Autoimmunity is intrinsically driven by memory T and B cell clones inappropriately targeted at self-antigens. Selective depletion or suppression of self-reactive T cells remains a holy grail of autoimmune therapy, but disease-associated T cell receptors (TCRs) and cognate antigenic epitopes remained elusive. A TRBV9-containing CD8 + TCR motif was recently associated with the pathogenesis of ankylosing spondylitis, psoriatic arthritis and acute anterior uveitis, and cognate HLA-B*27-presented epitopes were identified. Following successful testing in nonhuman primate models, here we report human TRBV9 + T cell elimination in ankylosing spondylitis. The patient achieved remission within 3 months and ceased anti-TNF therapy after 5 years of continuous use. Complete remission has now persisted for 4 years, with three doses of anti-TRBV9 administered per year. We also observed a profound improvement in spinal mobility metrics and the Bath Ankylosing Spondylitis Metrology Index (BASMI). This represents a possibly curative therapy of an autoimmune disease via selective depletion of a TRBV-defined group of T cells. The anti-TRBV9 therapy could potentially be applicable to other HLA-B*27-associated spondyloarthropathies. Such targeted elimination of the underlying cause of the disease without systemic immunosuppression could offer a new generation of safe and efficient therapies for autoimmunity.

Spondyloarthritis (SpA) comprises a number of inflammatory rheumatic diseases with overlapping clinical manifestations. Strong association with several HLA-I alleles and T cell infiltration into an inflamed joint suggest involvement of T cells in SpA pathogenesis. In this study, we performed high-throughput T cell repertoire profiling of synovial fluid (SF) and peripheral blood (PB) samples collected from a large cohort of SpA patients. We showed that synovial fluid is enriched with expanded T cell clones that are shared between patients with similar HLA genotypes and persist during recurrent synovitis. Using an algorithm for identification of TCRs involved in immune response we discovered several antigen-driven CD8+ clonal groups associated with risk HLA-B*27 or HLA-B*38 alleles. We further show that these clonal groups were enriched in SF and had higher frequency in PB of SpA patients vs healthy donors, implying their relevance to SpA pathogenesis. Several of the groups were shared among patients with different SpAs that suggests a common immunopathological mechanism of the diseases. In summary, our results provide evidence for the role of specific CD8+ T cell clones in pathogenesis of SpA.

Adoptive cell transfer (ACT) is a promising approach to cancer immunotherapy, but its efficiency fundamentally depends on the extent of tumor-specific T cell enrichment within the graft. This can be estimated via activation with identifiable neoantigens, tumor-associated antigens (TAAs), or living or lysed tumor cells, but these approaches remain laborious, time-consuming, and functionally limited, hampering clinical development of ACT. Here, we demonstrate that homology cluster analysis of T cell receptor (TCR) repertoires efficiently identifies tumor-reactive TCRs allowing to: (1) detect their presence within the pool of tumor-infiltrating lymphocytes (TILs); (2) optimize TIL culturing conditions, with IL-2low/IL-21/anti-PD-1 combination showing increased efficiency; (3) investigate surface marker-based enrichment for tumor-targeting T cells in freshly isolated TILs (enrichment confirmed for CD4+ and CD8+ PD-1+/CD39+ subsets), or re-stimulated TILs (informs on enrichment in 4-1BB-sorted cells). We believe that this approach to the rapid assessment of tumor-specific TCR enrichment should accelerate T cell therapy development.

Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.

Objective:The risk of AS is associated with genomic variants related to antigen presentation and specific cytokine signalling pathways, suggesting the involvement of cellular immunity in disease initiation/progression. The aim of the present study was to explore the repertoire of TCR sequences in healthy donors and AS patients to uncover AS-linked TCR variants.Methods:Using quantitative molecular-barcoded 5'-RACE, we performed deep TCR β repertoire profiling of peripheral blood (PB) and SF samples for 25 AS patients and 108 healthy donors. AS-linked TCR variants were identified using a new computational approach that relies on a probabilistic model of the VDJ rearrangement process.Results:Using the donor-agnostic probabilistic model, we reveal a TCR β motif characteristic for PB of AS patients, represented by eight highly homologous amino acid sequence variants. Some of these variants were previously reported in SF and PB of patients with ReA and in PB of AS patients. We demonstrate that identified AS-linked clones have a CD8+ phenotype, present at relatively low frequencies in PB, and are significantly enriched in matched SF samples of AS patients.Conclusion:Our results suggest the involvement of a particular antigen-specific subset of CD8+ T cells in AS pathogenesis, confirming and expanding earlier findings. The high similarity of the clonotypes with the ones found in ReA implies common mechanisms for the initiation of the diseases.

