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Objectives An important limitation in granulomatosis with polyangiitis (GPA) is the lack of disease activity markers. Immunoglobulin G4-positive (IgG4+) B cells and plasma cells are implicated in the pathogenesis of GPA. We hypothesized that the presence of these cells in peripheral blood could serve as disease activity parameter in GPA. Methods We included 35 proteinase 3-antineutrophil cytoplasmic antibodies-positive patients with GPA in a cross-sectional study. Active disease was defined as Birmingham Vasculitis Activity Score (BVAS) ≥ 3 (n = 15), remission as BVAS of 0 (n = 17), and low disease activity (LDA) as BVAS of 1–2 and clinical remission (n = 3). Healthy subjects (n = 10), patients with systemic lupus erythematosus (n = 24), and patients with rheumatoid arthritis (n = 19) functioned as control subjects. An additional longitudinal study was performed in ten patients with GPA. Using a validated qPCR test, we measured the IgG4:IgG RNA ratio in all groups and compared the results with known biomarkers. Results The median qPCR score was higher in active GPA (21.4; IQR 12.1–29.6) than in remission/LDA (3.3; IQR 1.6–5.6) (Mann-Whitney U test, p

Objective To gain more insight into the dynamics of lymphocyte depletion and develop new predictors of clinical response to rituximab in rheumatoid arthritis (RA). Methods RNA-based next-generation sequencing was used to analyse the B cell receptor (BCR) repertoire in peripheral blood and synovial tissue samples collected from 24 seropositive patients with RA treated with rituximab. Clonal expansion, mutation load and clonal overlap were assessed in samples collected before, at week 4 and at week 16 or 24 after treatment and correlated to the patients’ clinical response. Results After 4 weeks of rituximab-induced B cell depletion, the peripheral blood BCR repertoire of treated patients consisted of fewer, more dominant and more mutated BCR clones. No significant changes in the synovial tissue BCR repertoire were detected until week 16 post-treatment, when a reduced clonal overlap with baseline and an increased mutation load were observed. In patients who were non-responders at month 3 (n=5) using the European League Against Rheumatism response criteria, peripheral blood samples taken at week 4 after rituximab treatment showed more dominant clones compared with moderate responders (n=9) (median (IQR): 36 (27–52) vs 18 (16–26); p Conclusion Significant changes in BCR clonality are observed in peripheral blood of patients 4 weeks after rituximab treatment, while changes in synovial tissue were observed at later time points. Incomplete depletion of the dominant baseline peripheral blood BCR repertoire in the first month of treatment might predict clinical non-response at 3 months.

Genetic and immunological evidence clearly points to a role for T cells in the pathogenesis of rheumatoid arthritis (RA). Selective targeting of such disease-associated T cell clones might be highly effective while having few side effects. However, such selective targeting may only be feasible if the same T cell clones dominate the immune response at different sites of inflammation. We leveraged high-throughput technology to quantitatively assess whether different T cell clones dominate the inflammatory infiltrate at various sites of inflammation in this prototypic autoimmune disease. In 13 RA patients, we performed quantitative next-generation sequencing–based human TCRβ repertoire analysis in simultaneously obtained samples from inflamed synovial tissue (ST) from distinct locations within one joint, from multiple joints, and from synovial fluid (SF) and peripheral blood (PB). Identical TCRβ clones dominate inflammatory responses in ST samples taken from different locations within a single joint and when sampled in different joints. Although overall ST–SF overlap was comparable to higher ST–ST values, the overlap in dominant TCRβ clones in ST–SF comparisons was much lower than ST–ST and comparable to the low ST–PB overlap. In individual RA patients, a limited number of TCRβ clones dominate the immune response in the inflamed ST regardless of the location within a joint and which joint undergoes biopsy; in contrast, there is limited overlap of ST with SF or PB TCR repertoires. This limited breadth of the T cell response in ST of the individual RA patient indicates that development of immunotherapies that selectively modulate dominant T cell responses might be feasible.

