45 Inflamed joints share dominant B-cell clones prior to the arthroscopy in order to avoid contamination of SF by hemorrhagic fluid. In all patients, peripheral blood was drawn at the time of the arthroscopy and/or arthrocentesis. Linear amplification and next-generation sequencing The protocol used for linear amplification and next- generation sequencing of the BCR-heavy chain (BCR-heavy) was based on previously described methods [8,11,14– 16]. Sequencing was performed on the MiSeq (Illumina). To identify and quantify separate BCR-clones more accurately unique molecular identifiers (UMI) were used [17]. A unique clone was identified by the unique sequence of the CDR3-region in combination with the use of the (V)ariable and (J)oining genes. Based on earlier studies BCR-clones with a frequency ≥ 0.5% were considered dominant, and therefore called Highly Expanded Clones (HECs) [8,18]. BCR repertoire raw fast data are available on the Sequence Read Archive with the accession number BioProject PRJNA822925. Bioinformatics and statistics Values are either expressed as mean or median depending on the presence of a (non)-normal distribution of the data. For each sample an equal number of reads (N=9,736) was randomly drawn from the acquired reads to standardize comparisons. The Chao- modified Sørensen index was used to test for similarity between different samples. This biodiversity index measures dispersion and gives a value between 0 and 1. Values near 0 indicate no overlap between two locations, while values close to 1 show that the two repertoires are identical [19–22]. Because somatic hypermutation causes a broader, and more diverse repertoire we decided to only use the CDR3-sequence for the similarity analysis of the top clones and total repertoire. Differences between groups were analyzed using the unpaired Mann– Whitney U test, paired t test, one-way ANOVA or Tukey’s multiple comparison test where appropriate. p Values < 0.05 (two-sided) were considered statistically significant. R (version 3.5.1), package SpadeR (version 0.1.1) and Graphpad Prism (version 8.0) were used to perform the analyses. Results Different locations within an inflamed joint share dominant BCR clones When performing an arthroscopy the knee joint can be divided into two anatomic locations, the suprapatellar (SP) and infrapatellar (IP) region. For T-cells we know that synovial tissue biopsies from different locations within the same joint show substantial clonal overlap [11]. However, for B-cells this is still unknown. In this study 3
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