164 APPENDICES Furthermore, the predictive value of early depletion and early repopulation on clinical response was assessed to gain more insight into how this correlates with clinical response. We showed that B-cell deletion using rituximab and recurrence of B-cells carrying unmutated BCRs proved to be a sensitive marker for depletion and repopulation. Using the percentage of unmutated BCR clones, as a proxy for the fraction of naive B-cells, we observed that timing of depletion and repopulation does not predict response after 6 or 12 months. However, repopulation within the first 6 months did significantly correlate with decrease in disease activity in the subsequent period, in comparison with patients who achieved repopulation later or not at all. This might indicate that it is in fact the repopulation following rituximab – rather than depletion itself – that is able to “reset” the (pathological) B-cell compartment, leading to temporal improvement of the disease activity. Importantly, early repopulation of the B-cell compartment did not predict treatment outcome at 6 or 12 months, but was associated with improvement of disease activity shortly after repopulation. Moving forward on this, we focused on B-cells in individuals at-risk of developing RA (Chapter 6). We investigated changes in the BCR repertoire over time and the effects of a single dose of rituximab in at-risk individuals. For this, we used unique data from a randomized controlled trial, “Prevention of RA by Rituximab” (PRAIRI) study, and combined this with data from a longitudinal cohort, the “DOMINant clones in the Onset of RA” (DOMINO) study. In addition, a phenotypic analysis of B lineage cells was performed in a similar cohort of at-risk-individuals. The earlier-described PRAIRI study showed that rituximab treatment in the preclinical phase delayed the onset of RA. The current data demonstrated that the BCR repertoire of rituximab treated RA-risk individuals is fundamentally altered due to effective B-cell depletion for up to 6 months after a single infusion of rituximab. This is underlined by a decrease in the total number of clones, an increase in number and impact of dominant clones, a decrease in the diversity of the BCR repertoire and a subsequent increase of unmutated BCR clones over time, suggesting re-establishment of the BCR repertoire after B-cell repopulation. This re-establishment of the repertoire is best visible between 6 and 12 months post-rituximab-infusion. In the DOMINO study, no BCR repertoire related parameters, that would potentially associate with RA development, could be distinguished in the peripheral blood. The BCR repertoire turned out to be very unstable over time, with dominant clones continuously changing from timepoint to timepoint. Furthermore, no differences in dominant BCR clones could be found when comparing individuals who eventually developed RA versus individuals who did not progress towards the disease. However, we did find in the phenotypical analysis that once dominant clones, at screening or baseline visit, disappeared over time, they seemed to “switch” to a lower frequency with a memory B-cell phenotype.
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