Background Rituximab (RTX) induces more than 98% depletion of the CD20 +B cells in blood after a single injection, yet 35% to 50% of RA treated patients show a poor response to the therapy1. Despite the identification of many different biomarkers, mostly in the B cell compartment2, adequate prediction of response to RTX treatment is still quite challenging. Objectives To test the hypothesis that non-response to rituximab can be predicted by analysing B-cell receptor (BCR) repertoire characteristics before and shortly after rituximab therapy. Methods Paired peripheral blood (PB) samples and synovial tissue (ST) samples were available from a total of 21 patients before therapy with RTX, and at 4 and 16/24 weeks after treatment. Next-generation sequencing was used to analyse the BCR repertoire, and asses the frequency of high expanded clones (HECs:>0.5% of the sequenced reads)3 and load of somatic hypermutation (SHM). Clinical response was evaluated at 6 month following EULAR response criteria. Results In spite of the complete depletion of B cells (measured using CD19) with conventional flow cytometry, we detect a complete BCR repertoire at week 4 and 16/24 after RTX treatment. The post-treatment PB BCR repertoire is composed of fewer, but more expanded and more mutated clones compared to baseline (figure 1). Non-response associates with a higher number of HECs at week 4 (p Conclusions Incomplete depletion of the baseline BCR clonal repertoire in peripheral blood within the first month of treatment predicts poor clinical response at 6 months, revealing the persistence of “rituximab-resistant” BCR clonal signatures associated with treatment failure. In all patients the PB BCR repertoire at 4 weeks after rituximab is dominated by few but highly expanded and highly mutated BCR clones, most likely CD20-negative plasmablasts, while less pronounced and delayed effects are observed in the ST BCR repertoire. References [1] Cohen SB, et al. Arthritis Rheum2006;54:2793–806. [2] Benucci M, et al. Autoimmun Rev2010;9:801–3. [3] Doorenspleet ME, et al. Ann Rheum Dis2014;73:756–628. Disclosure of Interest None declared

Human cytomegalovirus (hCMV) infection is characterized by a vast expansion of resting effector-type virus-specific T cells in the circulation. In mice, interleukin-7 receptor α (IL-7Rα)-expressing cells contain the precursors for long-lived antigen-experienced CD8 + T cells, but it is unclear if similar mechanisms operate to maintain these pools in humans. Here, we studied whether IL-7Rα-expressing cells obtained from peripheral blood (PB) or lymph nodes (LNs) sustain the circulating effector-type hCMV-specific pool. Using flow cytometry and functional assays, we found that the IL-7Rα + hCMV-specific T cell population comprises cells that have a memory phenotype and lack effector features. We used next-generation sequencing of the T cell receptor to compare the clonal repertoires of IL-7Rα + and IL-7Rα − subsets. We observed limited overlap of clones between these subsets during acute infection and after 1 year. When we compared the hCMV-specific repertoire between PB and paired LNs, we found many identical clones but also clones that were exclusively found in either compartment. New clones that were found in PB during antigenic recall were only rarely identical to the unique LN clones. Thus, although PB IL-7Rα-expressing and LN hCMV-specific CD8 + T cells show typical traits of memory-type cells, these populations do not seem to contain the precursors for the novel hCMV-specific CD8 + T cell pool during latency or upon antigen recall. IL-7Rα + PB and LN hCMV-specific memory cells form separate virus-specific compartments, and precursors for these novel PB hCMV-specific CD8 + effector-type T cells are possibly located in other secondary lymphoid tissues or are being recruited from the naive CD8 + T cell pool. IMPORTANCE Insight into the self-renewal properties of long-lived memory CD8 + T cells and their location is crucial for the development of both passive and active vaccination strategies. Human CMV infection is characterized by a vast expansion of resting effector-type cells. It is, however, not known how this population is maintained. We here investigated two possible compartments for effector-type cell precursors: circulating acute-phase IL-7Rα-expressing hCMV-specific CD8 + T cells and lymph node (LN)-residing hCMV-specific (central) memory cells. We show that new clones that appear after primary hCMV infection or during hCMV reactivation seldom originate from either compartment. Thus, although identical clones may be maintained by either memory population, the precursors of the novel clones are probably located in other (secondary) lymphoid tissues or are recruited from the naive CD8 + T cell pool.