Unique molecular identifiers (UMIs) show outstanding performance in targeted high-throughput resequencing, being the most promising approach for the accurate identification of rare variants in complex DNA samples. This approach has application in multiple areas, including cancer diagnostics, thus demanding dedicated software and algorithms. Here we introduce MAGERI, a computational pipeline that efficiently handles all caveats of UMI-based analysis to obtain high-fidelity mutation profiles and call ultra-rare variants. Using an extensive set of benchmark datasets including gold-standard biological samples with known variant frequencies, cell-free DNA from tumor patient blood samples and publicly available UMI-encoded datasets we demonstrate that our method is both robust and efficient in calling rare variants. The versatility of our software is supported by accurate results obtained for both tumor DNA and viral RNA samples in datasets prepared using three different UMI-based protocols.

Bioluminescent fungi are spread throughout the globe, but details on their mechanism of light emission are still scarce. Usually, the process involves three key components: an oxidizable luciferin substrate, a luciferase enzyme, and a light emitter, typically oxidized luciferin, and called oxyluciferin. We report the structure of fungal oxyluciferin, investigate the mechanism of fungal bioluminescence, and describe the use of simple synthetic α-pyrones as luciferins to produce multicolor enzymatic chemiluminescence. A high-energy endoperoxide is proposed as an intermediate of the oxidation of the native luciferin to the oxyluciferin, which is a pyruvic acid adduct of caffeic acid. Luciferase promiscuity allows the use of simple α-pyrones as chemiluminescent substrates.

The accuracy with which DNA polymerase can replicate a template DNA sequence is an extremely important property that can vary by an order of magnitude from one enzyme to another. The rate of nucleotide misincorporation is shaped by multiple factors, including PCR conditions and proofreading capabilities, and proper assessment of polymerase error rate is essential for a wide range of sensitive PCR-based assays. In this paper, we describe a method for studying polymerase errors with exceptional resolution, which combines unique molecular identifier tagging and high-throughput sequencing. Our protocol is less laborious than commonly-used methods, and is also scalable, robust and accurate. In a series of nine PCR assays, we have measured a range of polymerase accuracies that is in line with previous observations. However, we were also able to comprehensively describe individual errors introduced by each polymerase after either 20 PCR cycles or a linear amplification, revealing specific substitution preferences and the diversity of PCR error frequency profiles. We also demonstrate that the detected high-frequency PCR errors are highly recurrent and that the position in the template sequence and polymerase-specific substitution preferences are among the major factors influencing the observed PCR error rate.

Background: Recently we proposed efficient method to exclude undesirable primers at any stage of amplification reaction, here termed NOPE (NOnsense-mediated Primer Exclusion). According to this method, added oligonucleotide overlapping with the 3'-end of unwanted amplification primer (NOPE oligo) simultaneously provides a template for its elongation. This elongation disrupts specificity of unwanted primer, preventing its further participation in PCR. The suggested approach allows to rationally manage the course of PCR reactions in order to facilitate analysis of complex DNA mixtures as well as to perform multistage PCR bypassing intermediate purification steps.Results: Here we apply NOPE method to DNA library preparation for the high-throughput sequencing (HTS) with the PCR-based introduction of unique molecular identifiers (UMI). We show that NOPE oligo efficiently neutralizes UMI-containing oligonucleotides after introduction of UMI into sample DNA molecules, thus allowing to proceed with further amplification steps without purification and associated loss of starting material. At the same time, NOPE oligo does not affect the efficiency of target PCR amplification.Conclusion: We describe a simple, robust and cheap modification of UMI-labeled HTS libraries preparation procedure, that allows to bypass purification step and thus to preserve starting material which may be limited, e.g. circulating tumor DNA, circulating fetal DNA, or small amounts of isolated cells of interest. Furthermore, demonstrated simplicity and robustness of NOPE method should make it popular in various PCR protocols.

