B. D. C. van Schaik

Inflamed target tissue provides a specific niche for highly expanded T-cell clones in early human autoimmune disease

Objective To profile quantitatively the T-cell repertoire in multiple joints and peripheral blood of patients with recent onset (early) or established rheumatoid arthritis (RA) using a novel next-generation sequencing protocol to identify potential autoreactive clones. Methods Synovium of patients with recent onset (early) RA (<6 months) (n=6) or established RA (>18 months) (n=6) was screened for T-cell clones by sequencing over 10 000 T-cell receptors (TCR) per sample. T cells from paired blood samples were analysed for comparison. From two patients synovial T cells were obtained from multiple inflamed joints. The degree of expansion of each individual clone was based on its unique CDR3 sequence frequency within a sample. Clones with a frequency of over 0.5% were considered to be highly expanded clones (HEC). Results In early RA synovium, the T-cell repertoire was dominated by 35 HEC (median, range 2–70) accounting for 56% of the TCR sequenced. The clonal dominance in the synovium was patient specific and significantly greater than in established RA (median of 11 HEC (range 5–24) in established RA synovium accounting for 9.8% of T cells; p<0.01). 34% (range 28–40%) of the most expanded T-cell clones were shared between different joints in the same patients, compared with only 4% (range 0–8%) between synovium and blood (p=0.01). Conclusions In RA, a systemic autoimmune disease, the inflamed synovium forms a niche for specific expanded T-cell clones, especially in early disease. This suggests that, at least in RA, autoreactive T cells should be addressed specifically in the inflamed tissue, preferably in the early phase of the disease.

Deep Sequencing of Antiviral T-Cell Responses to HCMV and EBV in Humans Reveals a Stable Repertoire That Is Maintained for Many Years

CD8+ T-cell responses against latent viruses can cover considerable portions of the CD8+ T-cell compartment for many decades, yet their initiation and maintenance remains poorly characterized in humans. A key question is whether the clonal repertoire that is raised during the initial antiviral response can be maintained over these long periods. To investigate this we combined next-generation sequencing of the T-cell receptor repertoire with tetramer-sorting to identify, quantify and longitudinally follow virus-specific clones within the CD8+ T-cell compartment. Using this approach we studied primary infections of human cytomegalovirus (hCMV) and Epstein Barr virus (EBV) in renal transplant recipients. For both viruses we found that nearly all virus-specific CD8+ T-cell clones that appeared during the early phase of infection were maintained at high frequencies during the 5-year follow-up and hardly any new anti-viral clones appeared. Both in transplant recipients and in healthy carriers the clones specific for these latent viruses were highly dominant within the CD8+ T-cell receptor Vβ repertoire. These findings suggest that the initial antiviral response in humans is maintained in a stable fashion without signs of contraction or changes of the clonal repertoire.

Rheumatoid arthritis synovial tissue harbours dominant B-cell and plasma-cell clones associated with autoreactivity

Objective To identify potential autoreactive B-cell and plasma-cell clones by quantitatively analysing the complete human B-cell receptor (BCR) repertoire in synovium and peripheral blood in early and established rheumatoid arthritis (RA). Methods The BCR repertoire was screened in synovium and blood of six patients with early RA (ERA) (<6 months) and six with established RA (ESRA) (>20 months). In two patients, the repertoires in different joints were compared. Repertoires were analysed by next-generation sequencing from mRNA, generating >10 000 BCR heavy-chain sequence reads per sample. For each clone, the degree of expansion was calculated as the percentage of the total number of reads encoding the specific clonal sequence. Clones with a frequency ≥0.5% were considered dominant. Results Multiple dominant clones were found in inflamed synovium but hardly any in blood. Within an individual patient, the same dominant clones were detected in different joints. The majority of the synovial clones were class-switched; however, the fraction of clones that expressed IgM was higher in ESRA than ERA patients. Dominant synovial clones showed autoreactive features: in ERA in particular the clones were enriched for immunoglobulin heavy chain gene segment V4–34 (IGHV4–34) and showed longer CDR3 lengths. Dominant synovial clones that did not encode IGHV4–34 also had longer CDR3s than peripheral blood. Conclusions In RA, the synovium forms a niche where expanded—potentially autoreactive—B cells and plasma cells reside. The inflamed target tissue, especially in the earliest phase of disease, seems to be the most promising compartment for studying autoreactive cells.

Genome Sequence of Neisseria meningitidis Serogroup B Strain H44/76

Neisseria meningitidis is an obligate human pathogen. While it is a frequent commensal of the upper respiratory tract, in some individuals the bacterium spreads to the bloodstream, causing meningitis and/or sepsis, which are serious conditions with high morbidity and mortality. Here we report the availability of the genome sequence of the widely used serogroup B laboratory strain H44/76.