Background Granulomatosis with Polyangiitis (GPA) is a form of vasculitis characterized by inflammation of blood vessels in lungs, kidneys and the ear, nose and throat region. Regular monitoring and treatment adjustments are common, as the disease activity tends to fluctuate over time. Unfortunately, good markers for disease activity are lacking. This leads to both over- and undertreatment. Immunoglobulin G4 positive (IgG4+) B-cells and plasma cells are implicated in the pathogenesis of GPA, but the level of serum IgG4 does not seem to be a good disease activity marker. Recently we developed a test that indirectly measures the presence of IgG4+ B-cells/plasma cells by measuring the IgG4:IgG RNA ratio 1 . We hypothesized that this test could be used as disease activity marker. Objectives To test the IgG4:IgG RNA ratio in peripheral blood as a disease activity marker in GPA. Methods 27 PR3+ ANCA+ positive GPA patients were included in this cross-sectional study. Mean age was 52 years, 56% were female, and 39% had active disease. For each patient the ESR, CRP, BVAS, and ANCA titre were measured and peripheral blood samples were obtained. Patients were defined as having active disease if the BVAS was ≥3. In addition we included 10 healthy controls, and 63 patients with other immune mediated inflammatory diseases (systemic lupus erythematosus (SLE) (n=24), rheumatoid arthritis (RA) (n=19), primary sclerosing cholangitis (PSC) (n=20)). A validated qPCR test was performed in all groups to measure the IgG4:IgG RNA ratio in peripheral blood samples 1 Results The median IgG4:IgG RNA ratio was significantly higher in the GPA cohort (5.7, IQR 2.6 – 19.7) compared to all control groups: 1.2 in SLE (0.7 - 3.3; p 2 =0.76, p Conclusions The IgG4:IgG RNA ratio distinguishes active GPA from GPA in remission with excellent specificity and sensitivity. Moreover the ratio shows a significant correlation with disease severity, in contrast to ESR, CRP and ANCA titre. Retesting in another, prospective study is indicated to validate the IgG4:IgG ratio as a novel, highly sensitive and specific marker of disease activity in GPA. References Doorenspleet ME et al. Hepatology 2016; 64(2): 501–7. Acknowledgements We thank D. van der Coelen for the technical assistance, the doctors of the vasculitis outpatient clinic, especially Dr. B.J.H. van der Born and Dr. A.E. Hak, for the contribution to patient inclusions, and all the patients that participated in this study. Disclosure of Interest None declared

Immunoglobulin repertoire sequencing has successfully been applied to identify expanded antigen-activated B-cell clones that play a role in the pathogenesis of immune disorders. One challenge is the selection of the Ag-specific B cells from the measured repertoire for downstream analyses. A general feature of an immune response is the expansion of specific clones resulting in a set of subclones with common ancestry varying in abundance and in the number of acquired somatic mutations. The expanded subclones are expected to have BCR affinities for the Ag higher than the affinities of the naive B cells in the background population. For these reasons, several groups successfully proceeded or suggested selecting highly abundant subclones from the repertoire to obtain the Ag-specific B cells. Given the nature of affinity maturation one would expect that abundant subclones are of high affinity but since repertoire sequencing only provides information about abundancies, this can only be verified with additional experiments, which are very labor intensive. Moreover, this would also require knowledge of the Ag, which is often not available for clinical samples. Consequently, in general we do not know if the selected highly abundant subclone(s) are also the high(est) affinity subclones. Such knowledge would likely improve the selection of relevant subclones for further characterization and Ag screening. Therefore, to gain insight in the relation between subclone abundancy and affinity, we developed a computational model that simulates affinity maturation in a single GC while tracking individual subclones in terms of abundancy and affinity. We show that the model correctly captures the overall GC dynamics, and that the amount of expansion is qualitatively comparable to expansion observed from B cells isolated from human lymph nodes. Analysis of the fraction of high- and low-affinity subclones among the unexpanded and expanded subclones reveals a limited correlation between abundancy and affinity and shows that the low abundant subclones are of highest affinity. Thus, our model suggests that selecting highly abundant subclones from repertoire sequencing experiments would not always lead to the high(est) affinity B cells. Consequently, additional or alternative selection approaches need to be applied.

T cells are key players in the chronic intestinal inflammation that characterises Crohn's disease. Here we aim to map the intestinal T-cell receptor [TCR] repertoire in patients with Crohn's disease, using next-generation sequencing technology to examine the clonality of the T-cell compartment in relation to mucosal inflammation and response to therapy. Biopsies were taken from endoscopically inflamed and uninflamed ileum and colon of 19 patients with Crohn's disease. From this cohort, additional biopsies were taken after 8 weeks of remission induction therapy from eight responders and eight non-responders. Control biopsies from 11 patients without inflammatory bowel disease [IBD] were included. The TCRβ repertoire was analysed by next-generation sequencing of biopsy RNA. Both in Crohn's disease patients and in non-IBD controls, a broad intestinal T-cell repertoire was found, with a considerable part consisting of expanded clones. Clones in Crohn's disease were more expanded [p = 0.008], with the largest clones representing up to as much as 58% of the total repertoire. There was a substantial overlap of the repertoire between inflamed and uninflamed tissue and between ileum and colon. Following therapy, responders showed larger changes in the T-cell repertoire than non-responders, although a considerable part of the repertoire remained unchanged in both groups. The intestinal T-cell repertoire distribution in Crohn's disease is different from that in the normal gut, containing profoundly expanded T-cell clones that take up a large part of the repertoire. The T-cell repertoire is fairly stable regardless of endoscopic mucosal inflammation or response to therapy

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 (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; pCONCLUSIONS: 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.