The diversity, architecture, and dynamics of the TCR repertoire largely determine our ability to effectively withstand infections and malignancies with minimal mistargeting of immune responses. In this study, we have employed deep TCRβ repertoire sequencing with normalization based on unique molecular identifiers to explore the long-term dynamics of T cell immunity. We demonstrate remarkable stability of repertoire, where approximately half of all T cells in peripheral blood are represented by clones that persist and generally preserve their frequencies for 3 y. We further characterize the extremes of lifelong TCR repertoire evolution, analyzing samples ranging from umbilical cord blood to centenarian peripheral blood. We show that the fetal TCR repertoire, albeit structurally maintained within regulated borders due to the lower numbers of randomly added nucleotides, is not limited with respect to observed functional diversity. We reveal decreased efficiency of nonsense-mediated mRNA decay in umbilical cord blood, which may reflect specific regulatory mechanisms in development. Furthermore, we demonstrate that human TCR repertoires are functionally more similar at birth but diverge during life, and we track the lifelong behavior of CMV- and EBV-specific T cell clonotypes. Finally, we reveal gender differences in dynamics of TCR diversity constriction, which come to naught in the oldest age. Based on our data, we propose a more general explanation for the previous observations on the relationships between longevity and immunity.

AbstractBackground:Fluorescence diffuse tomography (FDT) is the most accurate technique for the imaging of labeled tumors in the small animal body. However, the procedure for reconstruction of the spatial distribution of the fluorophore requires a high signal-to-noise ratio due to the ill-condition of the inverse problem. Therefore, the FDT technique is ineffective for imaging tumors of small size or with dim fluorophores because of the low intensity of their fluorescence compared with the high level of tissue autofluorescence. In these cases, the size and position of a marked tumor in the animal body can be estimated from two-dimensional fluorescence images obtained using trans- or epi-illumination techniques.Material and methods:A versatile system for small animal fluorescence imaging which combines planar epi- and trans-illumination geometries of the light source and of the fluorescence receiver was created and tested. For epi-illumination imaging, light-emitting diode sources were used to provide homogeneous and stable illumination of the experimental animal, in combination with a cooled CCD camera which covers the entire illuminated area. For trans-illumination imaging, mechanical raster-scanning devices modulated at a low frequency were used for the laser source, together with a cooled photomultiplier tube, which provided outstanding sensitivity.Results:Monitoring the orthotopic tumor growth in animal bodies has demonstrated the efficacy of trans-illumination imaging in comparison with the epi-illumination technique. The results obtained also showed that the effective use of the trans-illumination technique requires Born normalization of the fluorescence signal and the exclusion of lateral illumination by surrounding the animal with additional light absorption material using light-absorption pads on both sides of the body.

Most living cells maintain a continuous flow of electrons, which provides them with energy. Many compounds are present in a cell simultaneously in the oxidized and reduced states in the form of active redox couples. Some of the redox couples, such as NAD + /NADH, NADP + /NADPH, and oxidized/reduced glutathione (GSSG/GSH) are universal, as they are involved in the regulation of many cellular reactions. The ratio of the oxidized and reduced forms of these compounds is the important cellular redox parameter. Modern research approaches allow for revealing new functions of the main redox couples in complicated cellular processes. The review presents the information about the main cellular redox couples and their participation in various biological processes.

Leuchtende Pilze wurden von Aristoteles bereits im 4. Jahrhundert v. Chr. beschrieben. I. V. Yampolsky, J. I. Gitelson et al. decken in ihrer Zuschrift auf S. 8242 ff. die Struktur von Pilz-Luciferin auf, einer Verbindung, die für die Biolumineszenz von Pilzen verantwortlich ist. Leuchtende Pilze wurden von Aristoteles bereits im 4. Jahrhundert v. Chr. beschrieben. I. V. Yampolsky, J. I. Gitelson et al. decken in ihrer Zuschrift auf S. 8242 ff. die Struktur von Pilz-Luciferin auf, einer Verbindung, die für die Biolumineszenz von Pilzen verantwortlich ist. Krebsdiagnostik Ein Hypoxie erkennender Sauerstoffsensor für die Diagnose von Krebsmetastasierungen wird von X. Q. Jiang et al. auf S. 8212 ff. beschrieben. Nach systemischer Verabreichung des Sensors wird die Metastase von Krebszellen in die Lunge oder in Lymphknoten durch optische Ganzkörperbildgebung nachgewiesen.1 Elektroreduktion von NO Die entscheidende Rolle von Wasser für den Mechanismus der NO-Elektroreduktion auf Pt(111) untersuchen J. Greeley et al. in der Zuschrift auf S. 8373 ff. Wasser erleichtert den Elektronentransfer zu adsorbierten Oberflächenintermediaten mit sehr niedrigen kinetischen Barrieren.1 Porphyrinoide M. Bröring et al. präsentieren in ihrer Zuschrift auf S. 8331 ff. die erste radikalische freie Corrolbase. Der Verlust eines inneren H-Atoms des luftstabilen, leicht handhabbaren Porphyrinoids ergibt ein planares Molekül, das z. B. Zn2+ binden kann.1

