124 CHAPTER 6 (0-1.94) vs 4.24 (0.52-10.95), p=0.15 (Supplementary figure 1D-F). When comparing the RA-risk individuals to the healthy controls, a non-significant trend towards a higher number and impact of dominant clones was observed in the RA-risk untreated group (p=0.07) compared to healthy controls (Supplementary figure 1D-1F). A single dose of rituximab induces a depletion in the BCRh repertoire Several studies have investigated the effects of rituximab on the B cell repertoire in RA [8,14]. However, it is unknown if and how rituximab influences the B cell repertoire during the pre-clinical phase of the disease. To this end, we investigated the effects of rituximab and compared the effects of the intervention with placebo treated at-risk-individuals. Six months after rituximab treatment, the number of BCRh clones was significantly lower while the number and impact of dominant BCRh clones was significantly higher when compared to the screening and baseline visits (p<0.0001, p=0.002, p=0.008 respectively) (Figure 1A-1C). Further analysis on follow-up time points suggests a shift towards re-establishment of the pre-treatment BCR repertoire after B cell repopulation: The number and impact of dominant clones at baseline, after twelve months and at onset of arthritis were highly comparable (Figure 1A-1C). To investigate qualitative changes in the BCRh repertoire after rituximab treatment, we used the screening visit as control to determine to what extent the 25 most dominant (top 25) BCRh clones at screening were present in follow-up visits before and after rituximab treatment. Our analysis showed that compared with the screening visit, there was a relatively high top 25 clonal overlap with the baseline visit. However, this overlap was lost in further follow-up visits post-RTX infusion, on 6 months, 12 months and eventual arthritis onset (Figure 1D). We observed a trend towards an increase in the number of unmutated BCRh clones which became significant 12 months after treatment when compared to the baseline and screening visits, suggesting an onset of B cell repopulation at 12 months after treatment (Figure 1E). However, at 6 months some patients already showed a strong increase in the number of unmutated clones, while others showed almost a complete lack of unmutated clones, pointing to substantial heterogeneity in timing of depletion and repopulation. Finally, a diversity analysis using Simpson’s index revealed that RTX resulted in a marked decrease in the diversity of the BCRh repertoires 6 months after treatment when compared to the baseline and screening visits (Figure 1F). Again, we observed
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