Rebecca E. E. Esveldt

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.

Clonal Evolution of CD8+ T Cell Responses against Latent Viruses: Relationship among Phenotype, Localization, and Function

ABSTRACT 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.

Somatic Variation of T-Cell Receptor Genes Strongly Associate with HLA Class Restriction

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.

Profoundly Expanded T-cell Clones in the Inflamed and Uninflamed Intestine of Patients With Crohn's Disease

Background and Aim T cells are key players in the chronic intestinal inflammation that characterises Crohns disease. Here we aim to map the intestinal T-cell receptor [TCR] repertoire in patients with Crohns disease, using next-generation sequencing technology to examine the clonality of the T-cell compartment in relation to mucosal inflammation and response to therapy. Methods Biopsies were taken from endoscopically inflamed and uninflamed ileum and colon of 19 patients with Crohns 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. Results Both in Crohns 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 Crohns 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. Conclusions The intestinal T-cell repertoire distribution in Crohns 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.

Computational Model Reveals Limited Correlation between Germinal Center B-Cell Subclone Abundancy and Affinity: Implications for Repertoire Sequencing

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.

AB0021 In Psoriatic Arthritis Synovial Tissue Harbors Expanded T-Cell Clones Which Are not Fully Represented in Synovial Fluid or Blood Samples

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. Table 1 Patient 1 Patient 2 PB Right Knee Left Knee PB Knee Sample PB ST SF ST SF PB ST SF Number of reads 10515 7845 6789 7144 7575 7940 9691 8065 Number of clones 6151 2686 2877 3002 2493 3716 2105 2225 Expanded (#) 8 10 10 6 14 7 7 18 Number of expanded clones also present in:  SF 9 6 5  Expanded in SF 3 3 2  PB 6 9 5 13 6 8  Expanded in PB 1 4 1 4 1 0 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

In Rheumatoid Arthritis, Synovitis at Different Inflammatory Sites Is Dominated by Shared but Patient-Specific T Cell Clones

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.

FRI0303 The IGG4:IGG RNA ratio is a new and promising disease activity marker in granulomatosis with polyangiitis

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 ratio1. 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<0.01), 2.5 in RA (0.7 - 4.1; p<0.05), 1.6 in PSC (1.0 - 2.8; p<0.001) and 1.3 in HC (0.6 - 1.8, p<0.01). In addition, the median IgG4:IgG RNA ratio was significantly higher in patients with active disease (23.8; IQR 12.1 – 29.1) compared to patients in remission (3.5; IQR 2.0 – 5.5) (p<0.0001). The height of the IgG4:IgG RNA ratio significantly correlated with height of the BVAS (r2 =0.76, p<0.0001), while the ESR, CRP and ANCA titre did not. Interestingly, IgG4/IgG RNA ratios among patients with active disease were consistently above 9.3, and among patients in remission they were below this threshold. 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