127 B-cells during preclinical phase of rheumatoid arthritis group are not likely to be influenced by the vaccination(s) or methylprednisolone they received prior to follow-up visits. In order to investigate the stability of the BCRh repertoire during the at-risk phase, we tracked dominant clones over time, both in the PRAIRI-placebo-group and in the untreated-group. We calculated the mean number of dominant clones in these groups at the screening visit and determined its standard deviation (i.e. 5). Within each subject, increases or decreases in the number of dominant clones equal or larger than 5 (1 SD) were scored as relevant changes over time. In the PRAIRI-placebo group, 30% of individuals showed a relevant change in the number of dominant clones while 60% of individuals in the untreated group showed a relevant change between the screening and baseline visits (Figure 3E-F). The observed differences in the PRAIRI study occurred largely between screening and baseline visit and may be explained by the vaccinations and/or MPNS treatment given after screening. Additionally, using the screening visits as control, we analysed to what extent the 25 most dominant (top 25) BCRh clones were also present in follow-up visits. Regarding the placebo-group, a prolonged detectability of certain initial clones could be seen in follow-up visits, which significantly decreased over time (Figure 3G). Up to the arthritis visit, a mean of 3.1% (SD 4.6) of the top 25 clones from the screening visit were still detectable. In the arthritis visit of RA-developers (N=2) of the untreated RA-risk group, a mean of 2.0 % (SD 2.9) of the top 25 clones from the screening visit were still detectable. After 12 months, on average 2.3% (SD 3.9) of the initial top 25 clones were still detectable as top 25 clones (Figure 3H). These results suggest that the majority of the most expanded BCRh clones are replaced by new clones or that the frequency of these clones decreases overtime. These findings indicate that increases in number of dominant clones can occur without any intervention, suggesting a behavioural pattern in which dominant clones are either not persistently present or may rise/decrease in frequency over time. Dividing the placebo-group into individuals who either did not (n=22; 61%) or did develop arthritis (n=14, 39%), we did not see any significant differences in terms of the stability of dominant BCR clones during the at-risk phase (data not shown). 6
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