101 B-cell repopulation after rituximab in rheumatoid arthritis Figure 1 | Peripheral blood BCR repertoire in patients undergoing rituximab treatment Boxplots showing (A) clonal expansion: Gini index, (B) clonal diversity: Shannon index and (C) the average IGHV gene mutation load in samples obtained before (M0), and at one (M1), three (M3), six (M6) and twelve (M12) months after treatment with rituximab. Boxplots show the median, 25% and 75% interquartile range, error bars show the range, and single data points are depicted in grey (*p ≤ 0.05, ***p ≤ 0.001, using one-way ANOVA). Defining depletion and repopulation timepoint The trend to re-establish pre-treatment conditions, observed in the peripheral blood BCR repertoire 6 months post-treatment, might reflect the restoration of the B-cell compartment after B-cell depletion. It has been reported that repopulation of B cells after depletion starts with antigen naïve B cells with reduced somatic hypermutation in the IGHV genes [15,16]. We therefore speculated that the percentage of unmutated BCR clonotypes in the repertoire could be used as a proxy to monitor the fraction of naïve B cells in patients undergoing rituximab therapy. The percentage of unmutated BCR sequences in the total repertoire significantly decreased one month after treatment compared to baseline (p ≤ 0.001; 4.33 ± 3.98 vs. 40.0 ± 17.8, mean ± SD, figure 2A). This decrease was at its lowest at month 3, and persisted up to month 12. Of note, when we excluded re-treated patients, the percentage of unmutated BCRs at 12 months did not differ significantly from that at baseline (online supplementary figure S2D). From month 6 onwards we observed an increase in the percentage of unmutated BCR sequences, which at month 12 was significantly different compared to the dip at month 3 (p ≤ 0.01; 23.1 ± 26.5 vs. 2.54 ± 3.38, mean ± SD). Overall, it was clear that the observed decrease and increase in the percentage of unmutated BCRs in the repertoire was patient specific. To address this variability, we defined for each patient a post-depletion and a post-repopulation timepoint as the first timepoint at which the percentage of unmutated BCR in the repertoire respectively drops below and increases again above 4.4% 5
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