Characterization of peripheral and lesional single B cell autoreactivity in patients with Sjögren’s syndrome and rheumatoid arthritis

PhDSjögren’s syndrome (SS) and rheumatoid arthritis (RA) are characterised by breach of self-tolerance with high affinity circulating autoantibodies and peripheral B cell disturbances in the naïve and memory B cell compartments. In addition, both SS and RA develop functional ectopic B cell follicles in the respective target organs, i.e. the salivary glands and the joint synovium, whereby autoreactive B cell undergo antigen selection and affinity maturation. However, the exact stage at which errors in B cell tolerance checkpoints accumulate is unknown. In this PhD project, I amplified and sequenced Ig VH and VL gene transcripts from single B cells which were FACS sorted either from the peripheral blood of SS patients or from the RA synovium. Healthy donors (HD) were used as controls. Subsequently, I cloned and expressed recombinant monoclonal antibodies displaying identical antigenic specificity of the original B cells. Finally, I tested the poly- and autoreactivity profile of these antibodies against SS and RA-associated autoantigens. In SS, I analysed 353 VH and 293 VL sequences and obtained 114 recombinant antibodies from circulating naïve (n=66) and memory (n=48) B cells of 4 SS patients and compared their autoreactive and polyreactive profile to 45 naïve clones from 2 HD. Analysis of the VH and VL gene usage showed no significant differences between SS and HD. Conversely, I observed accumulation of circulating autoreactive naïve B cells in SS as demonstrated by Hep-2 cells, ENA, Ro/SSA and/or La/SSB reactivity. The elevated frequency of autoreactive naïve B cells in the circulation of SS patients supports the existence of early defects in B cell tolerance checkpoints in this condition In RA, I analysed the Ig gene repertoire and the VH gene somatic mutation rate of 139 VH and 175 VL sequences of synovial CD19+ B cells which demonstrated evidence of antigen selection and hypermutated alpha>gamma>mu VH chains with presence of intra-synovial clonal diversification. Recombinant antibodies from synovial B cell clones were then screened for reactivity towards citrullinated antigens with a plan for a wider analysis using autoantigen microarrays. Overall, these results highlighted the existence of B cell abnormalities and loss of tolerance for self-antigens both in the peripheral and/or lesional compartment of SS and RA. Further analysis of the fine specificity and pathogenicity of recombinant antibodies from autoreactive B cells will be invaluable in order to dissect the mechanisms and the antigens driving the development and the persistence of autoimmunity in RA and SS

Characterization of a Synovial B Cell-Derived Recombinant Monoclonal Antibody Targeting Stromal Calreticulin in the Rheumatoid Joints

Research grant from Arthritis Research UK (Grant 20858 to E.C.) and a short-term travel fellowship from the European Academy of Allergy and Clinical Immunology (to E.C.

Accumulation of Self-Reactive Naive and Memory B Cell Reveals Sequential Defects in B Cell Tolerance Checkpoints in Sjogren's Syndrome

This work was funded by grants number 18237 and 20089 from Arthritis Research UK (http://www.arthritisresearchuk.org) to MB and the William Harvey Research Foundation. EC was recipient of short-term travel fellowships from EMBO (ASTF 318-2010) and EFIS-IL

Hep-2 cell IFA self-reactivity of naïve and memory B cell antibodies from SS patients and HD.

<p>Naïve and memory B cell antibodies from SS patients and HD (naïve B cells) were tested for self-reactivity by Hep-2 cell IFA assay. (<b>A</b>) Examples of cytoplasmic, cytoplasmic and nuclear, and nuclear Hep-2 cell staining pattern are shown. (<b>B</b>) Graph bars summarize the frequency of Hep-2 cell reactive antibodies with cytoplasmic, nuclear and cytoplasmic, nuclear reactivity, and non Hep-2 cell reactive antibodies in SS patients (naïve and memory B cell antibodies) and HD (naïve B cell antibodies). <i>P</i> value compares ANA+ (cytoplasmic only and nuclear ± cytoplasmic reactive) versus ANA– (non Hep-2 reactive) naïve clones from SS patients and HD. The percentage of self-reactivity for each population is reported over each graph. The number of tested antibodies is indicated below each graph.</p

Isolation strategy of single naïve B cells and comparison of the frequencies of naïve, memory switched and unswitched B cells in SS patients and HD.