Provenance for distributed biomedical workflow execution

Scientific research has become very data and compute intensive because of the progress in data acquisition and measurement devices, which is particularly true in Life Sciences. To cope with this deluge of data, scientists use distributed computing and storage infrastructures. The use of such infrastructures introduces by itself new challenges to the scientists in terms of proper and efficient use. Scientific workflow management systems play an important role in facilitating the use of the infrastructure by hiding some of its complexity. Although most scientific workflow management systems are provenance-aware, not all of them come with provenance functionality out of the box. In this paper we describe the improvement and integration of a provenance system into an e-infrastructure for biomedical research based on the MOTEUR workflow management system. The main contributions of the paper are: presenting an OPM implementation using relational database backend for the provenance store, providing an e-infrastructure with a comprehensive provenance system, defining a generic approach to provenance implementation, potentially suitable for other workflow systems and application domains and demonstrating the value of this system based on use cases presenting the provenance data through a user-friendly web interface.

Complete T and B cell receptor repertoire analysis in rheumatoid arthritis using high throughput sequencing

T cells and B cells are likely to have important roles in the pathogenesis of rheumatoid arthritis (RA). Previous attempts to investigate the role of T and B cell clones in RA by screening the T/B cell receptor (TCR/BCR) repertoires were hampered by the sheer size and complexity of the repertoires. Here the authors use a novel high throughput sequencing-based protocol which overcomes current technological limitations and produces DNA sequences of >100 000 receptors in a single experiment. Using this technique, the authors …

OP0172 Expanded t-cell clones present in synovium at onset of rheumatoid arthritis are already present in the synovium in the seropositive “at risk” stage

Background T-cells are thought to be key players in the initiation and progression of rheumatoid arthritis (RA). Earlier we showed that already at the seropositive “at risk” stage uninflamed synovial tissue contains T-cell infiltrates1. In another study we showed that inflamed synovium selectively harbours expanded T-cell clones that are hardly present in paired blood samples2. Objectives Following up on these observations, we longitudinally investigated whether the same expanded T-cell clones found in the inflamed synovial tissue at onset of RA are already present in the synovium in the seropositive “at risk” stage. Methods Fifty-five individuals without arthritis but seropositive for IgM rheumatoid factor and/or anti-citrullinated protein antibody (ACPA) were prospectively followed. In five aCCP+ individuals synovial biopsies and paired blood samples at inclusion (“at risk” stage) and after development of RA (ACR2010 criteria; mean time to arthritis 27 months (range 11.7–47.3)) were available for analysis. T-cell clones were identified by their unique TCRβ sequence using RNA-based next generation sequencing3. For each sample, 3570 TCRβ sequences were analysed. Clones with a frequency of ≥0.5% were arbitrarily considered as highly expanded clones (HECs). ANOVA and t-test were used for statistical analysis. Results T-cell repertoires in “at risk” and RA synovium were similar (mean (± SD) number of clones 488±70 vs 567±204 respectively, p=0.46), number of HECs (37±7 vs 31±18, p=0.41) and the impact of HECs collectively on the TCR repertoire (mean 48% ± 13% vs 50% ± 20%, p=0.84). Interestingly, of the HECs present in the synovium at onset of arthritis 23% (±9%) were already present as HECs in the synovium at the seropositive “at risk” stage. This overlap was significantly higher than that with paired blood samples taken at the arthritis (3% ± 3%; p=0.01) or at the seropositive “at risk” stage (5% ± 7%; p=0.01; Figure 1a-c patient example; Figure 1d summary of results). Further characterization of the synovial CDR3 sequences (length, total charge, polar, aromatic and aliphatic side chains) showed no significant differences between RA HECs that were and those that were not expanded in the seropositive “at risk” stage. Conclusions Many T-cell clones found in early RA synovial tissue are already present in the pre-clinical “at risk” phase. The resemblance in TCR repertoires indicates that the process leading to disease – at least at the T-cell level – constitutes a smooth development. These clones, being already present in the very early stage of this disease and persisting as dominant clones during contraction of active arthritis, form attractive candidates for further characterization. References de Hair MJ et al. Arthritis Rheum. 2014. Klarenbeek PL et al. Ann Rheum Dis. 2012. Klarenbeek PL et al. Immunol Lett. 2010. Disclosure of Interest None declared

OP0203 In Rheumatoid Arthritis Synovitis Is Not Dominated by Polymorphic Local, but Rather by Uniform Systemic T Cell Responses