Glowing fungi were described by Aristotle as early as the fourth century B.C. In their Communication on page 8124 ff., I. V. Yampolsky, J. I. Gitelson, and co-workers unveil the structure of fungal luciferin, a compound that is responsible for fungal biolumescence. Glowing fungi were described by Aristotle as early as the fourth century B.C. In their Communication on page 8124 ff., I. V. Yampolsky, J. I. Gitelson, and co-workers unveil the structure of fungal luciferin, a compound that is responsible for fungal biolumescence. Cancer Diagnostics A hypoxia-sensitive sensor for tracking cancer metastasis is reported by X. Q. Jiang et al. on page 8094 ff. After systemic administration of the sensor, cancer cells metastasizing to the lungs or to the lymph nodes are detected by whole-body optical imaging.1 NO Electroreduction The crucial role of water in determining the mechanism of NO electroreduction to ammonia on Pt(111) surfaces is investigated by J. Greeley et al. in their Communication on page 8255 ff. Water facilitates proton transfer to adsorbed surface intermediates with very low kinetic barriers.1 Porphyrinoids M. Bröring et al. present the first free-base corrole radical in their Communication on page 8213 ff. Loss of an inner hydrogen atom from the air-stable, easy-to-handle porphyrinoid gives a planar molecule that can, for example, bind Zn2+.1

High-throughput sequencing of T cell (TCR) receptor repertoires is a promising approach that can be used to characterize the state and dynamics of adaptive immunity and, potentially, to deduce the antigen specificity of the immune response [1].

Background: SypHer is a genetically encoded fluorescent pH-indicator with a ratiometric readout, suitable for measuring fast intracellular pH shifts. However, the relatively low brightness of the indicator limits its use.Methods: Here we designed a new version of pH-sensor called SypHer-2, which has up to three times brighter fluorescence in cultured mammalian cells compared to the SypHer.Results: Using the new indicator we registered activity-associated pH oscillations in neuronal cell culture. We observed prominent transient neuronal cytoplasm acidification that occurs in parallel with calcium entry. Furthermore, we monitored pH in presynaptic and postsynaptic termini by targeting SypHer-2 directly to these compartments and revealed marked differences in pH dynamics between synaptic boutons and dendritic spines. Finally, we were able to reveal for the first time the intracellular pH drop that occurs within an extended region of the amputated tail of the Xenopus laevis tadpole before it begins to regenerate.Conclusions: SypHer2 is suitable for quantitative monitoring of pH in biological systems of different scales, from small cellular subcompartments to animal tissues in vivo.General significance: The new pH-sensor will help to investigate pH-dependent processes in both in vitro and in vivo studies. (C) 2015 Elsevier B.V. All rights reserved.

This work is aimed at the development of new approach to register intracellular pH with genetically encoded ratiometric sensor. Intracellular pH of cancer cells was studied in vitro and in vivo. Changes of intracellular pH under conditions of co-culturing with fibroblast were investigated.

Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

Of the various super-resolution techniques, stimulated emission depletion (STED) microscopy achieves the best temporal resolution at high spatial resolution, enabling live-cell imaging beyond the diffraction limit. However, STED and most other super-resolution imaging methods utilize a particular type of information extractable from the raw data, namely the positions of fluorophores. To expand on the use of super-resolution techniques, we report here the live-cell STED microscopy of a dynamic biosensor. Using the fluorescent H2O2 sensor HyPer2 for subdiffraction imaging, we were able not only to image filaments with superior resolution by localizing emission but also to trace H2O2 produced within living cell by monitoring brightness of the probe. STED microscopy of HyPer2 demonstrates potential utility of FP-based biosensors for super-resolution experiments in situ and in vivo.