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) (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.

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 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).

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., Author Summary Several viruses have found ways to evade the human immune system and cause latent infections. Examples include HIV and herpes-viruses. Most humans carry these herpes-viruses. The human immune system mounts continuous responses against these viruses to prevent them from causing disease. If this balance is disturbed, these viruses can cause extensive pathology. We do not know how the immune response against these viruses evolves over time. Understanding this response might help to understand why the immune system does not clear these viruses and might help in preventive and therapeutic strategies. Here we used a new technology that allowed us to track virus specific immune cells (CD8+ T cells) over time in a quantitative manner. When we used this technology to study the evolution of latent responses against herpes-viruses (from infection until 5 years later) we found that immune responses were very rigid and did not evolve over time. Collectively our data shows that – for these herpes-viruses – the initial immune response is maintained despite the fact that this does not result in clearance of the virus. Therefore, if a virus survives the initial response, it will not be cleared in the future. This is an important consideration in understanding latent infection and for vaccination-design.

Every person carries a vast repertoire of CD4+ T-helper cells and CD8+ cytotoxic T cells for a healthy immune system. Somatic VDJ recombination at genomic loci that encode the T-cell receptor (TCR) is a key step during T-cell development, but how a single T cell commits to become either CD4+ or CD8+ is poorly understood. To evaluate the influence of TCR sequence variation on CD4+/CD8+ lineage commitment, we sequenced rearranged TCRs for both α and β chains in naïve T cells isolated from healthy donors and investigated gene segment usage and recombination patterns in CD4+ and CD8+ T-cell subsets. Our data demonstrate that most V and J gene segments are strongly biased in the naïve CD4+ and CD8+ subsets with some segments increasing the odds of being CD4+ (or CD8+) up to five-fold. These V and J gene associations are highly reproducible across individuals and independent of classical HLA genotype, explaining ~11% of the observed variance in the CD4+ vs. CD8+ propensity. In addition, we identified a strong independent association of the electrostatic charge of the complementarity determining region 3 (CDR3) in both α and β chains, where a positively charged CDR3 is associated with CD4+ lineage and a negatively charged CDR3 with CD8+ lineage. Our findings suggest that somatic variation in different parts of the TCR influences T-cell lineage commitment in a predominantly additive fashion. This notion can help delineate how certain structural features of the TCR-peptide-HLA complex influence thymic selection.

Background Several lines of evidence support a role for the T cell receptor (TCR) in the pathogenesis of RA. Previously we demonstrated that many T cell clones are specifically present in the synovial tissue (ST) of patients with established RA (ESRA) suggesting TCR-driven involvement of T cells. To further unravel TCR-involvement in the development of RA we investigated the earliest stages of synovial inflammation, as defined clinically. We hypothesised that the synovial inflammation in RA leads to epitope spreading, reflected by accumulation of new clonal T cell expansions. Therefore, ST in early RA (ERA) should yield less, but more expanded T cell clones. Objective To compare the presence of T cell clones in ST and peripheral blood (PB) between patients with ERA and ESRA, using a new high-throughput sequencing protocol. Material and methods Three DMARD-naive ERA patients (disease duration 2 years) were included. All fulfilled the American College of Rheumatology1987 criteria for RA. mRNA was isolated from paired ST and PB samples. A linear amplification with primers for all V(ariable)-genes of the T cell receptor β-chain was performed. The amplified products contain the CDR3s of all T cells. Samples were processed using a Genome Sequencer (454) analysing up to 15 000 receptors per sample, The frequency of each clone was determined by its CDR3 frequency (% of all CDR3s analysed). Clones with a CDR3 frequency of >1% were arbitrarily considered as highly expanded clones (HECs). Results T cell clones were found with a frequency of up to 10–20% in the ST of ERA patients while this was only 1–3% in ESRA patients (samples had equal numbers of T cells). Only a minority of the HECs could also be identified in PB, and always in low frequencies. Surprisingly, we identified twice as many HECs in ERA-ST compared to ESRA-ST (39.5 clones (± 5.8) (SEM) vs 18.8 (± 3.6)). However, the impact of the HECs in ERA was far higher in ERA-ST. In ESRA only 17.3% (± 5.0) of the T cells analysed belonged to the HECs, whereas this was 77% (± 8.0) for the ERA samples. Conclusion The TCR-repertoire in ST of ERA patients appears to be dominated by a few HECs (oligoclonal), in contrast to observations in ESRA (polyclonal). This is consistent with the notion of epitope spreading. Further research should therefore focus on the earliest phases of RA to unravel the role of T cells in the initiation of RA.

