resident memory T lymphocytes. This suggests that a substantial proportion of bone marrow memory T lymphocytes are resident. It adds to previous evidence that bone marrow memory T cells are resting in terms of mobility and proliferation, and maintain exclusive long-term memory to distinct, systemic antigens.

Conflicting evidence has been provided as to whether induction of intestinal inflammation by adoptive transfer of naïve T cells into Rag-/- mice requires expression of the transcription factor T-bet by the T cells. Here, we formally show that the intestinal microbiota composition of the Rag-/- recipient determines whether or not T-bet-deficient Th cells can induce colitis and we have resolved the differences of the two microbiomes, permissive or non-permissive to T-bet-independent colitis. Our data highlight the dominance of the microbiota over particular T cell differentiation programs in the pathogenesis of chronic intestinal inflammation. © 2017 WILEY-VCH Verlag GmbH Co. KGaA, Weinheim.

Multiparametric fluorescence and mass cytometry offers new perspectives to disclose and to monitor the high diversity of cell populations in the peripheral blood for biomarker research. While high-end cytometric devices are currently available to detect theoretically up to 120 individual parameters at the single cell level, software tools are needed to analyze these complex datasets automatically in acceptable time and without operator bias or knowledge. We developed an automated analysis pipeline, immunoClust, for uncompensated fluorescence and mass cytometry data, which consists of two parts. First, cell events of each sample are grouped into individual clusters. Subsequently, a classification algorithm assorts these cell event clusters into populations comparable between different samples. The clustering of cell events is designed for datasets with large event counts in high dimensions as a global unsupervised method, sensitive to identify rare cell types even when next to large populations. Both parts use model-based clustering with an iterative expectation maximization algorithm and the integrated classification likelihood to obtain the clusters. A detailed description of both algorithms is presented. Testing and validation was performed using 1) blood cell samples of defined composition that were depleted of particular cell subsets by magnetic cell sorting, 2) datasets of the FlowCAP III challenges to identify populations of rare cell types and 3) high-dimensional fluorescence and mass-cytometry datasets for comparison with conventional manual gating procedures. In conclusion, the immunoClust-algorithm is a promising tool to standardize and automate the analysis of high-dimensional cytometric datasets. As a prerequisite for interpretation of such data, it will support our efforts in developing immunological biomarkers for chronic inflammatory disorders and therapy recommendations in personalized medicine. immunoClust is implemented as an R-package and is provided as source code from www.bioconductor.org.

Biochemical, molecular and genetic studies emphasize the role of the potato vacuolar invertase Pain-1 in the accumulation of reducing sugars in potato tubers upon cold storage, and thereby its influence on the quality of potato chips and French fries. Previous studies showed that natural Pain-1 cDNA alleles were associated with better chip quality and higher tuber starch content. In this study, we focused on the functional characterization of these alleles. A genotype-dependent transient increase of total Pain-1 transcript levels in cold-stored tubers of six different genotypes as well as allele-specific expression patterns were detected. 3D modelling revealed putative structural differences between allelic Pain-1 proteins at the molecule's surface and at the substrate binding site. Furthermore, the yeast SUC2 mutant was complemented with Pain-1 cDNA alleles and enzymatic parameters of the heterologous expressed proteins were measured at 30 and 4 °C. Significant differences between the alleles were detected. The observed functional differences between Pain-1 alleles did not permit final conclusions on the mechanism of their association with tuber quality traits. Our results show that natural allelic variation at the functional level is present in potato, and that the heterozygous genetic background influences the manifestation of this variation.

Context Antibody-mediated rejection (ABMR) after kidney transplantation (KTx) remains the crucial obstacle to successful long-term graft function. The identification of gene signatures involved in ABMR could grant the basis for better prevention and treatment strategies. Objective The identification of gene signatures in whole blood cells specific for ABMR after KTx. Materials and methods Total RNA from blood cells of 16 kidney-transplanted patients with ABMR, stable graft function (SGF), and with T-cell-mediated rejection (TCMR) was isolated. Gene expression was determined by high-throughput sequencing followed by validation and analyses of differentially expressed candidates on mRNA level and on protein level in a large patient cohort (n = 185) in patients with SGF, urinary tract infection (UTI), borderline rejection (BL), TCMR, ABMR, and interstitial fibrosis and tubular atrophy. Results From the 570 genes detected, 111 discriminated ABMR from SGF and TCMR. A distinct enrichment of interferon (IFN) type I and type II signature gene set was observed. The expression of candidate genes IFIT1, ETV7, and RSAD2 distinguished ABMR patients from patients with SGF and also TCMR, whereas ETV7 and RSAD2 differentiated ABMR also from BL. Conclusion The IFN-inducible genes ETV7 and RSAD2 represent specific biomarkers for ABMR episodes after KTx.

A nonribosomal peptide synthetase (NRPS) in Schizosaccharomyces pombe, which possesses an unusual structure incorporating three adenylation domains, six thiolation domains and six condensation domains, has been shown to produce the cyclohexapeptide siderophore ferrichrome. One of the adenylation domains is truncated and contains a distorted key motif. Substrate-binding specificities of the remaining two domains were assigned by molecular modelling to glycine and to N-acetyl-N-hydroxy-L-ornithine. Hexapeptide siderophore synthetase genes of Magnaporthe grisea and Fusarium graminearum were both identified and analyzed with respect to substrate-binding sites, and the predicted product ferricrocin was identified in each. A comparative analysis of these synthetase systems, including those of the basidiomycete Ustilago maydis, the homobasidiomycete Omphalotus olearius and the ascomycetes Aspergillus nidulans, Aspergillus fumigatus, Fusarium graminearum, Cochliobolus heterostrophus, Neurospora crassa and Aureobasidium pullulans, revealed divergent domain compositions with respect to their number and positioning, although all produce similar products by iterative processes. A phylogenetic analysis of both NRPSs and associated L-N5-ornithine monooxygenases revealed that ferrichrome-type siderophore biosynthesis has coevolved in fungi with varying in trans interactions of NRPS domains.