Background Genetic and immunological evidence clearly points to a pathogenic role of T cell clones in rheumatoid arthritis (RA). Previous studies of T cell receptor (TCR) repertoires in the synovial tissue (ST) of individual inflamed joints showed highly expanded T cell clones; however, these clones had background frequencies in peripheral blood (PB)1. Objectives To gain more insight into the potential role of these expanded T cell clones in RA, and to study whether single ST biopsies and synovial fluid (SF) samples are representative for the clonal T-cell disturbances observed, we analyzed the distribution of these highly expanded T cell clones in ST and SF samples from different joints in the same patient at the same time point. Methods In 7 RA-patients we simultaneously obtained ST biopsies from two inflamed joints via mini-arthroscopy together with paired PB and SF samples. ST biopsies were obtained from ankle or from the infrapatellar region of the knee. In 6 patients we took additional independent suprapatellar (SP) biopsies within the same knee joint. Samples were processed for RNA-based next generation sequencing. T cell clones were identified by their unique TCR β-chain sequence, the degree of expansion being expressed as a percentage of the total number of reads. To test for dissimilarity between different sites the Morisita index was used. This index originates from the field of biodiversity and gives a value between 0 and 1, values near 0 resemble no overlap between two locations while values close to 1 indicate similarity2. A One-tailed Mann-Whitney test was performed where applicable. Results We identified 1,114,386 clones in 7,890,571 TCR-sequences. Only a small proportion of the clones present in ST were retrieved in the same proportion in SF (Morisita Index 0.07, SD 0.20) and PB (0.006, SD 0.14). In contrast, considerable overlap was seen if we analyzed cellular infiltrate in ST biopsies from different regions of the same joint: 0.55 (SD 0.28). An identical overlap was seen if we analyzed ST from different joints in the same patient: 0.54 (SD 0.26). This overlap was significantly different from that between ST and PB (p<0.005) and ST and SF (p<0.05). Conclusions SF and PB T cells do not fully reflect the repertoire of dominant clones in the ST, suggesting that analysis of biopsies rather than SF or PB is preferred. ST biopsies taken from different locations within the same joint show substantial overlap suggesting that single biopsies, e.g. taken by ultrasound, show a representative snapshot of the ST T cell infiltrate. Interestingly, ST biopsies from different joints also show substantial overlap of the most dominant T cell clones in patients with active RA. This indicates that in individual patients a limited number of T cell clones dominate the adaptive immune response in the ST. Detailed cellular characterization of these clones seems warranted, and may help to devise more selective strategies for therapeutic targeting. References Klarenbeek P, et al. Ann. Rheum. Dis. 2012;71(6):1088–1093. Wolda H. Oecologia 1981;50:296–302. Disclosure of Interest A. Musters: None declared, P. Klarenbeek: None declared, M. Doorenspleet: None declared, R. Esveldt Employee of: BTCURE, a research project from the Innovative Medicines Initiative Joint Undertaking (grant No 115142–2)., B. van Schaik: None declared, A. Jongejan: None declared, S. Tas: None declared, A. van Kampen: None declared, F. Baas: None declared, N. de Vries Grant/research support from: BTCURE, a research project from the Innovative Medicines Initiative Joint Undertaking (grant No 115142–2).

Exploring Dynamic Enactment of Scientific Workflows Using Pilot-Abstractions

Current workflow abstractions in general lack: (a) an adequate approach to handle distributed data and (b) proper separation between logical tasks and data-flow from their mapping onto physical locations. As the complexity and dynamism of data and processing distribution have increased, optimized mapping of logical tasks to physical resources have become a necessity to avoid bottlenecks. We argue that the management of dynamic data and compute should become part of the runtime system of workflow engines to enable workflows to scale as necessary to address big data challenges and fully exploit distributed computing infrastructures (DCI). In this paper we explore how the P* model for pilot-abstractions, which proposes a clear separation between the logical compute and data units and their realization as a job or a file in some physical resource, could provide these capabilities for such a runtime environment. The Pilot-API provides a general-purpose interface to pilot-abstractions and the ability to assign compute and data resources to them. We share our experience of using the case study of a DNA sequencing pipeline, to re-implement the workflow using the Pilot-API. This first exercise, which resulted in a running application that is discussed here, illustrates the potential of this API to address (a) and (b). Our initial results indicate that the pilot abstractions (as captured by the P* model)offer an interesting approach to explore the design of a new generation of workflow management systems and runtime environments that are capable of intelligently deciding on application-aware late binding to physical resources.

hCMV-Specific CD8+ T Cells in Lymph Nodes from Renal Transplant Recipients Contain ‘True’ Memory Cells: 1440

Remmerswaal E.B.M.1,2, Klarenbeek P.M.3, Doorenspleet M.E.3, van Schaik B.D.C.4, van Donselaar K.A.M.I.2, Bemelman F.2, Esveldt R.E.E.3, van Kampen A.H.4, Baas F.5, ten Brinke A.6, van Lier R.A.W.1,6, de Vries N.3, ten Berge I.J.M.2 1AMC, Dept. of Exp. Immunology, Amsterdam, Netherlands, 2AMC, Renal Transplant Unit, Dept of Nephrology, Div of Int Medicine, Amsterdam, Netherlands, 3AMC, Dept of Clin Immunology and Rheumatology, Amsterdam, Netherlands, 4AMC, Dept of Clin Epidemiology, Amsterdam, Netherlands, 5AMC, Biostatistics and Bioinformatics, Dept of Genome Analysis, Amsterdam, Netherlands, 6Sanquin, Landsteiner Laboratory, Amsterdam, Netherlands