Background B cells may play an important role in the pathogenesis of systemic sclerosis (SSc), as suggested by reports in the effectiveness of rituximab (RTX). To find support for a role of clonal B cell activation in SSc we studied the presence of clonal B cell expansions in the involved skin using newly developed next generation sequencing protocols, which provide quantitative data on up to 1 000 000 clones in one run. Objective investigate whether expanded B cell clones are present in involved and uninvolved skin of SSc patients, and how they are influenced by RTX therapy. Methods mRNA was isolated from involved and uninvolved skin samples of 3 SSc patients (with active diffuse disease) taken before and after 12 months after RTX-therapy. A linear amplification with multiplex primers for all V(ariable)-families of the heavy-chain (BCR) was performed. The amplified products contain the CDR3 of the BCRs of all cells which can be used as ‘clonal fingerprint’ for each cell. The samples were analysed using a Genome Sequencer FLX (454). The frequency of each clone was determined by its CDR3 frequency (% of all CDR3s analysed). Clones with a frequency of >1% were arbitrarily considered as highly expanded clones (HECs). Results Both involved and uninvolved skin showed similar amounts of HECs at baseline (14.3±4.2 vs 15.0±0.0 respectively; p = 0.9). However, at baseline BCR repertoires found in involved skin were clearly different from those in uninvolved skin; BCR clonal sequences were not shared between both compartments. Strikingly, RTX treatment led to a non-significant decrease of the number of HECs only in the involved skin (14.3±4.2 vs 9.0±4.2; p = 0.22) before and after RTX respectively). The uninvolved skin did not show a decrease in HECs at all. Comparing the HECs in the involved skin before and after RTX, hardly any overlap could be detected between both time points. Similar results were found in the uninvolved skin samples. Conclusions This high-resolution technique provides the first detailed characterisation of the BCR repertoire in SSc. The number of highly expanded clones (HECs) was not different when comparing involved versus uninvolved skin. This suggests that here is no BCR driven clonal expansion in involved skin. RTX treatment had a marginal effect on number of B cell HECs in involved skin, but no effect on the number of HECs in uninvolved skin.

Background T-cells play a key role in psoriatic arthritis (PsA). Nevertheless, the distribution and exact role of these cells remains unclear. Here, we study whether expanded T-cell clones are present in synovial tissue (ST), and whether such clones are also detectable in peripheral blood (PB) and synovial fluid (SF). Methods ST and SF from inflamed knees was sampled in 2 PsA patients together with paired PB samples. Using next generation sequencing T-cell clones were identified by their unique T-cell receptor β-chain sequence. Clones with a frequency of ≥0.5% were arbitrarily considered to be expanded. Results ST and SF samples from the same joint shared many of the expanded clones (see Table 1). Ninety percent (median, range 71-100%) of the expanded clones in ST were retrieved in SF, 33% of these being expanded in both samples. ST clones accounted for 14% of the SF T-cell repertoire. In PB 83% of the expanded ST clones were retrieved, 17% being expanded in both samples. Analysis of both inflamed knees in patient 1 showed that 83% of the expanded ST clones in the left knee were retrieved in the right knee, 80% of these being expanded in both. Conclusions In PsA synovial tissue harbors expanded T-cell clones, which are not fully represented in synovial fluid or blood samples. Preliminary analysis indicates that different joints contain identical expanded T cell clones. Our results suggest that in PsA comprehensive analysis of inflammation-related expanded clones best focuses on ST, rather than PB and SF. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4456

