100 CHAPTER 5 normally distributed data or Mann-Whitney test and Kruskal-Wallis test followed by Dunn’s multiple comparisons post-test for not-normally distributed data. Contingency tables were evaluated with Fisher’s exact test. P-values less than 0.05 were considered statistically significant. Graphpad Prism 7 software (Graph Pad, San Diego, CA, USA) was used to perform the statistical tests. Results The impact of rituximab on the B cell receptor repertoire persists for up to one year It has been shown that 4 weeks after treatment with rituximab the peripheral blood BCR repertoire is characterized by more expanded and mutated BCR clones [3]. To confirm and extend these findings, we prospectively analyzed a cohort of 23 (see Methods section ‘Dealing with missing data’) rituximab-treated RA patients with one year follow-up after treatment. At one month after treatment, B cells were undetectable in all patients using classical cytometry. Using AIRR, at one month, clonal expansion was significantly higher (p < 0.001; 0.52 ± 0.08 vs. 0.27 ± 0.14, mean ± SD; figure 1A), while clonal diversity was significantly lower compared to baseline (p < 0.001; 4.75 ± 1.06 vs. 7.53 ± 0.49, mean ± SD; figure 1B). In addition, BCR clonotypes carried a significantly higher number of somatic hypermutations in the IGHV gene (p < 0.001; 16 ± 2 vs. 8 ± 3, mean ± SD; figure 1C). These data confirm that one month after treatment with rituximab the BCR repertoire is dominated by fewer, more expanded and more mutated BCR clones. Further analysis at the follow-up timepoints revealed a trend towards re-establishment of pre-treatment conditions from month 6 post-treatment onwards for all three analyzed indexes. Compared to month 1, the BCR repertoire at month 12 showed significantly higher clonal diversity (5.93 ± 1.20; p < 0.001) and less IGHV gene mutations (12 ± 5; p < 0.05). Of note, this effect was even more accentuated when excluding re-treated patients at month 6 (online supplementary figure S2). Taken together these results show that rituximab induces profound changes on the peripheral blood BCR clonality. In some patients these changes persist for up to 12 months after treatment while in others restoration of the pre-treatment conditions starts at month 6 after treatment.
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