<p>(<b>A</b>) PBMC from SS patients were surface labeled with fluorochrome-coupled anti-CD19, anti-CD3, anti-IgD and anti-CD27. CD19+CD3− cells were gated and analyzed for IgD and CD27 expression. (<b>B</b>) The frequencies of peripheral naïve (CD3−CD19+CD27−IgD+), memory switched (CD3−CD19+CD27+IgD–) and unswitched (CD3−CD19+CD27+IgD+) B cells among all CD19+ B cells are shown. Differences between patients and HD were statistically significant using the nonparametric Mann-Whitney U test (<i>p</i> value is reported over each graph). Error bars indicate standard error of the mean (SEM) for individual patient or control.</p

Polyreactivity of naïve and memory B cell antibodies from SS patients and HD.

<p>Naïve B cell antibodies from SS patients (n = 60), HD (n = 41) and memory B cells from SS patients (n = 39) were tested for reactivity with dsDNA (top left), ssDNA (top right), LPS (bottom left) and insulin (bottom right) by ELISA. Each graph shows the reactivity at a concentration of 1 µg/ml and it shows the result of two independent experiments. The cut-off OD (450 nm) at which antibodies were considered reactive is shown by the horizontal lines. Data points represent individual antibodies. Internal controls for polyreactivity (star dots) are shown in each graph and include mGO53 (negative; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114575#pone.0114575-Tiller1" target="_blank">[17]</a>), JB40 (low polyreactive; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114575#pone.0114575-Tiller1" target="_blank">[17]</a>), and ED38 (highly polyreactive; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114575#pone.0114575-Tiller1" target="_blank">[17]</a>).</p

Interference of antigen selection in memory B cells from SS patients.

<p>(<b>A</b>) Replacement:silent ratio (R/S ratios) were calculated for the complementary determining regions CDRs (black) and framework regions (white) for the heavy chain of both memory unswitched and switched B cells. A significant higher R/S ratio in the CDRs was observed in memory switched vs memory unswitched B cells. (<b>B</b>) The graphs show the ratio of replacement mutations in CDR1 and CDR2 (R_CDR) to the total number of mutations in V region (M_V) plotted against M_V for the memory unswitched and switched B cells from SS patients. The dark and the light grey area indicate the 90% and 95% confidence limits for the probability of random mutations, respectively. A data point outside these areas represents a sequence that was antigen selected. The data were obtained using the Immunoglobulin Analysis Tool software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114575#pone.0114575-Rogosch1" target="_blank">[22]</a>.</p

Ig gene analysis of naïve and memory B cells from SS patients and HD.

<p>Single naïve and memory unswitched and switched B cell antibodies from SS patients and HD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114575#pone.0114575-Mietzner1" target="_blank">[19]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114575#pone.0114575-Tiller2" target="_blank">[21]</a> were analyzed for Ig V_κ and J_κ (<b>A</b>) and Ig V_λ and J_λ (<b>B</b>) gene usage. The absolute number of sequences analyzed is reported over each graph. Error bars in bar graphs indicate standard error of mean (SEM) for individual patient or control.</p

SS patients analysed in this study.

<p>Demographic and clinical characteristics of the 12 patients with Sjögren’s syndrome (SS).</p>a<p>Patient SS7 is the same as patient SS13 but analysed at different time points.</p>b<p>pSS = primary Sjögren’s syndrome; sSS = secondary Sjögren’s syndrome; RA = rheumatoid arthritis.</p>c<p>HCQ = Hydroxychloroquine; MTX = Methotrexate; PDN = prednisolone; N = no treatment.</p><p>SS patients analysed in this study.</p