Background Autoreactive B-lymphocytes are thought to play an important role in rheumatoid arthritis (RA). B-cell depletion therapy by rituximab (RTX) has shown that targeting the humoral immune response can result in clinical improvement. Unfortunately, the depletion is transient and might result in disease relapse. Hence, analysis of the B-cell/plasma cell compartment in synovium (ST) in patients undergoing RTX therapy might help to identify autoreactive cells responsible for disease persistence and relapse. Objectives To compare the B-cell/plasma cell repertoire in ST at baseline, 4 weeks and 16 weeks after RTX treatment using a newly developed next-generation sequencing protocol. Methods Eleven RA patients were included and treated with two intravenous infusions of 1000mg RTX without additional methylprednisolone. At baseline, all patients fulfilled ACR criteria for RA, were ACPA+ and had a DAS28score >3.2. At week 24, nine patients demonstrated moderate to good EULAR responses. mRNA was isolated from consecutive samples and full-repertoire analysis of the immunoglobulin heavy-chain was performed with primers for all V(ariable)-genes. All amplified products encode the CDR3, a unique sequence that defines a unique clone. The number of sequences reflects the amount of immunoglobulins produced by that clone and can be used as a measure for “dominance” of that particular clone. The samples were analyzed using GS-FLX/454 and custom bioinformatics algorithms (>10,000 sequences/sample). Clones with a frequency of >0,5% were arbitrarily considered as dominant clones. Results Synovial clones were detected in all patients in equal numbers at baseline and 4 weeks after RTX. However, after 16 weeks the number of dominant clones significantly increased compared to baseline (mean +55,3%, p=0.04). In line, there was a significant decrease in the number of non-dominant clones compared to baseline (mean -47.7%, p=0.03). In 5 patients 17,9% of the dominant clones at baseline was detectable after 16 weeks (mean, SD 8,0%), half of these clones were dominant at both time points (mean 8,8%, SD 4,6%). In the remaining 6 patients dominant clones at baseline were not at all retrieved after 16 weeks. Interestingly, the latter group showed better treatment responses determined by a decrease in DAS28score (-0.84 (SD 0.48) and -2.1 (SD 0.88) DAS28 points resp., p=0.01). Conclusions Our observations suggest a disruption of dominant clones and a repopulation of distinctly different clones in synovium 16 weeks after rituximab. The persistence of dominant clones was associated with decreased treatment response. Further study of persisting dominant clones, especially during disease relapse, might help to identify and characterize disease-associated B-cells and/or plasma cells. Disclosure of Interest None Declared

Background B cells are thought to play an important role in the pathogenesis of rheumatoid arthritis (RA). We previously observed that highly expanded B-cell clones (HECs) were specifically present in inflamed synovial tissue (ST) compared to peripheral blood (PB) in a patient with established RA (ESRA). These findings suggest involvement of B cells via their B cell receptor (BCR), and local retention and/or proliferation of B cell clones in ST. Here we extend our patient group and include patients with early synovitis (ERA). Objective To analyse the presence of expanded B-cell clones in paired samples from PB and ST during different stages of disease using a newly developed High-Throughput Sequencing (HTS) protocol. Methods Six ACPA+ patients with active RA were included. Three patients were disease-modifying antirheumatic drug naive and had synovitis for less than 1 year (ERA). Three patients had active disease despite methotrexate treatment and had a disease duration of more than 10 years (ESRA). mRNA was isolated from paired samples (PB and ST biopsies from arthritic ankle/knee), and linear amplification of the B-cell receptor heavy chain was performed with primers for all V-gene families. The amplified products encode the CDR3 of all analysed B cells, used as a ‘fingerprint’ for each clone. The samples were analysed using a Genome Sequencer (454). The frequency of clones was determined by custom bioinformatics algorithms identifying the CDR3 of each receptor (up to 10.000 receptors per sample). Clones with a frequency of ≤1% were arbitrarily considered as HECs. Results HECs were detected in ST of both ERA and ESRA patients, with comparable frequencies in both stages (9 vs 11 clones resp.). Frequencies of HECs were as high as 53% in ERA, while this was 34% in ESRA. These HECs were either absent, or present in very low frequency in PB-samples. Surprisingly, HECs were also found in PB-samples of both groups (up to 30% in both groups), but the HECs recovered in PB were not found in ST. Conclusion This is the first analysis of B cell clones in ST- and PB-samples comparing RA patients with different disease duration, using novel HTS technology. Our data suggests that ST-specific HECs can already be detected in early stages of synovial inflammation, and that the number of HECs does not change during disease. These findings suggest that specific BCRs are involved in the early phase of RA and provide a rationale for early anti-B cell therapy.