122 CHAPTER 6 individuals [9,10]. The final list of clones obtained from the RESEDA pipeline were analyzed with in-house developed R scripts using R version 4.1 using R studio [11,12]. The frequency of each clone was calculated as percentage of the total number of reads with unique UMIs obtained from sequencing of that sample. Clones with a frequency greater than or equal to 0.5% of the total repertoire were labelled dominant or highly expanded clones (HECs) [8]. The impact of a clone was calculated as its UMI-corrected frequency in the repertoire, and the impact of a group of clones as their cumulative frequency. Determination of B cell repopulation after depletion After depletion with rituximab, repopulation of B cells in RA patients starts with reappearance in peripheral blood of antigen-naïve B cells with reduced somatic hypermutation in the IGHV genes [13,14]. We decided to use the percentage of unmutated BCRh clones in the repertoire as a proxy to monitor the fraction of naïve B cells in RA-risk individuals undergoing RTX therapy. For each patient, the percentage of unmutated BCRs was determined at all visits. After depletion, repopulation was taken to occur at the time-point that the percentage of unmutated BCRs increased again above the cut-off of 2.9%. This cut-off was calculated as the mean unmutated BCR percentage at screening minus two times the standard deviation. Statistics Data and statistical analysis was done with R software (version 3.4.3) and Graphpad Prism software (version 9.0). Differences between groups were analysed using the Kruskall Wallis test after testing for normality. P values <0.05 were considered statistically significant. Results Patient characteristics Patient characteristics at the screening visit from the 74 included RA-risk individuals in the randomized controlled trial (PRAIRI, RTX-group and placebo-group), the 10 RA-risk individuals in the untreated DOMINO group and 9 healthy controls are shown in Table 1. All RA-risk individuals were double-positive for IgM-RF and ACPA. CRP and ESR levels were low and did not differ significantly between the RA-risk groups. From the 74 individuals in either of the intervention groups, 38 were randomized to receive RTX (RTX-group) and 36 were randomized to receive placebo (placebo-group). In total, 29 RA-risk individuals developed RA over time. 27 had received an intervention with RTX or placebo and 2 were untreated. From the 27 treated RA-developers, 13 had been randomized into the RTX-group.
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