table_1.XLSX

<p>Previous studies revealed high incidence of acquired N-glycosylation sites acquired N-glycosylation sites in RNA transcripts encoding immunoglobulin heavy variable region (IGHV) 3 genes from parotid glands of primary Sjögren’s syndrome (pSS) patients. In this study, next generation sequencing was used to study the extent of ac-Nglycs among clonally expanded cells from all IGVH families in the salivary glands of pSS patients. RNA was isolated from parotid gland biopsies of five pSS patients and five non-pSS sicca controls. IGHV sequences covering all functional IGHV genes were amplified, sequenced, and analyzed. Each biopsy recovered 1,800–4,000 unique IGHV sequences. No difference in IGHV gene usage was observed between pSS and non-pSS sequences. Clonally related sequences with more than 0.3% of the total number of sequences per patient were referred to as dominant clone. Overall, 70 dominant clones were found in pSS biopsies, compared to 15 in non-pSS. No difference in percentage mutation in dominant clone-derived IGHV sequences was seen between pSS and non-pSS. In pSS, no evidence for antigen-driven selection in dominant clones was found. We observed a significantly higher amount of ac-Nglycs among pSS dominant clone-derived sequences compared to non-pSS. Ac-Nglycs were, however, not restricted to dominant clones or IGHV gene. Most ac-Nglycs were detected in the framework 3 region. No stereotypic rheumatoid factor rearrangements were found in dominant clones. Lineage tree analysis showed in four pSS patients, but not in non-pSS, the presence of the germline sequence from a dominant clone. Presence of germline sequence and mutated IGHV sequences in the same dominant clone provide evidence that this clone originated from a naïve B-cell recruited into the parotid gland to expand and differentiate locally into plasma cells. The increased presence of ac-Nglycs in IGHV sequences, due to somatic hypermutation, might provide B-cells an escape mechanism to survive during immune response. We speculate that glycosylation of the B-cell receptor makes the cell sensitive to environmental lectin signals to contribute to aberrant B-cell selection in pSS parotid glands.</p

Impact of sequence variation of the TCR α chain on CD4⁺/CD8⁺ propensity.

<p>We analyzed 19,501 and 28,572 unique TCR α transcripts of naïve CD4⁺ and CD8⁺ T cells, respectively, from 5 healthy donors. The odds ratio (OR) is plotted for (<b>A</b>) each Vα gene segment and (<b>B</b>) each Jα gene segment, with OR < 1 indicating a propensity towards CD8⁺ and OR > 1 indicating a propensity towards CD4⁺. Total number of observations for each gene is listed. Significant associations after Bonferroni correction are denoted with an asterisk. (<b>C</b>) Odds ratios were computed for TCRs as a function of the calculated CDR3α net charge (error bars reflect 95% confidence intervals). A histogram of the number of observations is also plotted. Negative charge increases propensity of T cell towards CD8⁺ whereas positive charge increases propensity of T cell towards CD4⁺.</p

Percentage of CD4+/CD8+ propensity explained by V-genes, J-genes and CDR3-net charge².

<p>¹Calculated as the difference between the Nagelkerke R² of the null regression model (which only accounts for individual-specific effects) and the Nagelkerke R² of an alternative model (which also includes the contribution of V and J genes, CDR3 net charge and/or CDR3 length).</p><p>² Variation between different donors is shown in Table K in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140815#pone.0140815.s001" target="_blank">S1 File</a>.</p><p>Percentage of CD4+/CD8+ propensity explained by V-genes, J-genes and CDR3-net charge<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140815#t001fn002" target="_blank">²</a>.</p

Association results and SNP annotations in the <i>1q23 CD84</i> locus.

<p>A) Regional association plots with ΔDAS (top panel) and with <i>CD84</i> expression (bottom panel), showing strengths of association (−Log10 P-value) versus position (Kb) along chromosome 1. B) Schematic of <i>CD84</i> gene structure (RefSeq gene model, box exons connected by diagonal lines, arrow indicates direction of transcription) with strong enhancer chromatin states (orange rectangles) and SNPs in high LD (r2>0.8) with rs6427528 (vertical ticks). SNPs in enhancers are labeled below. C) Annotations of strong-enhancer rs6427528 proxy SNPs; listed are SNP rs-ID (major and minor alleles), conservation score, cell line with DNAse footprint if present, and transcription factor binding sites altered. 1- Genomic evolutionary rate profiling (GERP) conservation score, where a score >2 indicates conservation across mammals. 2- DNase footprint data are compiled from publicly available experiments by HaploReg. 3- Position weight matrix logos show transcription factor consensus binding sites with nucleotide bases proportional to binding importance. SNP position is boxed. Note that the rs10797077 AIRE_2 and the rs6427528 SREBP_4 motifs are on the minus strand (base complements correspond to SNP alleles), with the SREBP motif shown upside down to align with the rs6427528 KROX motif on the positive strand. Data are from HaploReg.</p

<i>CD84</i> expression level and clinical features.

<p>Analyses are shown in RA patients from the BRASS and ABCoN registries, for baseline DAS (top panel, n = 210; R² = 0.02, p = 0.02) and ΔDAS (bottom panel, n = 31; R² = 0.001, p = 0.46). Best-fit linear regression lines are shown in black, with shaded regions showing linear regression model (slope and intercept) 95% confidence intervals. <i>CD84</i> expression levels were quantile normalized, and ΔDAS values were adjusted for age, gender and baseline DAS.</p

GWAS results for the ΔDAS phenotype.

<p>Shown are strengths of association (−Log10 P-value) for each SNP versus position along chromosomes 1 to 22. A) All samples (n = 2,706). B) Etanercept-treated patients (n = 733). C) Infliximab-treated patients (n = 894). D) Adalimumab-treated patients (n = 1,071).</p